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LANZKOWSKY’S MANUAL OF PEDIATRIC HEMATOLOGY AND ONCOLOGY SEVENTH EDITION This page intentionally left blank LANZKOWSKY’S MANUAL OF PEDIATRIC HEMATOLOGY AND ONCOLOGY SEVENTH EDITION Managing Editor: JONATHAN D. FISH, MD Head, Stem Cell Transplantation and Cellular Therapy Medical Director, Survivors Facing Forward Program Division of Pediatric Hematology-Oncology and Stem Cell Transplantation Cohen Children’s Medical Center of New York, Northwell Health, New Hyde Park, New York Associate Professor of Pediatrics Zucker School of Medicine at Hofstra Northwell, Hempstead, New York Editors: JEFFREY M. LIPTON, MD, PHD Chief, Division of Pediatric Hematology-Oncology and Stem Cell Transplantation Cohen Children’s Medical Center of New York, Northwell Health, New Hyde Park, New York Frances and Thomas Gambino Professor of Hematology-Oncology Professor of Pediatrics and Molecular Medicine Professor, Institute of Molecular Medicine, Feinstein Institutes for Medical Research Zucker School of Medicine at Hofstra Northwell, Hempstead, New York PHILIP LANZKOWSKY, MBCHB, MD, SCD (HONORIS CAUSA), FRCP, DCH, FAAP Chief Emeritus, Division of Pediatric Hematology-Oncology and Stem Cell Transplantation Chairman Emeritus, Department of Pediatrics Executive Director and Chief-of-Staff (Retired) Cohen Children’s Medical Center of New York, Northwell Health, New Hyde Park, New York Vice President, Children’s Health Network (Retired), Professor of Pediatrics Zucker School of Medicine at Hofstra Northwell, Hempstead, New York Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2022 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-821671-2 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Stacy Masucci Acquisitions Editor: Patricia Osborn Editorial Project Manager: Susan Ikeda Production Project Manager: Maria Bernard Cover Designer: Mark Rogers Typeset by MPS Limited, Chennai, India Dedication This book is dedicated To our parents, Vicky and Larry Fish, Thelma and Al Lipton, and Abe and Lily Lanzkowsky who instilled in us the importance of integrity, the rewards of industry and the primacy of being a mensch. To our wives, Leah Fish, Linda Lipton and Rhona Lanzkowsky, who understand that the study of medicine is a lifelong and consuming process. To our children and grandchildren, our pride and joy. And To our patients, students, pediatric house staff, fellows in pediatric hematology and oncology, and to our colleagues who have taught us so much over the years. Today he can discover the errors of yesterday and tomorrow he may obtain new light on what he thinks himself sure of today Moses Maimónides This page intentionally left blank Contents List of contributors About the editors Preface to the seventh edition Preface to the sixth edition Preface to the fifth edition Preface to the fourth edition Preface to the third edition Preface to the second edition Preface to the first edition Introduction: Historic perspective (1955!2015) 3. Classification and diagnosis of anemia in children and neonates xiii xvii xxi xxiii xxv xxvii xxix xxxi xxxiii 1. Molecular and genomic methodologies for clinicians 37 OMAR NISS AND CHARLES T. QUINN Classification and diagnosis Neonatal anemia Further reading and references 37 40 59 4. Nutritional anemias 61 JACQUELYN M. POWERS xxxv 1 Overview Iron-deficiency anemia Megaloblastic anemia Further reading and references 61 61 71 80 5. Lymphadenopathy and diseases of the spleen 81 JORDAN A. SHAVIT AND RAJEN J. MODY PHILIP LANZKOWSKY Clinical molecular and genomic methodologies: goals 1 Methods of genetic analysis 1 Interpretation of genetic variants obtained from next-generation sequencing 4 Applications of next-generation sequencing to oncology 4 Interpreting and evaluating the results from clinical genetic testing 6 Further reading and references 6 Lymphadenopathy Diseases of the spleen Further reading and references 81 86 89 6. Bone marrow failure 91 2. Hematologic manifestations of systemic illness ADRIANNA VLACHOS, MICHELLE NASH AND JEFFREY M. LIPTON 7 Aplastic anemia Inherited bone marrow failure syndromes Further reading and references 91 102 122 7 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects 125 BRIAN M. DULMOVITS AND LAWRENCE C. WOLFE Alterations to red blood cells related to organ-specific pathologies Alterations to white blood cells related to organ-specific pathologies Alterations to platelets and coagulation related to organ-specific pathologies General considerations for the hematologic sequelae of infection Viral and bacterial illnesses associated with marked hematologic sequelae Parasitic illnesses associated with marked hematologic sequelae Hemolytic uremic syndrome Autoimmune disease Anemia of inflammation Nutritional deficiencies and environmental exposures Marrow infiltrative disorders Further reading and references LIONEL BLANC AND LAWRENCE C. WOLFE 12 13 15 15 21 22 23 25 27 28 34 Clinical features of hemolytic disease Laboratory findings Membrane defects Paroxysmal nocturnal hemoglobinuria Enzyme defects Further reading and references 125 126 128 138 142 148 8. Extracorpuscular hemolytic anemia 151 ANSHUL VAGRECHA AND LAWRENCE C. WOLFE Immune hemolytic anemia Nonimmune hemolytic anemia Further reading and references vii 151 158 159 viii 9. Hemoglobinopathies Contents 161 EUGENE KHANDROS AND JANET L. KWIATKOWSKI 14. Vascular anomalies 341 RACHEL KESSEL, FRANCINE BLEI AND IONELA IACOBAS Sickle cell disease Sickle cell trait (heterozygous form, AS) Hemoglobin C Hemoglobin E Unstable hemoglobins Thalassemias Further reading and references 161 179 179 180 180 180 191 10. Primary and secondary erythrocytosis 193 TSEWANG TASHI AND JOSEF T. PRCHAL Introduction Vascular tumors Simple vascular malformations Diagnostic work-up for vascular lesions Management Evaluation and monitoring of a vascular hepatic tumor Vascular anomaly syndromes Further reading and references 341 341 343 344 347 348 350 356 15. Histiocytic disorders 357 OLIVE S. ECKSTEIN AND CARL E. ALLEN Erythrocytosis or polycythemia Primary erythrocytosis Secondary erythrocytosis Diagnostic approach to erythrocytosis Further reading and references 193 195 197 203 204 11. Disorders of white blood cells 207 Introduction Langerhans cell histiocytosis Other histiocytic disorders Hemophagocytic lymphohistiocytosis (hemophagocytic syndromes) Further reading and references 371 374 KELLY WALKOVICH AND JAMES A. CONNELLY 16. Lymphoproliferative disorders 377 Leukocytosis Leukopenia Neutrophil disorders Monocytes disorders Eosinophil disorders Basophil disorders Lymphocyte disorders Dedication Further reading and references DAVID T. TEACHEY 12. Disorders of platelets 207 208 208 217 220 226 228 235 235 237 CATHERINE MCGUINN AND JAMES B. BUSSEL Thrombocytopenia in the newborn Inherited thrombocytopenias Immune thrombocytopenic purpura Other causes of thrombocytopenia Thrombocytosis Qualitative platelet disorders Inherited vascular and connective tissue disorders Nonthrombocytopenic purpura Laboratory evaluation of platelets and platelet function Further reading and references 13. Disorders of coagulation 237 248 257 265 270 272 280 281 282 284 287 SUSMITA N. SARANGI AND SUCHITRA S. ACHARYA Hemostatic disorders Acquired coagulation factor disorders Inherited coagulation factor disorders Thrombotic disorders Antithrombotic therapy Further reading and references 287 297 299 315 332 339 Angioimmunoblastic lymphadenopathy with dysproteinemia Small lymphocytic infiltrates of the orbit and conjunctiva (ocular adnexal lymphoid proliferation, pseudolymphoma, benign lymphoma, atypical lymphocytic infiltrates) Angiocentric immunolymphoproliferative disorders Castleman disease (angiofollicular lymph node hyperplasia, benign giant lymph node hyperplasia, angiomatous lymphoid hamartoma) Epstein!Barr virus-associated Lymphoproliferative disorders in immunocompromised individuals X-linked lymphoproliferative syndrome Autoimmune lymphoproliferative syndrome Lymphomatoid papulosis in children Further reading and references 17. Myelodysplastic syndromes and myeloproliferative disorders 357 357 369 377 378 378 379 380 385 386 389 390 391 INGA HOFMANN, NOBUKO HIJIYA AND MOHAMED TAREK ELGHETANY Myelodysplastic syndromes Myeloid proliferations in children with Down syndrome (DS) Juvenile myelomonocytic leukemia Myeloproliferative neoplasms Further reading and references 391 398 398 403 409 18. Acute lymphoblastic leukemia 413 PALLAVI M. PILLAI AND WILLIAM L. CARROLL Incidence of ALL Etiology 413 413 Contents Clinical features of ALL Diagnosis Classification Cytogenetics and molecular genetics of ALL Prognostic factors Treatment Infant leukemia Philadelphia-positive ALL Ph-like ALL Down syndrome and ALL Relapse in children with ALL Immunotherapy for ALL Central nervous system relapse Long-term effects of ALL therapy Future drugs in ALL therapy Further reading and references 414 416 417 420 421 422 429 430 431 431 431 433 434 436 436 437 19. Acute myeloid leukemia 439 ARLENE REDNER AND RACHEL KESSEL ix Staging Prognosis Management Non-Hodgkin Lymphoma Subtypes Further reading and references 476 476 477 479 482 22. Central nervous system tumors 485 DEREK HANSON AND MARK P. ATLAS Pathology Clinical manifestations Diagnostic evaluation Treatment Specific CNS tumors Genetic syndromes related to brain tumors Further reading and references 485 486 488 490 492 503 504 23. Neuroblastoma 507 JULIE KRYSTAL, ELIZABETH SOKOL AND ROCHELLE BAGATELL Incidence and epidemiology Etiology and predisposing conditions Clinical features Diagnostic and monitoring studies Classification of AML Supportive care The treatment of newly diagnosed AML Prognosis of newly diagnosed AML Relapsed and refractory AML Novel therapeutic approaches Acute promyelocytic leukemia AML special subgroups Further reading and references 439 439 440 441 442 447 448 450 450 451 454 456 457 20. Hodgkin lymphoma 459 CHRISTINE M. SMITH AND DEBRA L. FRIEDMAN Etiology and epidemiology Risk factors Biology Pathology Clinical presentation Diagnostic evaluation and staging Prognostic factors Treatment Follow-up evaluations Further reading and references Web resources 459 459 460 460 461 464 465 466 471 471 472 21. Non-Hodgkin lymphoma 473 MARY S. HUANG AND HOWARD J. WEINSTEIN Introduction Incidence and epidemiology Pathologic classification Clinical features Diagnosis 473 473 474 475 475 Epidemiology Predisposition Pathology and biology Clinical features Diagnosis and staging Treatment modalities Prognosis, risk stratification, and therapy Neuroblastoma in the adolescent and young adult Further reading and references 507 507 507 508 510 511 513 522 522 24. Renal tumors 525 ANNE B. WARWICK AND JEFFREY S. DOME Wilms tumor Nephroblastomatosis Congenital mesoblastic nephroma Clear cell sarcoma of the kidney Rhabdoid tumor of the kidney Renal cell carcinoma Further reading and references 525 538 538 539 539 539 540 25. Rhabdomyosarcoma and other soft-tissue sarcomas 541 SEEMA AMIN AND CAROLYN FEIN LEVY Incidence and epidemiology Pathologic and genetic classification Clinical features Diagnostic evaluation Staging Prognosis Treatment Follow-up after completion of therapy Recurrent disease Future perspectives Further reading and references 541 542 545 547 549 551 552 558 559 560 561 x 26. Malignant bone tumors Contents 563 KATRINA WINSNES AND NOAH FEDERMAN Osteosarcoma Ewing sarcoma family of tumors Other bone tumors Further reading and references 563 572 578 581 27. Retinoblastoma 583 AMISH SHAH AND ANN LEAHEY Incidence Classification Risk for second malignant neoplasms Pathology Clinical features Differential diagnosis Patterns of spread Diagnostic procedures Classification Treatment Treatment of intraocular RB Treatment of recurrent RB Treatment of extraocular RB Posttreatment management Further reading and references 583 583 585 586 586 587 587 588 588 589 590 594 594 594 595 28. Extracranial germ cell tumors 597 ADRIANA FONSECA AND THOMAS A. OLSON 30. Hematopoietic stem cell transplantation and cellular therapy 623 HISHAM ABDEL-AZIM AND MICHAEL A. PULSIPHER Allogeneic stem cell transplantation HSC sources, collection, and manipulation Graft manipulation postcollection Medical evaluation of HSC donors Pretransplantation preparative regimens (conditioning) Engraftment Complications of HSCT Recent advances in HSCT Outcomes Further reading and references 623 628 630 630 631 635 636 652 655 657 31. Management of oncologic emergencies 659 JASON L. FREEDMAN, CAITLIN W. ELGARTEN AND SUSAN R. RHEINGOLD Metabolic emergencies Cardiothoracic emergencies Abdominal emergencies Renal emergencies Neurologic emergencies Endocrine emergencies Treatment-associated emergencies Further reading and references 659 663 664 666 668 669 671 673 32. Supportive care of patients with cancer 675 ANURAG K. AGRAWAL AND JAMES FEUSNER Epidemiology Biology Histology Molecular characteristics Clinical features Diagnostic evaluation Staging and risk stratification Treatment Risk-stratified therapeutic approaches Relapsed and resistant germ cell tumors Further reading and references 597 597 597 598 599 600 601 601 607 610 610 29. Hepatic tumors 613 KATHRYN S. SUTTON AND THOMAS A. OLSON Incidence Epidemiology Pathology Clinical features Diagnostic evaluation Staging and risk stratification Treatment Further reading and references 613 613 615 615 615 617 618 620 Management of infectious complications Recognition and management of nausea and vomiting Mucositis Pain management Nutritional status of the oncology patient Utilization of hematopoietic growth factors Management of acute radiation side effects Management of CVCs Posttreatment immunizations Palliative care Further reading and references 675 686 688 690 696 698 700 703 708 709 710 33. Evaluation, investigations, and management of late effects of childhood cancer 713 MIRIAM RADINSKY AND JONATHAN D. FISH Models of survivorship care Interventions and screening guidelines Organ system!specific late effects Future considerations Further reading and references 713 714 716 733 733 Contents 34. Psychosocial factors impacting children with cancer and their families 735 AMY NADEL Time of diagnosis Treatment initiation Illness stabilization During relapses and recurrences Treatment outcomes: the unsuccessful course Treatment outcomes: the successful course Conclusion Further reading and references 735 740 742 743 744 746 747 748 35. Pediatric blood banking principles and transfusion medicine practices 749 MAHA AL-GHAFRY AND CASSANDRA D. JOSEPHSON Introduction Immunohematology 749 749 Donor recruitment and testing Blood collection and processing Blood products Blood component modification and administration Transfusion reactions Special populations Therapeutic apheresis Patient blood management Further reading and references Appendix 1: Hematological reference values Index xi 750 751 753 756 758 761 763 764 765 767 781 This page intentionally left blank List of contributors Hisham Abdel-Azim Transplantation and Cellular Therapy, Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA, United States William L. Carroll Department of Pediatrics, Division of Pediatric Hematology-Oncology, Hassenfeld Children’s Hospital at NYU Langone Health, Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY, United States Suchitra S. Acharya Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States James A. Connelly Pediatric Hematology/Oncology, Monroe Carell Jr Children’s Hospital, Vanderbilt University Medical Center, Nashville, TN, United States Anurag K. Agrawal Department of Pediatrics, Division of Oncology, UCSF Benioff Children’s Hospital, Oakland, CA, United States Jeffrey S. Dome Center for Cancer and Blood Disorders, Children’s National Hospital, Washington, DC, United States; Department of Pediatrics, School of Medicine & Health Sciences, George Washington University, Washington, DC, United States Maha Al-Ghafry Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Brian M. Dulmovits Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Carl E. Allen Pediatric Hematology-Oncology, Texas Children’s Hospital/Baylor College of Medicine, Houston, TX, United States; Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States Olive S. Eckstein Pediatric Hematology-Oncology, Texas Children’s Hospital/Baylor College of Medicine, Houston, TX, United States; Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States Seema Amin Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Mark P. Atlas Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Rochelle Bagatell Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Lionel Blanc Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States; Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, NY, United States Caitlin W. Elgarten Department of Pediatrics, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States; Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Mohamed Tarek Elghetany Departments of Pathology & Immunology and Pediatrics, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX, United States Noah Federman Department of Pediatrics, Hematology/ Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States; Department of Orthopaedics, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States; Department of Pediatric Bone and Soft Tissue Sarcoma Program, UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA, United States; Clinical & Translational Research Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States Francine Blei Vascular Anomalies Program, Lenox Hill Hospital, Northwell Health, New York, NY, United States Carolyn Fein Levy Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States James B. Bussel Division of Hematology-Oncology, Department of Pediatrics, Weill Cornell Medical College, New York, NY, United States James Feusner Department of Pediatrics, Division of Oncology, UCSF Benioff Children’s Hospital, Oakland, CA, United States xiii xiv List of contributors Jonathan D. Fish Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States Janet L. Kwiatkowski Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States Adriana Fonseca Pediatric Haematology Oncology, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Philip Lanzkowsky Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States Jason L. Freedman Department of Pediatrics, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States; Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Debra L. Friedman Division of Pediatric Hematology/ Oncology, Vanderbilt University Medical Center, Nashville, TN, United States; Vanderbilt-Ingram Cancer Center, Nashville, TN, United States Derek Hanson Neuro-oncology, Children’s Cancer Institute, Joseph M. Sanzari Children’s Hospital, Hackensack University Medical Center, Hackensack, NJ, United States; Assistant Professor of Pediatrics and Neurology, Hackensack Meridian School of Medicine at Seton Hall Nobuko Hijiya Pediatric Hematology/Oncology/Stem Cell Transplantation, Columbia University, Irving Medical Center, New York, NY, United States Inga Hofmann Pediatric Hematology/Oncology, University of Wisconsin, Madison, WI, United States Mary S. Huang Pediatric Hematology and Oncology, Mass General Hospital for Children, Boston, MA, United States Ionela Iacobas Texas Children’s Hospital Vascular Anomalies Center, Baylor College of Medicine, Houston, TX, United States Cassandra D. Josephson Department of Pathology and Laboratory Medcine, Center for Transfusion and Cellular Therapies, Emory University School of Medicine, Atlanta, GA, United States; Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, United States Rachel Kessel Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Eugene Khandros Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States Julie Krystal Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Ann Leahey Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, The University of Pennsylvania School of Medicine, Philadelphia, PA, United States Jeffrey M. Lipton Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States; Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, NY, United States Catherine McGuinn Division of Hematology-Oncology, Department of Pediatrics, Weill Cornell Medical College, New York, NY, United States Rajen J. Mody Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Michigan, C.S. Mott Children’s Hospital, Ann Arbor, MI, United States Amy Nadel Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Michelle Nash Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States; Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, NY, United States Omar Niss Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States Thomas A. Olson Pediatrics, Emory University School of Medicine, Atlanta, GA, United States; Pediatric Hematology/Oncology, Aflac Cancer & Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, GA, United States; School of Medicine, Emory University, Atlanta, GA, United States; Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, GA, United States Pallavi M. Pillai Department of Pediatrics, Division of Pediatric Hematology-Oncology, Mount Sinai Kravis Children’s Hospital, Icahn School of Medicine at Mount Sinai, New York, NY, United States List of contributors Jacquelyn M. Powers Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States; Iron Disorders and Nutritional Anemias Program, Texas Children’s Hospital, Houston, TX, United States Josef T. Prchal Division of Hematology and Hematologic Malignancies, University of Utah, Salt Lake City, UT, United States; Division of Hematology/Oncology, VA Medical Center, Salt Lake City, UT, United States; Huntsman Cancer Center, University of Utah, Salt Lake City, UT, United States Michael A. Pulsipher Transplantation and Cellular Therapy, Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA, United States Charles T. Quinn Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States; Erythrocyte Diagnostic Laboratory Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States Miriam Radinsky Stern College for Women, Yeshiva University, New York, NY, United States Arlene Redner Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States Susan R. Rheingold Department of Pediatrics, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, United States; Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Susmita N. Sarangi Division of Pediatric Hematology Oncology, MedStar Georgetown University Hospital, Washington, DC, United States Amish Shah Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, The University of Pennsylvania School of Medicine, Philadelphia, PA, United States Jordan A. Shavit Division of Pediatric Hematology/ Oncology, Department of Pediatrics, University of Michigan, C.S. Mott Children’s Hospital, Ann Arbor, MI, United States Christine M. Smith Division of Pediatric Hematology/ Oncology, Vanderbilt University Medical Center, Nashville, TN, United States xv Elizabeth Sokol Division of Hematology, Oncology, Neuro-Oncology, and Stem Cell Transplant, Department of Pediatrics, Ann & Robert H. Lurie Children’s Hospital of Chicago and Feinberg School of Medicine, Northwestern University, Chicago, IL, United States Kathryn S. Sutton Pediatrics, Emory University School of Medicine, Atlanta, GA, United States; Pediatric Hematology/Oncology, Aflac Cancer & Blood Disorders Center, Children’s Healthcare of Atlanta, Atlanta, GA, United States Tsewang Tashi Division of Hematology and Hematologic Malignancies, University of Utah, Salt Lake City, UT, United States; Division of Hematology/Oncology, VA Medical Center, Salt Lake City, UT, United States David T. Teachey Divisions of Hematology and Oncology, Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Anshul Vagrecha Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States Adrianna Vlachos Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States; Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, NY, United States Kelly Walkovich Pediatric Hematology/Oncology, Department of Pediatrics, C.S. Mott Children’s Hospital, University of Michigan, Ann Arbor, MI, United States Anne B. Warwick Department of Pediatrics, Uniformed Services University, Bethesda, MD, United States Howard J. Weinstein Pediatric Hematology and Oncology, Mass General Hospital for Children, Boston, MA, United States Katrina Winsnes Department of Pediatrics, Oregon Health & Science University, Portland, OR, United States Lawrence C. Wolfe Division of Pediatric Hematology/ Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States; Zucker School of Medicine at Hofstra/ Northwell, Hempstead, NY, United States This page intentionally left blank About the Editors xvii PHILIP LANZKOWSKY, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP. About the Editors xix Philip Lanzkowsky Founding Author and Editor Dr. Philip Lanzkowsky was born in Cape Town on March 17, 1932 and graduated high school from the South African College. He obtained his MBChB degree from the University of Cape Town School of Medicine in 1954 and his Doctorate of Medicine (MD) degree in 1959 for his thesis on Iron Deficiency Anemia In Children. He completed a pediatric residency at the Red Cross War Memorial Children’s Hospital in Cape Town in 1960. In the same year, he received the Diploma in Child Health (DCH) from the Royal College of Physicians and Surgeons of London, and in 1961 he was awarded the prestigious MRCP degree from the Royal College of Physicians of Edinburgh. After working in Pediatrics at the University of Edinburgh and St Mary’s Hospital of the University of London, Dr. Lanzkowsky completed a pediatric Hematology-Oncology fellowship at Duke University School of Medicine and the University of Utah. In 1963 he was appointed Consultant Pediatrician and Pediatric Hematologist at the Red Cross War Memorial Children’s Hospital of the University of Cape Town and established Pediatric Hematology and Oncology as a distinct discipline from internal medicine. In 1965 he was appointed as Director of Pediatric Hematology and Associate Professor of Pediatrics at the New York Hospital-Cornell University School of Medicine. In 1968 he was scientific advisor to the Department of Pediatrics at the University of Chile in Santiago sponsored by the Pan American Health Organization. In 1970 he was appointed Professor of Pediatrics and Chairman of Pediatrics at Long Island Jewish Medical Center and established a division of Pediatric Hematology-Oncology which he directed until 2000. He was the founder of the Schneider Children’s Hospital (presently named Cohen Children’s Medical Center), which he developed, planned, and was the hospital’s Executive Director and Chief of Staff from its inception in 1983 until 2010. Dr. Lanzkowsky has received numerous honors and awards and has lectured extensively at various institutions and medical schools in the United States and around the world. In 1973 he was appointed a Fellow of the Royal College of Physicians of Edinburgh (FRCP). In the same year, he was an invited panelist and lecturer on Nutritional Deficiency secondary to Inborn Errors of Metabolism at the United States!Japan cooperative Medical Science Program in Sendai, Japan sponsored by the US State Department. In 1994 he received a Doctor of Science Degree (Honoris Causa) from St Johns University in New York for “his notable contribution to the field of pediatric medicine and to the children of the world.” Among many other awards, he was the recipient of the Joseph Arenow Prize for original postgraduate research in the field of Science, Medicine, and Applied Science from the University of Cape Town; the Cecil John Adams Memorial Traveling Fellowship, administered by the Nuffield Foundation, for his research in Pediatric Hematology; the Hill-Pattison-Struthers Bursary from the Royal College of Physicians of Edinburgh and the Sonia Mechanick Traveling Fellowship from the South African College of Medicine. Dr. Lanzkowsky was the author of Pediatric Hematology-Oncology (1980) and Pediatric Oncology (1983) by McGraw-Hill. He was the founding author and the editor of five editions of the Manual of Pediatric Hematology and Oncology, used by clinicians worldwide. He is also the author of How It All Began: The History of a Children’s Hospital and more than 280 scientific papers, abstracts, monographs, and book chapters. Dr. Lanzkowsky’s medical writings have been prodigious. His seminal contributions to the medical literature have included the first description of the relationship of pica to iron-deficiency anemia (Arch. Dis. Child., 1959), effects of timing of clamping of umbilical cord on infant’s hemoglobin level (Br. Med. J., 1960), normal oral Dxylose test values in children (New Engl. J. Med., 1963), normal coagulation factors in women in labor and in the newborn (Thromboses Diath. Hemorr., 1966), erythrocyte abnormalities induced by malnutrition (Br. J. Haematol., 1967), radiologic features in iron-deficiency anemia (Am. J. Dis. Child., 1968), isolated defect of folic acid absorption associated with mental retardation (Blood, 1969; Am. J. Med., 1970), [subsequently designated OMIM 229050], disaccharidase levels in iron deficiency (J. Pediatr., 1981), and Hexokinase “New Hyde Park” in a Chinese kindred (Am. J. Hematol., 1981). Dr. Lanzkowsky is Professor of Pediatrics at the Donald and Barbara Zucker School of Medicine at Hofstra Northwell; Life Trustee of the Board of Trustees of Northwell Health System; Member of the Board of Trustees of the Children’s Health Alliance of Israel, and Member of the Board of Trustees of the Israel Healthcare Foundation. xx About the Editors Jonathan D. Fish Managing Editor Jonathan D. Fish, MD, is the Head of Stem Cell Transplantation and Cellular Therapy, and the Medical Director of the Survivors Facing Forward Program in the Division of Pediatric Hematology/Oncology and Stem Cell Transplantation at Cohen Children’s Medical Center, Northwell Health, New Hyde Park, New York, and is Associate Professor of Pediatrics at the Donald and Barbara Zucker School of Medicine at Hofstra/Northwell. He holds a BA degree, with distinction, from the University of Western Ontario and an MD degree, magna cum laude, from the Upstate Medical University of the State University of New York. He did his pediatric training at the Schneider Children’s Hospital, New Hyde Park, New York (presently known as the Cohen Children’s Medical Center, Northwell Health, New Hyde Park, New York) and completed his Pediatric Hematology/Oncology fellowship training at the Children’s Hospital of Philadelphia. His clinical and research interests are in stem cell transplant and cellular therapy, as well as the late effects of childhood cancer treatment and survivorship care. Jeffrey M. Lipton Jeffrey M. Lipton, MD, PhD, is Chief of Hematology/Oncology and Stem Cell Transplantation at the Cohen Children’s Medical Center, Northwell Health, New Hyde Park, New York; Professor, Frances and Thomas Gambino Professor of Hematology/Oncology; Professor of Pediatrics and Molecular Medicine, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell; and Professor, Institute of Molecular Medicine, Feinstein Institutes for Medical Research. He holds a BA degree from Queens College, City University of New York; a PhD in Chemistry from Syracuse University; and an MD degree, magna cum laude, from Saint Louis University Medical School. He did his pediatric residency training at the Children’s Hospital, Boston, Massachusetts and his Pediatric Hematology/Oncology fellowship training at the Children’s Hospital and the Dana Farber Cancer Institute in Boston. Dr. Lipton is a Past-President of the American Society of Pediatric Hematology/Oncology (ASPHO). His clinical and research interest is bone marrow failure, in particular, Diamond Blackfan anemia. Preface to the seventh edition We are entering a new era in our understanding and management of childhood cancer and blood disorders. The 5 years between the sixth edition of Lanzkowsky’s Manual of Pediatric Hematology and Oncology and the seventh have witnessed seminal changes in the practice of both pediatric hematology and oncology. Cellular immunotherapy has emerged as a standard of care for relapsed and refractory leukemia, and gene therapy has been approved in Europe to treat thalassemia (soon to be followed by the United States), with sickle cell anemia not far behind. More advanced gene-editing approaches are on the horizon. Breakthroughs in cellular and molecular biology, genetics, and genomics have increased our understanding of pathophysiology, producing a burgeoning pharmacopeia of small molecule inhibitors, antibodies, and radiopharmaceuticals that have dramatically altered how we treat our patients. The seventh edition has undergone extensive rewriting to reflect the fast pace of change. Every effort has been made to retain the original style and clarity that have become the hallmark of the previous editions while providing comprehensive coverage of each topic. This has led to several chapters being combined or reorganized, and the remainder being broadly updated. The seventh edition contains 35 chapters, including a new chapter on vascular anomalies, and remains a practical, concise, up-to-date guide for all professional staff treating children with hematological and oncologic diseases. The book is replete with detailed tables, practical algorithms, and flow diagrams useful for teaching house staff, fellows, advanced care practitioners, nursing staff, and practicing physicians. While this edition has been built upon the foundation laid by the original author, editor, and visionary, Dr. Lanzkowsky, the complexity of the medicine, has grown dramatically, demanding expert involvement from across the disciplines of our specialty. As such, the seventh edition reflects contributions from 67 authors representing 26 institutions who are leaders in their respective fields. We express our appreciation for the time and effort that all of the authors have committed to ensuring that Lanzkowsky’s Manual of Pediatric Hematology and Oncology remains a preeminent resource. We hope you continue to find the Manual a helpful guide, keep it at hand, and reference it often. Jonathan D. Fish, MD, Managing Editor, Jeffrey M. Lipton, MD, PhD and Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxi This page intentionally left blank Preface to the sixth edition The sixth edition of Lanzkowsky’s Manual of Pediatric Hematology and Oncology has significant changes from previous editions. The title of the book, the editors, and its content have changed but the objective has remained unchanged and every effort has been made to retain the original style and clarity which have become the hallmark of the previous editions. The title has changed to include the name of the original and sole editor of the book in its various editions for the past 45 years. In addition, the list of editors has increased from single editorship to include two additional hematologist!oncologists to reflect advances in pediatric hematology and oncology over the years. Jeffrey Lipton MD, PhD and Jonathan Fish, MD have been selected as coeditors. The book has been expanded from 33 chapters in the fifth edition to 36 in the present edition. A chapter has been added on the burgeoning subject of diagnostic, molecular, and genomic methodologies for the hematologist!oncologist and a new chapter has been added on transfusion medicine. Lymphoproliferative disorders and myelodysplasia have been assigned separate chapters and lymphoid and myeloid leukemia have also been assigned distinct chapters. A number of new experts in particular fields have been added to the contributing-author panel. Despite these significant changes the book has retained the original objective, format, and clarity of the founding editor. It remains a practical, concise, up-to-date guide to all professional staff treating children with hematological and oncologic diseases. The book is replete with detailed tables, practical algorithms, and flow diagrams useful for teaching housestaff, fellows, nursing staff, and practicing physicians and essential for the day-to-day investigation and management of patients with hematologic and oncologic conditions. I would like to pay tribute to Drs. Gungor Karayalcin and Ashok Shende, my close associates for over 40 years who retired after a lifelong career in clinical practice and research in Pediatric Hematology-Oncology, for their major contribution to the first four editions of the book which formed the very foundation of all subsequent editions. Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxiii This page intentionally left blank Preface to the fifth edition The fifth edition of the Manual of Pediatric Hematology and Oncology differs considerably from previous editions but has retained the original intent of the author to offer a concise manual of predominantly clinical material culled from personal experience and to be an immediate reference for the diagnosis and management of hematologic and oncologic diseases. I have resisted succumbing to the common tendency of writing a comprehensive tome which is not helpful to the practicing hematologist!oncologist at the bedside. The book has remained true to its original intent. The information included at all times keeps “the eye on the ball” to ensure that pertinent, up-to-date, practical clinical advice is presented without extraneous information, however interesting or pertinent this information may be in a different context. The book differs from previous editions in many respects. The number of contributors has been considerably expanded drawing on the expertise of leaders in different subjects from various institutions in the United States. Increased specialization within the field of hematology and oncology has necessitated including this large a number of contributors in order to bring to the reader balanced and up-to-date information for the care of patients. In addition, the number of chapters has increased from 27, in the previous edition, to 33. The reason for this is that many of the chapters, such as hemolytic anemia and coagulation, had become so large and the subject so extensive that they were better handled by subdividing the chapter into a number of smaller chapters. An additional chapter on the psychosocial aspects of cancer for children and their families, not present in previous editions, has been added. Some chapters have been extensively revised and rewritten where advancement in knowledge has dictated this approach, for example, Hodgkin lymphoma, neuroblastoma and rhabdomyosarcoma and other soft-tissue sarcomas, whereas other chapters have been only slightly modified. In nearly all the chapters there has been significant change in the management and treatment section reflecting advances that have occurred in these areas. This edition has retained the essential format written and developed decades ago by the author and, with usage over the years, has proven to be highly effective as a concise, practical, up-to-date guide replete with detailed tables, algorithms and flow diagrams for investigation and management of hematologic and oncologic conditions. The tables?and flow diagrams included in the book have been updated using the latest information and the most recent protocols of treatment, which have received general acceptance and have become the standard of care, have been included. In a book with so many details, errors inevitably occur. I do not know where they are because if I did they would have been corrected. I apologize in advance for any inaccuracies that may have crept in inadvertently. The four previous editions of this book were published when the name of the hospital was the Schneider Children’s Hospital. Effective April 1, 2010 the name of the hospital was changed to the Steven and Alexandra Cohen Children’s Medical Center of New York. I would like to acknowledge Morris Edelman, MB, BCh, BSc (Laboratory Medicine) for his contribution in reviewing the pathology on Hodgkin disease. I thank Rose Grosso for her untiring efforts in the typing and coordination of the various phases of the development of this edition. Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxv This page intentionally left blank Preface to the fourth edition This edition of the Manual of Pediatric Hematology and Oncology is the fourth edition and the sixth book written by the author on pediatric hematology and oncology. The first book written by the author 25 years ago was exclusively on pediatric hematology and its companion book, exclusively on pediatric oncology, was written 3 years later. The book reviewers at the time suggested that these two books be combined into a single book on pediatric hematology and oncology and the first edition of the Manual of Pediatric Hematology and Oncology was published by the author in 1989. It is from these origins that this 4th edition arises—the original book written in its entirety by the author was 456 pages—has more than doubled in size. The basic format and content of the clinical manifestations, diagnosis and differential diagnosis has persisted with little change as originally written by the author. The management and treatment of various diseases have undergone profound changes over time and these aspects of the book have been brought up-to-date by the subspecialists in the various disease entities. The increase in the size of the book is reflective of the advances that have occurred in both hematology and oncology over the past 25 years. Despite the size of the book, the philosophy has remained unchanged over the past quarter century. The author and his contributors have retained this book as a concise manual of personal experiences on the subject over these decades rather than developing a comprehensive tome culled from the literature. Its central theme remains clinical as an immediate reference for the practicing pediatric hematologist!oncologist concerned with the diagnosis and management of hematologic and oncologic diseases. It is extremely useful for students, residents, fellows and pediatric hematologists and oncologists as a basic reference assembling in one place, essential knowledge required for clinical practice. This edition has retained the essential format written and developed decades ago by the author and, with usage over the years, has proven to be highly effective as a concise, practical, up-to-date guide replete with detailed tables, algorithms and flow diagrams for investigation and management of hematologic and oncologic conditions. The tables and flow diagrams have been updated with the latest information and the most recent protocols of treatment, that have received general acceptance and have produced the best results, have been included in the book. Since the previous edition, some 5 years ago, there have been considerable advances particularly in the management of oncologic disease in children and these sections of the book have been completely rewritten. In addition, advances in certain areas have required that other sections of the book be updated. There has been extensive revision of certain chapters such as on Diseases of the White Cells, Lymphoproliferative Disorders, Myeloproliferative Disorders and Myelodysplastic Syndromes and Bone Marrow Failure. Because of the extensive advances in thrombosis we have rewritten that entire section contained in the chapter on Disorders of Coagulation to encompass recent advances in that area. The book, like its previous editions, reflects the practical experience of the author and his colleagues based on half a century of clinical experience. The number of contributors has been expanded but consists essentially of the faculty of the Division of Hematology Oncology at the Schneider Children’s Hospital, all working together to provide the readers of the manual with a practical guide to the management of the wide spectrum of diseases within the discipline of pediatric hematology!oncology. I would like to thank Laurie Locastro for her editorial assistance, cover design, and for her untiring efforts in the coordination of the various phases of the production of this edition. I also appreciate the efforts of Lawrence Tavnier for his expert typing of parts of the manuscript and would like to thank Elizabeth Dowling and Patrician Mastrolembo for proof reading of the book to ensure its accuracy. Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxvii This page intentionally left blank Preface to the third edition This edition of the Manual of Pediatric Hematology and Oncology, published 5 years after the second edition, has been written with the original philosophy in mind. It presents the synthesis of experience of four decades of clinical practice in pediatric hematology and oncology and is designed to be of paramount use to the practicing hematologist and oncologist. The book, like its previous editions, contains the most recent information from the literature coupled with the practical experience of the author and his colleagues to provide a guide to the practicing clinician in the investigation and up-to-date treatment of hematologic and oncologic diseases in childhood. The past 5 years have seen considerable advances in the management of oncologic diseases in children. Most of the advances have been designed to reduce the immediate and long-term toxicity of therapy without influencing the excellent results that have been achieved in the past. This has been accomplished by reducing dosages, varying the schedules of chemotherapy, and reducing the field and volume of radiation. The book is designed to be a concise, practical, up-to-date guide and is replete with detailed tables, algorithms, and flow diagrams for investigation and management of hematologic and oncologic conditions. The tables and flow diagrams have been updated with the latest information, and the most recent protocols that have received general acceptance and have produced the best results have been included in the book. Certain parts of the book have been totally rewritten because our understanding of the pathogenesis of various diseases has been altered in the light of modern biological investigations. Once again, we have included only those basic science advances that have been universally accepted and impinge on clinical practice. I thank Ms. Christine Grabowski, Ms. Lisa Phelps, Ms. Ellen Healy and Ms. Patricia Mastrolembo for their untiring efforts in the coordination of the writing and various phases of the development of this edition. Additionally, I acknowledge our fellows, Drs. Banu Aygun, Samuel Bangug, Mahmut Celiker, Naghma Husain, Youssef Khabbase, Stacey Rifkin-Zenenberg, and Rosa Ana Gonzalez, for their assistance in culling the literature. I also thank Dr. Bhoomi Mehrotra for reviewing the chapter on bone marrow transplantation, Dr. Lorry Rubin for reviewing the sections of the book dealing with infection, and Dr. Leonard Kahn for reviewing the pathology. Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxix This page intentionally left blank Preface to the second edition This edition of the Manual of Pediatric Hematology and Oncology, published 5 years after the first edition, has been written with a similar philosophy in mind. The basic objective of the book is to present useful clinical information from the recent literature in pediatric hematology and oncology and to temper it with experience derived from an active clinical practice. The manual is designed to be a concise, practical, up-to-date book for practitioners responsible for the care of children with hematologic and oncologic diseases by presenting them with detailed tables and flow diagrams for investigation and clinical management. Since the publication of the first edition, major advances have occurred, particularly in the management of oncologic diseases in children, including major advances in recombinant human growth factors and bone marrow transplantation. We have included only those basic science advances that have been universally accepted and impinge on clinical practice. I would like to thank Dr. Raj Pahwa for his contributions on bone marrow transplantation, Drs. Alan Diamond and Leora Lanzkowsky-Diamond for their assistance with the neuroradiology section, and Christine Grabowski and Lisa Phelps for their expert typing of the manuscript and for their untiring assistance in the various phases of the development of this book. Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxxi This page intentionally left blank Preface to the first edition The Manual of Pediatric Hematology and Oncology represents the synthesis of personal experience of three decades of active clinical and research endeavors in pediatric hematology and oncology. The basic orientation and intent of the book is clinical, and the book reflects a uniform systematic approach to the diagnosis and management of hematologic and oncologic diseases in children. The book is designed to cover the entire spectrum of these diseases, and although emphasis is placed on relatively common disorders, rare disorders are included for the sake of completion. Recent developments in hematology!oncology based on pertinent advances in molecular genetics, cytogenetics, immunology, transplantation, and biochemistry are included if the issues have proven of value and applicability to clinical practice. Our aim in writing this manual was to cull pertinent and useful clinical information from the recent literature in pediatric hematology and oncology and to temper it with experience derived from active clinical practice. The result, we hope, is a concise, practical, readable, up-to-date book for practitioners responsible for the care of children with hematologic and oncologic diseases. It is specifically designed for the medical student and practitioner seeking more detailed information on the subject, the pediatric house officer responsible for the care of patients with these disorders, the fellow in pediatric hematology!oncology seeking a systemic approach to these diseases and a guide in preparation for the board examinations, and the practicing pediatric hematologist!oncologist seeking another opinion and approach to these disorders. As with all brief texts, some dogmatism and “matters of opinion” have been unavoidable in the interests of clarity. The opinions expressed on management are prudent clinical opinions; and although they may not be accepted by all, pediatric hematologists!oncologists will certainly find a consensus. The reader is presented with a consistency of approach and philosophy describing the management of various diseases rather than with different managements derived from various approaches described in the literature. Where there are divergent or currently unresolved views on the investigation or management of a particular disease, we have attempted to state our own opinion and practice so as to provide some guidance rather than to leave the reader perplexed. The manual is not designed as a tome containing the minutiae of basic physiology, biochemistry, genetics, molecular biology, cellular kinetics, and other esoteric and abstruse detail. These subjects are covered extensively in larger works. Only those basic science advances that impinge on clinical practice have been included here. Each chapter stresses the pathogenesis, pathology, diagnosis, differential diagnosis, investigations, and detailed therapy of hematologic and oncologic diseases seen in children. I would like to thank Ms. Joan Dowdell and Ms. Helen Witkowski for their expert typing and for their untiring assistance in the various phases of the development of this book. Philip Lanzkowsky, MBChB, MD, ScD (Honoris Causa), FRCP, DCH, FAAP xxxiii This page intentionally left blank Introduction: Historic perspective (1955!2015) Philip Lanzkowsky Reflection on 60 years of progress in pediatric hematology/oncology As the seventh edition of the Lanzkowsky’s Manual of Pediatric Hematology and Oncology is published, I have reflected on the advances that have occurred during the 60-year period that I practiced pediatric hematology/ oncology and since my first book on the subject was published by McGraw Hill in 1980. The present edition is more than four times the size of the original book. Our understanding of hematologic conditions has advanced considerably with the explosion of molecular biology, and the management of most hematologic conditions has kept pace with these scientific advances. Our understanding of the basic science of oncology, molecular biology, genetics, and the management of oncologic conditions has undergone a seismic change. The previous age of dismal and almost consistent fatal outcomes for most childhood cancers has been replaced by an era in which most childhood cancers are cured. This has been made possible not only because of advances in oncology but also because of the parallel development of radiology, radiologic oncology, and surgery as well as supportive care such as the preemptive use of antibiotics and blood component therapy. It has been a privilege to be a witness and participant in this great evolution over the past 60 years. Yet we still have a long way to go as current advances are superseded by therapy based upon the application of knowledge garnered from an accurate understanding of the fundamental biology of cancer. In the early days of hematology/oncology practice, hematology dominated and occupied most of the practitioner’s time because most patients with cancer had a short life span, and limited therapeutic modalities were available. Automated electronic blood-counting equipment has enabled valuable red cell parameters such as mean corpuscular volume (MCV) and red cell distribution width (RDW) to be applied in routine clinical practice. This advance permitted the reclassification of anemias based on MCV and RDW. Previously, these parameters were determined by microscopy with considerable observer variability. The attempt at a more accurate determination of any one of these parameters was a laborious, time-consuming enterprise relegated only as a demonstration in physiology laboratories. Rh hemolytic disease of the newborn and its management by exchange transfusion, which occupied a major place in the hematologists’ domain, has now become almost extinct in developed countries due to the use of Rh immunoglobulin. The description of the various genetic differences in patients with vitamin B12 deficiency has opened up new vistas of our understanding of cobalamin transport and metabolism. Similar advances have occurred with reference to folate transport and metabolism. Gaucher and other similar diseases have been converted from crippling and often disabling disorders to ones where patients can live a normal and productive life thanks to the advent of enzyme replacement therapy. Aplastic anemia has been transformed from a near-death sentence to a disease with hope and cure in 90% of patients thanks to immunosuppressive therapies, hematopoietic stem cell transplantation, and advanced supportive care. The emergence of clonal disease years later in patients treated medically with immunosuppressive therapy, however, does remain a challenge. The discovery of the various genes responsible for Fanconi anemia and other inherited bone marrow failure syndromes has revealed heretofore unimaginable advances in our understanding of DNA repair, telomere maintenance, ribosome biology, and other new fields of biology. The relationship of these syndromes to the development of various cancers may hold the key to our better understanding of the etiology of cancer as well as birth defects. xxxv xxxvi Introduction: Historic perspective (1955!2015) The hemolytic anemias, previously lumped together as a group of congenital hemolytic anemias, can now be identified as separate and distinct enzyme defects of the Embden!Meyerhof and hexose monophosphate pathways in intracellular red cell metabolism as well as various well-defined defects of red cell skeletal proteins due to advances in molecular biology and genetics. With improvement in electrophoretic and other biochemical as well as molecular techniques, hemoglobinopathies are being identified, which was not previously possible. Diseases requiring a chronic transfusion program to maintain a hemoglobin level for hemodynamic stability, such as in thalassemia major, frequently had marked facial characteristics with broad cheekbones and developed what was called “bronze diabetes,” a bronzing of the skin along with organ damage and failure, particularly of the heart, liver, beta-cells of the pancreas, and other tissues due to secondary hemochromatosis because of excessive iron deposition. The clinical findings attributed to extramedullary hematopoiesis are essentially of historic interest because of the development and widespread use of proper transfusion and chelation regimens. However, the full potential of the role of intravenous and oral chelating agents is yet to be realized due to the problems of compliance with difficult treatment regimens and also due to the failure of some patients to respond adequately. Advances in our understanding of the biology of iron absorption and transport at the molecular level hold out promise for further improvement in the management of these conditions. Curative therapy in thalassemia major and other conditions by hematopoietic stem cell transplantation in suitable cases is widely available today. Gene therapy looms on the horizon but will not, for some time, be available to patients in the developing world requiring the development of other approaches. In the treatment of idiopathic thrombocytopenic purpura, intravenous gamma globulin and anti-D immunoglobulin as well as thrombopoietin mimetic agents have been added to the armamentarium of management and are useful in specific indications in patients with this disorder. Major advances in the management of hemophilia have included the introduction of commercially available products for replacement therapy, which has saved these patients from a life threatened by hemorrhage into joints, muscles, and vital organs. Surgery has become possible in hemophilia without the fear of being unable to control massive hemorrhage during or after surgery. The devastating clinical history of tragic hemophilia outcomes has been relegated to the pages of medical history. Patients with inhibitors, however, remain a clinical challenge. The whole subject of factors associated with inherited thrombophilia such as mutations of factor V, prothrombin G20210A, and 5,10-methylenetetrahydrofolate reductase as well as the roles of antithrombin, protein C and S deficiency, and antiphospholipid antibodies in the development of thrombosis has opened new vistas of understanding of thrombotic disorders. Notwithstanding these advances, the management of these patients still presents a clinical challenge. There are few diseases in which advances in therapy have been as dramatic as in the treatment of childhood leukemia. In my early days as a medical student, the only available treatment for leukemia was blood transfusion. Patients never benefitted from remission and died within a few months. Steroids and single-agent chemotherapy, first with aminopterin, demonstrated the first remissions in leukemia and raised hope of a potential cure; however, relapse ensued in almost all cases and most patients died within the first year of diagnosis. In most large pediatric oncology centers, there were few patients with leukemia as the disease was like a revolving door—diagnosis and death. The development of multiple-agent chemotherapy for induction, consolidation, maintenance, CNS prophylaxis, and supportive care ushered in a new era of cure for patients with leukemia. These principles were refined over time by more accurate classification of acute leukemia using morphological, cytochemical, immunological, cytogenetic, and molecular criteria, which replaced the crude microscopic and highly subjective characteristics previously utilized for the classification of leukemia cells. These advances paved the way for the development of specific protocols of treatment for different types of leukemia. The management of leukemia was further refined by risk stratification, response-based therapy, and identification of minimal residual disease, all of which have led to additional chemotherapy or different chemotherapy protocols, resulting in an enormous improvement in the cure rate of acute leukemia. The results have been enhanced by modern supportive care, including antibiotic, antifungal, antiviral therapy, and blood component therapy. Those patients whose leukemia is resistant to treatment or who have recurrences can be successfully treated by advances that have occurred with the development of hematopoietic stem cell transplantation. The challenge of finding appropriate, unrelated transplantation donors has been ameliorated by molecular human leukocyte antigen (HLA)-typing techniques and the development of large, international donor registries. Emerging targeted and pharmacogenetic therapies hold great promise for the future. Already dramatic results are being reported exploiting surface antigens targeted by engineered cytotoxic T cells. Hodgkin disease, originally defined as a “fatal illness of the lymphatics,” is a disease that is cured in most cases today. Initially, Hodgkin disease was treated with high-dose radiation to the sites of identifiable disease Introduction: Historic perspective (1955!2015) xxxvii resulting in some cures but with major lifelong radiation damage to normal tissues because of the use of cobalt machines and higher doses of radiation than are currently used. The introduction of nitrogen mustard early on, as single-agent chemotherapy, improved the prognosis somewhat. A major breakthrough occurred with the staging of Hodgkin’s disease and the use of radiation therapy coupled with multiple-agent chemotherapy (MOPP). With time, this therapeutic approach was considerably refined to include a reduction in radiation dosage and field and a modification of the chemotherapy regimens designed to reduce the toxicity of high-dose radiation and some of the chemotherapeutic agents. These major advances in treatment ushered in a new era in the management and cure of most patients with this disease. The management of Hodgkin disease, however, did go through a phase of staging laparotomy and splenectomy with a great deal of unnecessary surgery and splenectomies being performed. There were considerable surgical morbidity and postsplenectomy sepsis, occasionally fatal, which occurred in some cases. With the advent of MRI and PET scans, surgical staging, splenectomy, and lymphangiography have become unnecessary. Non-Hodgkin lymphoma, previously considered a dismal disease, is another success story. Improvements in histologic, immunologic, and cytogenetic techniques have made the diagnosis and classification more accurate. The development of a staging system and multiagent chemotherapy was a major step forward in the management of this disease. This, together with enhanced supportive care, including the successful management of tumor lysis syndrome, has contributed to the excellent results that occur today. Brain tumors were treated by surgery and radiation therapy with devastating results due to primitive neurosurgical techniques and radiation damage. The advent of MRI scans has made the diagnosis and the determination of the extent of disease more accurate. Major technical advances in neurosurgery such as image guidance that allows 3D mapping of tumors, functional mapping, and electrocorticography which allow pre- and intraoperative differentiation of normal and tumor tissues, the use of ultrasonic aspirators, and neuroendoscopy have all improved the results of neurosurgical intervention and resulted in less surgical damage to normal brain tissues. These neurosurgical advances, coupled with the use of various chemotherapy regimens, have resulted in considerable improvements in outcomes for some. This field, however, remains an area begging for a better understanding of the optimum management of these devastating and often fatal tumors. Improved radiation techniques, including proton beam therapy, have led to more precise radiation fields, sparing normal brain. In the early days of pediatric oncology, Wilms tumor in its early stages was cured with surgery followed by radiation therapy. The diagnosis was made with an intravenous pyelogram and inferior venocavogram, and chest radiography was employed to detect pulmonary metastases. The diagnosis and extent of the disease were better defined when CT of the abdomen and chest became available. The development of the clinicopathological staging system and the more accurate definition of the histology into favorable and unfavorable histologic types allowed for more focused treatment with radiation and multiple chemotherapy agents, for different stages and histology of Wilms tumor, resulting in the excellent outcomes observed today. The success of the National Wilms Tumor Study Group, more than any other effort, provided the model for cooperative group therapeutic cancer trials, which in large measure has been responsible for advances in the treatment of Wilms tumor. The diagnosis of neuroblastoma and its differentiation histologically from other round blue cell tumors such as rhabdomyosarcoma, Ewing sarcoma, and non-Hodgkin lymphoma was difficult before neuron-specific enolase cytochemical staining, Shimada histopathology classification, N-myc gene status, vanillylmandelic acid (VMA) and homovanillic acid (HVA) determinations, and meta-iodobenzylguanidine (MIBG) scintigraphy were introduced. In the future, new molecular approaches will offer diagnostic tools to provide even greater precision for diagnosis. The existing markers coupled with a staging system have enabled neuroblastoma to be assigned to various risk group categories with specific multimodality treatment protocols for each risk group, which has improved the prognosis in this disease. Improvements in diagnostic radiology determining the extent of disease and modern surgical techniques have enhanced the advances in chemotherapy in this condition. With these advances and the addition of targeted immunologic approaches and radiopharmaceutical-linked therapy to our armamentarium, the progress for disseminated neuroblastoma appears to be improving. Major advances have occurred in rhabdomyosarcoma treatment over the years. Early on treatment of this disease was characterized by mutilating surgery, including amputation and a generally poor outcome. More accurate histologic diagnosis, careful staging, judicious surgery, combination chemotherapy, and radiotherapy have all contributed a great deal to the improved cure rates with significantly less disability. Malignant bone tumors had a terrible prognosis. They were generally treated by the amputation of the limb with the primary tumor; however, this was usually followed by pulmonary metastases and death. The major advance in the treatment of this disease came with the use of high-dose methotrexate and leucovorin rescue which, coupled with limb salvage treatment, has resulted in improved survival and quality-of-life outcomes. xxxviii Introduction: Historic perspective (1955!2015) Of note, however, improvements in outcomes for pediatric sarcomas have not kept pace with those for leukemia, lymphoma, and other tumors. The advances in the treatment of hepatoblastoma were made possible by safer anesthesia, more radical surgery, intensive postoperative management together with multiagent chemotherapy, and more recently the increased use of liver transplantation. These advances have allowed many patients to be cured compared to past years. Histiocytosis is a disease that has undergone many name changes from Letterer!Siwe disease, Hand!Schüller!Christian disease, and eosinophilic granuloma to the realization that these entities are one disease, renamed histiocytosis X (to include all three entities) to its present name of Langerhans cell histiocytosis (LCH) due to the realization that these entities have one pathognomonic pathologic feature that is the immunohistochemical presence of Langerhans cells defined in part by expression of CD1a or langerin (CD207), which induces the formation of Birbeck granules. Advances have occurred in the management of this disease by an appreciation of risk stratification depending on the number and type of organs involved in this disease process as well as by early response to therapy. Once this was established, systemic therapy was developed for the various risk groups, which led to appropriate and improved primary and salvage therapy and the introduction of new agents with better overall results. Recent advances describing stereotypic mutations in LCH offer hope for new targeted approaches. Until a final prevention or cure for cancer in children is at hand, hematopoietic stem cell transplantation must be viewed as a major advance. Improved methods for tissue typing, the use of umbilical and peripheral blood stem cells, and improved preparative regimens, including intensity-reduced approaches and better management of graft-versus-host disease, have made this an almost routine treatment modality for many metabolic disorders, hemoglobinopathies, and malignant diseases following ablative chemotherapy in chemotherapy-sensitive tumors. Posttransplantation support with antibiotic, antifungal, antiviral, hematopoietic growth factors, and judicious use of blood component therapy has made this procedure safer than it was in years gone by. The recognition of severe and often permanent damage to organs and life-threatening complications from chemotherapy and radiation therapy has, over the years, led to regimens consisting of combination chemotherapy at reduced doses and reduction in dose and field of radiation with improved outcomes. An entirely new scientific discipline, survivorship, has arisen because of the near 80% overall cure rate for childhood cancer. Focusing on the improvement of the quality of life of survivors coupled with research in this new discipline gives hope that many of the remaining long-term effects of cancer chemotherapy in children will be mitigated and possibly eliminated. Major advances have occurred in the management of chemotherapy-induced vomiting and pain management because of the greater recognition and attention to these issues and the discovery of many new, effective drugs to deal with these symptoms. The availability of symptom control and palliative care has provided a degree of comfort for children undergoing chemotherapy, radiation, and surgery that did not exist only a few years ago. Hematologist/oncologists today are privileged to practice their specialty in an era in which most oncologic and many hematologic diseases in children are curable and at a time when national and international cooperative groups are making major advances in the management of these diseases and when basic research is at the threshold of making major breakthroughs. The present practice is grounded in evidence-based research that has been and is still being performed by hematologists/oncologists and researchers that form the foundation for ongoing advances. Today we stand on the shoulders of others, which permits us to see future advances unfold to benefit generations of children. While we bask in the glory of past achievements, we should always be cognizant that much work remains to be done until the permanent cure of all childhood malignancies and blood diseases is at hand. This book encompasses the advances in the management of childhood cancer and blood diseases, which have been accomplished to date and which have become the standard of care. C H A P T E R 1 Molecular and genomic methodologies for clinicians Jordan A. Shavit and Rajen J. Mody Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Michigan, C.S. Mott Children’s Hospital, Ann Arbor, MI, United States Over the last decade, molecular diagnostic testing in patients with hematologic and oncologic disorders has become increasingly sophisticated and prevalent. While in the past focused genetic tests were performed, in recent years the widespread use of genomic and molecular approaches in both research and clinical settings has refined diagnostics and therapeutics for pediatric blood disorders and cancer. This chapter provides an overview of the currently used molecular and genomic methods, with the goal of introducing these new technologies for trainees and clinicians without extensive laboratory experience. This is not meant to be a comprehensive review of the topic and will primarily focus on genetic methods that are currently used in clinical settings. Clinical molecular and genomic methodologies: goals The goal of genetic and genomic analysis is the identification of molecular lesions underlying patient disease and using the information to inform clinical care. These include: 1. Identification of the causative gene or mutation, usually by sequencing a gene or panel of genes known to be associated with a specific disorder such as thrombocytopenia, thalassemia, or familial cancer predisposition syndromes. 2. Evaluation of genomic or molecular markers in multiple affected and unaffected individuals (related or unrelated) through indirect approaches. These often identify markers that may be coinherited with a disease but do not cause the disease itself. Markers may be single nucleotide variants (SNVs) or structural variations such as insertions, deletions, and copy number variation. 3. Genome-wide evaluation of genes and markers for the purpose of diagnosis of a disease or directing treatment, typically through the use of next-generation sequencing (NGS) technologies. These include whole exome and genome sequencing (WES and WGS) and are currently being applied increasingly for clinical applications in diagnostics and therapeutics. Table 1.1 lists the commonly used genetic testing methodologies, along with the types of molecular lesions that they are able to identify. Methods of genetic analysis Markers of genetic defects While most methods are now focused on identifying the precise molecular cause of disease through sequencing, indirect tests have been quite useful in the past and are still used today, particularly for mapping Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00036-2 1 © 2022 Elsevier Inc. All rights reserved. 2 1. Molecular and genomic methodologies for clinicians TABLE 1.1 Overview of molecular and genomic diagnostic methodologies. Method Linkage analysis (using markers such as short tandem repeats) Common Rare Copy point point number mutations mutations variants X Uniparental disomy Balanced inversions or Repeat Examples of use in pediatric translocations expansions hematology/oncology X Fluorescence in situ hybridization X Array comparative genomic hybridization X Family pedigree with history of hereditary spherocytosis and interest in identifying the causal gene X Acquired monosomy in myelodysplastic syndrome X Testing for microdeletion in patient with hematologic and syndromic phenotype Genome-wide single nucleotide variant microarrays X X Testing for small copy number variants in pediatric leukemia Targeted polymerase chain reaction analysis X X Sanger sequencing X X Molecular diagnosis of a patient with pyruvate kinase deficiency Gene panel sequencing X X Hematologic and solid tumors, severe congenital neutropenia Whole genome or exome sequencing X X X X Testing for JAK2 V617F mutation in patient with a myeloproliferative disorder Hematologic and solid tumors, unknown bone marrow failure syndrome Modified from Sankaran, V. 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. causes of a disease in a family. These are found throughout the genome, include SNVs or short tandem repeats (two to five base long repetitive elements with varying numbers of repeats), and have been extremely useful as a way to identify likely causal genes. These markers may be located in close proximity to the causal mutation and thus within a family should only be found in affected members, suggesting that the causal mutation is located nearby. This approach, a process called linkage analysis, is particularly useful when causative mutations reside in regulatory regions such as enhancers or promoters, rather than protein coding sequences. Genomewide single nucleotide polymorphism (SNP) microarrays accelerated the use of these approaches prior to large-scale sequencing. These methods have been used as an initial screen in large families or populations with suspected cancer predisposition syndromes to guide in-depth analysis of certain regions of the genome, prior to NGS being available. However, as costs decrease, genome-wide approaches such as NGS can be used initially, with efforts subsequently directed to linkage analysis if sequencing fails to detect the disease-causing mutation. Other technologies are useful for assessing large-scale structural chromosome defects. Fluorescence in situ hybridization (FISH) is a cytogenetic technique that queries whether chromosomes or chromosomal fragments are duplicated or deleted and has been particularly useful for mapping gene locations and classifying malignant tumors. Polymerase chain reaction (PCR)-based methods have been used with increasing frequency as a replacement for FISH. Another method that has been highly used in recent years for examination of copy number variation and structural defects is array comparative genomic hybridization (CGH), although this may be supplanted by NGS. Despite the increased use of PCR and NGS, FISH remains one of the best clinically available methods to detect classic cytogenetic changes that are diagnostic and implicated in a number of pediatric cancers such as translocations that are frequently seen in leukemia and certain solid tumors. As with sequencing (see later), all of these technologies may detect changes that are of unclear significance and should be interpreted with caution. Array genotyping technologies are most commonly used by direct-toconsumer services that report questionable relative risk information to individuals who request such services. However, as large-scale NGS has become more affordable, arrays have been used with decreasing frequency. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Methods of genetic analysis 3 Sequencing approaches Sanger sequencing Often a single mutation confers significant disease risk or occurs in a substantial proportion of disease cases, such as factor V Leiden or JAK2 V617F, which are examples of germline variation and somatic mutation, respectively. Targeted PCR analysis continues to be used for mutation detection in such cases. These tests can be done at low cost and relatively rapidly because of their focused nature and output of the presence/absence of a single mutation, but they are only useful for known variants. It should be kept in mind that while Sanger sequencing can be very sensitive to detect point mutations, copy number or structural changes in genes will often be missed using this approach. Therefore targeted sequencing of a disease gene in the past was complemented using array CGH or SNP array-based approaches to look for deletions that may be implicated in a subset of cases of a particular disease. For diseases where there are multiple different known variants in either single or multiple genes, PCR can be performed more broadly across multiple exons or genes, followed by Sanger sequencing. However, this approach is cumbersome and costly; thus the Sanger sequencing step has largely been supplanted by NGS, enabling disease-specific multigene panels. Next-generation sequencing technologies and applications Although Sanger methodology was once the primary means of sequencing DNA, the development of highthroughput NGS platforms over the past decade has enabled rapid and exponentially decreasing costs for WES and WGS. Multiple competing technologies have led to an era in which a whole genome can be sequenced for under $1000. Using various systems, NGS can sequence millions to billions of DNA strands in parallel to yield substantially more throughput than Sanger sequencing. In earlier and current NGS technologies, genomic DNA was broken into short fragments, which were then enriched and the sequences of all fragments read on NGS platforms. For WES, only exons are captured from these genomic fragments and sequenced, which in the early years of NGS was significantly cheaper and faster than the whole genome. In recent years with continuing decreases in costs, WGS is being used with increasing frequency. Newer technologies can read long stretches of single DNA molecules rather than short fragments, which simplifies alignment of the reads and may allow determining the phase of closely linked variants (i.e., whether they are on one of the two copies of the same chromosome). The details of these approaches are beyond the scope of this chapter and vary depending upon the technology or platform used, each with its own advantages and disadvantages. More information can be gleaned from the references. NGS platforms have been used with increasing regularity over the last decade to sequence selected candidate genes, whole exomes, or whole genomes of patients with particular disorders in both clinical and research settings. This technology has taken a strong hold over the last few years in a number of areas, most commonly for rare undiagnosed disorders and malignancies. Numerous panels composed of tens of associated candidate genes are now routinely available for multiple disorders with results available within days to weeks, which would have taken months at much greater expense using Sanger sequencing. Even though WES and WGS are ordered in the clinical setting for the detection of rare variants in patients with inherited single-gene disorders after known candidates have been eliminated, perhaps one of the biggest impacts of NGS technology has been on the practice of clinical oncology. These are now the backbone of precision pediatric oncology and could eventually replace the majority of the aforementioned technologies. A number of large academic centers have published their experiences, and several commercial companies are now offering WES or selective oncology panels. Currently, WGS or WES are ordered in the clinical setting for the detection of rare variants in patients with a phenotype that is suspected to be due to a single-gene disorder, after known single-gene candidates have either been eliminated or when a multigene panel testing approach is prohibitively expensive. Before such tests are ordered, it is important that clinicians gather a thorough family history, fully evaluate a patient’s phenotype, and obtain appropriate informed consent. The latter is critical as results may often introduce more confusion when new variants are discovered that may or may not underlie the disease. Often WGS/WES will be ordered on family members as well, which can help to narrow mutations based on those who are affected or unaffected by the disorder. There is little doubt that as WGS and WES are increasingly employed in clinical settings, specific guidelines for when it is best to use these tests for patients with particular groups of diseases will continue to emerge. It is important for clinicians to be aware of the significant limitations of WGS and WES. Although NGS can sequence nonrepetitive and repetitive regions of the genome, sequencing data from the latter are often uninterpretable. Since some diseases occur in repetitive DNA (e.g., fragile X syndrome), those diagnoses will be missed using such approaches. In addition, the greater the final sequencing “coverage,” that is, the average number of reads that align to the reference genome, the better the accuracy. Higher coverage means that there are fewer gaps in the Lanzkowsky’s Manual of Pediatric Hematology and Oncology 4 1. Molecular and genomic methodologies for clinicians assembled sequence. However, there is a trade-off between this accuracy and cost. If the sequencing is performed at low coverage (i.e., fewer reads) in order to reduce expenditures, NGS may miss copy number variants, insertion!deletion variants, or chromosomal translocations. It is likely that with improvements in technology to allow for greater sequencing depth at lower costs, longer NGS reads, and better computational methods, these types of variants could be routinely detected using these approaches in the future. Another limitation is that even when a mutation is identified in a regulatory region of a gene, its effect on the gene implicated in that disease may not be immediately apparent. Interpretation of genetic variants obtained from next-generation sequencing One important consideration in using NGS approaches is that since the entire genome (or a substantial portion) is sequenced, there are two types of incidental findings that can cause confusion or distress. The first is the identification of potentially pathogenic variants in known genes that do not contribute to the disease that initially motivated the NGS. More commonly, variants of uncertain significance (VUS) are discovered in the target or other genes. This is nucleotide variation, often causing an amino acid substitution, which is unclear to have any deleterious effect. Such results trigger two debates. The former case raises the ethical question of whether the ordering physician is required to inform the patient. In the case of VUS the clinician and patient are left with uncertainty of whether this result is an indication for further investigation or intervention. There are varying opinions on the types of circumstances in which such incidental findings can or should be reported to patients, and studies are exploring the impact of delivering such information. There has been substantial debate in the community regarding the transmission and broad impact of the information derived from NGS on patients, physicians, and society. Undoubtedly, this is an area that will continue to evolve in the coming years. Since numerous potential causal variants may be identified using such broad-based sequencing approaches, the American College of Medical Genetics has developed recommendations regarding categories to which variants should be assigned and these are helpful for clinicians to be aware of when interpreting results from WES or WGS (although they apply to other sequencing approaches as well). • Pathogenic: a sequence variant that directly contributes to the development of disease • Likely pathogenic: variant with greater than 90% certainty to be disease-causing • Uncertain significance: not enough information to determine whether a variant is disease-causing or benign • Likely benign: variant with greater than 90% certainty to be benign • Benign: a sequence variant that does not cause disease While these categories are useful when results are reported, they are largely dependent upon databases of prior variants. As more sequencing data are being reported, it is important to bear in mind that some variants previously thought to be pathogenic are being reclassified as benign, and the reverse could occur as well. It is very likely that the categorization for a particular variant may evolve over time and therefore it is useful to evaluate the prior reports of any genetic variants identified in such studies on an individual basis. In some cases, it is important to bear in mind that in many single-gene disorders, variable penetrance or expressivity (where patients with a particular mutation may or may not have the disease or may have varying severities of the disease) may have a significant and underestimated impact. In practice, most clinical sequencing groups employ centralized sequence variant databases, such as the Clinical Genome Resource (ClinGen) for precision medicine, ClinVar for genomic variation and human health, or bioinformatic algorithms for prediction of pathogenic variants such as PolyPhen-2, as well as expert opinion. Applications of next-generation sequencing to oncology Clinical interpretation of sequence variants in cancer Determination of the clinical importance of somatic sequence variants in cancer remains a work in progress. Determining their true significance requires intimate knowledge of large genomic, cancer-specific, population, and constitutional variant databases and requires a combination of in silico (computational) algorithms and manual curation to assign significance. These variants can have a spectrum of clinical utility, including diagnosis, prognosis, therapeutics, and monitoring of therapy, although strong evidence linking genomic alterations to FDA-approved cancer Lanzkowsky’s Manual of Pediatric Hematology and Oncology Applications of next-generation sequencing to oncology 5 therapies exists only for a few pediatric cancers. Peer-reviewed literature, clinical practice guidelines, and large-scale cancer mutation databases remain primary resources for evidence needed to effectively assess clinical significance of a particular variant. Based on available evidence, somatic cancer variants are classified into the following categories: Tier I: Variants with strong clinical significance: • Level-A: FDA-approved therapy available • Level-B: well-powered clinical studies with consensus from experts Tier II: Variants with potential clinical significance: • Level-C: availability of FDA-approved therapy for different tumor types, several smaller studies with some consensus, or a few case reports • Level-D: preclinical studies without consensus Tier III: Variants with unknown clinical significance: variants not observed at a significant allele frequency in a pan- or specific cancer database or published evidence of cancer association Tier IV: Variants that are benign or likely benign: observed at a significant allele frequency in a general or specific subpopulation database and no existing evidence of cancer association Recent NGS-based precision pediatric oncology investigations have shown several important findings, as well as some limitations of this technology. Studies have identified the ability to detect potential clinically relevant genomic alterations in a substantial fraction of pediatric cancer patients. Pediatric tumors are distinguished by relatively few SNVs as compared to adults, but an overall higher prevalence of driving gene fusions or other structural changes are observed, especially in leukemias, soft tissue sarcomas, and low-grade glial brain tumors. These features make a strong case for including RNA sequencing as an additional strategy. Finally, across all studies, around 10% of patients demonstrate pathogenic germline mutations and familial cancer syndromes. Taken together, these findings suggest that comprehensive integrative clinical sequencing panels targeted for pediatric cancer patients should include a combination of tumor and normal tissue DNA sequencing, as well as tumor RNA sequencing. Multiinstitutional studies of National Cancer Institute!Children’s Oncology Group are currently testing the ability of such comprehensive testing to guide precision oncology. Given the rapid decrease in the cost of NGS and highly efficient bioinformatic resources, analyses can be completed in weeks, with the potential that this could be routinely available for clinical care. Despite these exciting advances, in its current state, NGS still has many limitations. Liquid tumor biopsies Tumors continuously evolve under the pressure of therapy and it is important to repeat tumor sequencing in order to accurately map evolving genomic events driving tumor growth and/or resistance. However, it is not practical or feasible to perform invasive tumor biopsies and discover every event. Recent studies have shown that circulating tumor cells (CTCs) and/or cell-free DNA (cfDNA) are present in the blood of the majority of pediatric cancer patients and may offer an opportunity to evaluate tumor biology noninvasively. CTCs and cfDNA offer the advantage of frequent monitoring during and after treatment, potentially helping with early detection of relapse or resistance to therapy. Methods to isolate circulating tumor DNA are challenging but are increasingly clinically feasible. Epigenetic sequencing Ewing sarcoma, ependymoma, and many other pediatric tumors are driven primarily by epigenetic modification, which is a change in gene expression without alteration of the DNA sequence. DNA methylation sequencing or other forms of epigenomic studies may be more appropriate for these tumors but is still primarily used in research settings. However, the field of epigenomics is evolving rapidly, and the coming decade is likely to see a much more widespread clinical application of this important tool. Single-cell sequencing Conventional “bulk” sequencing of tumors that are made of multiple different cell types has several limitations, including loss of critical information due to averaging over the molecular phenotype of individual cells. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 6 1. Molecular and genomic methodologies for clinicians Single-cell multiomics has the capability to generate high-resolution molecular phenotype information and provide the means for quantitative analysis of several key properties of tumors, including intratumor heterogeneity, cell types, cellular hierarchies, and cell states. Quantitative characterization of tumor heterogeneity, in particular, including detection of rare subclones of cells with possible drug resistance, has the potential to be translated to the clinic in the future. However, the single-cell sequencing (SCS) methodology is still evolving. It is very expensive currently and has issues with low coverage and low patient numbers, especially for pediatric tumors. But once these issues are resolved in the coming years, SCS from primary and CTCs holds great promise and has the potential to replace conventional bulk sequencing. Interpreting and evaluating the results from clinical genetic testing A key role of any pediatric hematologist/oncologist will be interpreting and evaluating the results obtained from clinical diagnostic genetic testing. As newer methods are used, different limitations of each approach need to be understood and addressed. For each abovementioned diagnostic approach, we have addressed some specific uses and/or limitations. Depending upon the genetic lesion under consideration, this may alter what tests are performed. For example, while large chromosomal alterations or translocations are readily detected using RNA sequencing or FISH, smaller deletions might only be detected using array CGH or SNP arrays. Therefore depending upon the type of lesion expected, the various tests may be used in different ways or combinations. Another example is that while WES or WGS can be useful for looking for many causes of a disease, they may miss deletions that could result in a subset of cases of a particular disease. Therefore it may be useful to run WES and CGH or a SNP array on a particular patient if the disease can be caused by deletions in some cases. Finally, whenever a pathogenic germline variant is identified in a patient, it is very important to follow that up with a formal referral to genetic specialists in order to fully assess the risk to other family members. Further reading and references Abbou, S.D., Shulman, D.S., DuBois, S.G., Crompton, B.D., 2019. Assessment of circulating tumor DNA in pediatric solid tumors: the promise of liquid biopsies. PBC 66 (5), e27595. Artin, M.G., Stiles, D., Kiryluk, K., Chung, W.K., 2019. Cases in precision medicine: when patients present with direct-to-consumer genetic test results. Ann. Intern. Med. 170 (9), 643!650. Bisecker, L.G., Green, R.C., 2014. Diagnostic clinical genome and exome sequencing. N. Engl. J. Med. 370, 2418!2425. Feero, W.G., Green, E.D., 2011. Genomics education for health care professionals in the 21st century. JAMA 306, 989!990. Harris, M.H., DuBois, S.G., Glade Bender, J.L., et al., 2016. Multicenter feasibility study of tumor molecular profiling to inform therapeutic decisions in advanced pediatric solid tumors: the individualized cancer therapy (iCat) study. JAMA Oncol. 2. Johnsen, J.M., Nickerson, D.A., Reiner, A.P., 2013. Massively parallel sequencing: the new frontier of hematologic genomics. Blood 122, 3268!3275. Katsanis, S.H., Katsanis, N., 2013. Molecular genetic testing and the future of clinical genomics. Nat. Rev. Genet. 14, 415!426. Li, M.M., et al., 2017. Standards and guidelines for the interpretation and reporting of sequence variants in cancer—a joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J. Mol. Diagn. 19 (1), 4!23. PMCID: PMC5707196, PMID: 27993330. Lindsley, R.C., Ebert, B.L., 2013. The biology and clinical impact of genetic lesions in myeloid malignancies. Blood 122, 3741!3748. MacArthur, D.G., Manolio, T.A., 2014. Guidelines for investigating causality of sequence variants in human disease. Nature 508, 469!476. Mody, R.J., Wu, Y.M., Lonigro, R.J., et al., 2015. Integrative clinical sequencing in the management of refractory or relapsed cancer in youth. JAMA 314 (9), 913!925. Mody, R.J., Prensner, J.R., Everett, J., Parsons, D.W., Chinnaiyan, A.M., 2017. Precision medicine in pediatric oncology: lessons learned and next steps. Pediatr. Blood Cancer 64 (3). Available from: https://doi.org/10.1002/pbc.26288. Mullighan, C.G., 2013. Genome sequencing of lymphoid malignancies. Blood 122, 3899!3907. Parsons, D.W., Roy, A., Yang, Y., et al., 2016. Diagnostic yield of clinical tumor and germline whole-exome sequencing for children with solid tumors. JAMA Oncol. 2. Richards, S., Aziz, N., et al., 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405!424. Sankaran, V.G., Gallagher, P.G., 2013. Applications of high-throughput DNA sequencing to benign hematology. Blood 122, 3575!3582. Zhang, J., Walsh, M.F., Wu, G., et al., 2015. Germline mutations in predisposition genes in pediatric cancer. N. Engl. J. Med. 373 (24), 2336!2346. Lanzkowsky’s Manual of Pediatric Hematology and Oncology C H A P T E R 2 Hematologic manifestations of systemic illness Brian M. Dulmovits1,2 and Lawrence C. Wolfe1,2 1 Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States 2Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States A variety of systemic illnesses, including acute and chronic infections, neoplastic diseases, connective tissue disorders, and storage diseases, are associated with hematologic manifestations. The hematologic manifestations are the result of the following mechanisms: • bone marrow dysfunction, • anemia or erythrocytosis, • thrombocytopenia or thrombocytosis, • leukopenia or leukocytosis, • hemolysis, • immune cytopenias, • alterations in hemostasis, • acquired inhibitors to coagulation factors, • acquired von Willebrand disease, • acquired platelet dysfunction, and • alterations in leukocyte function. Alterations to red blood cells related to organ-specific pathologies Cardiovascular system Anemia • Intravascular hemolytic anemia may occur following cardiac procedures using prosthetic valves or synthetic patches for correction of valvular disease and anatomic defects, respectively. Aberrant mechanical forces generated by regurgitant flow, impaired endothelialization, or valve calcification mediate red cell lysis. • Laboratory findings indicative of intravascular hemolysis include elevated serum lactate dehydrogenase (LDH), reduced serum haptoglobin, hyperbilirubinemia with an increased indirect component, and hemoglobinuria. • Chronic intravascular hemolysis may lead to iron deficiency secondary to shedding of hemosiderin within renal tubular cells into the urine. • Treatment relies on surgical repair of dysfunctional prosthesis in cases of severe hemolysis. • An uncommon cause of autoimmune hemolytic anemia occurs following cardiac surgeries such as heart transplantation and may also be associated with additional immune cytopenias including acquired Glanzmann thrombasthenia and idiopathic thrombocytopenic purpura (ITP). Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00022-2 7 © 2022 Elsevier Inc. All rights reserved. 8 2. Hematologic manifestations of systemic illness • A 4-week course of weekly intravenous (IV) rituximab 375 mg/m2 demonstrated efficacy in a series of pediatric transplant patients with autoimmune cytopenias postcardiac transplant. • Infective endocarditis mediates anemia through intravascular hemolysis and anemia of inflammation. Rarely, infective endocarditis may cause pancytopenia. • Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by easy bruisability, epistaxis, and respiratory and gastrointestinal (GI) bleeding due to abnormal blood vessels (i.e. telangiectatic lesions). Chronic blood loss from these lesions may result in an iron deficiency anemia (IDA) and infrequently (approximately 5% of patients) causes severe hemorrhage manifesting as a normocytic anemia. Treatment for HHT-associated anemia includes: • Oral or IV iron supplementation; • Trials are underway exploring medical treatments, including the vascular endothelial growth factor (VEGF) inhibitor bevacizumab; and • Packed red blood cell (RBC) transfusion as indicated. Erythrocytosis • Congenital heart disease (CHD)-associated hypoxemia produces a compensatory elevation in erythropoietin (EPO) and secondary erythrocytosis (see Chapter 10: Primary and Secondary Erythrocytosis). • Secondary erythrocytosis predisposes patients to cerebrovascular accidents secondary to hyperviscosity as well as symptomatic hypoglycemia (especially in the neonatal period). • The use of partial exchange transfusion has been suggested, although the long-term value of exchange has been challenged. • In general, serious consideration for dehydration and iron deficiency must be considered. Unlike many situations relieved by therapeutic phlebotomy, iron deficiency secondary to phlebotomy may actually increase the chance of viscosity-related injury and must be avoided. Any therapeutic phlebotomy in these patients should be performed in well-hydrated and iron-replete patients with no sign of iron deficient maturation defect in their red cells. • Hydroxyurea at an initial dose of 10!13 mg/kg/day escalated by 5 mg/kg/day every 8 weeks resulted in symptomatic relief in a small series of patients with persistent CHD. However, large studies are lacking, and the potential for myelosuppression should be considered. Qualitative changes to RBC morphology • Cardiac anomalies, particularly situs inversus, may be associated with hyposplenism and the blood smear may show Howell!Jolly and Pappenheimer bodies. Lungs Anemia • Idiopathic pulmonary hemosiderosis is a rare chronic disease characterized by recurrent intraalveolar microhemorrhages with pulmonary dysfunction, hemoptysis, and hemosiderin-laden macrophages, which results in IDA. • Idiopathic pulmonary hemosiderosis associated with celiac disease is referred to as Lane!Hamilton syndrome. • Variant associated with hypersensitivity to cows’ milk (Heiner’s syndrome) and one that occurs with a progressive glomerulonephritis (Goodpasture syndrome). • Bronchoscopy with bronchoalveolar lavage or gastric aspirates containing siderophages establishes the diagnosis. • The mainstray of treatment involves protecting hemoglobin levels with transfusion when necessary and iron supplementation. Alternative therapies are also being used to prevent further hemorrhage, including high-dose steroids, rituximab, and chemotherapy immunosuppression such as cyclophosphamide. Erythrocytosis • Hypoxia secondary to pulmonary disease results in secondary erythrocytosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Alterations to red blood cells related to organ-specific pathologies 9 Gastrointestinal tract Anemia • Pediatric IDA after the age of 5 years must suggest the possibility of GI blood loss. IDA may occur as a manifestation of gastroesophageal reflux disease, Meckel’s diverticulum, inflammatory bowel disease (IBD), polyps, or HHT, and therefore, endoscopy may be required in unexplained iron deficiency. • Chronic atrophic gastritis causes iron deficiency and may lead to megaloblastic anemia secondary to associated vitamin B12 malabsorption. Parietal cells play an important role in vitamin B12 absorption by producing intrinsic factor (IF). • Pernicious anemia is a subtype of megaloblastic anemia resulting from autoantibodies against parietal cells and IF (see Chapter 4: Nutritional Anemias). • Gastric resection may result in iron deficiency or in vitamin B12 deficiency. • Zollinger!Ellison syndrome (increased parietal cell production of hydrochloric acid) may cause iron deficiency through mucosal ulceration. • Helicobacter pylori infection, although atypical in children, may cause chronic gastritis as well as initiate IDA and vitamin B12 deficiency. • Standard treatment includes amoxicillin, clarithromycin, and a proton pump inhibitor. • Short bowel syndrome as a result of significant bowel resection may impair micronutrient absorption leading to iron, folate, and vitamin B12 deficiencies. • Celiac disease or tropical sprue may cause malabsorption of iron and folate. Other hematologic manifestations of celiac disease are discussed in later sections and in Table 2.1. • IBD may cause anemia of chronic inflammation and iron deficiency from blood loss. Ulcerative colitis tends to appear as pure IDA, while Crohn’s disease often has a component of the anemia of inflammation. • Peutz!Jeghers syndrome (intestinal polyposis and mucocutaneous pigmentation) predisposes to adenocarcinoma of the colon. • Diarrheal illnesses of infancy can produce life-threatening methemoglobinemia. Most patients are at or below the 10th percentile for weight at the time the syndrome is discovered. There is evidence that this responds to methylene blue. Pancreas Anemia • Hemorrhagic pancreatitis produces an acute normocytic, normochromic anemia. It may also be associated with disseminated intravascular coagulation (DIC). TABLE 2.1 Hematologic manifestations of celiac disease. Problem Frequency Comments Anemia: iron deficiency, folate deficiency, vitamin B12 deficiency, and other nutritional deficiencies Common The anemia is most commonly secondary to iron deficiency but may be multifactorial in etiology. Low serum levels of folate and vitamin B12 without anemia are frequently seen. Anemia due to other deficiencies appears to be rare Thrombocytopenia Rare May be associated with other autoimmune phenomena Thrombocytosis Common May be secondary to iron deficiency or hyposplenism Thromboembolism Uncommon Etiology is unknown, but may be related to elevated levels of homocysteine or other procoagulants Leukopenia/neutropenia Uncommon May be autoimmune or secondary to deficiencies of folate, vitamin B12, or copper Coagulopathy Uncommon Malabsorption of vitamin K Hyposplenism Common Rarely associated with infections IgA deficiency Common May be related to anaphylactic transfusion reactions Lymphoma Uncommon The risk is highest for intestinal T-cell lymphomas Modified from Halfdanarson TR, Litzow MR, Murray JA., 2007. Hematologic manifestations of celiac disease. Blood. 109(2):412!21. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 10 2. Hematologic manifestations of systemic illness • Shwachman!Diamond syndrome (see Chapter 6: Bone Marrow Failure, and Chapter 11: Disorders of White Blood Cells) is characterized by congenital exocrine pancreatic insufficiency, metaphyseal bone abnormalities, and neutropenia. There may also be some degree of anemia and thrombocytopenia. • Pearson syndrome is characterized by exocrine pancreatic insufficiency and severe sideroblastic anemia. Liver Anemia • Anemias of diverse etiologies occur in acute and chronic liver disease. Red cells are frequently macrocytic [mean corpuscular volume (MCV) of 100!110 fL] having acquired additional surface area from lipid accumulation. Target cells and acanthocytes (spur cells) are frequently seen. Some of the pathogenic mechanisms of cirrhosis-related anemia include: • Reduced red cell survival and red cell fragmentation (spur cell anemia) often occur in later-stage cirrhosis in the presence of dyslipidemia. • Hypersplenism with splenic sequestration in the presence of secondary portal hypertension. • IDA due to chronic blood loss or normocytic anemia secondary to acute hemorrhage from esophageal varices in portal hypertension. • Normochromic normocytic anemia secondary to chronic illness. • Megaloblastic anemia secondary to folate deficiency in malnourished individuals. • Aplastic anemia following acute hepatitis (typically seronegative) in certain immunologically predisposed hosts. • Chronic hemolysis, which is the presenting manifestation in 5!6% of patients with Wilson disease, induced by copper accumulation in RBC. Kidneys Anemia • Renal insufficiency and chronic kidney disease (CKD) are frequently associated with anemia (and sometimes pancytopenia), and occur through multiple pathogenic mechanisms, including reduced serum EPO concentrations mediated by loss of EPO-producing interstitial cells in response to renal inflammation and fibrosis (90% of EPO synthesis occurs in the kidney), diminished circulating red cell lifespan, and decreased bone marrow erythropoiesis secondary to uremic toxins, altered iron homeostasis emanating from reduced reneal clearance if hepcidin, and iron and folate deficiencies induced by dialysis. • Laboratory findings include: ! hemoglobin as low as 4!5 g/dL; ! normochromic and normocytic red cell morphology; ! low reticulocyte count; and ! decreased erythroid precursors in bone marrow aspirate. • Evaluation and treatment [as per Kidney Disease: Improving Global Outcomes(KDIGO) guidelines]: ! In children ,15 years of age, hemoglobin concentrations ,11.0 g/dL (0.5!5 years), ,11.5 g/dL (5!12 years), and ,12.0 g/dL (12!15 years) warrant an anemia workup. ! In adolescents .15 years of age, hemoglobin ,13 g/dL in males and ,12 g/dL in females warrant an anemia workup. ! Recombinant human EPO (rHuEPO) administration (Fig. 2.1) There is a black box Food and Drug Administration warning for the use of EPO: In contrast to adults, there is no exact hemoglobin threshold for rHuEPO initiation in children, but rather, is initiated based on clinical judgment and possible benefits to patient quality of life. However, one study demonstrated increased mortality in patients with hemoglobin ,11 g/dL. Initial dosing relies on the type of rHuEPO: 20!50 IU/kg three times per week for epoetin alfa/beta, and 0.45 µg/kg once weekly or 0.75 µg/kg every 2 weeks for darbepoetin-alfa. Target hemoglobin increase is 1!2 g/dL over a 4-week period with optimal hemoglobin range between 11 and 12 g/dL (not to exceed 14 g/dL). Titrate the dose: if no response, increase rHuEPO up to 300 unit/kg/day subcutaneous (SC) three times a week; Lanzkowsky’s Manual of Pediatric Hematology and Oncology Alterations to red blood cells related to organ-specific pathologies 11 FIGURE 2.1 Approach to rHuEPO administration in pediatric kidney disease. Abbreviations: Hct, hematocrit; Hgb, hemoglobin; rHuEPO, recombinant human erythropoietin. if hematocrit (Hct) reaches 40%, stop rHuEPO until Hct is 36% and then restart at 75% dose; if Hct increases very rapidly ( . 4% in 2 weeks), reduce dose by 25%. Adverse effects from rHuEPO treatment include hypertension secondary to increased viscosity (30% of patients), increased risk of thrombosis, and increased mortality with high-dose erythropoiesis stimulating agent (ESA) (mean ESA equivalent dose of .6000 IU/m2/week). ! Iron administration and iron status monitoring: For anemic CKD patients not on iron supplementation or rHuEPO, oral iron should be initiated in patients with a ferritin ,100 ng/mL and transferrin saturation (TSAT) ,20% or a soluble transferrin index .2. IV iron is used for patients on hemodialysis. ! Folic acid 1 mg/day is recommended because folate is dialyzable. ! Packed red cell transfusion is rarely required. • Experimental ESAs include EPO mimetic peptides, EPO receptor modulators, molecules that prevent hydroxylation and subsequent degradation of hypoxia induced factor, hepcidin regulators, and EPO gene therapy. ! These novel ESAs have shown efficacy in preclinical cellular and animal models, and many are currently in human clinical trials. Endocrine system Anemia • Anemia is frequently present in overt and subclinical hypothyroidism due to the importance of thyroid hormone signaling in erythroid precursor differentiation. • Laboratory findings include: ! Normochromic and normocytic anemia, but may present as hypochromic or macrocytic secondary to an associated iron or vitamin B12 deficiency, respectively ! Bone marrow is characterized as fatty and hypocellular Lanzkowsky’s Manual of Pediatric Hematology and Oncology 12 2. Hematologic manifestations of systemic illness • Of note, hypothyroidism with concomitant macrocytic anemia and megaloblastic bone marrow warrants investigation of underlying autoimmune processes (i.e., juvenile pernicious anemia with polyendocrinopathies) • In Addison disease, some degree of anemia is also present, but may be masked by coexisting hemoconcentration. The association between Addison disease and megaloblastic anemia raises the possibility of an inherited autoimmune disease directed against multiple tissues (i.e., juvenile pernicious anemia with polyendocrinopathies). Erythrocytosis • Hypercortisolism (i.e., Cushing syndrome) and congenital adrenal hyperplasia may produce secondary erythrocytosis mediated by excess androgens, which stimulate erythropoiesis. Skin Eczema and psoriasis • Patients with extensive eczema and psoriasis commonly have anemia. • The anemia is typically normochromic and normocytic (anemia of inflammation), and mild in most cases, but severely affected individuals can have hemoglobin levels less than 9 g/dL. Dermatitis herpetiformis • Macrocytic anemia secondary to malabsorption. • Hyposplenism: Howell!Jolly bodies may be present on blood smear. Dyskeratosis congenita • This disease is characterized by ectodermal dysplasia and aplastic anemia (see Chapter 6: Bone Marrow Failure). • The aplastic anemia is associated with high MCV, thrombocytopenia, and elevated fetal hemoglobin. This may occur before the onset of skin manifestations. Alterations to white blood cells related to organ-specific pathologies Cardiovascular system • Infective endocarditis may present with leukopenia or leukocytosis. Gastrointestinal tract • Peripheral eosinophilia is observed in upwards of 50% of patients with eosinophilic esophagitis. Spleen • Asplenia resulting from splenectomy or patients with functional asplenia may exhibit neutrophilia. Endocrine system • Increased cortisol levels arising from acute stress or Cushing syndrome increase neutrophil counts. Decreased cortisol levels may lead to peripheral eosinophilia. • Obesity has been shown to cause isolated leukocytosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Alterations to platelets and coagulation related to organ-specific pathologies 13 Skin Mast cell disease • Mast cell disease or mastocytosis is associated with an abnormal accumulation of mastocytes (more closely related to monocytes or macrophages rather than to basophils) in the dermis (cutaneous mastocytosis) or in an internal organ (systemic mastocytosis). • The systemic form is rare in children. • In children, this condition is more common under 2 years of age. • It typically presents either as a solitary cutaneous mastocytoma or more commonly, as urticaria pigmentosa. Involvement beyond the skin is unusual in children, but splenomegaly and bone lesions have been reported. • No reports of bone marrow disease in either acquired or congenital mastocytosis have been reported. Alterations to platelets and coagulation related to organ-specific pathologies Cardiovascular system • Prosthetic valves or synthetic patches may cause thrombocytopenia secondary to platelet adhesion to abnormal surfaces as well as microangiopathic hemolysis. • Dysfunctional prosthetic valves may cause an acquired von Willebrand syndrome. However, this is rare, and acquired von Willebrand syndrome appears more commonly with congenital heart defects. In both cases, abnormal shear forces permit cleavage of high molecular weight von Willebrand factor multimers. • Infective endocarditis is a rare cause of thrombocytopenia. • A coagulopathy exists in some patients with cyanotic CHD. The coagulation abnormalities correlate with the extent of the erythrocytosis. Hyperviscosity may lead to tissue hypoxemia, which may trigger DIC. Cyanotic CHD may also cause thrombocytopenia and aberrant platelet aggregation. • Marked derangements in coagulation such as DIC, thrombocytopenia, thrombosis, and fibrinolysis can accompany surgery involving cardiopulmonary bypass. Heparinization must be strictly monitored. • Patients with DiGeorge syndrome (chromosome 22q11.2 deletion) can have platelet abnormalities, including a Bernard!Soulier-like syndrome due to haploinsufficiency of the gene for GP1BB, and thrombocytopenia due to autoimmunity. • Vascular tumors such as tufted angioma and kaposiform hemangioendothelioma are associated with a rare coagulopathy, Kasabach!Merritt phenomenon. The abnormal vasculature housed within these tumors generates a hypercoagulable state resulting in severe thrombocytopenia, hypofibrinogenemia, and possible anemia (see Chapter 12: Disorders of Platelets, and Chapter 14: Vascular Anomalies). Gastrointestinal tract • H. pylori infections are associated with autoimmune thrombocytopenia and platelet aggregation defects (adenosine diphosphate [ADP]-like defect). • Cystic fibrosis produces malabsorption of fat-soluble vitamins (e.g., vitamin K) with impaired prothrombin production. TABLE 2.2 Coagulation abnormalities in liver disease. Hemorrhage Thrombosis (1) Thrombocytopenia/platelet dysfunction due to hypersplenism, altered TPO production (1) Decreased anticoagulant—AT-III proteins C and S (2) Decreased liver synthesis of procoagulant factors (2) Portal hypertension!portal vein thrombosis (3) Impaired carboxylation of vitamin K factors (4) Dysfibrinogenemia (5) Hyperfibrinolysis due to increased tPA and decreased PAI, α2 antiplasmin Abbreviations: PAI, Plasminogen activator inhibitor; tPA, tissue plasminogen activator; TPO, thrombopoietin. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 14 2. Hematologic manifestations of systemic illness TABLE 2.3 Tests to differentiate coagulopathies of different etiologies. Procoagulant factors Liver Vitamin K DIC Factor V Decreased (late) Normal Decreased Factor VII Decreased (early) Decreased Decreased Factor VIII Normal/increased Normal Decreased Abbreviation: DIC, Disseminated intravascular coagulation. Liver Thrombocytopenia • The liver represents the predominant source of thrombopoietin (TPO), and therefore, thrombocytopenia is commonly encountered in liver disease. • Thrombocytopenia in pediatric liver transplant patients portends adverse outcomes highlighting the importance of hepatic TPO on bone marrow megakaryopoiesis. Coagulation abnormalities • The liver is involved in the synthesis of most of the coagulation factors. Liver dysfunction can be associated with either hyper- or hypocoagulable states because both procoagulant and natural anticoagulant syntheses are impaired. Table 2.2 lists the various coagulation abnormalities seen in liver disease, and Table 2.3 lists the tests to differentiate between the coagulopathy of liver disease and other etiologies (see Chapter 13: Disorders of Coagulation). • Factor I (fibrinogen) ! Fibrinogen levels are generally normal in liver disease. Low levels may be seen in fulminant acute liver failure. • Factors II, VII, IX, and X (vitamin K!dependent factors) ! These factors are reduced in liver disease secondary to impaired synthesis. Factor VII is the most sensitive. • Factor V ! Factor V does not require vitamin K for synthesis, and is highly representative of actual liver function. ! Factor V levels at 36 hours post!liver injury have been used as a stand-alone marker for the possible need for transplant in patients with early liver failure. • Factor VIII ! The procoagulant activity of factor VIII is generally normal in liver disease, which permits use of factor VIII levels as an important diagnostic mechanism to distinguishing between DIC and severe liver disease in a patient with abnormal coagulation tests and thrombocytopenia. If there is associated DIC, factor VIII will be markedly depressed, whereas in severe liver disease factor VIII remains close to or normal. Traditionally, factor VII, factor V, and factor VIII levels are measured along with prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen to distinguish liver disease from DIC. • Protein C, protein S, and antithrombin III ! These natural anticoagulants are decreased in liver disease, and proteins C and S are the most sensitive to vitamin K deficiency. ! In many cases, the fall in natural anticoagulant levels creates a sensitive balance between loss of procoagulant activity and natural anticoagulant activity. Bleeding or thrombosis may appear quickly when additional illness (e.g., infection) upsets this balance. • Tissue plasminogen activator (tPA) and alpha-2-antiplasmin ! tPA is cleared by the liver, and as liver disease progresses persistent tPA activity increases. ! Alpha-2-antiplasmin is also suppressed by liver disease, creating increased plasmin activity and ultimately, the syndrome of hyperfibrinolysis with a tendency toward severe bleeding. • α2-Macroglobulin and plasmin activator inhibitor ! These opponents of plasmin activity are still present in liver disease. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Viral and bacterial illnesses associated with marked hematologic sequelae 15 Skin Ehlers!Danlos syndrome • This condition may be associated with platelet dysfunction: reduced aggregation with ADP, epinephrine, and collagen. • An unusual sensitivity to aspirin is described in type IV Ehlers!Danlos syndrome. General considerations for the hematologic sequelae of infection Anemia • Acute infection, particularly viral infection, can produce transient bone marrow aplasia or selective transient erythrocytopenia. • Chronic infection is associated with the anemia of inflammation. • Many viral and bacterial illnesses may be associated with hemolysis. White cell alterations • Viral infections can produce leukopenia and neutropenia. • Neutrophilia with an increased band count and left shift frequently results from bacterial infection. • Neonates, particularly premature infants, may not develop an increase in white cell count in response to infection. Neonatal neutropenia may be serious and requires investigation and treatment. Granulocyte colonystimulating factor (G-CSF) has been found to be helpful in randomized clinical trials. • Eosinophilia may develop in response to parasitic infections. Clotting abnormalities • Severe infections, for example, Gram-negative sepsis, can produce DIC. • Polymicrobial sepsis (including both aerobic and anaerobic organisms) in the head and neck region may cause thrombosis of major vessels. • When this occurs in the jugular veins, it leads to a constellation of findings called Lemierre’s syndrome (suppurative thrombophlebitis with inflammation starting in the pharynx and spreading to the lateral parapharyngeal tissues in association with jugular vein thrombosis). Thrombocytopenia • Infection can produce thrombocytopenia through decreased marrow production, immune destruction, or DIC. Viral and bacterial illnesses associated with marked hematologic sequelae Parvovirus • Parvovirus B19 has a peculiar predilection for both erythroid progenitors and precursors with the highest tropism for colony forming unit-erythroid and early precursors (proerythroblast and basophilic erythroblast) in the bone marrow. • High tropism for the red cell lineage is attributed to the expression of P antigen, the primary receptor for parvovirus B19, and associated coreceptors on these stages of erythroid differentiation. • Bone marrow aspirate shows decreased or arrested maturation of erythroid precursors and the pathognomonic “giant pronormoblasts.” • Parvovirus infection is associated with a transient erythroblastopenic crisis, particularly in individuals with an underlying hemolytic disorder such as sickle cell disease or hereditary spherocytosis. • Erythroblastopenic crisis can produce a rapid fall in hemoglobin with anemia and reticulocytopenia; there may be an associated neutropenia and less commonly, thrombocytopenia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 16 2. Hematologic manifestations of systemic illness • Parvovirus B19 may produce thrombocytopenia, neutropenia, and a hemophagocytic syndrome. • In immunocompromised individuals, parvovirus B19 infection can produce prolonged aplasia. Epstein!Barr virus • Epstein!Barr virus (EBV) infection is associated with: • Atypical lymphocytosis • Acquired immune hemolytic anemia • Agranulocytosis • Lymphadenopathy and splenomegaly • Immune thrombocytopenia • Hepatitis and uncommonly, hyperbilirubinemia • Aplastic anemia, rarely • EBV infection also has immunologic and oncologic associations (see Chapter 16: Lymphoproliferative Disorders). • A list of EBV-associated lymphoproliferative disorders are given in Table 2.4. TORCHES infections • This is a group of congenital infections, including: • Toxoplasma • Rubella • Cytomegalovirus (CMV) • Herpes simplex virus • Syphilis • All of these congenital infections may cause neonatal anemia, jaundice, thrombocytopenia, and hepatosplenomegaly. • These infections possess significant sequelae, and therefore, prevention through prenatal screening and early identification and treatment are required. Salmonella typhi • Typhoid fever usually produces profound leukopenia and neutropenia in the initial stages of the illness, and is often accompanied by thrombocytopenia. • Bone marrow examination may show marrow suppression as well as hemophagocytosis. • Diminished absolute eosinophil counts may be a clue to the diagnosis. TABLE 2.4 EBV-associated lymphoproliferative disorders. EBV-associated B-cell lymphoproliferative disorders 1. Classic Hodgkin lymphoma 2. Burkitt lymphoma 3. Posttransplantation lymphoproliferative disorders 4. HIV-associated lymphoproliferative disorders a. Primary CNS lymphoma b. Diffuse large B-cell lymphoma, immunoblastic c. HHV-8-positive primary effusion lymphoma d. Plasmablastic lymphoma EBV-associated T/NK-cell lymphoproliferative disorders 1. Peripheral T-cell lymphoma, unspecified 2. Angioimmunoblastic T-cell lymphoma 3. Extranodal nasal T/NK-cell lymphoma Abbreviations: EBV, Epstein!Barr virus; HHV-8, human herpes virus-8; NK, natural killer. Modified from Carbone A, Gloghini A, Dotti G., 2008. EBV-associated lymphoproliferative disorders: classification and treatment. Oncologist. 13(5):577!85. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Viral and bacterial illnesses associated with marked hematologic sequelae 17 Acute infectious lymphocytosis • Acute infectious lymphocytosis is caused by a coxsackievirus and is a rare benign, self-limiting childhood condition. • It is associated with a low-grade fever, diarrhea, and marked lymphocytosis (50,000/µL). Lymphocytes are mainly CD4 T cells. • The condition resolves in 2!3 weeks without treatment. Bartonellosis • Bartonellosis is caused by a Gram-negative bacillus, Bartonella bacilliformis, confined to the mountain valleys of the Andes. • The vector is a local sand fly. • Oroya fever is a fatal syndrome of severe hemolytic anemia with fever caused by B. bacilliformis. • Bartonella henselae causes cat scratch fever. It is associated with a regional lymphadenitis, and thrombocytopenia may occur in this condition. Tuberculosis • Tuberculosis is caused by Mycobacterium tuberculosis. • Hematologic manifestations include leukemoid reaction mimicking chronic myelogenous leukemia (CML), monocytosis, and rarely, pancytopenia from diffuse granulomatous marrow infiltration (often associated with leukoerythroblastosis). Leptospirosis (Weil disease) • This disease is caused by a leptospira, Leptospira icterohemorrhagiae. • A coagulopathy occurs which is complex and can be corrected with vitamin K administration. • Thrombocytopenia commonly occurs, which may predispose patients to bleeding. Severe acute respiratory syndrome coronavirus 2 • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive stranded RNA virus responsible for the coronavirus disease 2019 pandemic. • SARS-CoV-2 binds to angiotensin converting enzyme 2, and Transmembrane Serine Protease 2 (TMPRSS2), a serine protease that cleaves viral spike protein, permitting entry into the cell. • In the pediatric population, presentation is variable with upper respiratory infection and pneumonia symptomology, GI symptoms without respiratory symptoms, and cutaneous manifestations. • Typically, pediatric disease is less severe than in adults. • Hematologic manifestations include leukopenia, lymphopenia, and elevated inflammatory markers. In adults, SARS-CoV-2 is associated with a hypercoagulable state. • There are limited reports of a similar hypercoagulability in children, and therefore, anticoagulation therapy should be initiated only in cases where there is a high clinical suspicion for thrombosis. • A severe sequela of SARS-CoV-2 in children is a Kawasaki disease!like condition referred to as multisystem inflammatory syndrome in children (MIS-C) associated with elevated inflammatory markers, lymphopenia, and thrombocytopenia. Human immunodeficiency virus • The main pathophysiology of human immunodeficiency virus (HIV) infection is a constant decline in CD4 lymphocytes due to HIV tropism for this T lymphocyte subset, which leads to immune failure and death. Other hematopoietic lineages are also affected as HIV disease [acquired immunodeficiency syndrome (AIDS)] progresses. • HIV infection possesses numerous hematologic and oncologic manifestations that are discussed later. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 18 2. Hematologic manifestations of systemic illness • At presentation of AIDS, lymphopenia is the most frequent cytopenia, followed by neutropenia, anemia, and thrombocytopenia. Thrombocytopenia • Thrombocytopenia incidence varies among studies from 10 to 14% possibly related to comorbidities. • Initially, the clinical findings resemble those of immune ITP. Some degree of splenomegaly is common, and the platelet-associated antibodies are often in the form of immune complexes that may contain antibodies with anti-HIV specificity. • Megakaryocytes are normal or increased, and production of platelets is reduced in the bone marrow. • Thrombotic thrombocytopenia purpura (TTP) is also associated with HIV disease. This occurs in advanced AIDS. Anemia and neutropenia • HIV-infected individuals develop progressive cytopenia as immunosuppression advances. • Anemia occurs in approximately 70!80% of patients and neutropenia in 50%. • Cytopenias in advanced HIV disease are often of complex etiology and include the following: • A production defect in the marrow appears to be the most common. • Antibody and immune complexes associated with red and white cell surface antigens may contribute. ! Up to 40% have erythrocyte-associated antibodies with specific antibodies against i and U antigens occasionally have been noted. ! Approximately 70% of patients with AIDS have neutrophil-associated antibodies. • Infections: myelosuppression is frequently caused by the involvement of the bone marrow by infecting organisms (e.g., mycobacteria, CMV, parvovirus, fungi, and, rarely, Pneumocystis jirovecii). • Neoplasms: non-Hodgkin lymphoma (NHL) in AIDS patients is associated with infiltration of the bone marrow in up to 30% of cases. This is particularly prominent in the small noncleaved histologic subtype of NHL. • Medications: widely used antiviral agents in AIDS patients are myelotoxic, which is related to drug dose and progression of HIV disease. ! Zidovudine (AZT) causes anemia in approximately 29% of patients. ! Ganciclovir and trimethoprim/sulfamethoxazole or pyrimethamine/sulfadiazine may cause neutropenia. ! Importantly, the other nucleoside analogs of anti-HIV compounds (e.g., dideoxycytidine, dideoxyinosine, stavudine, or lamivudine) are usually not associated with significant myelotoxicity. • Nutrition: poor caloric intake is common in advanced HIV disease and is occasionally accompanied by poor absorption. ! Vitamin B12 levels may be significantly decreased in HIV infection resulting from malabsorption and abnormalities in vitamin B12-binding proteins. Coagulation abnormalities • Dysregulation of immunoglobulin production may affect the coagulation cascade through antibody-mediated effects. The dysregulation of immunoglobulin production may also occasionally result in beneficial effects, as in the resolution of antifactor VIII antibodies in HIV-infected patients with hemophilia. • Lupus-like anticoagulant (antiphospholipid antibodies) or anticardiolipin antibodies occur in 82% of patients. The titers or specificities have not led to thrombosis in most patients. • Low levels of protein S occur in 73% of patients, which may predispose HIV patients to thrombosis. Role of hematopoietic growth factors in treatment of AIDS-associated cytopenia • rHuEPO results in a significant improvement in the Hct and reduces transfusion requirements while the patient receives AZT. • Newer antiretroviral therapy protocols do not lead to severe anemia, and rHuEPO is used primarily for its well-known indication in renal dialysis. • G-CSF in a dose of 5 mg/kg/day SC is the most widely used growth factor for neutropenia. • Granulocyte-macrophage colony stimulating factor (GM-CSF) at doses starting at 250 µg/day is also effective. Yet, GM-CSF possesses more side effects than G-CSF, and therefore, is used less frequently. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Viral and bacterial illnesses associated with marked hematologic sequelae 19 TABLE 2.5 AIDS-related neoplasms in children. 1. Classic Hodgkin lymphoma (lymphocyte depleted) 2. Non-Hodgkin lymphoma a. Burkitt lymphoma b. Central nervous system lymphoma c. Diffuse large B-cell lymphoma d. MALT-type lymphoma 3. Leiomyoma and leiomyosarcoma 4. Kaposi’s sarcoma 5. Acute leukemias 6. Miscellaneous tumors—isolated cases of hepatoblastoma, fibrosarcoma of liver, embryonal rhabdomyosarcoma of the biliary tree, and Ewing’s tumor of the bone Abbreviation: MALT, Mucosa-associated lymphoid tissue. Modified from Balarezo FS, Joshi VV., 2002. Adv Anat Pathol. 9(6):360!70. TABLE 2.6 Spectrum of systemic lymphoproliferative lesions in children with AIDS. 1. Hyperplasia involving a. Lymph nodes b. Peyer’s patches of the ileum c. Lymphoid nodules in the esophagus and colon d. Thymus e. PLH f. Lymphoplasmacytic infiltrates in g. Lungs (LIP) h. Salivary glands i. Liver j. Thymitis and multilocular thymic cyst 2. Polyclonal PBLD involving a. Lungs b. Liver, spleen, and lymph nodes c. Kidneys d. Salivary glands e. Muscle, periadrenal fat f. Myoepithelial sialadenitis g. Myoepithelial sialadenitis with focal lymphoma h. MALT lymphoma (involving nodal and extranodal sites) i. Non-MALT lymphoma (involving nodal and extranodal sites) Abbreviations: LIP, Lymphoid interstitial pneumonitis; MALT, mucosa-associated lymphoid tissue; PBLD, polymorphic B-cell lymphoproliferative disorder; PLH, pulmonary lymphoid hyperplasia. Modified from Balarezo FS, Joshi VV., 2002. Adv Anat Pathol. 9(6):360!70. Cancers in children with human immunodeficiency virus infection • Malignancies in children with HIV infection are not as common as in adults. Table 2.5 lists the AIDS-related neoplasms in children with HIV infection and Table 2.6 lists the spectrum of lymphoproliferative lesions in children with AIDS. HIV-associated lymphoma • NHL is the most common malignancy secondary to HIV infection in children. • It is usually of B-cell origin as in Burkitt’s (small noncleaved cell) or immunoblastic (large cell) NHL. • The mean age at presentation of malignancy in congenitally transmitted disease is 35 months, with a range of 6!62 months. • In transfusion-transmitted disease, the latency from the time of HIV seroconversion to the onset of lymphoma is 22!88 months. • The CD4 lymphocyte count is less than 50/µL at the time of diagnosis of the malignancy. • The presenting manifestations include: • Fever, weight loss, and extranodal manifestations [e.g., hepatomegaly, jaundice, abdominal distention, bone marrow involvement, or central nervous system (CNS) symptoms]. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 20 2. Hematologic manifestations of systemic illness • Some patients will already possess lymphoproliferative diseases such as lymphocytic interstitial pneumonitis or pulmonary lymphoid hyperplasia; these children usually have advanced (stage III or IV) disease at the time of presentation. • Children with CNS lymphomas present with developmental delay, loss of developmental milestones, or encephalopathy (dementia, cranial nerve palsies, seizures, or hemiparesis). • Differential diagnosis includes infections such as toxoplasmosis, cryptococcosis, or tuberculosis. • Contrast-enhanced computed tomography studies of the brain show hyperdense mass lesions that are usually multicentric or periventricular. • CNS lymphomas in AIDS are fast growing and often have central necrosis and a “rim of enhancement” as in an infectious lesion. • A stereotactic biopsy will provide a definitive diagnosis. • Treatment consists of standard protocols as described in Chapter 21, Non-Hodgkin Lymphoma, on NHL. • In addition, a concomitant approach to improving HIV viral load is critical in achieving positive survival outcomes in infected patients. • Treatment of CNS lymphomas is more difficult. Intrathecal therapy is indicated even for those without evidence of meningeal or mass lesions at diagnosis of NHL. • Radiation therapy may be a helpful adjunct for CNS involvement. • The following are more favorable prognostic features in NHL secondary to AIDS: • CD4 lymphocyte count above 100/µL, • Normal serum LDH level, • No prior AIDS-related symptoms, and • Good Karnofsky score (80!100). Proliferative lesions of mucosa-associated lymphoid tissue • Mucosa-associated lymphoid tissue (MALT) shows reactive lymphoid follicles with prominent marginal zones containing centrocyte-like cells, lymphocytic infiltration of the epithelium (lymphoepithelial lesion), and the presence of plasma cells under the surface epithelium. • These lesions may be associated with the mucosa of the GI tract, Waldeyer’s ring, salivary glands, respiratory tract, thyroid, and thymus. • Proliferative lesions of MALT can be benign or malignant (i.e., lymphomas), and represent a continuum extending from reactive to neoplastic lesions. • Neoplastic lesions are usually of low grade, but may progress into high-grade MALT lymphomas. • MALT lymphomas characteristically remain localized, but if dissemination occurs, they are usually confined to the regional lymph nodes and other MALT sites. • MALT lesions represent a category of pediatric HIV-associated disease that may arise from a combination of viral etiologies, including HIV, EBV, and CMV. • Treatment of low-grade MALT lymphoma include: • α-Interferon: 1 million units/m2 SC three times a week (continued until regression of disease or severe toxicity occurs) • Rituximab (monoclonal antibody-anti-CD20): 375 mg/m2 IV weekly for 4 weeks (courses may be repeated as clinically indicated). Some patients may not require any treatment because of the indolent nature of the disease. Leiomyosarcomas and leiomyomas • Malignant or benign smooth muscle tumors, leiomyosarcomas (LS) and leiomyomas (LM), respectively, are the second most common type of tumor in children with HIV infection. • The incidence in HIV patients is 4.8% (in non-HIV children, it is 2 per million). • The most common sites of presentation are the lungs, spleen, and GI tract. • Patients with endobronchial LM or LS often have multiple nodules in the pulmonary parenchyma. • Bloody diarrhea, abdominal pain, or signs of obstruction may signal intraluminal bowel lesions. • EBV infection may play a role in the pathogenesis of LS and LM as studies using in situ hybridization and quantitative polymerase chain reaction (PCR) demonstrated high copy numbers of EBV present in these tumors. • The EBV receptor (CD21/C3d) is present on tumor tissue at very high concentrations, but conversely, is present at lower concentrations in normal smooth muscle. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Parasitic illnesses associated with marked hematologic sequelae 21 • In AIDS patients the EBV receptor may be upregulated, allowing EBV to enter the muscle cells and cause their transformation. • Treatment consists of: • complete surgical resection when technically feasible, • radiation therapy, • decreasing viral load in patients if they present with an acceleration, and • possible use of immunomodulators or chemotherapy. Kaposi sarcoma • Kaposi sarcoma (KS) is rare in children, and constitutes the third most common malignancy in pediatric AIDS patients. Of note, it occurs in 25% of adults with AIDS. • KS occurs only in those HIV-infected children who were born to mothers with HIV. • The lymphadenopathic form of KS is seen mostly in Haitian and African children and may represent the epidemic form of KS unrelated to AIDS. • The cutaneous form is a true indicator of the disease related to AIDS. • Visceral involvement has not been pathologically documented in children with AIDS. • With early highly active antiretroviral treatment intervention, the incidence of KS is falling as KS is an AIDS-defining cancer. Leukemias • Leukemia represents the fourth most common malignancy in children with AIDS. • HIV-associated leukemia is predominantly of B-cell origin. • The clinical presentation and biologic features are similar to those found in non-HIV children. • Treatment involves chemotherapy designed for B-cell leukemia and lymphomas as well as lowering viral load where necessary. Miscellaneous tumors • There is no increase in Hodgkin disease in children with AIDS. • Children with AIDS rarely develop hepatoblastoma, embryonal rhabdomyosarcoma, fibrosarcoma, and papillary carcinoma of the thyroid suggesting that the occurrence of these tumors is likely unrelated to the HIV infection. Parasitic illnesses associated with marked hematologic sequelae Malaria • The etiology of anemia in acute infections is multifactorial: • Intracellular parasite metabolism alters negative charges on the RBC membrane, which causes altered permeability with increased osmotic fragility. • Spleen removes the damaged RBC or the parasites are “pitted” during their passage through the spleen, which results in microspherocytes of RBC. • Autoimmune hemolytic anemia may also occur. An immunoglobulin G (IgG) antibody is formed against the parasite, and the resulting immune complex attaches nonspecifically to RBC thereby activating complement. Red cell destruction ensues. ! Positive Coombs test due to IgG is found in 50% of patients with Plasmodium falciparum malariae. • Thrombocytopenia without DIC is common. IgG antimalarial antibody binds to the platelet-bound malaria antigen, and the IgG platelet parasite complex is removed by the reticuloendothelial system. • Exchange transfusion has been used in severe cases. Babesiosis • Babesiosis is caused by several species from the genus Babesia that colonizes erythrocytes. It is a zoonotic disease transmitted by the Ixodes tick, and has similar clinical features to malaria. • The clinical features include fever, myalgia, and arthralgia with hepatosplenomegaly and hemolysis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 22 2. Hematologic manifestations of systemic illness • Blood smear may reveal intraerythrocytic trophozoites arranged in the form of a “Maltese cross.” • Hemolysis can be very severe and require exchange transfusion especially in sickle cell disease, other asplenic patients, and immunosuppressed patients. Leishmaniasis • The protozoal species Leishmania causes progressive splenomegaly and subsequent pancytopenia (anemia, neutropenia, and thrombocytopenia). • The bone marrow is usually hypercellular with hemophagocytosis. Some children may possess a coagulopathy. Hookworm • Worldwide hookworm is a major cause of IDA. • Two species infest humans: • Ancylostoma duodenale is found in the Mediterranean region, North Africa, and the west coast of South America. • Necator americanus is found in most of Africa, Southeast Asia, Pacific islands, and Australia. • Hookworms penetrate exposed skin, usually soles of bare feet, and migrate through the circulation to the right side of the heart, then lungs (causing hypereosinophilic syndrome), through the airway down to the esophagus. They mature in the small intestine and attach their mouthparts to the mucosa. • Each adult A. duodenale consumes about 0.2 mL/day, and therefore, heavily infested children may present with profound IDA, hypoproteinemia, and marked eosinophilia. Tapeworm • Diphyllobothrium latum is a fish tapeworm that is acquired by eating uncooked freshwater fish. • This worm infestation in the intestine results in vitamin B12 deficiency. Trypanosomiasis • This disease may cause immune-mediated anemia and less often, thrombocytopenia and neutropenia. • Early diagnosis is important for survival, and trypanosomes are more likely to be seen early in the illness on classic thick smears. Hemolytic uremic syndrome • Hemolytic uremic syndrome (HUS) describes the classical clinical triad of hemolytic anemia, thrombocytopenia, and acute kidney injury resulting from a thrombotic microangiopathy. • Although clinically defined by this triad, HUS itself consists of a number of subtypes that reflect the various pathogenic mechanisms driving HUS development. The differential diagnosis based on HUS subgroup is detailed next. • Infection-induced HUS: shiga toxin!producing Escherichia coli (STEC), Streptococcus pneumoniae, influenza A, H1N1, and HIV. Of note, STEC-induced HUS is the most common cause of pediatric HUS representing 85!90% of cases. • Atypical HUS: diacylglycerol kinase ε (DGKE) and complement mutations (CFH, CFI, MCP, C3, CFB, THBD), complement inhibitor antibodies (anti-CFH), or HUS of unknown etiology. • Cobalamin C HUS due to defects in cobalamin metabolism. • Secondary HUS: hematopoietic stem cell (HSC) or solid organ transplantation, autoimmune disease, underlying nephropathy, malignant hypertension, malignancy, and drugs. • Thrombotic microangiopathy can be seen post stem cell transplant. • TTP should be considered in the differential of HUS. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Autoimmune disease 23 • The pathophysiology of HUS is multifactorial due to the diverse upstream mechanisms that underlie each subgroup of HUS. Yet, all forms of HUS ultimately activate complement and cause injury to the renal microvasculature, which induces procoagulant and inflammatory pathways in endothelial cells. • HUS manifests clinically as oliguria, pallor, dyspnea, and fatigue, and in pediatric populations, encephalopathy may occur in upward of 30% patients. • Historical information, including age of onset and preceding events, may help to narrow underlying cause of HUS. ! Age of onset is variable, particularly, among cases of complement-mediated atypical HUS. Conversely, infection-induced HUS typically occurs in younger children, and atypical HUS arising from DGKE mutations presents within the first year of life. ! HUS symptomology following bloody diarrheal illness suggests STEC as an underlying etiology, whereas pulmonary infection or meningitis may indicate S. pneumoniae. ! HUS in the setting of acute stressors (e.g., illness, immunization, medications) favors atypical forms of the disease. • Initial laboratory evaluation for suspected HUS includes complete blood count (CBC) and blood smear, LDH, haptoglobin, BMP, and urinalysis. • Laboratory findings indicative of HUS include thrombocytopenia and anemia with shistocytes on peripheral smear, elevated blood urea nitrogen (BUN) and creatinine with possible proteinuria and hematuria. • Following the diagnosis of HUS, further testing is required to elucidate the underlying etiology as clinically indicated. ! Infection-induced: stool studies and shiga toxin PCR, S. pneumoniae antigen/PCR testing, and direct antiglobulin test (DAT). ! Colbalamin defects: methylmalonic acid and homocysteine levels. ! Atypical: DGKE sequencing and/or plasma C3, C4, CFH, and CFI levels. Sequencing for complement mutations could be considered as well. ! To rule out TTP, obtain plasma a disintegrin and metalloprotease with thrombospondin type 1 repeats13 (ADAMST13) activity. • Treatment is dictated by the pathogenic mechanism underlying HUS development. • Acute management of infection-induced HUS is largely supportive, including hemodynamic monitoring and ensuring adequate fluid status. In addition to supportive measures, S. pneumoniae!induced HUS should also be treated with amoxicillin or third-generation cephalosporin (e.g., S. pneumoniae meningitis). • Hydroxocobalamin, folinic acid, betaine, and carnitine treat HUS resulting from defective colbalamin metabolism. • Atypical HUS is treated with plasma exchange or eculizumab. • Eculizumab, a humanized monoclonal antibody that targets complement C5 protein thereby inhibiting terminal complement (C5!9) activation, has been used in both atypical and STEC-induced HUS. ! A number of clinical trials in adults demonstrate that eculizumab improves outcomes in atypical HUS not caused by DGKE mutation. ! A subsequent study in children provide evidence that eculizumab is well tolerated and also improves platelet counts and glomerular filtration rate (GFR) in atypical HUS. ! Eculizumab has also been used for STEC-induced HUS with severe neurologic sequelae. Despite its efficacy in atypical HUS, the evidence for its use in STEC-HUS is equivocal with benefits restricted to a limited number of patients. Additional studies are needed as large, controlled trials are currently lacking. ! An adverse effect of eculizumab, particularly with long-term use, is increased risk of meningococcal meningitis. Therefore, immunization and antibiotic prophylaxis should be undertaken during treatment. Autoimmune disease Rheumatoid arthritis • Hematologic manifestations of rheumatoid arthritis (RA) include: • anemia of inflammation (normocytic, normochromic); • high incidence of iron deficiency; • leukocytosis and neutropenia common in exacerbations of juvenile RA (JRA); and Lanzkowsky’s Manual of Pediatric Hematology and Oncology 24 2. Hematologic manifestations of systemic illness • thrombocytosis associated with a high level of interleukin (IL)-6 occurs in many patients, although there may be transient episodes of thrombocytopenia. Felty syndrome • Characterized by the triad of RA, splenomegaly, and neutropenia. • Patients may be at risk for life-threatening bacteremia. • Splenic dysfunction resulting in infection with encapsulated organisms has been observed. • Treatment involves controlling the RA, antibiotics, and growth factors: • Controlling RA often leads to anemia improvement. • Administration of parenteral antibiotics with coverage for encapsulated organisms for febrile episodes is recommended. • G-CSF may be used in urgent situations. However, case reports of spontaneous splenic rupture in Felty syndrome exist, raising concerns for G-CSF-induced splenic rupture. Systemic lupus erythematous • Hematologic manifestations of systemic lupus erythematous (SLE) include: • Anemia of inflammation (normocytic, normochromic) and acquired, autoimmune DAT-positive hemolytic anemia; both of these are common types of anemia encountered in SLE patients. • Neutropenia is common as a result of decreased marrow production and immune-mediated destruction. • Lymphopenia with abnormalities of T-cell function. • Immune thrombocytopenia. • Antiphospholipid antibodies may be present, which prolong the activated partial thromboplastin time (aPTT), but are associated with severe thrombosis (lupus anticoagulant). Polyarteritis nodosa • Microangiopathic hemolytic anemia may be associated with renal disease or hypertensive crises. • Prominent eosinophilia. Wegener granulomatosis • This autoimmune disorder is rare in children. Hematological features include: • anemia: normocytic; RBC fragmentation with microangiopathic hemolytic anemia, • leukocytosis with neutrophilia, • eosinophilia, and • thrombocytosis. Kawasaki syndrome • This syndrome is characterized by: • mild normochromic, normocytic anemia with reticulocytopenia; • leukocytosis with neutrophilia and toxic granulation of neutrophils and vacuoles; • decreased T-suppressor cells; • high C3 levels; • increased cytokines IL-1, IL-6, IL-8, interferon-α (IFN-α), and tumor necrosis factor (TNF); • marked thrombocytosis (mean platelet count 700,000/µL); and • DIC. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Anemia of inflammation 25 FIGURE 2.2 Overview of the pathophysiology of anemia of inflammation. Inflammation, infection, and cell injury induce the release of cytokines, DAMP, and PAMP that alter processes essential for physiologic erythropoiesis. These inflammatory molecules increase myeloid commitment of hematopoietic stem cells, apoptosis of erythroid progenitors and precursors, iron sequestration affecting hemoglobinization in erythroid precursors, and erythrophagocytosis, and conversely, inflammation inhibits renal production of EPO leading to reduced survival of EPO-sensitive erythroid progenitors and precursors. Abbreviations: BFU, burst forming unit; CFU, colony forming unit; DAMP, Damage-associated molecular patterns; EPO, erythropoietin; HMGB, high mobility group box; HSC, hematopoeitic stem cell; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; PAMP, pathogenassociated molecular patterns; RBC, red blood cell; TNF, tumor necrosis factor. Henoch!Schönlein purpura • Henoch!Schönlein purpura (HSP), also called anaphylactoid purpura, is associated with systemic vasculitis characterized by unique palpable, erythema multiforme!like purpuric lesions, transient arthralgias or arthritis (especially affecting knees and ankles), colicky abdominal pain, and nephritis. • Anemia occasionally occurs as a result of GI bleeding or decreased RBC production caused by renal failure. • Transient decreased factor XIII activity may occur, which may play a role in either GI bleeding or HSP. • Vitamin K deficiency from severe vasculitis-induced intestinal malabsorption has been reported. Anemia of inflammation • Anemia of inflammation (previously referred to as the anemia of chronic disease) is the anemia that arises from inflammation in the setting of chronic illness or prolonged inflammatory processes such as cancer, IBD, autoimmune diseases, chronic infection, and sepsis. • Anemia develops downstream of inflammatory cytokine signaling that induces multiple pathogenic mechanisms, including altered iron homeostasis, increased erythrophagocytosis by reticuloendothelial macrophages, reduced kidney EPO production, and direct inhibition of erythropoiesis (Fig. 2.2). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 26 2. Hematologic manifestations of systemic illness TABLE 2.7 Laboratory tests to differentiate anemia of chronic disease from iron-deficiency anemia. Variable (serum levels) Anemia of inflammation Iron-deficiency anemia Anemia of inflammation and iron-deficiency anemia Iron Reduced Reduced Reduced Transferrin Reduced to normal Increased Reduced Transferrin saturation Normal to mildly reduced Reduced Reduced Ferritin Normal to increased Reduced Reduced to normal sTfR Normal Increased Increased sTfR index Normal Increased Increased Signs of inflammation Present Absent Present Abbreviation: sTfR, Soluble transferrin receptor. TABLE 2.8 Therapeutic options for the treatment of anemia of chronic disease. Treatment Anemia of chronic disease Anemia of chronic disease with true iron deficiency Treatment of underlying disease Yes Yes Transfusionsa Yes Yes Iron supplementation Nob Yes Erythropoietin agents Yes Yes, in patients who do not have a response to iron therapy a This treatment is for the short-term correction of severe or life-threatening anemia. Potentially adverse immunomodulatory effects of blood transfusions are controversial. Although iron therapy is indicated for the correction of anemia of chronic disease in association with absolute iron deficiency, no data from prospective. b • Altered iron homeostasis occurs through hepcidin and erythroferrone signaling pathways. IL, particularly IL-6 along with endotoxin, induce hepcidin synthesis in the liver. ! Hepcidin binds to ferroportin, an Fe-exporter, expressed in the GI tract and reticuloendothelial system leading reduced plasma iron levels and increased iron sequestration. ! Typically, in patients with anemia of inflammation the serum iron will be low, but there will also be a low level of transferrin iron-binding capacity secondary to a suppression of protein synthesis. This leads to normal or slightly diminished iron saturation. Serum ferritin is paradoxically elevated secondary to the hepcidin-induced sequestration and inflammation itself. ! Increased erythrophagocytosis by reticuloendothelial macrophages. ! Inhibition of EPO release from the kidney [especially by IL-1β and TNF-alpha (TNF-α)] leads to reduced EPO-stimulated hematopoietic proliferation. ! Direct inhibition of the proliferation of erythroid progenitors as well as hematopoietic stem and progenitor lineage skewing from erythroid to myelomonocytic lineages (TNF-α, IFN-γ, and IL-1β). ! Novel inflammatory mediators, including high mobility group box protein 1 and IL-33, have been identified as causative factors in the development of anemia of inflammation in preclinical animal models. • The anemia has the following characteristics: • normochromic, normocytic, occasionally microcytic; • usually mild, characterized by decreased plasma iron and normal or increased reticuloendothelial iron; • impaired flow of iron from reticuloendothelial cells to the bone marrow; • decreased sideroblasts in the bone marrow; and • refractory to exogenous and endogenous EPO in some cases. • Laboratory studies for anemia of inflammation diagnosis are listed in Table 2.7 and summarized next: • a CBC and smear; • iron studies, including serum iron, total iron-binding capacity, ferritin, transferrin, and TSAT; and • inflammatory markers (e.g., C-reactive protein and erythrocyte sedimentation rate) are not necessary for diagnosis, but are typically obtained during clinical work up for systemic inflammatory processes. • Diagnostic evaluation of anemia of inflammation also necessitates investigation for a coexisting IDA as its presence carries additional treatment considerations. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Nutritional deficiencies and environmental exposures 27 • Detection of concomitant IDA represents a diagnostic challenge as inflammation alters markers of body iron status. For example, ferritin is an acute phase reactant, and therefore, elevated in anemia of inflammation. • The addition of soluble transferrin receptor (sTfR) and calculation of the sTfR index have been shown to help detect IDA in the setting of the anemia of inflammation (Table 2.7). ! sTfR is elevated in iron deficiency, and unlike other markers of iron status, not influenced by inflammation. ! sTfR index 5 sTfR/log ferritin; this index is also elevated in iron deficient states. ! The use of sTfR index alone carries an 81% sensitivity and 83% specificity for IDA, whereas use of ferritin, sTfR, and sTfR index possesses a high sensitivity but poor specificity (92% and 49%, respectively). ! Elevated sTfR ( . 1.55 mg/L) and sTfR ( . 1.03) index with underlying anemia of inflammation should raise suspicion of concurrent IDA. • Therapeutic options for the treatment of anemia in chronic disease are outlined in Table 2.8. • If sTfR and sTfR index suggest concurrent IDA, an IV iron challenge may be pursued. Parenteral administration provides greater bioavailability compared to oral iron due to impaired intestinal iron absorption resulting from downregulation of ferroportin. If there is a component of IDA, anemia will demonstrate some improvement over 7!10 days. • Novel therapeutic options, including hepcidin signaling axis modulators and anti-bone morphogenic protein (BMP)-6 antibodies, have demonstrated efficacy in preclinical animal models of anemia of inflammation. Inflammatory bowel disease as a model for anemia of inflammation • In both Crohn’s disease and ulcerative colitis, the anemia of chronic illness is often seen—sometimes before GI symptomatology manifests. • It is often associated with concomitant iron deficiency due to bleeding from the involved bowel. • The patient may present with mild normochromic anemia or severe microcytic anemia. • As mentioned earlier, inflammation complicates the identification of IDA. In an older child or adolescent presenting with iron deficiency, a detailed history of GI symptoms must be pursued with particular detail to clinical suggestion of anemia of inflammation. • If sTfR and sTfR index indicate the presence of both IDA and anemia of inflammation: • IV iron preparations are superior to oral preparation as these bypass the intestinal block resulting from the action of hepcidin and also diminish the additional GI toxicity of oral iron. • However, the administration of iron alone may not ameliorate anemia as patients may simultaneously possess a profound inhibitory effect on erythropoiesis; this may require rHuEPO administration. ! It should be noted that there are certain cases of anemia of inflammation (e.g., sepsis, critical illness) that are refractory to exogenous EPO. • These considerations are identical to those faced in other conditions with profound ongoing inflammation (e.g., JRA). Nutritional deficiencies and environmental exposures Protein!calorie malnutrition • Protein deficiency in the presence of adequate carbohydrate caloric intake (kwashiorkor) is associated with mild normochromic, normocytic anemia secondary to reduced RBC production despite normal, or increased EPO levels as well as reduced red cell survival (also see Chapter 4: Nutritional Anemias). • Protein!calorie malnutrition is also associated with impaired leukocyte function. Scurvy • Mild anemia is common. • There is a bleeding tendency due to loss of vascular integrity, which may result in petechiae, subperiosteal, orbital, or subdural hemorrhages. Hematuria and melena may occur. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 28 2. Hematologic manifestations of systemic illness Vitamin B12 deficiency • Vitamin B12 deficiency causes a megaloblastic anemia, which commonly arises in an acquired fashion secondary to malabsorption or reduced dietary intake. Although called “megaloblastic anemia,” the deficiency is more often characterized by anemia, neutropenia, and thrombocytopenia. • Rare causes of vitamin B12 deficiency have been identified and arise from mutations in IF, cubilin (CUBN), or amnionless (AMN) genes. Anorexia nervosa • Anorexia nervosa is associated with hematologic changes which may be helpful in diagnosis: • Red cell morphology is striking for the unusual morphology of acanthocytosis, which results from acquired hypobetalipoproteinemia secondary to nutritional failure. • Mild anemia (macrocytic), neutropenia, and thrombocytopenia. • Mild predisposition to infection associated with neutropenia. • Gelatinous changes of bone marrow that may become severely hypoplastic. Lead intoxication • The most striking hematologic stigmata of lead intoxication is basophilic stippling (coarse basophilia) of red cells. It is caused by precipitation of denatured mitochondria secondary to the inhibition of prymidine-50 nucleotidase. This is a major peripheral blood differentiator of this cause of anemia from iron deficiency and thalassemia trait (which has fine basophilic stippling). • Lead also produces ring sideroblasts in the marrow, and it is associated with hypochromic microcytic anemia and markedly elevated free erythrocyte protoporphyrin levels. Marrow infiltrative disorders • The bone marrow may be infiltrated by nonneoplastic disease (e.g., storage disease), granulomata from infection, sarcoid or rheumatologic disease, or neoplastic disease. These disease processes are discussed later (Table 2.9 lists the diseases that may infiltrate the marrow). • In storage disease a diagnosis is made on the basis of the family history, clinical picture, enzyme assays of white cells or cultured fibroblasts, and bone marrow aspiration revealing the characteristic cells of the disorder. Mutation analysis, where available, is the standard for diagnosis. Differential diagnosis of granulomatous conditions begins with recognition of the granulomas by bone marrow morphology (with TABLE 2.9 Diseases invading bone marrow. 1. Nonneoplastic a. Storage diseases b. Gaucher disease c. Niemann!Pick disease d. Cystine storage disease e. Marble bone disease (osteopetrosis) f. Langerhans cell histiocytosis (see Chapter 20, Hodgkin Lymphoma) 2. Neoplastic a. Primary b. Leukemia (see Chapter 18: Lymphoid leukemias, and Chapter 19: Myeloid leukemias) c. Secondary d. Neuroblastoma (see Chapter 24: Renal Tumors) e. Non-Hodgkin lymphoma (see Chapter 22: Central Nervous System Tumors) f. Hodgkin lymphoma (see Chapter 21: Non-Hodgkin Lymphoma) g. Wilms tumor (rarely) (see Chapter 25: Rhabdomyosarcoma and Other Soft-Tissue Sarcomas) h. Retinoblastoma (see Chapter 27: Retinoblastoma) i. Rhabdomyosarcoma (see Chapter 25: Rhabdomyosarcoma and Other Soft-Tissue Sarcomas) Lanzkowsky’s Manual of Pediatric Hematology and Oncology Marrow infiltrative disorders 29 culture if suspicious at the time of bone marrow examination). Neoplastic disease may arise de novo in the marrow (leukemias) or invade the marrow as metastases from solid tumors (neuroblastoma or rhabdomyosarcoma). Neoplastic disease • In addition to manifestations of marrow infiltration, neoplastic disease can be associated with the following hematologic alterations: • hemorrhage; • nutritional deficiency states; • dyserythropoietic anemias (including erythroid hypoplasia, sideroblastic anemia, and anemia similar to that seen in anemia of inflammation); • defect in EPO production; • hemodilution; • hemolysis; • pancytopenia secondary cytotoxic therapy; • acquired von Willebrand disease as in Wilms tumor; • hypercoagulable states as in NHL; and • coagulopathy as in acute promyelocytic leukemia. • Marrow infiltration may cause pancytopenia and is suspected when leukoerythroblastic anemia develops. • Leukoerythroblastic anemia signifies the presence of myelocytes, normoblasts, and teardrop-shaped red cells with anemia, thrombocytopenia, and neutropenia. • This presentation is due to extramedullary erythropoiesis that occurs when the marrow is infiltrated, permitting the escape of early myeloid and erythroid cells into the circulation. • Normal blood findings, however, do not exclude marrow infiltration. • Bone marrow examination frequently demonstrates infiltration with tumor cells in the presence of pancytopenia. • Since metastatic bone marrow involvement from solid tumors may be patchy, a single aspiration is not diagnostic, and at least two aspirates and two biopsies should be performed. • The hematologic alterations associated with malignancy should be managed supportively, and will resolve if the underlying neoplasms can be successfully treated. Infantile malignant osteopetrosis (marble bone disease) • Osteopetrosis is a hereditary disorder that may be present in either a severe or a mild form. • Severe form: • Inherited in an autosomal recessive manner. • The marrow space is progressively obliterated by excessive osseous growth. • The difficulty in obtaining marrow by aspiration is a diagnostic clue. • Radiologic changes are characteristic and diagnostic, consisting of generalized osteosclerosis. • The cranial foramina progressively narrow resulting in blindness due to optic atrophy, deafness, and other cranial nerve lesion. • The hematologic characteristics include the following: ! progressive pancytopenia due to encroachment on the hematopoietic marrow by the overgrowth of bone; ! compensatory extramedullary hematopoiesis with resultant leukoerythroblastic anemia (circulating normoblasts, teardrop-shaped poikilocytosis, and early myelocytes), hepatosplenomegaly, and lymphadenopathy; ! bone marrow hypoplasia; and ! hemolysis due to splenic sequestration of red cells, and perhaps, general overactivity of the reticuloendothelial system. • Treatment involves allogeneic stem cell transplantation by providing unaffected multipotent HSC, which serve as a source of normal osteoclasts. • Mild form: • Inherited in an autosomal recessive manner. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 30 TABLE 2.10 2. Hematologic manifestations of systemic illness Clinical manifestations of subtypes of Gaucher disease. Type I Type II Type III Characteristic Symptomatic Asymptomatic Infantile Neonatal IIIa IIIb IIIc Most common genotype 1226G compound heterozygous 1226G (IN370S) homozygous None Two null mutations None 1448C (L444P) homozygous 1342C (D409H) homozygous Ethnicity Ashkenazi Jews Ashkenazi Jews None None None Norbottnians (northern Sweden) Palestinian Arab Japanese Clinical manifestations Hepatosplenomegaly hypersplenism bleeding bone pains None OMA strabismus opisthotonus trismus Hydrops fetalis congenital ichthyosis OMA myoclonic seizures OMA hepatosplenomegaly growth retardation Cardiac valve calcification Central nervous system involvement None None Yes Yes (lethal) Progressive OMA, progressive OMA Bone involvement Yes None None None Yes Yes Yes (minimal) Lung involvement Variable None Yes Yes Variable Variable Minimal Enzyme replacement therapy Indicated Not indicated Not indicated Not indicated Indicated for visceral features only Life expectancy Unchanged Unchanged Lethal prior to age 2 years Neonatal lethal Childhood death Variable with possible survival to adulthood Adolescence Abbreviation: OMA, Oculomotor apraxia. Modified from Balarezo FS, Joshi VV., 2002. Adv Anat Pathol. 9(6):360!70. • Pathologic fractures occur in sclerotic bone. • Nerve entrapment syndromes may also be present. Gaucher disease • Gaucher disease is the most common lysosomal storage disease, resulting from deficient activity of β-glucocerebrosidase. • It is inherited in an autosomal-recessive manner, and more than 200 mutations have been identified in the β-glucocerebrosidase gene located on 1q21, including point mutations, crossovers, and recombinations. • The presence of the 1226G (N370S) mutation on one allele is synonymous with type-I disease (i.e., it is apparently protective against neurologic involvement), whereas homozygosity for the allele 1488C (L444P) is invariably correlated with neurological disease. • However, there is marked heterogeneity in clinical course and prognosis among similar genotypes highlighting a lack of clear genotype!phenotype relationships. • Deficiency of β-glucocerebrosidase prevents the catabolism of glucocerebroside. Lysosomes accumulate glucocerebroside, and this pathologic accumulation in macrophages and Kupffer cells of the reticuloendothelial system of the spleen and liver leads to hepatosplenomegaly. • Hypersplenism produces anemia and thrombocytopenia. Glucocerebroside accumulation in the bone marrow results in osteopenia, lytic lesions, pathologic fractures, chronic bone pain, bone infarcts, osteonecrosis, and acute excruciating bone crises. • Gaucher disease is classified into three types based on the presence and degree of neuronal involvement (Table 2.10 outlines the clinical manifestations of the three types of Gaucher disease). • Patients with type 1 Gaucher disease (nonneuropathic), which accounts for 90% of all cases of Gaucher disease, present with: Lanzkowsky’s Manual of Pediatric Hematology and Oncology Marrow infiltrative disorders 31 • Asymptomatic splenomegaly (rarely, portal hypertension develops). Splenic infarction is common, and presents with pain, rigid abdomen, and fever. Splenic nuclide scanning is helpful in the presence of an acute abdomen. • Pancytopenia secondary to hypersplenism (rarely from infiltration of the bone marrow with Gaucher cells). • Skeletal manifestations include bone marrow infiltration with Erlenmeyer flask deformity from bone marrow expansion, generalized bone mineral loss, and infarction on radiographs. The resultant osteopenia and infarction can lead to pathologic fractures. • Bone crises characterized by fever and excruciating local pain most frequently along femurs. • Growth delay: 50% of symptomatic children are at or below the third percentile for height and another 25% are shorter than expected based on their mid-parental height. • Typical Gaucher cells in the bone marrow. • Decreased glucocerebrosidase activity in leukocytes. • Characteristic mutations of the β-glucocerebrosidase gene on chromosome 1 on DNA analysis. • Diagnosis and further evaluation relies on a series of DNA sequencing, cellular assays, and laboratory and imaging studies: • DNA evaluation for β-glucocerebrosidase gene abnormalities in patient, parents, and siblings. • β-Glucocerebrosidase assay on leukocytes or cultured skin fibroblasts is the most efficient method of diagnosis in the absence of molecular testing. ! The typical child with type 1 Gaucher disease will have enzyme activity that is 10!30% of normal. • CBC: often, if a patient presents with no prior diagnosis, pancytopenia, splenomegaly, and leukoerythroblastosis lead to a concern for leukemia. • Serum chemistry with liver function tests. • Acid phosphatase level. • Angiotensin-converting enzyme. • Chitotriosidase. • Liver: spleen volume. • MRI of femora. • Bone density of the spine and hips (Dual-energy X-ray absorptiometry). • Chest radiograph. • The mainstay of Gaucher disease treatment is enzyme replacement therapy (ERT) or substrate reduction therapy (SRT): • ERT is recommended for the treatment of symptomatic type 1 patients. ! Recombinant human macrophage-targeted human glucocerebrosidase (imiglucerase, Cerezyme) is used for ERT. ! The initial dose is 30!60 unit/kg IV every 2 weeks, and must be individualized for each patient based on disease severity and rate of progression. ! The maintenance dose is 15!60 unit/kg IV every 2 weeks. ! Children who require treatment need to continue therapy indefinitely to maintain their clinical improvement. Prolonged periods without therapy are not appropriate. ! ERT does not cross the blood!brain barrier precluding its use for neurologic sequelae. • SRT is available using miglustat (Zavesca; Actelion Pharmaceuticals, Allschwill, Switzerland) an inhibitor of glucosylceramide (GlcCer) synthase. ! Unlike Cerezyme, Zavesca is given orally and does cross the blood!brain barrier. ! Of note, Zavesca causes a number of side effects. ! Currently, the role of substrate reduction is still evolving. ! The earliest response to therapy is an improvement in hematologic parameters. A progressive decrease in hepatosplenomegaly is regarded as a positive response. Skeletal response occurs more slowly (after 2!4 years), along with a decrease in pain and bone crises. • Approximately 5% of patients develop hypersensitivity to ERT. ! These reactions respond to interruption of infusion and administration of antihistamine and glucocorticoids. ! Reducing the initial rate of infusion to no more than 10 unit/min typically prevents subsequent reactions. ! These reactions commonly occur during the first 12 months of treatment, and necessitate direct supervision by a physician during treatment sessions. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 32 2. Hematologic manifestations of systemic illness TABLE 2.11 Recommendations for monitoring children with type 1 Gaucher disease (minimal evaluations only). Patients not receiving enzyme therapy Every 12!24 months Patients receiving enzyme therapy Every 3 monthsa Every 12 monthsa At time of dose change All patients, baseline Every 12 months Hemoglobin X X X X Platelet count X X X X Acid phosphatase (total, nonprostatic), angiotensin converting enzyme, chitotriosidaseb X X X X Hematologic Visceralc Spleen volume (volumetric MRI or CT) X X X X Liver volume (volumetric MRI or CT) X X X X X X X X Radiograph: AP view of entire femora and view of lateral spine X X X X Dual-energy X-ray absorptiometry X X Every 12!24 months d Skeletal MRI (coronal; T1- and T2-weighted) of entire femorae e Quality of lifef Patient reported functional health and well-being X X X a For patients who have reached clinical goals and for whom there has been no change in dose, the frequency of monitoring can be decreased to every 12!24 months. One or more of these markers should be consistently monitored (at least once every 12 months) in conjunction with other clinical assessments of disease activity and response to treatment. Of the three currently recommended biochemical markers, chitotriosidase activity, when available as a validated procedure from an experienced laboratory, may be the most sensitive indicator of changing disease activity and is therefore preferred. c Obtain contiguous transaxial 10-mm-thick sections for the sum of region of interest. d Additional skeletal assessments that are optional include bone age for patients # 14 years old. Follow-up is recommended if baseline is abnormal. e Optimally, obtain hips to below knees. As an alternative, obtain hips to distal femur. f Ideally, quality of life should be assessed every 6 months using a standard and valid instrument. Abbreviations: AP, anteroposterior; CT, Computed tomography; DEXA, dual energy X-ray absorptiometry. Modified from Charrow J, Andersson HC, Kaplan P, Kolodny EH, Mistry P, et al., 2004. Enzyme replacement therapy and monitoring for children with type 1 Gaucher disease: consensus recommendations. J Pediatr. 144(1):112!20. b ! Therapy administered after 1 year may be administered at home by home nursing services. ! The nonneutralizing IgG antibodies that develop in up to 13% of patients are not clinically relevant. • Iron therapy in Gaucher patients with anemia is not recommended because Gaucher cells avidly take up iron, which leads to hemochromatosis and decreased iron availability for erythropoiesis. • See Table 2.10 for ERT in the other types of Gaucher disease. • Recommendations for monitoring of children with type 1 Gaucher disease receiving and not receiving ERT are outlined in Table 2.11. Niemann!Pick disease • Niemann!Pick disease types A and B result from deficient activity of acid sphingomyelinase, encoded by a gene on chromosome 11. • The defect results in accumulation of sphingomyelin in the monocyte!macrophage system. • The progressive deposition of sphingomyelin in the CNS leads to type A, and in nonneuronal tissues it leads to type B. • Type C is a neuronopathic form that results from the defective cholesterol transport. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Marrow infiltrative disorders 33 • Clinical manifestations of Niemann!Pick disease vary by the clinical subtype, and are characterized by classic signs, including hepatosplenomegaly, cherry red spot in the macula, psychomotor deterioration, reticular pulmonary infiltrates, and foamy cells in the bone marrow. • Diagnosis involves examining leukocytes or cultured fibroblasts to determine sphingomyelinase activity. • There is no specific treatment for Niemann!Pick disease; however, there is limited evidence that Miglustat (a GlcCer synthase inhibitor) or bone marrow transplant provides significant benefits, although bone marrow transplant has helped a few specific subtypes of patients (type B). • Splenectomy in type B patients frequently causes progression of pulmonary disease, and should be avoided if possible. “Foam Cells” in bone marrow • The differential diagnosis for foam cells, cells with numerous uniform intracellular vacuoles often described as having a “honeycomb” appearance, in the bone marrow is as follows: • Niemann!Pick disease (types A, B, C, D) • Gm1 gangliosidosis (type 1) • Gm2 gangliosidosis (Sandhoff variant) • Lactosyl ceramidosis • Sialidosis I • Sialidosis II, late infantile type • Mucolipidosis II • Mucolipidosis III • Mucolipidosis IV • Fucosidosis • Mannosidosis • Neuronal ceroid-lipofuscinosis • Farber’s disease • Wolman’s disease • Cholesteryl ester storage disease • Cerebrotendinous xanthomatosis • Chronic hyperlipidemia • Chronic corticosteroid therapy • Hematologic malignancies (e.g., Hodgkin disease, leukemia, and myeloma) • Hematologic disease (e.g., aplastic anemia and ITP) • Diagnostic workup includes careful history (including ethnic and family history), physical examination, examination of bone marrow using phase electron microscopy, and special stains and enzyme assays on leukocytes or cultured skin fibroblasts, and liver biopsy for biochemical analysis. Cystinosis • An autosomal-recessive defect, cystinosis, is associated with generalized deposits of cystine in the tissues. • Cystinosis occurs in the first year of life with the following manifestations: • thermal instability, polydipsia, polyuria; • failure to thrive; • recurrent episodes of vomiting and dehydration; • dwarfism and rickets, often prominent, and • early renal involvement with tubular dysfunction manifesting as a secondary Fanconi syndrome, leading to chronic renal failure. • Diagnosis relies on the presence of cystine crystals in the bone marrow and elevated cystine levels in leukocytes or fibroblasts. • Treatment includes cystine-depleting therapy [cysteamine bitartrate (Cystagon or Procysbi)] and symptomatic management. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 34 2. Hematologic manifestations of systemic illness Further reading and references Al-Ghafry, M., Aygun, B., Appiah-Kubi, A., Vlachos, A., Ostovar, G., Capone, C., et al., 2020. Are children with SARS-CoV-2 infection at high risk for thrombosis? Viscoelastic testing and coagulation profiles in a case series of pediatric patients. Pediatr. Blood Cancer 67 (12), e28737. Available from: https://doi.org/10.1002/pbc.28737. PMID: 33098753; PMCID: PMC7645957. Avila, M.L., Lee, K.J., Bouskill, V., Rand, M.L., James, P., Carcao, M., 2015. Acquired von Willebrand syndrome in paediatric patients with congenital heart disease: challenges in the diagnosis and management of this rare condition. Haemophilia 21, e89!e92. Babitt, J.L., Lin, H.Y., 2012. Mechanisms of anemia in CKD. J. Am. Soc. Nephrol. 23, 1631!1634. Baltimore, R.S., Gewitz, M., Baddour, L.M., Beerman, L.B., Jackson, M.A., Lockhart, P.B., et al., 2015. Infective endocarditis in childhood: 2015 update: a scientific statement from the American Heart Association. Circulation 132, 1487!1515. Bergamaschi, G., Di Sabatino, A., Corazza, G.R., 2018. Pathogenesis, diagnosis and treatment of anaemia in immune-mediated gastrointestinal disorders. Br. J. Haematol. 182, 319!329. Borzych-Duzalka, D., Bilginer, Y., Ha, I.S., Bak, M., Rees, L., Cano, F., et al., 2013. Management of anemia in children receiving chronic peritoneal dialysis. J. Am. Soc. Nephrol. 24, 665!676. Bunyavanich, S., Do, A., Vicencio, A., 2020. Nasal gene expression of angiotensin-converting enzyme 2 in children and adults. JAMA 323. Cerfolio, R.J., Orszulak, T.A., Daly, R.C., Schaff, H.V., 1997. Reoperation for hemolytic, anaemia complicating mitral valve repair. Eur. J. Cardiothorac. Surg. 11, 479!484. Chiappini, E., Berti, E., Gianesin, K., Petrara, M.R., Galli, L., Giaquinto, C., et al., 2014. Pediatric human immunodeficiency virus infection and cancer in the highly active antiretroviral treatment (HAART) era. Cancer Lett. 347, 38!45. Cohee, L.M., Laufer, M.K., 2017. Malaria in children. Pediatr. Clin. North Am. 64, 851!866. Connors, J.M., Levy, J.H., 2020. COVID-19 and its implications for thrombosis and anticoagulation. Blood 135, 2033!2040. Decker, M., Leslie, J., Liu, Q., Ding, L., 2018. Hepatic thrombopoietin is required for bone marrow hematopoietic stem cell maintenance. Science 360, 106!110. Dong, Y., Mo, X., Hu, Y., Qi, X., Jiang, F., Jiang, Z., et al., 2020. Epidemiology of COVID-19 among children in China. Pediatrics 145. Elmonem, M.A., Veys, K.R., Soliman, N.A., van Dyck, M., van den Heuvel, L.P., Levtchenko, E., 2016. Cystinosis: a review. Orphanet J. Rare Dis. 11, 47. Erdogan, M., Kosenli, A., Ganidagli, S., Kulaksizoglu, M., 2012. Characteristics of anemia in subclinical and overt hypothyroid patients. Endocr. J. 59, 213!220. Fakhouri, F., Zuber, J., Fremeaux-Bacchi, V., Loirat, C., 2017. Haemolytic uraemic syndrome. Lancet 390, 681!696. Friedman, A.D., 1996. Hematologic manifestations of viral infections. Pediatr. Ann. 25, 555!560. Ganaie, S.S., Qiu, J., 2018. Recent advances in replication and infection of human parvovirus B19. Front. Cell Infect. Microbiol. 8, 166. Ganz, T., 2019. Anemia of inflammation. N. Engl. J. Med. 381, 1148!1157. Grigg, C., Anderson, D., Earnshaw, J., 2017. Diagnosis and treatment of hereditary hemorrhagic telangiectasia. Ochsner J. 17, 157!161. Herishanu, Y., Rogowski, O., Polliack, A., Marilus, R., 2006. Leukocytosis in obese individuals: possible link in patients with unexplained persistent neutrophilia. Eur. J. Haematol. 76, 516!520. Honda, M., Yamamoto, H., Hayashida, S., Suda, H., Ohya, Y., Lee, K.J., et al., 2011. Factors predicting persistent thrombocytopenia after living donor liver transplantation in pediatric patients. Pediatr. Transpl. 15, 601!605. KDIGO, 2013. Summary of recommendation statements. Kidney Int. Suppl. (2011) 2, 283!287. Khosla, J., Yeh, A., Spitzer, T., et al., 2018. Hematopoietic stem cell transplant-associated thrombotic microangiopathy: current paradigm and novel therapies. Bone Marrow Transpl. 53, 129!137. Available from: https://doi.org/10.1038/bmt.2017.207. Koury, M.J., 2014. Abnormal erythropoiesis and the pathophysiology of chronic anemia. Blood Rev. 28, 49!66. Kritharis, A., Al-Samkari, H., Kuter, D.J., 2018. Hereditary hemorrhagic telangiectasia: diagnosis and management from the hematologist’s perspective. Haematologica 103, 1433!1443. Legendre, C.M., Licht, C., Muus, P., Greenbaum, L.A., Babu, S., Bedrosian, C., et al., 2013. Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N. Engl. J. Med. 368, 2169!2181. Loirat, C., Fakhouri, F., Ariceta, G., Besbas, N., Bitzan, M., Bjerre, A., et al., 2016. An international consensus approach to the management of atypical hemolytic uremic syndrome in children. Pediatr. Nephrol. 31, 15!39. Mahajan, P., Margolin, J., Iacobas, I., 2017. Kasabach!Merritt phenomenon: classic presentation and management options. Clin. Med. Insights Blood Disord. 10, 1179545X17699849. Mahat, U., Matar, R.B., Rotz, S.J., 2019. Use of complement monoclonal antibody eculizumab in Shiga toxin producing Escherichia coli associated hemolytic uremic syndrome: a review of current evidence. Pediatr. Blood Cancer 66, e27913. Marks, P.W., 2013. Hematologic manifestations of liver disease. Semin. Hematol. 50, 216!221. Nairz, M., Theurl, I., Wolf, D., Weiss, G., 2016. Iron deficiency or anemia of inflammation?: differential diagnosis and mechanisms of anemia of inflammation. Wien. Med. Wochenschr. 166, 411!423. Nielsen, M.J., Rasmussen, M.R., Andersen, C.B., Nexo, E., Moestrup, S.K., 2012. Vitamin B12 transport from food to the body’s cells—a sophisticated, multistep pathway. Nat. Rev. Gastroenterol. Hepatol. 9, 345!354. Oustamanolakis, P., Koutroubakis, I.E., Messaritakis, I., Niniraki, M., Kouroumalis, E.A., 2011. Soluble transferrin receptor-ferritin index in the evaluation of anemia in inflammatory bowel disease: a case!control study. Ann. Gastroenterol. 24, 108!114. Park, S., Han, C.R., Park, J.W., Zhao, L., Zhu, X., Willingham, M., et al., 2017. Defective erythropoiesis caused by mutations of the thyroid hormone receptor alpha gene. PLoS Genet. 13, e1006991. Percheron, L., Gramada, R., Tellier, S., Salomon, R., Harambat, J., Llanas, B., et al., 2018. Eculizumab treatment in severe pediatric STEC-HUS: a multicenter retrospective study. Pediatr. Nephrol. 33, 1385!1394. Petzer, V., Tymoszuk, P., Asshoff, M., Carvalho, J., Papworth, J., Deantonio, C., et al., 2020. A fully human anti-BMP6 antibody reduces the need for erythropoietin in rodent models of the anemia of chronic disease. Blood 136. Pollack, E.S., Pollack Jr., C.V., 1994. Incidence of subclinical methemoglobinemia in infants with diarrhea. Ann. Emerg. Med. 24, 652!656. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 35 Qian, S., Fu, F., Li, W., Chen, Q., de Sauvage, F.J., 1998. Primary role of the liver in thrombopoietin production shown by tissue-specific knockout. Blood 92, 2189!2191. Reiss, U.M., Bensimhon, P., Zimmerman, S.A., Ware, R.E., 2007. Hydroxyurea therapy for management of secondary erythrocytosis in cyanotic congenital heart disease. Am. J. Hematol. 82, 740!743. Revel-Vilk, S., Szer, J., Mehta, A., Zimran, A., 2018. How we manage Gaucher disease in the era of choices. Br. J. Haematol. 182, 467!480. Skikne, B.S., Punnonen, K., Caldron, P.H., Bennett, M.T., Rehu, M., Gasior, G.H., et al., 2011. Improved differential diagnosis of anemia of chronic disease and iron deficiency anemia: a prospective multicenter evaluation of soluble transferrin receptor and the sTfR/log ferritin index. Am. J. Hematol. 86, 923!927. Tubman, V.N., Smoot, L., Heeney, M.M., 2007. Acquired immune cytopenias post-cardiac transplantation respond to rituximab. Pediatr. Blood Cancer 48, 339!344. Vishnu, P., Aboulafia, D.M., 2015. Haematological manifestations of human immune deficiency virus infection. Br. J. Haematol. 171, 695!709. Warady, B.A., Silverstein, D.M., 2014. Management of anemia with erythropoietic-stimulating agents in children with chronic kidney disease. Pediatr. Nephrol. 29, 1493!1505. Weill, O., Peyre, M., Vergnat, M., Cazavet, A., Stos, B., Belli, E., et al., 2015. Repeat mitral valve repair for haemolysis in children. Arch. Cardiovasc. Dis. 108, 118!121. Whitehead, K.J., Sautter, N.B., McWilliams, J.P., Chakinala, M.M., Merlo, C.A., Johnson, M.H., et al., 2016. Effect of topical intranasal therapy on epistaxis frequency in patients with hereditary hemorrhagic telangiectasia: a randomized clinical trial. JAMA 316, 943!951. Whittaker, E., Bamford, A., Kenny, J., Kaforou, M., Jones, C.E., Shah, P., et al., 2020. Clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2. JAMA 324. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., et al., 2006. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin. Infect. Dis. 43, 1089!1134. Xu, Z., Berry, B.R., 2019. Oxidative hemolysis due to Wilson disease. Blood 134, 657. Yang, C.F., Duro, D., Zurakowski, D., Lee, M., Jaksic, T., Duggan, C., 2011. High prevalence of multiple micronutrient deficiencies in children with intestinal failure: a longitudinal study. J. Pediatr. 159, 39!44.e31. Yu, J.C., Shliakhtsitsava, K., Wang, Y.M., Paul, M., Farnaes, L., Wong, V., et al., 2019. Hematologic manifestations of nutritional deficiencies: early recognition is essential to prevent serious complications. J. Pediatr. Hematol. Oncol. 41, e182!e185. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 3 Classification and diagnosis of anemia in children and neonates Omar Niss1,2 and Charles T. Quinn2,3 1 Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States 2Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, United States 3Erythrocyte Diagnostic Laboratory Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States Classification and diagnosis Anemia is defined as a reduction in hemoglobin concentration, hematocrit, or red cell mass by more than two standard deviations below the mean for age and sex for the normal population. The normal ranges are also affected by geographic ancestry; African Americans have lower hemoglobin concentration on average (by B0.5 g/dL) than people of European ancestry. As a result, 2.5% of the general population and up to 10% of African Americans will be classified as anemic, especially if race-specific normal ranges are not used. Also note that children with cyanotic congenital heart disease, chronic respiratory insufficiency, arteriovenous pulmonary shunts, or hemoglobinopathies that alter oxygen affinity can be functionally anemic with hemoglobin levels in the normal or reference range. • Anemia can be an isolated abnormality or be a part of multiple cell line abnormalities (red cells, white cells, and platelets). Abnormalities of two- or three-cell lines may indicate one of the following: • bone marrow involvement (e.g., infections, aplastic anemia, leukemia, toxicity from medications); • autoimmune disorders (e.g., connective tissue disease, Evans syndrome); • sequestration (e.g., hypersplenism) or intravascular trapping and destruction (e.g., thrombotic microangiopathy). • Anemia can be classified based on morphology (e.g., size and shape of RBCs) or physiology (i.e., mechanism of anemia). Physiologically, anemia can be categorized into: • disorders of decreased red cell formation: this can be due to failure of erythropoiesis in which there is an absolute erythroblastopenia and reticulocytopenia (e.g., marrow failure diseases) or due to ineffective erythropoiesis in which the marrow has many erythroblasts but with defective maturation (e.g., thalassemias); • disorders of erythrocyte destruction (hemolysis); • loss of red blood cells (hemorrhage). Table 3.1 presents an etiologic classification of anemia and the diagnostic features. • The blood smear is an essential first step in the diagnosis of anemia. Anemia can be classified based on RBC morphology and RBC size into microcytic [decreased mean corpuscular volume (MCV)], normocytic (normal MCV), and macrocytic (high MCV). The mean corpuscular hemoglobin (MCH) and MCH concentration (MCHC) are calculated values and have complementary diagnostic value. The MCH usually parallels the MCV. The MCHC is a measure of cellular hydration status. A high value ( . 35 g/dL) due to membrane loss is characteristic of spherocytosis, and a low value is commonly associated with iron deficiency. Fig. 3.1 Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00011-8 37 © 2022 Elsevier Inc. All rights reserved. 38 3. Classification and diagnosis of anemia in children and neonates TABLE 3.1 Etiologic classification and major diagnostic features of anemia in children. Etiologic classification Diagnostic features 1. Impaired red cell formation a. Nutritional deficiency i. Decreased dietary intake [e.g., excessive cows’ milk (iron-deficiency anemia), vegan (vitamin B12 deficiency)] ii. Increased demand [e.g., growth (iron); hemolysis (folic acid)] iii. Decreased absorption 1. specific: intrinsic factor (vitamin B12) 2. generalized: malabsorption syndrome (e.g., folic acid, iron) iv. Impairment in red cell formation can result from one of the following deficiencies: a. Iron deficiency Hypochromic, microcytic red cells; low MCV, low MCH, low MCHC, high RDW,a low serum ferritin, high FEP b. Folate deficiency Macrocytic red cells, high MCV, high RDW, megaloblastic marrow, low serum, and red cell folate, high homocysteine and normal methylmalonic acids Macrocytic red cells, high MCV, high RDW, megaloblastic marrow, low c. Vitamin B12 deficiency serum B12, decreased gastric acidity, high homocysteine, and high methylmalonic acids d. Vitamin C deficiency Clinical scurvy e. Protein deficiency Kwashiorkor Hypochromic red cells, sideroblastic bone marrow, high serum ferritin f. Vitamin B6 deficiency g. Thyroxine deficiency Clinical hypothyroidism, low free T4, high TSH b. Bone marrow failure i. Failure of a single cell line • Megakaryocytes Amegakaryoctic thrombocytopenic purpura with Limb abnormalities, thrombocytopenia, absent megakaryocytes absent radii (TAR) • Red cell precursors Congenital red cell aplasia Absent red cell precursors (Diamond!Blackfan anemia) Acquired red cell aplasia Absent red cell precursors (TEC) • White cell precursors Congenital neutropenias Neutropenia, recurrent infection ii. Failure of multiple cell lines (characterized by pancytopenia and acellular or hypocellular marrow) • Congenital Fanconi anemia Multiple congenital anomalies, chromosomal breakage Familial without anomalies Familial history, no congenital anomalies Dyskeratosis congenita Mucosal and cutaneous abnormalities • Acquired Idiopathic No identifiable cause Secondary History of exposure to drugs, radiation, household toxins, infections (parvovirus B19, HIV) associated with immunologic disease iii. Infiltration • Benign (e.g., osteopetrosis, storage diseases) • Malignant primary (e.g., leukemia, myelofibrosis) • Secondary (e.g., neuroblastoma, lymphoma) Bone marrow: morphology, cytochemistry, immunologic markers, cytogenetics, molecular features VMA, imaging studies, skeletal survey, bone marrow iv. Dyshematopoietic anemias (decreased erythropoiesis, decreased iron utilization) • Anemia of chronic disease Evidence of systemic illness • Renal failure and hepatic disease kidney and liver function tests • Disseminated malignancy Clinical evidence • Connective tissue diseases Rheumatoid arthritis • Malnutrition Clinical evidence • Sideroblastic anemias Hypochromic anemia, ring sideroblasts 2. Blood loss Overt or occult blood positive 3. Hemolytic anemia a. Corpuscular (intrinsic) i. Membrane defects (spherocytosis, elliptocytosis) ii. Enzymatic defects (pyruvate kinase, G6PD) Splenomegaly, jaundice Morphology, osmotic fragility Enzyme assays (Continued) Lanzkowsky’s. Manual of Pediatric Hematology and Oncology Classification and diagnosis 39 TABLE 3.1 (Continued) Etiologic classification Diagnostic features iii. Hemoglobin defects • Heme • Globin Qualitative (e.g., sickle cell) Quantitative (e.g., thalassemia) b. Extracorpuscular (extrinsic) i. Immune • Isoimmune • Autoimmune Idiopathic Secondary Immunologic disorder (e.g., lupus) One-cell line (e.g., red cells) Multiple cell line (e.g., white blood cells, platelets) Hb electrophoresis Quantitative HbF, HbA2 content Direct antiglobulin test (Coombs’ test) Direct antiglobulin test, antibody identification Decreased C3, C4, CH50, positive ANA Anemia—direct antiglobulin test positive Evans syndrome: neutropenia—autoimmune neutropenia, thrombocytopenia—ITP ii. Nonimmune (idiopathic, secondary) a RDW—coefficient of variation of the RBC distribution width (normal between 11.5% and 14.5%). Abbreviations: ANA, Anti-nuclear antibody; FEP, free erythrocyte protoporphyrin; G6PD, glucose-6-phosphate dehydrogenase; Hb, hemoglobin; ITP, idiopathic thrombocytopenic purpura; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell; RDW, red cell distribution width (see definition); TAR, thrombocytopenic purpura with absent radii; TEC, transient erythroblastopenia of childhood; VMA, vanillylmandelic acid. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. delineates the causes of anemia based on peripheral blood morphology, and Table 3.2 shows the cause of anemia based on MCV and red cell distribution width (RDW), an index of the variation in red cell size (anisocytosis). • In addition, the blood smear may show specific morphologic abnormalities suggestive of red cell membrane disorders (e.g., spherocytosis, stomatocytosis, or elliptocytosis) or hemoglobinopathies (e.g., sickle cell disease and thalassemia). Table 3.3 lists the differential diagnosis of anemia based on the specific red cell morphological abnormalities. • The reticulocyte count is an important indicator of the physiology of anemia (Fig. 3.2). An elevated reticulocyte count suggests blood loss or hemolysis, while a normal or decreased count suggests impaired red cell formation. With acute blood loss or sequestration, the elevation of the reticulocyte count may take many hours to become apparent and several days (4!5) to reach its maximum. The reticulocyte count must be interpreted in the context of the degree of anemia, using either the absolute reticulocyte count or reticulocyte index, to assess the adequacy of erythropoiesis. In patients with bleeding or hemolysis, the reticulocyte index should be at least 3%, whereas in patients with anemia due to decreased production of red cells, the reticulocyte index is ,3% and frequently ,1.5%. Table 3.4 lists various laboratory studies helpful in the investigation of a patient with anemia. • The investigation of anemia entails the following steps: • Detailed history. Table 3.1 lists the various causes of anemia with the associated diagnostic laboratory and clinical features. • Complete blood count to establish whether anemia is isolated or part of a multilineage abnormality (abnormality of red cell count, white blood cell count, and platelet count). Determination of the morphologic characteristics of the anemia based on blood smear (Fig. 3.1 and Table 3.3) and consideration of the MCV (Fig. 3.2 and Table 3.2) and RDW (Table 3.2) and white blood cell and platelet morphology. • Reticulocyte count as a reflection of erythropoiesis (Fig. 3.2). • Determination of whether there is evidence of a hemolytic process by: ! consideration of the clinical features suggesting hemolytic disease (Table 3.5), ! laboratory demonstration of the presence of hemolysis (Table 3.4), and ! determination of the precise cause of the hemolytic anemia by special hematologic investigations (Table 3.4). Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 40 3. Classification and diagnosis of anemia in children and neonates Blood smear Hypochromic microcytic Macrocytic+ Normocytic Specific MCV low (red cell size <70 fL) MCV high (red cell size >85 fL) MCV normal (red cell size 72–79 fL) See Table 3.3 Iron-deficiency anemia Thalassemia, ! or " Sideroblastic anemia Chronic disease Infection Cancer Inflammation Renal disease Lead toxicity Hemoglobin E trait Atransferrinemia Inborn errors of iron metabolism Copper deficiency Severe malnutrition Normal newborn Increased erythropoiesis* Postsplenectomy Liver disease** Obstructive jaundice** Aplastic anemia Hypothyroidism Megaloblastic anemias Down syndrome Syndromes with elevated Hb F Myelodysplastic syndromes Diamond–Blackfan anemia Fanconi anemia Pearson syndrome Paroxysmal nocturnal hemoglobinuria Drugs (methotrexate, mercaptopurine, phenytoin) Acute blood loss Infection Renal failure Connective tissue disorder Liver disease Disseminated malignancy Early iron deficiency Aplastic anemia Bone marrow infiltration Dyserythropoietic anemia Hemolysis RBC enzyme deficiency RBC membrane defects Hypersplenism Drugs FIGURE 3.1 An approach to the diagnosis of anemia by examination of the blood smear. Abbreviations: MCV, Mean corpuscular volume. Spurious macrocytosis (high MCV) may be caused by agglutinated red cells (e.g., Mycoplasma pneumoniae and autoimmune hemolytic anemia). *Increased number of reticulocytes. **On the basis of increased membrane resulting in an increased membrane/volume ratio. Increased membrane results from exchanges between red cell lipids and altered lipid balance in these conditions. Source: From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. 1 • Bone marrow aspiration and biopsy, if required, to examine erythroid, myeloid, and megakaryocytic morphology to determine whether there is normoblastic, megaloblastic, or sideroblastic erythropoiesis and to exclude marrow pathology (e.g., aplastic anemia, leukemia, and benign or malignant infiltration of the bone marrow) (Fig. 3.3). • Determination of underlying cause of anemia by additional tests (Table 3.4). Neonatal anemia Anemia during the neonatal period can be caused by: • hemorrhage: acute or chronic; • hemolysis: congenital hemolytic anemias or due to immune hemolytic anemias; and • hypoplasia: failure of red cell production in inherited bone marrow failure syndromes, for example, Diamond!Blackfan anemia (pure red cell aplasia) or congenital infections. Table 3.6 lists the causes of anemia in the newborn. Hemorrhage Blood loss may occur during the prenatal, intrapartum, or postnatal periods. Prenatal blood loss may be transplacental, intraplacental, or retroplacental or may be due to a twin-to-twin transfusion. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 41 Neonatal anemia TABLE 3.2 Classification of nature of the anemia based on mean corpuscular volume (MCV) and red cell distribution width (RDW). RDW normal MCV low MCV normal MCV high Microcytic homogeneous Normocytic homogeneous Macrocytic homogeneous Heterozygous thalassemia Normal Inherited bone marrow failure syndromes Chronic disease Chronic disease "Preleukemia" or myelodysplastic syndrome Chronic liver disease Chemotherapy Chronic myelocytic leukemia Hemorrhage Hereditary spherocytosis RDW high Microcytic heterogeneous Normocytic heterogeneous Macrocytic heterogeneous Iron deficiency Early iron or folate deficiency Folate deficiency HbS/β-thalassemia Mixed deficiencies Vitamin B12 deficiency Hemoglobin H Hemoglobinopathy (e.g., Hb SS) Immune hemolytic anemia Red cell Myelofibrosis Cold agglutinins Fragmentation disorders Sideroblastic anemia Abbreviations: Hb, hemoglobin; HbS/β-thalassemia, sickle-beta-thalassemia; Hb SS, homozgous sickle cell anemia. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. TABLE 3.3 Specific red cell morphologic abnormalities. Target cells—Increased surface/volume ratio (generally does not affect red cell survival) 1. Thalassemic syndromes 2. Hemoglobinopathies a. Hb C trait or Hb C disease b. Sickle cell disease c. Hb E trait or Hb E disease d. Hb D trait 3. Liver disease 4. Postsplenectomy or hyposplenic states 5. Severe iron deficiency 6. LCAT deficiency: congenital disorder of lecithin/cholesterol acyltransferase deficiency (corneal opacifications, proteinuria, target cells, moderately severe anemia) 7. Abetalipoproteinemia Spherocytes—Decreased surface/volume ratio, hyperdense ( . MCHC) 1. Hereditary spherocytosis 2. ABO incompatibility: antibody-coated fragment of RBC membrane removed 3. Autoimmune hemolytic anemia: antibody-coated fragment of RBC membrane removed 4. G6PD deficiency 5. MAHA: fragment of RBC lost after impact with abnormal surface 6. Sickle cell disease: fragment of RBC removed in reticuloendothelial system 7. Hypersplenism 8. Burns: fragment of damaged RBC removed by spleen 9. Posttransfusion 10. Pyruvate kinase deficiency Acanthocytes (spur cells)—Cells with 5!10 spicules of varying length; spicules irregular in space and thickness, with wide bases; appear smaller than normal cells because they assume a spheroid shape 1. Liver disease 2. Disseminated intravascular coagulation (and other MAHA) 3. Postsplenectomy or hyposplenic state 4. Vitamin E deficiency (Continued) Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 42 3. Classification and diagnosis of anemia in children and neonates TABLE 3.3 (Continued) 5. Hypothyroidism 6. Abetalipoproteinemia: rare congenital disorder; 50!100% of cells acanthocytes; associated abnormalities (fat malabsorption, retinitis pigmentosa, neurologic abnormalities) 7. Malabsorptive states Echinocytes (burr cells)—10!30 spicules equal in size and evenly distributed over RBC surface; caused by alteration in extracellular or intracellular environment 1. Artifact 2. Renal failure 3. Dehydration 4. Liver disease 5. Pyruvate kinase deficiency 6. Peptic ulcer disease or gastric carcinoma 7. Immediately after red cell transfusion 8. Rare congenital anemias due to decreased intracellular potassium Pyknocytes—Distorted, hyperchromic, contracted RBC; can be similar to echinocytes and acanthocytes Schistocytes—Helmet, triangular shapes, or small fragments. Caused by fragmentation upon impact with abnormal vascular surface (e.g., fibrin strand, vasculitis, artificial surface in circulation) 1. DIC; 2. severe hemolytic anemia (e.g., G6PD deficiency); 3. MAHA 4. hemolytic uremic syndrome; 5. prosthetic cardiac valve, abnormal cardiac valve, cardiac patch, coarctation of the aorta; 6. connective tissue disorder (e.g., SLE); 7. Kasabach!Merritt syndrome; 8. purpura fulminans; 9. renal vein thrombosis; 10. burns (spheroschistocytes as a result of heat); 11. thrombotic thrombocytopenia purpura; 12. homograft rejection; 13. uremia, acute tubular necrosis, glomerulonephritis; 14. malignant hypertension; 15. systemic amyloidosis; 16. liver cirrhosis; and 17. disseminated carcinomatosis. Elliptocytes—Elliptical cells, normochromic; seen normally in less than 1% of RBCs; larger numbers occasionally seen in a normal patient 1. hereditary elliptocytosis; 2. iron deficiency (increased with severity, hypochromic); 3. sickle cell disease; 4. thalassemia major; 5. severe bacterial infection; 6. sickle cell trait; 7. leukoerythroblastic reaction; 8. megaloblastic anemias; 9. any anemia may occasionally present with up to 10% elliptocytes; and 10. malaria. Teardrop cells—Shape of drop, usually microcytic, often also hypochromic 1. Newborn 2. Thalassemia major 3. Leukoerythroblastic reaction 4. Myeloproliferative syndromes 5. Bone marrow infiltration Stomatocytes—Has a slit-like area of central pallor 1. Normal (in small numbers) 2. Hereditary stomatocytosis/xerocytosis 3. Artifact 4. Thalassemia 5. Acute alcoholism 6. Rh null disease (absence of Rh complex) 7. Liver disease 8. Malignancies (Continued) Lanzkowsky’s. Manual of Pediatric Hematology and Oncology Neonatal anemia 43 TABLE 3.3 (Continued) Nucleated red blood cells—Not normal in the peripheral blood beyond the first week of life 1. Newborn (first 3!4 days) 2. Intense bone marrow stimulation a. Hypoxia (especially postcardiac arrest) b. Acute bleeding c. Severe hemolytic anemia (e.g., thalassemia, sickle cell disease) 3. Congenital infections (e.g., sepsis, congenital syphilis, CMV, rubella) 4. Postsplenectomy or hyposplenic states: spleen normally removes nucleated RBC 5. Leukoerythroblastic reaction: seen with extramedullary hematopoiesis and bone marrow replacement; most commonly leukemia or solid tumor—fungal and mycobacterial infection may also do this; leukoerythroblastic reaction is also associated with teardrop red cells, 10,000!20,000 WBC with small-to-moderate numbers of metamyelocytes, myelocytes, and promyelocytes; thrombocytosis with large bizarre platelets 6. Megaloblastic anemia 7. Dyserythropoietic anemias Blister cells—Red cell area under membrane free of hemoglobin, appearing like a blister 1. G6PD deficiency or unstable hemoglobinopathy (during hemolytic episode) 2. Sickle cell disease (rare) 3. Pulmonary emboli (rare) Basophilic stippling—Coarse or fine punctate basophilic inclusions that represent aggregates of ribosomal RNA 1. Hemolytic anemias 2. Iron deficiency anemia 3. Lead poisoning Howell!Jolly bodies—Small, well-defined, round, densely stained nuclear-remnant inclusions; 1 mm in diameter; centric in location 1. Postsplenectomy or hyposplenia 2. Newborn 3. Megaloblastic anemias 4. Dyserythropoietic anemias 5. A variety of types of anemias (rarely iron-deficiency anemia, hereditary spherocytosis) Cabot’s ring bodies—Nuclear-remnant ring configuration inclusions 1. Pernicious anemia 2. Lead toxicity Heinz bodies—Denatured aggregated hemoglobin 1. Normal in newborn 2. Thalassemia 3. Asplenia 4. Chronic liver disease 5. Heinz body hemolytic anemia Abbreviations: CMV, cytomegalovirus; Hb, hemoglobin; SLE, systemic lupus erythematosus. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Prenatal blood loss Fetomaternal, intraplacental, and retroplacental hemorrhage Fetal red blood cells can be demonstrated in the maternal circulation in up to 50% of pregnancies; however, a clinically significant hemorrhage ( . 30 mL) is seen in only 1!2% of pregnancies. Significant fetomaternal hemorrhage is commonly seen following procedures such as diagnostic amniocentesis or external cephalic version. Also, intraplacental blood loss from the fetus may occur when there is a tight umbilical cord around the neck or body or when there is delayed cord clamping. Retroplacental bleeding from placental abruption is diagnosed by ultrasound or intraoperatively. Fetomaternal blood loss may be acute or chronic. Table 3.7 lists the characteristics of acute and chronic blood loss in the newborn. Fetomaternal hemorrhage is diagnosed by demonstrating fetal red cells by flow cytometry using an antibody against HbF (fetal hemoglobin)] in the maternal circulation. Less commonly, an older, differential acid elution technique (Kleihauer!Betke method) may still be used. Diagnosis of fetomaternal hemorrhage may be missed in situations in which red cells of the mother and infant have incompatible ABO blood groups. In such instances the infant’s incompatible red blood cells are rapidly cleared from the maternal circulation by maternal anti-A or anti-B antibodies. In these cases an increase in maternal immune anti-A or anti-B titers in the weeks after Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 44 3. Classification and diagnosis of anemia in children and neonates (Decreased hemoglobin and hematocrit) Anemia MCV Low Normal High Iron deficiency Thalassemia Lead poisoning Chronic disease Folate deficiency Vitamin B12 deficiency Aplastic anemia Preleukemia Immune hemolytic anemia Liver disease Reticulocyte count High Low Bilirubin White cell and platelet count Normal High Hemorrhage Hemolytic anemia Direct antiglobulin test Low Bone marrow depression Malignancy Aplastic anemia Congenital Acquired Negative Positive (a) Corpuscular Extracorpuscular Hemoglobinopathies Hemoglobin electrophoresis Enzymopathies Enzyme assays Membrane defects Morphology, Autohemolysis, Osmotic fragility Normal Increased Pure red cell aplasia Infection Diamond–Blackfan anemia Transient erythroblastopenia of childhood (TEC) Autoimmune hemolytic anemia Primary Secondary (e.g., connective tissue disease, drugs) Isoimmune hemolytic disease Rh, ABO, mismatched transfusion (b) Extracorpuscular Idiopathic Secondary (drugs, infection, microangiopathic) FIGURE 3.2 Approach to the diagnosis of anemia by MCV and reticulocyte count. Abbreviations: MCV, Mean corpuscular volume. Source: From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 45 Neonatal anemia TABLE 3.4 Laboratory studies in the investigation of a patient with anemia. Usual initial studies 1. Hemoglobin and hematocrit 2. Erythrocyte count and red cell indices, including MCV, MCH, MCHC and RDW 3. Reticulocyte count (absolute and relative) 4. Peripheral blood smear 5. Leukocyte count and differential count 6. Platelet count Suspected iron deficiency 1. Serum ferritin, iron, and TIBC levels 2. Stool for occult blood 3. Meckel’s diverticulum scan—if indicated 4. Endoscopy (upper and lower bowel)—if indicated Suspected vitamin B12 or folic acid deficiency 1. Serum vitamin B12 level 2. RBC and serum folate level 3. Serum methylmalonic acid level 4. Serum homocysteine level 5. Bone marrow examination—if indicated Suspected hemolytic anemia 1. Evidence of hemolysis a. blood smear—red cell fragments (schistocytes), spherocytes, target cells; b. increased unconjugated bilirubin; c. lower or absent serum haptoglobin; d. raised plasma free hemoglobin level; e. increased urinary urobilinogen; f. hemoglobinuria; g. hemosiderinuria (due to sloughing of iron-laden tubular cells into urine); h. increased methemoglobin level; 2. Evidence of increased erythropoiesis (in response to hemoglobin reduction) a. reticulocytosis—frequently up to 10!20%; rarely, as high as 80%; b. increased MCV due to the presence of reticulocytosis and increased RDW as the hemoglobin level falls; c. NRBCs in peripheral blood (beyond the third day of life); d. specific morphologic abnormalities—sickle cells, target cells, basophilic stippling, irregularly contracted cells (schistocytes), and spherocytes; e. erythroid hyperplasia of the bone marrow—erythroid/myeloid ratio in the marrow increasing from 1:5 to 1:1; f. expansion of marrow space in chronic hemolysis resulting in: i. prominence of frontal bones; ii. broad cheekbones; iii. widened intertrabecular spaces, hair-on-end appearance of skull radiographs; iv. biconcave vertebrae with fish-mouth intervertebral spaces. 3. Evidence of type of intrinsic hemolytic anemia a. membrane defects i. blood smear: spherocytes, ovalocytes, pyknocytes, stomatocytes; ii. osmotic gradient ektacytometry; iii. RBC cation studies. b. hemoglobin defects i. blood smear: sickle cells, target cells; ii. hemoglobin analysis (e.g., electrophoresis, HPLC); iii. quantitative hemoglobin F determination; iv. F-cell analysis by flow cytometry; v. heat-stability test for unstable hemoglobin; vi. globin gene analysis. c. enzymes defects i. Heinz body preparation; ii. specific enzyme assay. iii. genetic testing. 4. Evidence of type of extrinsic hemolytic anemia a. immune i. direct antiglobulin test: IgG (gamma), C (complement), or both ii. flow cytometric analysis of red cells with monoclonal antibodies to GPI-linked surface antigens for PNH; (Continued) Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 46 3. Classification and diagnosis of anemia in children and neonates TABLE 3.4 (Continued) iii. Donath!Landsteiner antibody; iv. ANA. Suspected aplastic anemia or leukemia 1. bone marrow (aspiration and biopsy)—cytochemistry, immunologic markers, chromosome analysis; Other tests often used especially to diagnose the primary disease 1. Viral testing, for example, HIV 2. ANA, complement profile 3. Renal, hepatic, and thyroid testing 4. Tissue biopsy (skin, lymph node, liver)!if indicated Abbreviations: ANA, antinuclear antibody; GPI, glycosylphosphatidylinositol; HPLC, high performance liquid chromatography; MCV, mean corpuscular volume; NRBC, nucleated red blood cell; PNH, paroxysmal nocturnal hemoglobinuria; RDW, red cell distribution width; TIBC, total iron binding capacity. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. TABLE 3.5 The clinical features suggestive of hemolysis. • Ethnogeographic factors: high incidence of sickle trait in people of African ancestry, high incidence of thalassemia trait in people of Mediterranean ancestry, and high incidence of G6PD deficiency among Sephardic Jews. • Age factors: anemia and jaundice in an Rh-positive infant born to a mother who is Rh negative or a group A or group B infant born to a group O mother (setting for a hemolytic anemia) • History of anemia, jaundice, or gallstones in family • Persistent or recurrent anemia associated with reticulocytosis • Anemia unresponsive to iron or vitamin supplements • Intermittent bouts or persistent indirect hyperbilirubinemia • Splenomegaly • Hemoglobinuria • Presence of gallstones • Chronic leg ulcers • Development of anemia or hemoglobinuria after exposure to certain drugs • Dark urine due to dipyrroluria: unstable hemoglobins, thalassemia, and ineffective erythropoiesis Abbreviation: G6PD, Glucose-6-phosphate dehydrogenase. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Bone marrow erythroid series Normoblastic Iron-deficiency anemia Infection Renal disease Malignancy Connective tissue disorders Hemolytic anemia FIGURE 3.3 Causes of normoblastic, meg- Megaloblastic Sideroblastic Vitamin B12 deficiency Folic acid deficiency Miscellaneous Congenital disorders in DNA synthesis Acquired disorders in DNA synthesis Drug induced Hereditary/congenital Acquired clonal Acquired reversible aloblastic, and sideroblastic bone marrow morphology. Source: From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. delivery, if measured, may be a diagnostic clue. The optimal timing for demonstrating fetal cells in maternal blood is within 2 hours of delivery and no later than 24 hours following delivery. These techniques are not reliable when maternal HbF is raised for other reasons (e.g., maternal thalassemia, sickle cell anemia, or hereditary persistence of HbF). In the presence of these conditions, other techniques based on differential agglutination have been used, but the fetomaternal hemorrhage is usually a clinical diagnosis (a diagnosis of exclusion) in such cases. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology Neonatal anemia 47 TABLE 3.6 Causes of anemia in the newborn. 1. Hemorrhage a. Prenatal i. Transplacental fetomaternal (spontaneous, traumatic amniocentesis, external cephalic version) ii. Intraplacental iii. Retroplacental iv. Twin-to-twin transfusion b. Intrapartum i. Umbilical cord abnormalities • rupture of normal cord (unattended precipitous labor); • rupture of varix or aneurysm of cord; • hematomas of cord or placenta; • rupture of anomalous aberrant vessels of cord (not protected by Wharton’s jelly); • vasa previa (umbilical cord is presenting part); and • inadequate cord tying ii. Placental abnormalities • multilobular placenta (fragile communicating veins to main placenta); • placenta previa—fetal blood loss predominantly; • abruptio placentae—maternal blood loss predominantly; • accidental incision of placenta during cesarean section; • traumatic amniocentesis; and • placental chorioangioma iii. Hemorrhagic disorders • coagulation factor deficiency; • thrombocytopenia c. Postnatal i. External • bleeding from umbilicus; • bleeding from gut; and • iatrogenic (diagnostic venipuncture, postexchange transfusion) ii. Internal • cephalohematoma; • subgaleal (subaponeurotic) hemorrhage; • subdural or subarachnoid hemorrhage; • intracerebral hemorrhage; • intraventricular hemorrhage; • intraabdominal hemorrhage; • retroperitoneal hemorrhage (may involve adrenals); • subcapsular hematoma or rupture of liver; • ruptured spleen; • pulmonary hemorrhage 2. Hemolytic anemia (see Chapter 7: General Considerations of Hemolytic Diseases, Red Cell Membrane, and Enzyme Defects,and Chapter 8: Extracorpuscular Hemolytic Anemia) a. Congenital erythrocyte defects i. Membrane defects (with characteristic morphology) • Hereditary spherocytosis • Hereditary elliptocytosis • Hereditary stomatocytosis • Hereditary xerocytosis • Infantile pyknocytosis •. Hereditary pyropoikilocytosis ii. Hemoglobin defects • α-Thalassemia syndromes three α-globin gene deletion/mutation [hemoglobin Barts (γ4)]; four α-globin gene deletion/mutation (hydrops in utero and increased risk of death). • γ β-Thalassemia, others • Unstable hemoglobins (Hb Köln, Hg Zürich, HbF Poole, Hb Hasharon) (see Chapter 9: Hemoglobinopathies) iii. Enzyme defects • Glycolytic pathway pyruvate kinase deficiency; other enzymes, for example, glucose phosphate isomerase deficiency. • Hexose-monophosphate shunt G6PD deficiency with or without drug exposure; enzymes concerned with glutathione reduction or synthesis (Continued) Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 48 3. Classification and diagnosis of anemia in children and neonates TABLE 3.6 (Continued) b. Acquired erythrocyte defects i. Immune • maternal autoimmune hemolytic anemia; • isoimmune hemolytic anemia: Rh disease, ABO, minor blood groups (M, S, Kell, Duffy, Luther) ii. Nonimmune • infections (cytomegalovirus, toxoplasmosis, herpes simplex, rubella, adenovirus, malaria, syphilis, and bacterial sepsis); • microangiopathic hemolytic anemia with or without disseminated intravascular coagulation: disseminated herpes simplex, coxsackie B infections, Gram-negative septicemia, and renal vein thrombosis; • toxic exposure (drugs, chemicals) 6 G6PD 6 prematurity: synthetic vitamin K analogs, maternal thiazide diuretics, antimalarial agents, sulfonamides, naphthalene, aniline-dye marking ink, and penicillin; • vitamin E deficiency; and • metabolic disease (galactosemia, osteopetrosis). 3. Failure of red cell production a. Congenital (Chapter 6: Bone Marrow Failure) i. Diamond!Blackfan anemia (pure red cell aplasia) ii. Fanconi anemia iii. Mitochondriopathies (e.g., Pearson syndrome) iv. Sideroblastic anemia v. Congenital dyserythropoietic anemia b. Acquired i. Viral infection (hepatitis, HIV, CMV, rubella, syphilis, parvovirus B19) ii. Malaria iii. Anemia of prematurity Abbreviations: G6PD, Glucose-6-phosphate dehydrogenase; Hb, hemoglobin. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Twin-to-twin transfusion syndrome Significant twin-to-twin transfusion occurs in at least 15% of monochorionic twins. Velamentous cord insertions are associated with increased risk of twin-to-twin transfusion. The hemoglobin level differs by 5 g/dL and the hematocrit by 15% or more between individual twins compared to discrepancy # 3.3 g/dL in cord blood hemoglobin between dizygotic twins. The donor twin is anemic, pale, and smaller and may have evidence of oligohydramnios and show evidence of congestive heart failure and shock. The recipient is polycythemic and larger, with evidence of polyhydramnios, and may show signs of hyperviscosity syndrome: hypoglycemia, central nervous system injury, hypocalcemia, disseminated intravascular coagulation, hyperbilirubinemia, and congestive heart failure. Intrapartum blood loss Hemorrhage may occur during birth as a result of various obstetric accidents, malformations of the umbilical cord or the placenta, or a bleeding disorder (e.g., coagulation factor deficiency or thrombocytopenia; Table 3.6). Postnatal blood loss Postnatal hemorrhage may occur from a number of sites and may be internal (enclosed) or external. Hemorrhage may be due to: • traumatic deliveries (resulting in intracranial or intraabdominal hemorrhage) • coagulation factor deficiencies (see Chapter 13: Disorders of Coagulation) • congenital—hemophilia or other coagulation factor deficiencies • acquired—vitamin K deficiency, disseminated intravascular coagulation • thrombocytopenia (see Chapter 12: Disorders of Platelets) • congenital—Wiskott!Aldrich syndrome, Fanconi anemia, thrombocytopenia absent radius syndrome • acquired—neonatal alloimmune thrombocytopenia, maternal immune thrombocytopenia, sepsis • rare causes—neonatal adenovirus infection, fetal cytomegalovirus infection, vascular malformations When hemoglobin is catabolized in a resorbing hematoma, hyperbilirubinemia may develop after several days. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 49 Neonatal anemia TABLE 3.7 The characteristics of acute and chronic blood loss in the newborn. Characteristic Acute blood loss Chronic blood loss Clinical Venous pressure Acute distress; pallor; shallow, rapid, and often irregular respiration; tachycardia; weak or absent peripheral pulses; low or absent blood pressure; no hepatosplenomegaly Marked pallor disproportionate to evidence of distress. On occasion signs of congestive heart failure may be present, including hepatomegaly Low Normal or elevated Laboratory Hemoglobin May be normal initially; then drops quickly during the first concentration 24 h of life Low at birth Red cell morphology Normochromic and normo- or macrocytic Hypochromic and microcytic, anisocytosis, and poikilocytosis Serum iron Normal at birth Low at birth Course Prompt treatment of anemia and shock Generally uneventful Treatment Intravenous fluids or packed red blood cells. If indicated, iron therapy Iron therapy. Packed red blood cells on occasion Adapted from Brugnara C., Oski F.A., Nathan D.G., 2015. Diagnostic approach to the anemic patient. In: Orkin, S.H., Fisher, D.E., Look, T., Lux, S.E., Ginsburg, D., Nathan, D.G., et al. (Eds.), Nathan and Oski’s Hematology and Oncology of Infancy and Childhood, eighth ed., WB Saunders, Philadelphia, PA. p. 293. Clinical and laboratory findings of anemia due to hemorrhage The clinical and laboratory manifestations of hemorrhage depend on the volume of the hemorrhage and the rapidity with which it occurs. 1. Anemia—pallor, tachycardia, and hypotension (if severe, e.g., $ 20 mL/kg blood loss). Nonimmune hydrops can occur in severe anemia. 2. Liver and spleen not enlarged (except in chronic transplacental bleed). 3. Jaundice absent (except after several days in entrapped hemorrhage). 4. Laboratory findings: a. reduced hemoglobin (as low as 2 g/dL has been observed), b. increased reticulocyte count, c. polychromatophilia, d. nucleated RBCs raised, e. fetal cells in maternal blood (in fetomaternal bleed), and f. direct antiglobulin test (DAT) negative. Treatment 1. Severely affected a. Transfusion of packed red blood cells. b. Crossmatch blood with the mother. If unavailable, use group O Rh-negative blood or intravenous fluids, temporarily for shock, while awaiting available blood. 2. Mild anemia due to chronic blood loss a. Ferrous sulfate (4!6 mg elemental iron/kg body weight per day) for 3 months. Hemolysis Hemolytic anemia in the newborn Hemolytic anemia in the newborn is usually associated with an abnormally low hemoglobin level, an increase in the reticulocyte count, and unconjugated hyperbilirubinemia. The degree of reticulocytosis may be less than Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 50 3. Classification and diagnosis of anemia in children and neonates anticipated depending on the age of neonate or infant, because any hemolytic anemia will be superimposed on the physiologic anemia of infancy. The hemolytic process is often first detected as a result of investigation for jaundice during the first week of life. The causes of hemolytic anemia in the newborn are listed in Table 3.6. Congenital erythrocyte defects Congenital erythrocyte defects involving the red cell membrane, hemoglobin, and enzymes are listed in Table 3.6 and discussed in Chapter 7, General Considerations of Hemolytic Diseases, Red Cell Membrane, and Enzyme Defects,and Chapter 8, Extracorpuscular Hemolytic Anemia. Any of these conditions may occur in the newborn and manifest clinically with: • hemolytic anemia (low hemoglobin, reticulocytosis, increased nucleated red cells, morphologic changes); • unconjugated hyperbilirubinemia; and • DAT negative. Infantile pyknocytosis The cause of this condition has not been clearly defined and should only be contemplated when other established causes of pyknocytes in the blood have been excluded such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, pyruvate kinase deficiency, microangiopathic hemolytic anemia, neonatal hepatitis, vitamin E deficiency, neonatal infections, and hemolysis caused by drugs and toxic agents. It is a congenital but not a constitutional disorder; that is, one is born with it (congenital) but it is not an ongoing, lifelong, or necessarily genetic condition (not constitutional). Infantile pyknocytosis is characterized by: • Hemolytic anemia—DAT negative (nonimmune). • Distortion of as many as 50% of red cells!dense, contracted cells (pyknocytes) with several to many spiny projections (up to 6% of cells may be distorted in normal infants). Abnormal morphology is extracorpuscular in origin, and transfused red blood cells from normal donors can acquire pyknocytosis. • Disappearance of pyknocytes and hemolysis by the age of 6 months. This is a self-limiting condition. • Hepatosplenomegaly. Hemoglobinopathies The developmental changes in globin proteins uniquely impact the presentation of hemoglobinopathies in neonates. For example, anemia due to gamma-hemoglobinopathies resolves spontaneously, whereas beta hemoglobinopathies are clinically inapparent at birth and manifest only after a few months. Anemia from alphahemoglobinopathies can occur throughout life (prenatal and postnatal) (see Chapter 9: Hemoglobinopathies). Gamma globin defects These variants spontaneously resolve as gamma chain production decreases in the newborn and generally produce no hematologic symptoms. Some mutations cause hypochromia and mild anemia may occur at birth. Rarely, marked in utero and neonatal hemolysis can occur. Some variants can be identified during newborn screening. Beta globin defects Beta globin defects generally produce no clinical issues in the newborn due to predominance of HbF. In sickle cell anemia (homozygosity for HbS), for example, there is no anemia at birth, but anemia and splenic infarction (hence, functional hyposplenism and increased risk of bacterial sepsis) can occur by 2!3 months of age, depending on the rapidity of the decline in HbF. In beta-thalassemia syndromes, hematologic findings at birth are normal and only manifest after 6 months of life. Alpha chain defects A large spectrum of alpha-thalassemia syndromes occurs in the newborn and they are discussed in the chapter on hemoglobinopathies (Chapter 9: Hemoglobinopathies). Acquired erythrocyte defects Acquired erythrocyte defects may be due to immune (DAT positive) or nonimmune (DAT negative) causes. The immune causes are due to blood group incompatibility between the fetus and the mother, for example, Rh Lanzkowsky’s. Manual of Pediatric Hematology and Oncology Neonatal anemia 51 (D), ABO, or minor blood group incompatibilities such as anti-c, Kell, Duffy, Luther, anti-C, anti-Cw, anti-E, and anti-Jk(b) causing isoimmunization. Kell antigen is second to Rh (D) in its immunizing potential. Immune hemolytic anemia Rh isoimmunization Clinical features 1. Anemia, mild-to-severe (if severe, may be associated with hydrops fetalis). 2. Jaundice (unconjugated hyperbilirubinemia). a. presents during first 24 hours. b. Kernicterus may occur if the bilirubin level in full-term infants rises to, or exceeds, 20 mg/dL and is an indication for exchange transfusion. Certain factors predispose to the development of kernicterus at lower levels of bilirubin such as prematurity, hypoproteinemia, metabolic acidosis, drugs (sulfonamides, caffeine, and sodium benzoate), and hypoglycemia. See Table 3.8 for a list of various causes of unconjugated hyperbilirubinemia. Fig. 3.4 outlines an approach to the diagnosis of unconjugated (indirect) hyperbilirubinemia. 3. Hepatosplenomegaly—varies with severity. 4. Petechiae (only in severely affected infants)—hyporegenerative thrombocytopenia and neutropenia may occur. 5. Hydrops fetalis, stillbirth, or death in utero and delivery of a macerated fetus may occur with severe illness. 6. Late hyporegenerative anemia with decreased reticulocyte count—this may occur during the second to the fifth weeks and is due to a diminished population of erythroid progenitors (serum concentration of erythropoietin (EPO) is low and the marrow numbers of erythroid precursors are not elevated). Laboratory findings 1. Serologic abnormalities (incompatibility between infant and mother blood groups), with a positive DAT in the infant. The antibodies can be detected in the mother’s serum by the indirect antiglobulin test. 2. Decreased hemoglobin level, elevated reticulocyte count, smear: increased nucleated red cells, marked polychromasia, and anisocytosis. 3. Raised indirect bilirubin level. Severity of disease is predicted by: • history indicating the severity of hemolytic disease of the newborn in previous infants; • the type of RBC antigen mismatch (e.g., hemolytic disease due to Rh mismatch is generally more severe than ABO mismatch); • maternal antibody titers; • fetal ultrasonography; and • percutaneous fetal blood sampling. Management Antenatal Mothers should be screened at their first antenatal visit for anti-D and other antibodies. If a likely pathogenic antibody is detected in the mother’s serum, proper management includes the following: • Detailed past obstetric history and outcome of previous pregnancies, including neonates who required exchange transfusion or intravenous immunoglobulin, hydrops fetalis, or stillbirth. • History of prior blood transfusions. • Blood group and indirect antiglobulin test (to determine the presence and titer of irregular antibodies). Most irregular antibodies can cause erythroblastosis fetalis; therefore screening of maternal serum is important. Titers should be determined at various weeks of gestation. The frequency depends on the initial or subsequent rise in titers. Theoretically, any blood group antigen (with the exception of Lewis and I, which are not present on fetal erythrocytes) may cause erythroblastosis fetalis. Anti-Lea, Leb, M, H, P, S, and I are IgM antibodies and rarely, if ever, cause erythroblastosis fetalis and need not cause concern. • Determination of zygosity of the father: if the mother is D negative and the father is D positive, the father’s zygosity becomes critical. If he is homozygous, all children of this couple will be D positive. If the father is heterozygous, there is a 50% chance that the fetus will be D negative and unaffected. Fetal RhD genotype can be accurately determined using cell-free DNA in maternal plasma. • Screening for severe fetal anemia using Doppler ultrasonography to measure peak systolic velocity in the middle cerebral artery of the fetus (MCA-PSV). This more accurate and noninvasive method has replaced the Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 52 3. Classification and diagnosis of anemia in children and neonates TABLE 3.8 Causes of unconjugated hyperbilirubinemia. 1. “Physiologic” jaundice: Jaundice of hepatic immaturity 2. Hemolytic anemia a. Congenital erythrocyte defect i. membrane defects: hereditary spherocytosis, ovalocytosis, stomatocytosis, and infantile pyknocytosis ii. enzyme defects (nonspherocytic) • glycolytic pathway: pyruvate kinase, triose phosphate isomerase, etc. • hexose-monophosphate shunt (reduction potential): G6PD iii. hemoglobin defects • alpha thalassemia (HbH disease) • unstable hemoglobins b. Acquired erythrocyte defect i. immune: alloimmunization (Rh, ABO, Kell, Duffy, Lutheran) ii. nonimmune • infection bacterial: E. coli, streptococcal septicemia viral: Cytomegalovirus, rubella, herpes simplex protozoal: toxoplasmosis spirochetal: syphilis • drugs • metabolic: asphyxia, hypoxia, shock, acidosis, vitamin E deficiency in premature infants, and hypoglycemia 3. Erythrocytosis (see Chapter 10: Primary and Secondary Erythrocytosis for more complete list of causes) a. Placental hypertransfusion i. twin-to-twin transfusion ii. maternal!fetal transfusion iii. delayed cord clamping b. Placental insufficiency i. small for gestational age ii. postmaturity iii. toxemia of pregnancy iv. placenta previa c. Endocrinological causes i. congenital adrenal hyperplasia ii. neonatal thyrotoxicosis iii. maternal diabetes mellitus d. Miscellaneous i. Down syndrome ii. hyperplastic visceromegaly (Beckwith!Wiedemann syndrome), associated with hypoglycemia 4. Hematoma Cephalohematoma, subgaleal, subdural, intraventricular, intracerebral, subcapsular hematoma of liver; bleeding into gut 5. Conjugation defects a. Reduction in bilirubin glucuronyl transferase i. severe (type I): Crigler!Najjar (autosomal-recessive) ii. mild (type II): Crigler!Najjar (autosomal-dominant) iii. Gilbert disease b. Inhibitors of bilirubin glucuronyl transferase i. drugs ii. breast milk iii. familial: transient familial hyperbilirubinemia 6. Metabolic Hypothyroidism, maternal diabetes mellitus, and galactosemia 7. Gut obstruction (due to increased enterohepatic recirculation of bilirubin)(e.g., pyloric stenosis, annular pancreas, and duodenal atresia) 8. Maternal indirect hyperbilirubinemia (e.g., sickle cell anemia) 9. Idiopathic Abbreviation: G6PD, Glucose-6-phosphate dehydrogenase. From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 53 Neonatal anemia Unconjugated (indirect reacting) bilirubin Direct antiglobulin test Positive ABO Rh Kell Duffy Luther Negative Reticulocyte count Raised Normal Blood smear Red cell morphology Hemoglobin Abnormal Normal Low Normal High Spherocytes Ovalocytes Pyknocytes Stomatocytes Schistocytes Enzyme defects Infections Hematomata Examine infant Conjugation defects Metabolic defects Gut obstruction Polycythemia Placental hypertransfusion Placental insufficiency Endocrinologic Miscellaneous Examine infant Examine infant FIGURE 3.4 Approach to investigation of indirect hyperbilirubinemia in the newborn. Abbreviations: G6PD, Glucose-6-phosphate dehydrogenase; MCV, mean corpuscular volume. Source: From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. use of serial spectrophotometric assessments of amniotic fluid for increased bilirubin concentration [i.e., the change in optical density at a wavelength of 450 nm (∆OD450)] to screen for severe fetal anemia. • Once critical titers of causative antibodies are reached, MCA-PSV values are used to determine need and timing for fetal blood sampling. • The need for intrauterine transfusion should be made by multidisciplinary teams in fetal medicine centers, and specific definitions of moderate-to-severe fetal anemia differ across centers. • There is limited evidence to support the use of intravenous immunoglobulin (IVIG), either alone or in combination with therapeutic plasma exchange, to decrease the risk of fetal hydrops or death in severely Rhsensitized mothers, and the decision to use these treatments are best made by multidisciplinary teams in fetal medicine centers. • Despite intrauterine transfusion, there is an approximately 20% risk of premature delivery and most will need exchange transfusion to prevent kernicterus. • Multiple intrauterine transfusions are associated with suppression of erythropoiesis in the newborn and late or prolonged postnatal anemia that requires transfusion support. Postnatal For the hydropic infant at birth, in addition to phototherapy, the following measures are employed or considered: • adequate ventilation must be established; • drainage of pleural effusions and ascites to improve ventilation; • use of resuscitation fluids and drugs, surfactant, and glucose infusions to counteract hyperinsulinemic hypoglycemia should be employed; • partial exchange transfusion may be necessary to correct severe anemia; and • double-volume exchange transfusion may be required later. Hyperbilirubinemia is the most frequent problem and can be managed by exchange transfusion. Phototherapy is an adjunct rather than the first line of therapy in hyperbilirubinemia due to erythroblastosis fetalis. Postnatal Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 54 3. Classification and diagnosis of anemia in children and neonates management and criteria for exchange transfusion have changed over the years and still remain somewhat controversial. Institutional, professional, and other current guidelines should be used to guide treatment decisions. In general: • Exchange transfusion should be performed for any signs of acute bilirubin encephalopathy (hypertonia, arching, retrocollis, opisthotonos, fever, high-pitched cry). • Exchange transfusion is recommended when the total serum bilirubin exceeds limits on age-specific nomograms despite intensive phototherapy. • Exchange transfusion should be considered when the bilirubin-to-albumin ratio exceeds cutoff values based on gestational age and comorbidities such as G6PD deficiency and isoimmune hemolytic anemia. • The blood for exchange transfusion should be ABO compatible and for anti-D hemolytic disease of the newborn, D negative. If the mother is alloimmunized to an antigen other than D, the blood should not have that antigen. It should be crossmatch compatible with the mother’s serum. Ideally, the blood should be leukocyte depleted and be negative for Kell antigen (to avoid sensitizing the infant) and be hemoglobin S negative. If the initial exchange transfusion is carried out using the group O blood, any further exchange transfusions should use O blood. Otherwise, brisk hemolysis and jaundice due to ABO incompatibility may become a further complication. Graft-versus-host disease occurs rarely after exchange transfusion, so blood must be irradiated to prevent this complication in neonates and infants. Prevention of Rh hemolytic disease Rh hemolytic disease can be prevented by the use of Rh immunoglobulin, which is indicated in the following circumstances: • For all D-negative or partial D-negative mothers who are unimmunized to D. In these patients, Rh immunoglobulin is given as a single large dose at 28 weeks’ gestation or as 2 smaller doses at 28 and 34 weeks’ gestation, and within 72 hours of delivery of D-positive newborn. Antenatal administration of Rh immunoglobulin is safe for the mother and the fetus. • For all unimmunized D-negative mothers who have undergone spontaneous (1.5!2% risk of sensitization) or induced abortion (5% risk of immunization). If surgical evacuation is done, Rh immunoglobulin should be given (the dose may differ based on indication or procedure). The D antigen is detectable on embryonic red cells by 38 days after conception, and Rh immunoglobulin should be given beyond the seventh or eighth weeks of gestation in these circumstances. The precise risk of alloimmunization associated with these events is less clear compared to the risk after delivery. • After ruptured tubal pregnancies in unimmunized D-negative mothers. • Following any event during pregnancy that may lead to transplacental hemorrhage such as external version, amniocentesis, or antepartum hemorrhage in unimmunized D-negative women. • Following tubal ligation or hysterotomy after the birth of a D-positive child in unimmunized Rh-negative women. • Following chorionic villus sampling at 10!12 weeks’ gestation. The dose of Rh immunoglobulin may differ based on indication or procedure. • In these potentially sensitizing events, fetomaternal hemorrhage should be tested and additional doses of Rh immunoglobulins may be needed in proportion to the amount of fetal blood. • Acute drug-induced hypersensitivity and delayed transfusion-related reaction, including anaphylaxis with deleterious effects on the mother and the fetus, have been described. ABO isoimmunization ABO incompatibility is milder than hemolytic disease of the newborn caused by other antibodies. Clinical features 1. Jaundice (indirect hyperbilirubinemia) usually within first 24 hours; rarely, it may be of sufficient severity to cause kernicterus. 2. Anemia at birth is usually absent or moderate and late anemia is rare. 3. Possible hepatosplenomegaly. Table 3.9 lists the clinical and laboratory features of isoimmune hemolysis due to Rh and ABO incompatibility. Diagnosis 1. Hemoglobin decreased 2. Smear: spherocytosis in 80% of infants, reticulocytosis, marked polychromasia Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 55 Neonatal anemia 3. Elevated indirect bilirubin level 4. Demonstration of incompatible blood group a. Group O mother and an infant who is group A or B. b. Rarely, mother may be A and baby B or AB or mother may be B and baby A or AB. 5. DAT of infant’s red cells often positive 6. Demonstration of antibody in infant’s serum a. When free anti-A is present in a group A infant or anti-B is present in a group B infant, ABO hemolytic disease may be presumed. These antibodies can be demonstrated by the indirect antiglobulin test in the infant’s serum using adult erythrocytes possessing the corresponding A or B antigen. This is a proof that the antibody has crossed from the mother’s to the baby’s circulation. b. Antibody can be eluted from the infant’s red cells and identified. 7. Demonstration of antibodies in maternal serum Treatment In ABO hemolytic disease, unlike Rh disease, antenatal management or premature delivery is not required. After delivery, management of an infant with ABO hemolytic disease is directed toward controlling hyperbilirubinemia by frequent determination of unconjugated bilirubin levels, with a view to the need for phototherapy or exchange transfusion. The principles and methods are the same as those described for Rh hemolytic disease. Group O blood of the same Rh type as that of the infant should be used. Whole blood or reconstituted red blood cells in fresh frozen plasma can be used to permit maximum bilirubin removal by albumin. TABLE 3.9 Clinical and laboratory features of immune hemolysis caused by Rh and ABO incompatibility. Feature Rh disease ABO incompatibility Frequency Rare (since the use of Rh-Ig) Common Occurrence in first born 5% 40!50% Predictably severe in subsequent pregnancies Usually No Stillbirth and/or hydrops Occasional Rare Pallor Marked Minimal Jaundice Marked Minimal (occasionally marked) Hepatosplenomegaly Marked Minimal Incidence of late anemia Common Uncommon Blood type, mother Rh negative O (occasionally A or B) Blood type, infant Rh positive A or B or AB Direct antiglobulin test Positive Usually positive Indirect antiglobulin test Positive Usually positive Hemoglobin level Moderately or severely low Mildly or moderately low Serum bilirubin Markedly elevated Variably elevated Red cell morphology Nucleated RBCs, polychromasia, and spherocytes Spherocytes, variable polychromasia Need for antenatal management Yes No Exchange transfusion Often needed Uncommonly needed Donor blood type D-negative group specific, when possible Rh same as infant group O only Clinical evaluation Laboratory findings Treatment From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 56 3. Classification and diagnosis of anemia in children and neonates Late-onset anemia in immune hemolytic anemia Infants with hemolytic anemia of the newborn due to isoimmunization and particularly in those infants who have had severe isoimmunization treated with intrauterine transfusion and in those infants who do not require an exchange transfusion for hyperbilirubinemia following immune hemolytic anemia may develop severe anemia during the first 6 weeks of life that can last to 4!6 months of life. This is at least partly due to persistent maternal IgG antibodies that clear circulating red blood cells and reticulocytes and also suppression of erythropoiesis due to intramedullary destruction of erythroblasts. Additional mechanisms of late suppression of erythropoiesis have also been postulated. For this reason, hemoglobin and reticulocyte counts should be monitored regularly for 4!6 weeks, or until evidence of resolution, which can take several months. Treatment with EPO or darbepoetin may be needed to decrease the need for transfusion. Nonimmune hemolytic anemia The causes of nonimmune hemolytic anemia are listed in Table 3.6. Vitamin E deficiency Premature infants (,36 weeks’ gestation or weighing less than 2000 g) are susceptible to vitamin E deficiency because of decreased absorption of the vitamin. With improvement in infant formulas recognizing this propensity, this condition has virtually disappeared. Clinical findings 1. Hemolytic anemia and reticulocytosis. Hemolytic anemia develops under the following conditions: diets high in polyunsaturated fatty acids supplemented with iron, which is a powerful oxidant; prematurity; oxygen administration. 2. Thrombocytosis. 3. Pyknocytes (acanthocytes), small number of spherocytes, and fragmented red cells. 4. Peripheral edema. 5. Neurologic signs: a. cerebellar degeneration, b. ataxia, and c. peripheral neuropathy. Diagnosis Mostly historically, the peroxide hemolysis test: red cells are incubated with small amounts of hydrogen peroxide and the amount of hemolysis is measured. This test is not readily available as a clinical assay and has been replaced, indirectly, by the measurement and interpretation of serum tocopherol levels in the context of consistent clinical and laboratory features. Hypoplasia Congenital The inherited bone marrow failure syndromes are discussed in Chapter 6, Bone Marrow Failure. Acquired Viral diseases Viral medullary toxicity (e.g., CMV and HIV) with fetal hematopoiesis may cause anemia, leukopenia, and thrombocytopenia in the newborn, sometimes with evidence of extramedullary hematopoiesis. Anemia of prematurity This anemia is due to impaired EPO production and characterized by reduced bone marrow erythropoietic activity (hypoproliferative anemia) with low reticulocyte count and low serum EPO levels. It may be compounded by folic acid, vitamin E, and iron availability, and frequent blood sampling. • The low hemoglobin concentration is due to: • preterm infants deprived of third-trimester hematopoiesis and iron transport; • decreased marrow erythroid elements and red cell production: premature infants have low EPO levels that reach a nadir between days 7 and 50, independently of weight at birth, and are less responsive to EPO; Lanzkowsky’s. Manual of Pediatric Hematology and Oncology Neonatal anemia 57 • shorter red cell lifespan; • increased blood volume with growth; and • marked blood loss from phlebotomy to monitor problems related to prematurity. The nadir of the hemoglobin level is at 4!8 weeks and is 8 g/dL in infants weighing less than 1500 g. However, small-for-gestational-age infants who have had intrauterine hypoxia exhibit increased erythropoiesis. The anemia of prematurity rarely occurs in association with cyanotic congenital heart disease or with respiratory insufficiency, indicating that higher oxygen-carrying capacities can be maintained in infants in the first few weeks of life if the need arises. Clinical features Tachycardia, increased apnea, and bradycardia increased oxygen requirement, poor weight gain. The anemia is normocytic and normochromic. Treatment Delaying cord clamping for 30!60 seconds in infants who do not require immediate resuscitation can be considered to reduce the severity of anemia of prematurity. In addition, limiting blood loss by phlebotomy is important. • Recombinant human EPO (rHuEPO) has been used to increase reticulocyte counts and raise hemoglobin. Darbepoetin can be used instead. It takes about 2 weeks to raise the hemoglobin to a biologically significant degree, which limits its usefulness when a prompt response is needed. A potential advantage of rHuEPO is the associated right shift in the oxyhemoglobin dissociation curve, most likely due to the increased erythrocyte 2,3-bisphosphoglycerate (2,3-BPG) content. Although many studies have shown a reduction in need for transfusions in premature infants who receive rHuEPO early (before day 8 of life) or late (between days 8 and 28 of life), a recent systematic review concluded that the overall benefit may not be clinically important as exposure to blood transfusions was unavoidable in many patients, especially when rHuEPO was started late. The incidence of necrotizing enterocolitis, intraventricular hemorrhage, and periventricular leukomalacia has been shown to be lower in very-low-birth-weight infants treated with rHuEPO, but whether this impacts the neurodevelopmental outcomes is controversial. Also, there are conflicting data about increased risk of severe retinopathy of prematurity (ROP) (stage 3 or higher) in patients who receive early rHuEPO. Based on these uncertainties surrounding rHuEPO, the routine early use of EPO in preterm infants is not recommended and more studies are needed to properly assess its effect on neurodevelopmental outcomes, ROP, and necrotizing enterocolitis. • Supplemental oral iron in a dose of at least 2 mg/kg/day or intravenous iron supplementation may also be required to prevent the development of iron deficiency, especially in combination with rHuEPO. Adequate intake of folate, vitamin E, and protein are important to support erythropoiesis. • The criteria for transfusion of preterm infants vary considerably among different institutions. All transfusions should be provided from a single donor, be less than 7!10 days old and be leukodepleted. Volume depletion may be needed depending on the hematocrit of donor packed red blood cells and the neonate’s clinical status. Low-risk cytomegalovirus blood products (cytomegalovirus-negative or leukodepleted red cells) should be used only for neonates with birth weight less than 1200 g who are cytomegalovirus negative or have unknown cytomegalovirus status. As a general rule, hemoglobin values should be maintained above 12 g/dL during the first 2 weeks of life. After that period the indication for transfusion should not be based on hemoglobin concentration alone but on available tissue oxygen which is determined by: • hemoglobin concentration; • position of the oxyhemoglobin dissociation curve (usually inferred or guessed, but it can be measured in some laboratories); • arterial oxygen saturation; and • infant’s clinical condition that includes: • weight gain, • fatigue during feeding, • tachycardia, • tachypnea, and • evidence of hypoxemia by an increase in blood lactic acid concentration. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology 58 3. Classification and diagnosis of anemia in children and neonates Physiologic anemia Physiologic anemia is a developmental response of the infant’s erythropoietic system. • In utero the oxygen saturation of the fetus is 70% (hypoxic levels) and this stimulates EPO, produces a reticulocytosis (3!7%), and increases red cell production causing a high hemoglobin at birth. After birth the oxygen saturation is 95%, EPO is undetectable, and red cell production by day 7 is 10% of the level in utero. As a result, the hemoglobin level falls to 11.4 6 0.9 g/dL at the nadir at 8!12 weeks (physiologic anemia). At this point, oxygen delivery becomes limiting, EPO is stimulated, and red cell production increases. Infants born prematurely experience a more marked decrease in hemoglobin concentration that may start earlier and last longer than full-term infants. Premature infants weighing less than 1500 g have a hemoglobin level of 8 g/ dL at age 4!8 weeks. Diagnostic approach to anemia in the newborn Fig. 3.5 is a flow diagram of the investigation of anemia in the newborn that stresses the importance of the DAT, reticulocyte count, MCV, and the blood smear as key investigative tools in elucidating the cause of the anemia, and Table 3.10 lists the clinical and laboratory evaluations required in anemia in the newborn. Positive Isoimmunization (ABO, Rh, minor blood group, e.g., Keil) Direct antiglobulin test Reticulocyte count Negative Subnormal Diamond–Blackfan anemia (pure red cell aplasia) Normal or elevated MCV Low Normal or high Chronic intrauterine blood loss α-Thalassemia syndromes Blood smear Rare miscellaneous causes Hexokinase deficiency Galactosemia Blood loss Iatrogenic (sampling) Fetomaternal/fetoplacental Cord problems Twin-to-twin Internal hemorrhage Normal Abnormal Infection e.g., HIV Toxoplasmosis CMV Rubella Syphilis Hereditary spherocytosis Hereditary elliptocytosis Hereditary stomatocytosis Infantile pyknocytosis Pyruvate kinase deficiency G6PD deficiency Disseminated intravascular coagulation Vitamin E deficiency FIGURE 3.5 Approach to the diagnosis of anemia in the newborn. Abbreviations: CMV, cytomegalovirus; HIV, human immunodeficiency virus. Source: From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology Further reading and references TABLE 3.10 59 Clinical and laboratory evaluation in anemia in the newborn. HISTORY Obstetric history Family history PHYSICAL EXAMINATION LABORATORY TESTS Complete blood count Reticulocyte count Blood smear Antiglobulin test (direct and indirect) Blood type of baby and mother Bilirubin level Flow cytometry of maternal blood (fetal red cells in maternal blood) Studies for neonatal infection Ultrasound of abdomen and head (if indicated) Red cell enzyme assays (if clinically indicated) Bone marrow (if clinically indicated) From Lanzkowsky, P., 2016. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Further reading and references American Academy of Pediatrics Subcommittee on Hyperbilirubinemia, 2004. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 114 (1), 297!316. Available from: https://doi.org/10.1542/peds.114.1.297. Bishara, N., Ohls, R.K., 2009. Current controversies in the management of the anemia of prematurity. Semin. Perinatol. 33 (1), 29!34. Brugnara, C., Oski, F.A., Nathan, D.G., 2015. Diagnostic approach to the anemic patient. In: Orkin, S.H., Fisher, D.E., Look, T., Lux, S.E., Ginsburg, D., Nathan, D.G., et al.,Nathan and Oski’s Hematology and Oncology of Infancy and Childhood, eighth ed. WB Saunders, Philadelphia, PA, p. 293. Christensen, R.D., Yaish, H.M., Lemons, R.S., 2014. Neonatal hemolytic jaundice: morphologic features of erythrocytes that will help you diagnose the underlying condition. Neonatology 105, 243!249. Committee on Practice, Bulletins-Obstetrics, 2017. Practice bulletin no. 181: Prevention of Rh D alloimmunization. Obstet. Gynecol. 130, e57!e70. Fasano, R.M., 2016. Hemolytic disease of the fetus and newborn in the molecular era. Semin. Fetal Neonatal. Med. 21, 28!34. Ford, J., 2013. Red blood cell morphology. Int. J. Lab. Hematol. 35, 351!357. Gallagher, P.G., 2015. The neonatal erythrocyte and its disorders. In: Orkin, S.H., Fisher, D.E., Look, T., Lux, S.E., Ginsburg, D., Nathan, D.G. (Eds.), Nathan and Oski’s Hematology and Oncology of Infancy and Childhood, eighth ed. WB Saunders, Philadelphia, PA, p. 52. Hermiston, M.L., Mentzer, W.C., 2002. A practical approach to the evaluation of the anemic child. Pediatr. Clin. North Am. 49, 877!891. Howarth, C., Banerjee, J., Aladangady, N., 2018. Red blood cell transfusion in preterm infants: current evidence and controversies. Neonatology 114, 7!16. McDaniel, J.K., Sorge, C.E., 2019. Diagnostic approach to anemia in childhood and adolescents. In: Means Jr., R. (Ed.), Anemia in the Young and Old. Springer, Cham. Available from: https://doi.org/10.1007/978-3-319-96487-4_2. Nassin, M.L., Vergilio, J.A., Heeney, M.M., LaBelle, J.L., 2017. Neonatal anemia: revisiting the enigmatic pyknocyte. Am. J. Hematol. 92, 717!721. Oepkes, D., Seaward, P.G., Vandenbussche, F.P., Windrim, R., Kingdom, J., Beyene, J., et al., 2006. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N. Engl. J. Med. 355, 156!164. Ohlsson, A., Aher, S.M., 2020. Early erythropoiesis-stimulating agents in preterm or low birth weight infants. Cochrane Database Syst. Rev. 2, CD004863. Prefumo, F., Fichera, A., Fratelli, N., Sartori, E., 2019. Fetal anemia: diagnosis and management. Best Pract. Res. Clin. Obstet. Gynaecol. 58, 2!14. Pilgrim, H., Lloyd-Jones, M., Rees, A., 2009. Routine antenatal anti-D prophylaxis for Rh-D-negative women: a systematic review and economic evaluation. Health Technol. Assess. 13 (10), 1!103. Sandler, S.G., Flegel, W.A., Westhoff, C.M., et al., 2015. It’s time to phase in RHD genotyping for patients with a serologic weak D phenotype. College of American Pathologists Transfusion Medicine Resource Committee Work Group. Transfusion 55, 680!689. Society for Maternal-Fetal, Medicine, Simpson, L.L., 2013. Twin-twin transfusion syndrome. Am. J. Obstet. Gynecol. 208, 3!18. Walters, M.C., Abelson, H.T., 1996. Interpretation of the complete blood count. Pediatr. Clin. North. Am. 43, 599!622. Widness, J.A., 2008. Pathophysiology of anemia during the neonatal period, including anemia of prematurity. Neoreviews 9, e520. Lanzkowsky’s. Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 4 Nutritional anemias Jacquelyn M. Powers1,2 1 Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States 2Iron Disorders and Nutritional Anemias Program, Texas Children’s Hospital, Houston, TX, United States Overview Nutritional anemias result from deficiencies of micronutrients that are essential for hematopoiesis and clinically defined by the presence of anemia with an inappropriately low reticulocyte count response by the marrow to the degree of anemia. Nutritional anemias are often grouped by size or mean corpuscular volume (MCV), with microcytic most commonly resulting in iron-deficiency anemia (IDA) and macrocytic or megaloblastic anemia due to either vitamin B12 or folate deficiency. Iron-deficiency anemia Introduction Iron deficiency is the most common nutritional deficiency in children and is worldwide in distribution, affecting 2 billion people, predominantly women and children. While patients of any age may be affected, pediatric patients are characteristically between 6 months and 3 years or 11 and 17 years of age because of the rapid growth that occurs during these periods. Prevalence The incidence of IDA is high in young children. It is estimated that 40!50% of children under age 5 years in low- and low middle!income countries are iron deficient. In the United States, approximately 3% of children age 1!2 years have IDA. Prevalence rates are twice as high in high-risk groups such as Latino children. Iron deficiency typically occurs during this age-group due to a combination of rapid growth and insufficient dietary iron (Table 4.1). A second peak is seen during adolescence with up to 16% of adolescent girls being iron deficient, with African-American and Latino girls disproportionately affected. Adolescents also experience rapid growth and occasionally suboptimal iron intake. Such risk factors are exacerbated in females due to the onset of menarche, particularly in the context of abnormal uterine or heavy menstrual bleeding. Etiology Growth Growth during childhood is particularly rapid during infancy and puberty. Blood volume and body iron are directly related to body weight throughout life. IDA can occur at any time when rapid growth outstrips the ability of diet and body stores to supply iron requirements. In the first year of life, body weight triples and circulating hemoglobin mass doubles. Each kilogram gain in weight requires an increase of 35- to 45-mg body iron. The Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00024-6 61 © 2022 Elsevier Inc. All rights reserved. 62 4. Nutritional anemias amount of iron in the newborn is 75 mg/kg. If no iron is present in the diet or blood loss occurs, the iron stores present at birth will be depleted by 6 months in a full-term infant and by 3!4 months in a premature infant. Diet The commonest cause of IDA is inadequate intake during the rapidly growing years of infancy and childhood (Table 4.2). Iron requirements of infancy In normal infants, 1 mg/kg/day (assuming 10% absorption) is required to support normal growth. In premature or low-birth-weight infants, infants with anemia during the neonatal period, and those who have experienced significant blood loss, 2 mg/kg/day initiated by 2 weeks of age is recommended. Dietary iron content and requirements Newborn infants predominantly receive breast milk or iron-fortified formula as their primary source of nutrition. Breast milk and cow’s milk each contain less than 1.5-mg iron per 1000 calories (0.5!1.5 mg/L). Although both forms of milk are equally poor in iron, the bioavailability of iron in breast milk is greater. Breastfed infants absorb 20!80% of the iron, in contrast to about 10% absorbed from cow’s milk. After 6 months of age, however, breastfeeding does not provide sufficient iron to support ongoing growth, and a supplemental source of dietary or medicinal iron is required for optimal iron nutrition. Full-term, formula-fed infants do not need additional iron as infant formulas for over the last two decades in the United States contain 12 mg of iron/L. Table 4.3 lists iron content of infant foods. Young children, 1!3 years of age, should have an intake of iron of 7 mg/day. In the transition from infant foods to more standard table foods, infants may take less iron-fortified formula and cereal, but gain a variety of naturally iron-containing foods, such as meats and some vegetables. During this time period, children with excessive cow milk intake are at particularly high risk for developing iron deficiency. In severe cases, cow milk can result in an exudative enteropathy associated with chronic gastrointestinal (GI) TABLE 4.1 Causes of iron-deficiency anemia. Deficient intake 1. Breastfeeding without supplemental iron 2. Excessive cow milk intake 3. Low iron diet (vegan, vegetarian diet without appropriate supplementation) Inadequate/impaired absorption 1. Antacid therapy or high gastric pH 2. Gastrointestinal disorder (inflammatory bowel disease, celiac disease) 3. Intestinal failure (malabsorption) /resection 4. Infection (prolonged diarrhea; Helicobacter pylori infection-associated gastritis) Increased demand 1. Low birth weight, prematurity, twins 2. Growth (infancy, adolescence, and pregnancy) 3. Cyanotic congenital heart disease Blood loss 1. Perinatal a. Placental b. Umbilicus c. Postnatal 2. Gastrointestinal tract a. Hypersensitivity to whole cow’s milk? Due to heat-labile protein, resulting in blood loss and exudative enteropathy (leaky gut syndrome) (Table 3.4) b. Anatomic gut lesions, including substantial intestinal resection c. Gastritis d. Intestinal parasites [e.g., hookworm (Necator americanus or Ancylostoma duodenale) and whipworm (Trichuris trichiura)] 3. Menstrual (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology Iron-deficiency anemia 63 TABLE 4.1 (Continued) 4. Other a. Recurrent epistaxis b. Idiopathic pulmonary hemosiderosis c. Renal disease: infectious cystitis, microangiopathic hemolytic anemia, nephritic syndrome (urinary loss of transferrin), Berger disease, Goodpasture syndrome, and chronic intravascular hemolysis (hemoglobinuria) d. Extracorporeal (hemodialysis, trauma) Inadequate presentation to erythroid precursors (atransferrinemia; antitransferrin receptor antibodies) Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). TABLE 4.2 Infants at high risk for iron deficiency. Increased iron needs 1. Low birth weight 2. Prematurity 3. Multiple gestation 4. High growth rate 5. Chronic hypoxia—high altitude, cyanotic heart disease 6. Fetal anemia/anemia at birth Blood loss 1. Perinatal bleeding 2. Iatrogenic Dietary factors 1. Early cow’s milk intake 2. Early solid food intake 3. Rate of weight gain greater than average 4. Low-iron formula 5. Frequent tea intakea 6. Low vitamin C intakeb 7. Low meat intake 8. Breastfeeding .6 months without iron supplements a Tea inhibits iron absorption. Vitamin C enhances iron absorption Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). b TABLE 4.3 Iron content of infant foods. Food Iron, mg Unit Milk 0.5!1.5 Liter Eggs 1.2 Each Cereal, fortified 3.0!5.0 Ounce Yellow 0.1!0.3 Ounce Green 0.3!0.4 Ounce Beef, lamb, liver 0.4!2.0 Ounce Pork, liver, bacon 6.6 Ounce 0.2!0.4 Ounce VEGETABLES (STARCHED) MEATS (STRAINED) FRUITS (STRAINED) Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 64 4. Nutritional anemias TABLE 4.4 Classification of iron-deficiency anemia (IDA) in relationship to gut involvement. Severea Mild or severe Pathogenesis Cow’s milk Gut changes None Effect No blood loss Cow’s milk Anatomic lesions or inflammation Leaky gut syndrome Loss of: Red cells only Loss of: Red cells Plasma protein Albumin Immune globulin Copper Calcium Malabsorption syndrome Impaired absorption of iron only Impaired absorption of xylose, fat, and vitamin A Duodenitis Result IDA IDA, guaiac-positive IDA, exudative enteropathy IDA, refractory to oral iron IDA, transient enteropathy Treatment Oral iron Modify diet Oral iron Discontinue cow milk Oral iron Discontinue cow milk Intravenous iron Intravenous iron a Can occur in severe chronic IDA from any cause. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). blood loss resulting in iron deficiency. Long-standing iron deficiency may also induce an enteropathy or leaky gut syndrome. In this condition a number of blood constituents, in addition to red cells, are lost in the gut (Table 4.4). Whole cow’s milk should be considered the cause of IDA under the following clinical circumstances: 1. $ 24 ounces or more of whole cow’s milk consumed per day 2. Iron deficiency accompanied by hypoalbuminemia (with or without edema), which is associated with hypotransferrinemia due to gut leakage of this similarly sized protein 3. IDA unexplained by low birth weight, poor iron intake, or excessively rapid growth 4. Suboptimal response to oral iron in IDA or IDA recurring after a satisfactory hematologic response following iron therapy 5. Positive stool guaiac tests in the absence of gross bleeding and other evidence of intestinal lesions; return of GI function and prompt correction of anemia on cessation of cow’s milk School-age children, 4!13 years of age, should have an intake of at least 8!10 mg/day of iron. Transitioning to adolescence increases iron requirements as this group has an increase in hemoglobin mass, increase in muscle mass, and menstrual loss in adolescent girls. For these reasons the recommended daily allowance for iron in the adolescent age-group is 11 mg for males and 15 mg for females. Most environmental iron exist as insoluble salts and gastric acidity assists in converting it to an absorbable form. Any factors reducing gastric acidity (e.g., drugs—histamine-2 blockers, acid pump blockers; surgical procedures) impair iron absorption from nonheme sources. The iron present in plant products is limited both by low solubility and the presence of natural chelators, for example, phytates. Heme iron derived from animal sources is the most readily absorbed iron and is independent of gastric pH. The exact mechanisms of heme iron absorption are unknown. Menstrual blood loss Post menarche, girls have increased iron requirements to maintain overall iron balance. In this age-group, menstrual blood loss is an important cause of iron deficiency. Menstrual blood loss may average approximately 40 mL (20-mg iron) per cycle or more in adolescents with abnormal uterine or heavy menstrual bleeding. Adolescent girls with underlying bleeding disorders are at even higher risk for the development of irondeficiency and/or severe anemia. Gastrointestinal blood loss and impaired absorption While blood loss from the GI tract can occur at any age, it should be formally evaluated and ruled out in any child presenting with IDA outside the typical young child and adolescent female patient populations. School-age children and adolescent males should have stool guaiac assessments performed and be Lanzkowsky’s Manual of Pediatric Hematology and Oncology Iron-deficiency anemia 65 considered for referral to gastroenterology to assess for underlying pathology (i.e., inflammatory bowel disease). Impaired iron absorption due to a generalized malabsorption syndrome (e.g., celiac disease) occurs at higher rates in adults but should be considered a cause for IDA in children, particularly when poor growth or other GI symptoms are present. Iron-refractory iron-deficiency anemia Iron-refractory iron-deficiency anemia (IRIDA) is an extremely rare autosomal recessive disorder caused by a mutation of TMPRSS6, the gene encoding transmembrane protease, serine 6, also known as matriptase-2, which inhibits the signaling pathway that activates hepcidin, the iron regulatory hormone. It is characterized by striking microcytosis, extremely low transferrin saturation, normal or borderline-low ferritin levels, and high hepcidin levels. The diagnosis is confirmed by sequencing of TMPRSS6. IRIDA occurs in less than 1% of cases of IDA seen in medical practice. Most cases of iron resistance are due to an uncorrected underlying etiology, poor adherence to prescribed oral iron administration, or disorders in the GI tract (see Table 4.1). Stages of iron depletion 1. Iron depletion: this occurs when tissue stores are decreased without a change in hematocrit or serum iron levels; serum ferritin levels will be first marker to decrease. 2. Iron-deficient erythropoiesis: this occurs when reticuloendothelial macrophage iron stores are depleted. Serum iron level drops, and the total iron-binding capacity increases without a change in the hematocrit. Erythropoiesis begins to be limited by a lack of available iron and soluble transferrin receptor (STfR) levels increase. Reticulocyte hemoglobin content or equivalent (Ret-He, CHr) will become low. 3. IDA: this is associated with erythrocyte microcytosis, hypochromia, increased red cell distribution width (RDW), and elevated levels of free erythrocyte protoporphyrin (FEP). It is detected when iron deficiency has persisted long enough that a large proportion of circulating erythrocytes were produced after iron became limiting. Anemia is the final stage in iron deficiency. Note that initiation of iron therapy will result in improvement of iron and hematologic parameters in the reverse order to which they initially developed (i.e., anemia will resolve first). Nonhematological manifestations of iron deficiency Iron deficiency is a systemic disorder involving multiple systems rather than exclusively a hematologic condition associated with anemia. Table 4.5 lists important iron-containing compounds in the body and their function and Table 4.6 lists the tissue effects of iron deficiency. Clinical features IDA is chronic, frequently asymptomatic, and likely undiagnosed. In mild anemia, minimal symptoms may be reported until treatment initiation and response occurs. In severe deficiency, pallor, irritability, anorexia, listlessness, fatigue, and pica (craving for nonfood items such as sand, dirt, ice, and clay) may occur. Symptoms of pica are often reversed in a few days on iron treatment before the anemia corrects itself. Restless leg syndrome, a condition that causes discomfort of the lower extremities that is relieved with movement, has been associated with iron deficiency, with a subset of patients having symptomatic improvement in response to iron therapy. It is theorized that this condition results from tissue iron deficiency within parts of the central nervous system related to movement. Differential diagnosis Although iron deficiency is one of the most common causes of microcytic anemia, other conditions should be considered, particularly in patients with a suboptimal response to a trial of oral iron therapy or history without significant risk factors for iron deficiency. Note that in patients with iron deficiency, hemoglobin A2 is decreased. Therefore the diagnosis of β-thalassemia trait/minor (in which hemoglobin A2 is increased) cannot typically be made until after the iron deficiency is corrected, if present. In patients with refractory or persistent microcytic anemia, it may be necessary to do additional investigations, such as determination of serum ferritin, STfR levels, hemoglobin analysis, and, rarely, the examination of the bone marrow for stained iron, in order to establish the cause of the hypochromia. Table 4.7 lists the investigations employed in the differential diagnosis of microcytic anemias. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 66 4. Nutritional anemias TABLE 4.5 Important iron-containing compounds and their function. Compound Function α-Glycerophosphate dehydrogenase Work capacity Catalase RBC peroxide breakdown Cytochromes ATP production, protein synthesis, electron transport Ferritin Iron storage Hemoglobin Oxygen delivery Hemosiderin Iron storage Mitochondrial dehydrogenase Electron transport Monoamine oxidase Catecholamine metabolism Myoglobin Oxygen storage for muscle contraction Peroxidase Bacterial killing Ribonucleotide reductase Lymphocyte DNA synthesis, tissue growth Transferrin Iron transport Xanthine oxidase Uric acid metabolism Abbreviation: RBC, Red blood cell. From Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Laboratory parameters consistent with iron-deficiency anemia 1. Hemoglobin: below the acceptable level for age (Appendix 1); final stage of iron deficiency. 2. Red cell indices: lower than normal MCV; widened RDW. In general, though not absolute, the RDW is high ( . 14.5%) in iron deficiency and normal in thalassemia (,13%). Decrease in MCV generally parallels decreases in hemoglobin. 3. Reticulocyte count: relative number of reticulocytes often increased, but when corrected for anemia the reticulocyte count is usually normal. In severe IDA associated with bleeding, a reticulocyte count of 3!4% may occur. 4. Reticulocyte hemoglobin content/equivalent (Ret-He, CHr): low, occurs prior to a drop in hemoglobin; one of the first parameters to correct with initiation of iron therapy. 5. Platelet count: varies from thrombocytopenia to thrombocytosis; thrombocytopenia more common in severe IDA. 6. Blood smear: red cells are hypochromic and microcytic with anisocytosis and poikilocytosis; thrombocytosis may also be noted. 7. Bone marrow: not indicated to diagnose iron deficiency. If performed, shows hypercellularity of red cell precursors; distortion of normoblast nuclei may occur. Little or no iron is shown in normoblast and reticulum cells by Prussian blue staining. 8. FEP: incorporation of iron into protoporphyrin represents the ultimate stage in the biosynthetic pathway of heme; failure of iron supply will result in an accumulation of free protoporphyrin not incorporated into heme synthesis and the release of erythrocytes into the circulation with high FEP levels. This process occurs prior to the development of microcytic anemia: a. Normal FEP level is 15.5 6 8.3 mg/dL (upper limit 40 mg/dL). b. Elevated in both iron deficiency and lead poisoning but much higher in lead poisoning; normal in thalassemia trait. c. Elevated FEP level, an indication for iron therapy even when anemia and microcytosis have not yet developed. 9. Red blood cell (RBC) zinc protoporphyrin/heme ratio: increased when there is disruption of normal heme production. Nonspecific—raised in iron deficiency, lead poisoning; markedly raised in protoporphyria, congenital erythropoietic porphyria. In these conditions, zinc substitutes for iron in protoporphyrin IX and the concentration of zinc protoporphyrin relative to heme increases. False-positive results may occur in hyperbilirubinemia and falsely low results if the specimen is not protected from light. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Iron-deficiency anemia 67 TABLE 4.6 Tissue effects of iron deficiency. Gastrointestinal tract Central nervous system Cardiovascular system Musculoskeletal system Immunologic system Cellular changes Anorexia: common and an early symptom Atrophic glossitis with flattened, atrophic, lingual papillae, which makes the tongue smooth and shiny Dysphagia Esophageal webs (Kelly!Paterson syndrome) Exudative enteropathy/leaky gut syndrome Generalized malabsorption Beeturia Decreased cytochrome oxidase activity, succinic dehydrogenase Decreased disaccharidases Increased absorption of cadmium and lead Increased intestinal permeability index Irritability Fatigue and decreased activity Reduced cognitive performance; lower mental/motor developmental test scores Decreased attentiveness, shorter attention span Breath-holding spells Papilledema Increase in exercise and recovery heart rate and cardiac output Cardiac hypertrophy Increase in plasma volume Increased minute ventilation values Increased tolerance to digitalis Deficiency of myoglobin and cytochrome C Decreased physical performance in both brief, intense exercise and prolonged endurance work Rapid development of tissue lactic acidosis on exercise Impaired rate of recovery from illness; increased frequency of respiratory infections Impaired leukocyte transformation; impaired granulocyte killing and nitroblue tetrazolium reduction by granulocytes Decreased myeloperoxidase in leukocytes and small intestine Decreased cutaneous hypersensitivity Transferrin inhibition of bacterial growth by binding iron so that no free iron is available for growth of microorganisms Red cells Ineffective erythropoiesis; decreased red cell survival Increased autohemolysis; increased red cell rigidity Increased susceptibility to sulfhydryl inhibitors Decreased heme production; decreased globin and α-chain synthesis Precipitation of α-globin monomers to cell membrane Decreased glutathione peroxidase and catalase activity Inefficient H2O2 detoxification; greater susceptibility to H2O2 hemolysis Oxidative damage to cell membrane; increased cellular rigidity Increased rate of glycolysis-glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, 2,3DPG, and glutathione Increase in NADH-methemoglobin reductase Increase in erythrocyte glutamic oxaloacetic transaminase Increase in free erythrocyte protoporphyrin Impairment of DNA and RNA synthesis in bone marrow cells Other tissues Reduction in heme-containing enzymes (cytochrome C, cytochrome oxidase) and iron-dependent enzymes (succinic dehydrogenase, aconitase) Reduction in monoamine oxidase Increased excretion of urinary norepinephrine Reduction in tyrosine hydroxylase (enzyme converting tyrosine to dihyroxyphenylalanine) Alterations in cellular growth, DNA, RNA, and protein synthesis Reduction in plasma zinc 2,3-DPG, 2,3-Diphosphoglycerate. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). 10. Serum ferritin: reflects the level of body iron stores; it is quantitative, reproducible, specific, and sensitive and requires only a small blood sample. A concentration of less than 15 ng/mL is considered diagnostic of iron deficiency. Low serum ferritin is always consistent with iron deficiency. Normal ferritin levels, however, can exist in iron deficiency when bacterial or parasitic infection, malignancy, or chronic inflammatory conditions Lanzkowsky’s Manual of Pediatric Hematology and Oncology 68 4. Nutritional anemias TABLE 4.7 Summary of laboratory studies in microcytic anemias. Iron deficiency 1 Ethnic origin Hb Serum MCV RDW FEP Ferritin iron Bone TIBC marrow iron Hb analysis Other features Any k k m m k k m k N Dietary deficiency β-Thalassemia β trait (heterozygous) Mediterranean Slightk k N N N orm N N N A2 raised Normal examination F N orm β0 (Homozygous) Mediterranean k k N m m m m m F raised Transfusion dependent (60!90%) Trait (α-thal-1) Asians, N or k Blacks, slightlyk Mediterranean N N N orm N N N N Hemoglobin H disease k k m N N orm N orm N m Hgb H (2!40%) N or m k N or m N or m N N orm N ork m N Anemia of chronic infection Any k N N m Sideroblastic Any k N m N N orm orm Variable hemolytic anemia; RBC inclusion bodies Abbreviations: FEP, Free erythrocyte protoporphyrin; Hb, hemoglobin; MCV, mean corpuscular volume; N, normal; RBC, red blood cell; RDW, red cell distribution width; TIBC, total iron-binding capacity, m, abnormally high; k, abnormally low. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). coexist because ferritin is an acute-phase reactant and its synthesis increases in acute or chronic infection or inflammation. 11. Serum iron and iron saturation percentage: marker of circulating iron; limitations as diagnostic tool for iron deficiency. Reflects balance between several factors, including iron absorbed, iron used for hemoglobin synthesis, iron released by red cell destruction, and size of iron stores. Wide range of normal, varies significantly with age (see Appendix 1) and is subject to marked circadian changes (as much as 100 mg/dL during the day) and recent ingestion of iron. May be helpful to assess iron absorption. 12. Soluble transferrin receptor (STfR) levels: sensitive measure of iron deficiency; correlates with hemoglobin and other laboratory parameters of iron status. STfR is increased in instances of hyperplasia of erythroid precursors such as IDA and thalassemia. It is unaffected by infection and inflammation, unlike serum ferritin. It is, therefore, of great value in distinguishing iron deficiency from the anemia of chronic disease and in identifying iron deficiency in the presence of chronic inflammation or infection. With erythroid hypoplasia or aplasia, for example, aplastic anemia, red cell aplasia, or chronic renal failure, the STfR concentration is decreased. 13. STfR/log ferritin ratio: calculating the ratio of STfR to the logarithm of serum ferritin concentration provides the highest sensitivity and specificity in the presence of chronic inflammation or infection. Values of less than 2.2 mg/L exclude iron deficiency and values of more than 2.9 mg/L confirm iron deficiency. 14. Serum hepcidin: may help identify patients in whom a response to oral iron is probable (those with low hepcidin levels) and those in whom it is not probable (those with normal or elevated hepcidin levels); not readily accessible as a diagnostic tool. Treatment Correct underlying etiology In addition to correctly diagnosing IDA, its etiology must be addressed to ensure treatment success. The history should include conditions resulting in low iron stores at birth, dietary history, and consideration of all factors leading to blood loss. In adolescent girls a detailed menstrual history is imperative, and hormone therapy may be indicated to support recovery of anemia and prevention of recurrence. Aside from menstrual blood loss the most Lanzkowsky’s Manual of Pediatric Hematology and Oncology Iron-deficiency anemia 69 common site of bleeding is into the bowel. If examination of the stool for occult blood is positive, its cause should be evaluated further. Negative guaiac tests may occur if occult blood loss is intermittent or in very small amounts (,5 mL). For this reason, occult bleeding should be tested multiple times when GI bleeding is suspected. Epistaxis, renal blood loss (hematuria or hemoglobinuria from paroxysmal nocturnal hemoglobinuria), and, on rare occasions, bleeding into the lung (idiopathic pulmonary hemosiderosis and Goodpasture syndrome) may all be causes of IDA. Bleeding into these areas requires specific investigations designed to detect the underlying cause. Dietary counseling Infants and young children 1. Promote breastfeeding for at least 6 months, if possible. 2. An alternate to breastfeeding is iron-fortified infant formula until 1 year of age. Avoid cow’s milk until after the first year of age because of the poor bioavailability of iron in cow’s milk and because the protein in cow’s milk can cause occult GI bleeding. 3. Provide supplemental iron (2 mg/kg/day) to premature infants by 2 weeks of age. 4. Provide supplemental iron (1 mg/kg) to breastfed infants by 4!6 months of age. 5. Introduction of table foods with iron is imperative in young children. Dietary counseling: School-age children/adolescents Iron-rich foods should be provided to children in all age-groups to support growth and meet the recommended daily allowance as described previously. Facilitators of iron absorption such as vitamin C!rich foods (citrus, tomatoes, and potatoes), meat, fish, and poultry should be included in the diet. Inhibitors of iron absorption such as tea, phosphate, and phytates common in vegetarian diets should be minimized in children with a diagnosis of IDA. Oral iron medication The goal of therapy is both correction of the hemoglobin level and replenishment of body iron stores. The most reliable criterion of IDA is the hemoglobin response to an adequate therapeutic trial of oral iron. Ferrous sulfate, in a dose of 3 mg/kg/day, is given for 1 month. A reticulocytosis with a peak occurring between the 5th and 10th days followed by a significant rise in hemoglobin level occurs (1 g/dL in 1 month; .2 g/dL in 1 month in moderate-to-severe IDA). 1. Product: ferrous iron (e.g., ferrous sulfate, ferrous gluconate, ferrous ascorbate, ferrous lactate, ferrous succinate, ferrous fumarate, or ferrous glycine sulfate) is effective. Ferric irons are often better tasting but absorbed less efficiently. 2. Dose: a. infants and young children: 3-mg/kg elemental iron, administered once daily; b. older children: 65- to 130-mg elemental iron given once daily; and c. children with significant GI side effects or resolution of anemia: iron once every other day may be better tolerated with good effect. 3. Duration: a minimum of 3 months of iron therapy is needed in order to replete the iron stores. Assessment of a serum ferritin level prior to iron discontinuation can assist with determining whether further iron therapy is indicated. 4. Response: the more severe the anemia (i.e., lower the hemoglobin to start), the higher the reticulocyte response, and more rapid the rise in hemoglobin: a. Peak reticulocyte count on days 5!10 following initiation of iron therapy. b. Following peak reticulocyte level, hemoglobin rises on average by 0.25!0.4 g/dL/day or hematocrit rises 1%/day during first 7!10 days. c. Thereafter, hemoglobin rises slower: 0.1!0.15 g/dL/day. 5. Failure to respond to oral iron: the following reasons should be considered: a. Poor adherence: failure or irregular administration of oral iron b. Inadequate iron dose, ineffective iron preparation, or insufficient duration c. Persistent or unrecognized blood loss d. Incorrect diagnosis: thalassemia, sideroblastic anemia e. Coexistent disease that interferes with absorption or utilization of iron (e.g., chronic inflammation, inflammatory bowel disease) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 70 4. Nutritional anemias f. Congenital IRIDA (see “Iron-refractory iron-deficiency anemia” section)—rare, least likely reason 6. Side effects: constipation, diarrhea, abdominal cramps, nausea, and metallic taste. Note: Although stools may be dark, oral iron does not produce false-positive results on tests for occult blood. Intravenous iron therapy Indications 1. Nonadherence, poor tolerance of oral iron (i.e., failure of oral iron therapy). 2. Severe bowel disease (e.g., inflammatory bowel disease) where the use of oral iron might aggravate the underlying disease of the bowel or iron absorption is compromised, after gastrectomy or duodenal bypass surgery, atrophic gastritis, and celiac disease. 3. Chronic hemorrhage (e.g., congenital coagulation disorders, hereditary telangiectasia, menorrhagia, and chronic hemoglobinuria from prosthetic heart valves). Losing blood at a rate too rapid for oral intake to compensate for the loss. 4. Severe iron deficiency requiring rapid replacement of iron stores. 5. Concomitant iron deficiency and inflammation, resulting in poor iron absorption. 6. Patients anemic after receiving erythropoietin therapy (e.g., renal dialysis and in patients receiving chemotherapy) to ensure an ample and steady supply of iron. 7. Congenital IRIDA. 8. Substitution for blood transfusion when not accepted by patient for religious reasons. 9. Iron deficiency in heart failure. Dose The Ganzoni formula can be utilized to estimate a patient’s total iron deficit. The Ganzoni formula incorporates the patient’s weight as well as current and target Hgb to determine the amount of iron needed to restore the patient to a normal hemoglobin value. It can also be used to estimate the amount of additional iron needed to replenish iron storage sites. ! " ! " ! # $" ! " Iron deficit mg 5 weight kg 3 target ! actual Hgb g=dL 3 2:4 1 15 3 weight kg After determining a patient’s iron deficit and selecting a parenteral iron formulation, the total number of necessary infusions can be determined based on the maximum amount that may be delivered in a single infusion, which varies per product. Formulations Iron formulations vary based on the strength of the iron!carbohydrate complex and immunogenic potential. The former affects the amount of iron that may be safely administered in a single infusion while minimizing the risk of hypersensitivity reactions that result from free iron release. The concentration of iron in each preparation varies slightly: 1. ferric gluconate (12.5 mg/mL), 2. iron sucrose (20-mg/mL elemental iron), 3. low-molecular-weight iron!dextran 50-mg elemental iron/mL, 4. ferumoxytol 30 mg/mL, 5. ferric carboxymaltose 50 mg/mL, 6. ferric derisomaltose 100 mg/mL. Side effects Infusion-related reactions such as flushing, headache, muscle and joint pain, nausea, dizziness, rashes, fever, and chills may occur with parenteral iron. A very small number of patients experience anaphylaxis requiring emergency treatment. Contraindications to parenteral iron therapy 1. Anemias not due to iron deficiency 2. Iron overload 3. History of hypersensitivity to parenteral iron preparations and/or severe allergy or anaphylactic reactions Lanzkowsky’s Manual of Pediatric Hematology and Oncology Megaloblastic anemia 71 4. Clinical or biochemical evidence of liver damage 5. Acute or chronic infection due to concern for “feeding” iron to pathogenic bacteria Intramuscular iron Intramuscular iron therapy was previously utilized in the United States for the treatment of patients with poor or limited iron absorption. This is no longer recommended in settings where intravenous iron therapy is available due to the side effects of intramuscular iron, which include pain and iron staining at the injection site, along with variable absorption. It may continue to be used, however, in low resource settings globally for patients with severe malabsorption when intravenous iron is unavailable. Blood transfusion A packed red cell transfusion should be given in severe anemia requiring correction more rapidly than is possible with oral iron or parenteral iron or because of the presence of certain complicating factors. This should be reserved for debilitated children with infection, especially when signs of cardiac dysfunction are present and the hemoglobin level is 4 g/dL or less. A partial exchange transfusion may be considered in the management of a severely anemic child who requires emergent surgery, when a final hemoglobin of 9!10 g/dL should be attained to permit safe anesthesia, or when anemia is associated with congestive heart failure, in which case it is sufficient to raise the hemoglobin to 4!5 g/dL to correct the immediate anoxia. Summary Iron deficiency is the most common nutritional anemia worldwide. Within pediatrics, young children and adolescent girls are at the highest risk for iron deficiency due to a combination of risk factors such as low iron diet, rapid growth, and menstrual blood loss in the latter. Children with underlying GI disorders that result in poor absorption or blood loss are also at risk for developing iron deficiency. A variety of laboratory tests are available to confirm the diagnosis of iron deficiency. Appropriate therapy includes addressing the underlying etiology and providing iron therapy. Oral iron therapy may be dosed once daily. Intravenous iron therapy may be considered in children who have failed oral iron therapy, have poorly controlled blood loss, or are affected by underlying conditions that limit absorption. Megaloblastic anemia Introduction Megaloblastic anemias result from impaired DNA synthesis in hematopoietic cells and are characterized by macrocytosis with marked variation in the size and shape of RBCs, appropriately low reticulocyte count, hypersegmented neutrophils, and occasionally pancytopenia. Megaloblastic changes in the marrow result from the dyssynchrony between nuclear and cytoplasmic maturation and include hypercellular marrow with an erythroid predominance (reversed myeloid:erythroid ratio), presence of giant pronormoblasts and metamyelocytes. Elevated lactic dehydrogenase, elevated unconjugated bilirubin, low haptoglobin, and occasionally red cell fragments on peripheral smear may be seen. In more than 95% of cases, megaloblastic anemia is a result of folate and vitamin B12 deficiency (Table 4.8). Overview of vitamin B12 (cobalamin) absorption and metabolism Dietary vitamin B12 is acquired mostly from animal sources such as meat and milk. It is absorbed in a series of steps that includes exposures to pancreatic proteases followed by binding to the gastric secretory protein intrinsic factor (IF) within the proximal ileum to form the IF!Cbl complex. Recognition of this complex allows for transport across ileal cells in the presence of calcium ions and release into the portal circulation bound to transcobalamin II (TC II)—the serum protein that carries newly absorbed B12 throughout the body. Fig. 4.1 shows the pathway of cobalamin absorption, transport, and cellular uptake. Cobalamin is converted into the two required coenzyme forms, adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). Causes of vitamin B12 deficiency are listed in Table 4.9. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 72 4. Nutritional anemias Causes of vitamin B12 deficiency Etiologies of cobalamin deficiency fall into four categories: inadequate intake, defective absorption, defective transport, and defective metabolism. Inadequate intake/vitamin B12 nutritional deficiency The recommended dietary allowance of vitamin B12 for children is 0.9!2.4 µg/day. The most common cause of Cbl deficiency in infants results from maternal dietary deficiency either during pregnancy or while breastfeeding. Mothers following vegetarian, vegan, macrobiotic, and other special diets are at particular risk. Cbl in breast milk parallels that in serum and is deficient when the mother is a vegan or has unrecognized pernicious anemia, has had previous gastric bypass surgery or short gut syndrome. Defective B12 absorption Malabsorption of vitamin B12 results in low serum B12 levels with elevations in methylmalonic acid and homocysteine. Table 4.10 lists the features of congenital and acquired defects of vitamin B12 absorption. Gastric acidity and peptic activity deficiency Acid pH and peptic activity are required to release cobalamin from its protein-bound state in food. Impaired gastric function, including atrophic gastritis and partial gastrectomy, therefore, impairs cobalamin absorption. TABLE 4.8 Causes of megaloblastosis. Vitamin B12 (cobalamin) deficiency (see Table 4.9) Folate deficiency (see Table 4.12) Drug-induced • Purine analogs (e.g., 6-mercaptopurine, azathioprine, and thioguanine) • Pyrimidine analogs (5-fluorouracil, 6-azauridine) • Inhibitors of ribonucleotide reductase (cytosine arabinoside, hydroxyurea) Acquired defects in DNA synthesis • • • • Liver disease Sideroblastic anemias Leukemia, especially acute myeloid leukemia (M6) Aplastic anemia (congenital or acquired) Congenital disorders in DNA synthesis • • • • • Orotic aciduria (uridine-responsive) pyrimidine biosynthesis is interrupted Thiamine-responsive megaloblastic anemiaa Congenital familial megaloblastic anemia requiring massive doses of vitamin B12 and folate Associated with congenital dyserythropoietic anemia Lesch!Nyhan syndrome (adenine-responsive) purine nucleotide regeneration is blocked a Associated in some cases with diabetes and sensorineural hearing impairment; wide clinical heterogeneity of this rare disorder; only anemia responsive to high doses of thiamine. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Imerslund Gräsbeck syndrome Intrinsic factor (IF) deficiency HC H+ Protein -bound Cbl Cbl Diet Proteases Cbl/HC H+ Endocytosis Cbl HC Stomach Transcobalamin (TC) deficiency Cbl/MF IF Cbl Uptake by CUBAM Intestine (Ileum) Cbl/TC Intra-cellular Cbl/TC Blood Tissues TC Enterocytes FIGURE 4.1 Summary of cobalamin absorption, transport, and cellular uptake. Abbreviations: Cbl, cobalamin; Cbl/HC, cobalamin!haptocorrin complex; Cbl/IF, cobalamin!intrinsic factor complex; Cbl/TC, cobalamin!transcobalamin complex; CUBAM, ileal receptors made up of cubilin and amnionless proteins; HC, haptocorrin; TC, transcobalamin; 1, intrinsic factor deficiency; 2, Imerslund!Gräsbeck syndrome; 3, transcobalamin deficiency. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Megaloblastic anemia 73 TABLE 4.9 Causes of vitamin B12 deficiency. Inadequate vitamin B12 intake 1. Dietary (,2 mg/day): lacto-ovo vegetarianism, low animal-source food intake, veganism, malnutrition, and poorly controlled phenylketonuria diet 2. Maternal deficiency leading to B12 deficiency in breast milk Defective vitamin B12 absorption (Table 4.10) 1. Congenital intrinsic factor deficiency (gastric mucosa normal) (OMIM 261000) 2. Juvenile pernicious anemia (autoimmune)a 3. Gastric mucosal disease, gastritis, gastric atrophy (i.e., Helicobacter pylori), gastrectomy 4. Failure of absorption in small intestine 5. Specific vitamin B12 malabsorption 6. Defective cobalamin transport by enterocytes (Imerslund!Gräsbeck syndrome) 7. Intestinal failure/resection; intestinal disease causing generalized malabsorption 8. Celiac disease (gluten enteropathy), tropical sprue 9. Infection: HIV infection, parasites (Giardia lamblia, Diphyllobothrium latum, Plasmodium falciparum, and Strongyloides stercoralis) 10. Competition for vitamin B12 11. Small-bowel bacterial overgrowth (e.g., small-bowel diverticulosis, anastomoses and fistulas, blind loops and pouches, multiple strictures, scleroderma, achlorhydria, and gastric trichobezoar) Defective vitamin B12 transport 1. Congenital TC II deficiency (OMIM 275350) 2. Transient deficiency of TC II 3. Partial deficiency of TC I, haptocorrin deficiency (OMIM 193090) Disorders of vitamin B12 metabolism Congenital 1. Adenosylcobalamin deficiency CblA and CblB diseases 2. Deficiency of methylmalonyl-CoA mutase (mut, mut2) 3. Methylcobalamin deficiency CblE and CblG diseases 4. Combined adenosylcobalamin, methylcobalamin deficiencies: CblC, CblD, CblF diseases Acquired 1. Liver disease 2. Protein malnutrition (kwashiorkor, marasmus) 3. Drugs associated with impaired absorption and/or utilization of vitamin B12 (e.g., p-aminosalicylic acid, colchicine, neomycin, ethanol, oral contraceptive agents, and metformin) Pernicious anemia is the final stage of an autoimmune disorder in which autoantibodies against H1K1-adenosine triphosphatase destroy parietal cells in the stomach. Abbreviations: CoA, Coenzyme A; OMIM, Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/omim/); TC I, transcobalamin I; TC II, transcobalamin II. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). a Intrinsic factor deficiency Patients with absent or defective IF (also known as S-binder) present with low serum B12 and megaloblastic anemia. This autosomal recessive disorder usually appears early in the second year of life but may be delayed until adolescence or adulthood. Some patients have no detectable IF, whereas others have IF that can be detected immunologically but lack function. Absorption can be corrected by mixing the vitamin with a source of normal IF. Imerslund!Gräsbeck syndrome: Defective cobalamin transport by ileal enterocyte receptors for the intrinsic factor!cobalamin complex This is a rare autosomal recessive disorder occurring on chromosome 10 that results in a selective defect in ileal cobalamin absorption, which is not corrected by treatment with IF. Patients usually present with a low serum B12 but normal gastric IF levels. There are no IF antibodies present, and the ileal intestinal morphology is normal. The ileal receptor for IF!cobalamin complex may be absent or present. Patients present with pallor, weakness, anorexia, failure to thrive, delayed development, recurrent infections, and GI symptoms, typically within the first 2 years of life. Most known patients are of Scandinavian, Saudi Arabian, or Sephardic Jewish ancestry. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 74 TABLE 4.10 4. Nutritional anemias Features of congenital and acquired defects of vitamin B12 absorption. Stomach Serum antibodies Condition Hydrochloric Histology IFa acid (HCl) IF Parietal cell Associated features Congenital pernicious anemia N A N A A None; relatives may exhibit defective vitamin B12 absorption Juvenile pernicious anemia (autoimmune) Atrophy A Achlorhydria P P (10%) (90%) Autoimmune conditions (diabetes mellitus, hypothyroid, Addison disease, hypoparathyroidism) Enterocyte B12 malabsorption (Imerslund!Gräsbeck) N P N A A Benign proteinuria, aminoaciduria, no generalized malabsorptionb Generalized malabsorption N P N A A Intestinal failure/resection, Crohn’s disease, lymphoma a Either absent secretion of IF or protein that is physiologically inactive; latter includes patients whose IF has reduced affinity for the ileal IF receptor, reduced affinity for cobalamin, or increased susceptibility for proteolysis. b Rare cases associated with generalized malabsorption, reversed by vitamin B12 administration; others without proteinuria or aminoaciduria. Abbreviations: A, Absent; IF, intrinsic factor; N, normal; P, present. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Defective B12 transport Table 4.11 lists the clinical manifestations, laboratory findings, and treatment of autosomal recessive inborn errors of cobalamin transport and metabolism. Disorders of B12 metabolism Congenital Once vitamin B12 has been taken up into cells, it must be converted to an active coenzyme in order to act as a cocatalyst for the two vitamin B12!dependent apoenzymes: methylmalonyl-coenzyme A (CoA) mutase and N5-methyltetrahydrofolate homocysteine methyltransferase. 1. Methylmalonyl-CoA mutase, which requires AdoCbl, catalyzes the conversion of methylmalonyl-CoA to succinyl CoA. Decreased activity of methylmalonyl-CoA mutase results in elevated amounts of methylmalonic acid. 2. Lack of MeCbl leads to deficient activity of N5-methyltetrahydrofolate homocysteine methyltransferase, reducing its ability to methylate homocysteine, and resulting in hyperhomocysteinemia and homocystinuria. Patients with inborn errors of cobalamin utilization present with methylmalonic acidemia and hyperhomocysteinemia, either alone or in combination. Those disorders causing methylmalonic aciduria are characterized by severe metabolic acidosis, with the accumulation of large amounts of methylmalonic acid in blood, urine, and cerebrospinal fluid. All the Cbl metabolism disorders are autosomal recessive with an estimated incidence of 1:61,000. The classification has relied on somatic cell complementation studies in cultured fibroblasts or prenatal detection in cultured amniotic cells in combination with chemical determinations on amniotic fluid or maternal urine. In several cases, in utero cbl therapy has been successfully attempted. Table 4.11 lists the clinical manifestations, laboratory findings, and treatment of autosomal recessive inborn errors of cobalamin metabolism. Acquired In protein malnutrition (kwashiorkor, marasmus) and liver disease, impaired utilization of vitamin B12 has been reported. Certain drugs are also associated with impaired absorption or utilization of vitamin B12 (see Table 4.9). Folic acid deficiency Overview of folate absorption and metabolism Folate in food occurs in the polyglutamate form, which must be hydrolyzed in the brush border of the intestine then absorbed in the duodenum and upper small intestine. It is then transported to the liver to become 5-methyltetrahydrofolate, the principal circulating folate form. Folate binds to and acts as a coenzyme for enzymes that mediate single-carbon transfer reactions. 5,10-Methylenetetrahydrofolate is used unchanged for the synthesis of thymidylate, reduced to 5-methyltetrahydrofolate for the synthesis of methionine, or oxidized to 10formyltetrahydrofolate for the synthesis of purines. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 75 Megaloblastic anemia TABLE 4.11 Clinical manifestations, laboratory findings, and treatment of the autosomal recessive inborn errors of cobalamin transport and metabolism. Condition OMIM no. Defect Typical clinical manifestations Typical onset Laboratory findings Treatment and response TC II deficiency 275350 Defective/absent TC II Failure to thrive, megaloblastic anemia, later neurologic features and immunodeficiency Early infancy 3!5 weeks Usually normal serum Cbl; elevated serum MMA, homocysteine; absent/defective TC II High doses of IM Cbl; good response to treatment if begun early TC I (haptocorrin or R-binder) deficiency 193090 Deficiency/absence of TCI in plasma, saliva, leukocytes Characterized by myelopathy; no megaloblastic hematologic features Unclear if symptoms are related to condition Low serum Cbl, normal TC II!Cbl levels. No increase in MMA or homocysteine Cbl therapy does not appear to be of benefit Defective synthesis of AdoCbl: cblA, cblB cblA (251100), cblB (251110) Defective synthesis of AdoCbl-cobalaminresponsive methylmalonic aciduria Lethargy, failure to thrive, recurrent vomiting, dehydration, hypotonia, ketoacidosis hypoglycemia First weeks Normal serum Cbl, or months of homocysteine, and life methionine; elevated MMA, ketones, glycine, ammonia; leukopenia, thrombocytopenia, anemia Pharmacologic doses of Cbl, dietary protein restriction, oral antibiotics. Treatment response for cblA better than for cblB Defects in methylmalonylCoA mutase apoenzyme formation Well at birth, rapid Initiation of deterioration on protein protein feeding (lethargy, failure feeding to thrive, basal ganglia infarcts) Methylmalonic aciduria, ketoacidosis, ammonia, hypoglycemia, leukopenia, thrombocytopenia Do not respond to B12 therapy. Protein restriction; oral antibiotics carnitine if carnitine deficient MethylmalonylCoA mutase (mut, mut2) deficiency Defective synthesis of MeCbl: cblE, cblG cblE (236270), cblG (250940) Defective synthesis of MeCbl Vomiting, poor feeding, lethargy, severe neurologic dysfunction (hypertonia, blindness, seizures, ataxia), megaloblastic anemia Most in the first 2 years of life Megaloblastic anemia; Normal serum Cbl and folate; homocystinuria, hypomethioninemia Pharmacologic doses of Cbl, betaine; good treatment response in some patients treated early Defective synthesis of AdoCbl and MeCbl: cblC, cblD, cblF cblC (277400) cblD (277410) cblF (277380) Impaired synthesis Failure to thrive, of both AdoCbl and developmental delay, MeCbl neurologic dysfunction, megaloblastic anemia, retinal findings Neonatal period to adolescence; majority neonatal onset Normal serum Cbl, TC II; methylmalonic aciduria, homocystinuria, hypomethioninemia Pharmacologic doses of hydroxocobalamin, moderate protein restriction, betaine treatment Abbreviations: AdoCbl, Adenosylcobalamin; Cbl, cobalamin; CoA, coenzyme A; IM, infectious mononucleosis; MeCbl, methylcobalamin; MMA, methylmalonic acid; OMIM, Online Mendelian Inheritance in Man; TC I, transcobalamin I; TC II, transcobalamin II. Modified from (Rasmussen et al., 2001).with permission; Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier. Causes of folate deficiency As with cobalamin deficiency, etiologies of folic acid deficiency include inadequate intake, defective absorption, defective transport, and defective metabolism (Table 4.12). Inadequate intake/dietary folate deficiency Folate deficiency, next to iron deficiency, is one of the commonest micronutrient deficiencies worldwide. The recommended dietary allowance of folate increases from 150 to 400 µg/day from age 1 to 18 years. Low daily folate during pregnancy intake is associated with a twofold increased risk for preterm delivery and low infant birth weight. Replete folate status is also necessary for the prevention of neural tube defects and is best achieved by prenatal vitamins that include folate to mothers during the periconceptional period. Folate requirements are increased in the following physiologic and pathophysiologic conditions: 1. rapid growth in the first few weeks of life, 2. pregnancy (due to the requirements of the growing fetus) and lactating women, 3. unfortified goat’s milk diet, 4. medication ingestion (e.g., antiepileptic medications, some oral contraceptive pills), Lanzkowsky’s Manual of Pediatric Hematology and Oncology 76 4. Nutritional anemias 5. diseases of the small intestine causing malabsorption, 6. hemolytic anemia with rapid red cell turnover (e.g., pyruvate kinase deficiency), and 7. chronic infections such as hepatitis, HIV infection. Inborn errors of folate transport and metabolism These include hereditary folate malabsorption, methylenetetrahydrofolate reductase (MTHFR) deficiency, and glutamate formiminotransferase deficiency. In addition to these rare severe deficiencies, polymorphisms in the MTHFR gene have been implicated with neural defects and vascular thrombosis. Table 4.13 lists the clinical manifestations, laboratory findings, and treatment of inherited defects of folate metabolism. Other megaloblastic anemias 1. Thiamine-responsive anemia in DIDMOAD (Wolfram) syndrome: this is a rare autosomal recessive disorder of thiamine transport due to mutations in a gene on chromosome 1q23. Pancytopenia with megaloblastic changes is present. Sideroblastic anemia with ringed sideroblasts may also be present. Other features include diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. The anemia responds to daily thiamine but megaloblastic changes persist. Insulin requirements may decrease. 2. Hereditary orotic aciduria: rare autosomal recessive defect of pyrimidine synthesis with failure to convert orotic acid to uridine resulting in orotic aciduria, sometimes with crystals. It is associated with severe megaloblastic anemia, neutropenia, and failure to thrive. Physical and mental retardation are frequently present. Treatment includes oral uridine. Anemia is refractory to vitamin B12 and folic acid. TABLE 4.12 Causes of folic acid deficiency. Inadequate intake Increased requirements Defective absorption Disorders of folic acid metabolism Unfortified goat’s milk feeding (6-mg folate/L) Malnutrition (marasmus, kwashiorkor) Special diets for phenylketonuria or maple syrup urine disease Rapid growth (e.g., prematurity, pregnancy) Chronic hemolytic anemia, especially with ineffective erythropoiesis (e.g., thalassemia, pyruvate kinase deficiency) Dyserythropoietic anemias Hypermetabolic states (e.g., infection, hyperthyroidism) Extensive skin disease (e.g., dermatitis herpetiformis, psoriasis) Cirrhosis Tropical sprue Anatomic/surgical (partial or total gastrectomy, multiple diverticula of small intestine, Jejunal resection) Whipple’s disease Intestinal lymphoma Drugs (broad-spectrum antibiotics); drugs associated with impaired absorption and/or utilization of folic acid, for example, methotrexate, diphenylhydantoin (Dilantin), primidone, barbiturates, oral contraceptive agents, cycloserine, metformin, ethanol, dietary amino acids (glycine, methionine), sulfasalazine, and pyrimethamine Post!bone marrow transplantation (total body irradiation, drugs, intestinal GVH disease) Post!bone marrow transplantation Congenital Methylenetetrahydrofolate reductase deficiency (OMIM 236250) Glutamate formiminotransferase deficiency (OMIM 229100) Functional N5-methyltetrahydrofolate: homocysteine methyltransferase deficiency caused by cblE (OMIM 236270) or cblG (OMIM 250940) disease Dihydrofolate reductase deficiency (less well established) Methenyltetrahydrofolate cyclohydrolase (less well established) Primary methyltetrahydrofolate: homocysteine methyltransferase deficiency (less well established) Acquired Folate antagonists (e.g., methotrexate, pyrimethamine, trimethoprim, and pentamidine) Vitamin B12 deficiency Liver disease (acute and chronic) (e.g., chronic dialysis, vitamin B12 deficiency, liver disease, and heart disease) Increased excretion Abbreviations: GVH, Graft-versus-host; OMIM, Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/omim/). Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 77 Megaloblastic anemia 3. Lesch!Nyhan syndrome: this condition presents with mental retardation, self-mutilation, and choreoathetosis due to impaired synthesis of purines from lack of hypoxanthine phosphoribosyltransferase. Megaloblastic anemia occurs in some patients and responds to adenine therapy. Clinical features of cobalamin and folate deficiency 1. Insidious onset: pallor, lethargy, fatigue, anorexia, sore red tongue and glossitis, and diarrhea. 2. History: limited or restricted diet (goat milk), maternal vitamin B12 or folate deficiency; maternal folate deficiency results in neural tube defects, prematurity, fetal growth retardation, and fetal loss. History of similarly affected siblings (inborn errors of metabolism) results in failure to thrive, neurologic disorders, unexplained anemias, or cytopenias. 3. Vitamin B12 deficiency: a. Infants may show signs of developmental delay, weakness, irritability, and loss of developmental milestones, particularly motor development (head control, sitting); athetoid movements, hypotonia, and loss of reflexes occur. b. Older children may develop subacute combined degeneration of the spinal cord, resulting in signs of degeneration of the posterior and lateral columns with associated peripheral nerve loss. Loss of vibration and position sense with an ataxic gait and positive Romberg’s sign. Paresthesia in the hands or feet, difficulty in walking and/or use of the hands may occur due to peripheral neuropathy. c. Long-term cognitive outcomes are irreversible following B12 treatment. d. MRI findings include increased signals on T2-weighted images of the spinal cord, brain atrophy, and retarded myelination. 4. Increased risk of vascular thrombosis due to hyperhomocysteinemia may occur. Diagnosis of cobalamin and folate deficiency Age of presentation is an important consideration for confirming the diagnosis and underlying etiology. Table 4.14 lists disorders giving rise to megaloblastic anemia in early life and likely age at presentation. The following laboratory findings may assist in the diagnostic approach: 1. Red cell changes. a. Hemoglobin: anemia with inappropriately low absolute reticulocyte count. b. Red cell indices: MCV increased for age, up to 110!140 fl; increased RDW. 2. White blood cell: leukopenia to 1500!4000/mm3. 3. Platelet count: moderately reduced to 50,000!180,000/mm3. Function may be impaired. 4. Blood smear: neutrophils show hypersegmentation (4!5% with .5 lobed nuclei); RBCs show macrocytes and macro-ovalocytes; marked anisocytosis and poikilocytosis with teardrop cells, presence of Cabot rings, Howell!Jolly bodies, and punctate basophilia. RBCs have a shorter survival time. TABLE 4.13 metabolism. Clinical manifestations, laboratory findings, and treatment of the autosomal recessive defects of folate transport and Condition OMIM no. Defect Typical clinical manifestations Typical onset Laboratory findings Treatment and response Hereditary folate malabsorption 229050 Loss of function mutation in protein-coupled folate transporter gene affecting intestines and choroid plexus (pcft/SLC46A1) Megaloblastic anemia, First few diarrhea, mouth sores, months of life failure to thrive, neurologic deterioration Low serum, red cell, and cerebrospinal fluid folate levels Oral folic acid versus parenteral doses; If oral ineffective, systemic therapy with reduced folates may be tried MTHFR deficiency 236250 MTHFR gene on chromosome 1p36.3; MTHFR assists with conversion of homocysteine to methionine Development delay, neurologic (hypotonia, gait abnormalities, strokes, seizures) Elevated plasma homocysteine, homocystinuria, decreased methionine levels Resistant to treatment; betaine therapy after prenatal diagnosis results in best outcomes Glutamate formiminotransferase deficiency 229100 Inability to transfer formimino group to tetrahydrofolate Infancy to Some asymptomatic; others severe mental/ adulthood physical retardation; megaloblastic findings Early infancy to adolescence; poor prognosis in early onset Formiminoglutamic acid excretion Abbreviations: MTHFR, Methylenetetrahydrofolate reductase; OMIM, Online Mendelian Inheritance in Man. Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 78 4. Nutritional anemias 5. Bone marrow: megaloblastic appearance; hyperplastic due to increased levels of erythropoietin acting on erythroid progenitor cells. a. Large cells, nucleus with open, stippled, or lacy appearance due to its retarded condensation. Cytoplasm comparatively more mature than the nucleus; this nuclear!cytoplasmic dissociation is best seen in more mature red cell precursors; Orthochromatic cells may be present with nuclei that are still not fully condensed. b. Mitoses frequent, sometimes abnormal; nuclear remnants, Howell!Jolly bodies, bi- and trinucleated cells and dying cells are evidence of gross dyserythropoiesis. c. Giant metamyelocytes with large horseshoe-shaped nucleus; hypersegmented polymorphs may be seen; and megakaryocytes show increase in nuclear lobes. 6. Markers of ineffective erythropoiesis: functional pathophysiology of megaloblastic anemia is ineffective erythropoiesis (manifest by increased levels of lactate dehydrogenase, indirect bilirubin, ferritin, serum iron and transferrin saturation, and low serum haptoglobin levels) due to programmed cell death or apoptosis of megaloblastic cells during maturation (rather than when they are mature), resulting in a predominance of younger erythroid cells in the bone marrow. 7. Serum vitamin B12 level: not most sensitive diagnostic test though levels ,80 pg/mL almost always indicative of vitamin B12 deficiency (normal values 200!800 pg/mL). 8. Serum and red cell folate levels: wide variation in normal range and reference ranges; serum levels ,3 ng/mL indicate low level, 3!5 ng/mL borderline level, and .5 ng/mL normal level; red cell folate levels ,160 ng/ mL considered low. 9. Homocysteine: nonspecific but elevated in both folate and vitamin B12 deficiency. TABLE 4.14 Megaloblastic anemia in early life and typical age at presentation. Age at presentation (months) 2!6 7!24 .24 Folate deficiency Inadequate supply Prematurity 1 Dietary (e.g., goat’s milk) 1 1 Chronic hemolysis Defective absorption Congenital 1 Anticonvulsant drugs 1 Celiac disease/sprue 1 Cobalamin deficiency Inadequate supply 1 Maternal cobalamin deficiency 1 Nutritional Defective absorption Congenital absence of intrinsic factor 1 6 Congenital malabsorption 1 6 1 Juvenile pernicious anemia Defective metabolism 1 Other Orotic aciduria 1 Thiamine responsive 1 Lesch!Nyhan syndrome 1 Modified from Lanzkowsky, P. Lanzkowsky’s Manual of Pediatric Hematology and Oncology, sixth ed. Elsevier (2016). Lanzkowsky’s Manual of Pediatric Hematology and Oncology Megaloblastic anemia 79 10. Methylmalonic acid: more specific test for cobalamin deficiency. Elevated levels more sensitive to functional B12 deficiency. Variable published thresholds (210 and 480 nmol/L) but majority consider .280 nmol/L as elevated. If vitamin B12 is suspected as the cause of megaloblastic anemia, a detailed dietary history and GI history should be undertaken, including assessing for factors that may affect gastric acidity or absorption (i.e., previous intestinal surgery). If dietary etiologies are ruled out (i.e., no evidence of insufficient vitamin B12 intake), then additional evaluations by a GI and/or metabolic specialist may be indicated based on the clinical history and presentation. Laboratory evaluations should include assessment of serum methylmalonic acid and homocysteine in addition to serum B12 and folate levels, if not already obtained. GI evaluation may include assessment for parietal cell antibodies and/or endoscopy. The Schilling urinary excretion was previously utilized to measure both IF availability and intestinal absorption of vitamin B12 (via a combination of radioactive labeled and nonradioactive B12) but is no longer available at many centers. If folic acid is suspected as a cause of the megaloblastic anemia, a detailed dietary and drug history should be performed, including review of antibiotics and antiepileptic medications. GI symptoms (e.g., malabsorption, diarrhea) should prompt referral to a GI subspecialist for further evaluation of diseases associated with malabsorption. As with suspected vitamin B 12 deficiency, laboratory evaluations should include assessment of serum methylmalonic acid and homocysteine in addition to serum B 12 and folate levels, if not already obtained. Additional laboratory assessments may include gluten autoantibodies or evaluation by endoscopy. Enzyme assays may be sent to diagnose congenital disorders of folate metabolism. Treatment Vitamin B12 deficiency Prevention In conditions under which there is a risk of developing vitamin B12 deficiency (e.g., autoimmune gastritis, ileal resection), prophylactic vitamin B12 should be prescribed. Active treatment Once the diagnosis is confirmed, and depending on the severity of the anemia, daily doses of cyanocobalamin (25!100 µg) in either oral, intramuscular, or deep subcutaneous may be used as initial therapy. After a week of daily dosing, weekly dosing may be given followed by transition to monthly doses. Alternatively, in view of the ability of the body to store vitamin B12 for long periods, maintenance therapy can be started with monthly intramuscular injections in doses between 200- and 1000-µg cyanocobalamin. Depending on the cause, lifelong vitamin B12 supplementation may be required. Patients with defects of intestinal absorption of vitamin B12 (abnormalities of IF or of ileal uptake) will respond to 100 µg of B12 injected subcutaneously monthly. Patients with complete TC II deficiency respond only to large amounts of vitamin B12 with doses of 1000-µg infectious mononucleosis two or three times weekly required to maintain adequate levels. Patients with methylmalonic aciduria with defects in the synthesis of cobalamin coenzymes are likely to benefit from high doses of vitamin B12. Response to vitamin B12 treatment In vitamin B12!responsive megaloblastic anemia, reticulocytes begin to increase on the third or fourth days, rise to a maximum on the sixth to eighth days, and fall gradually to normal at roughly 3 weeks. As in iron deficiency, the increment of the reticulocyte count is inversely proportional to the degree of anemia. Beginning bone marrow reversal from megaloblastic to normoblastic cells occurs rapidly and is usually complete within 3 days. Patients’ alertness and responsiveness may improve within 48 hours. Though some improvement in developmental delays may occur in young infants, permanent neurologic sequelae often occur. The use of oral folic acid is contraindicated in vitamin B12 deficiency. Folic acid supplementation may result in a hematologic response but has no effect on neurologic manifestations and may actually precipitate or accelerate their development. Folic acid deficiency Treatment Prior to initiation of folic acid, vitamin B12 deficiency must be excluded. Depending on the underlying cause of folate deficiency, improved diet and/or management of primary underlying GI disorder is critical and allows for only a limited duration of folic acid supplementation. Oral folic acid 1!5 mg/day is typically Lanzkowsky’s Manual of Pediatric Hematology and Oncology 80 4. Nutritional anemias sufficient even in patients with malabsorption. Patients with chronic malabsorption or with chronic hemolytic anemias (e.g., pyruvate kinase deficiency) may require lifelong supplementation. Response to folic acid treatment The clinical and hematologic response to folic acid is prompt. Clinical symptoms may improve within a few days. A rise in reticulocytes occurs at 2!4 days, reaching a peak at 4!7 days. This is followed by a return of hemoglobin levels to normal in 2!6 weeks. Leukocytes and platelets increase and megaloblastic changes in the marrow diminish within 1!2 days. Folic acid is usually administered for several months until a new population of red cells has been formed. Folinic acid is reserved for treating the toxic effects of dihydrofolate reductase inhibitors (e.g., methotrexate, pyrimethamine). Cases of hereditary dihydrofolate reductase deficiency respond to N-5-formyl tetrahydrofolic acid, not to folic acid. Further reading and references Allen, L.H., 2008. Causes of vitamin B12 and folate deficiency. Food Nutr. Bull. 29 (2 Suppl.), S20!S34. Carmaschella, C., 2015. Iron deficiency anemia. N. Engl. J. Med. 372, 1832!1843. Chaparro, C.M., 2008. Setting the stage for child health and development: prevention of iron deficiency in early infancy. J. Nutr. 138 (12), 2529!2533. Dallman, P.R., 1990. Progress in the prevention of iron deficiency in infants. Acta Paediatr. Scand. 365 (Suppl), 28!37. Dallman, P.R., Reeves, J.D., 1984. Laboratory diagnosis of iron deficiency. Iron nutrition in infancy and childhood. In: Steckel, A. (Ed.), Iron Nutrition in Infancy and Childhood. Raven Press, New York, p. 11. Hvas, A.M., Nexo, E., 2006. Diagnosis and treatment of vitamin B12 deficiency ! an update. Haematologica 91 (11), 1506!1512. Lanzkowsky, P., 1978. Iron metabolism and iron deficiency anemia. In: Miller, D.R., Pearson, M.A., Baehner, R.L., McMillan, C.W. (Eds.), Blood Diseases of Infancy and Childhood. CV Mosby, Saint Louis, MO. Lanzkowsky, P., 1987a. The megaloblastic anemias: vitamin B12 cobalamin deficiency and other congenital and acquired disorders. Clinical, pathogenetic and diagnostic considerations of vitamin B12 (cobalamin) deficiency and other congenital and acquired disorders. In: Nathan, D.G., Oski, F.A. (Eds.), Hematology of Infancy and Childhood. WB Saunders, Philadelphia, PA. Lanzkowsky, P., 1987b. The megaloblastic anemias: folate deficiency II. Clinical, pathogenetic and diagnostic considerations in folate deficiency. In: Nathan, D.G., Oski, F.A. (Eds.), Hematology of Infancy and Childhood. WB Saunders, Philadelphia, PA. Lozoff, B., 2007. Iron deficiency and child development. Food Nutr. Bull. 28, S560!S571. Oski, F.A., 1993. Iron deficiency in infancy and childhood. N. Engl. J. Med. (329), 190!193. Powers, J.M., Buchanan, G.R., 2019. Disorders of iron metabolism: New diagnostic and treatment approaches to iron deficiency. Hematol. Oncol. Clin. N. Am. 33 (3), 393!408. Powers, J.M., Buchanan, G.R., Adix, L., Zhang, S., Gao, A., McCavit, T.M., 2017. Effect of low dose ferrous sulfate versus iron polysaccharide complex on hemoglobin concentration in young children with nutritional iron deficiency anemia: a randomized clinical trial. JAMA 317 (22), 2297!2304. Rasmussen, S.A., Fernhoff, P.M., Scanlon, K.S., 2001. Vitamin B12 deficiency in children and adolescents. J. Pediatr. 138 (2001), 110. Rosenblatt, D.S., 1995. Inherited disorders of folate transport and metabolism. In: Scriver, C.R., Beaudet, A., Sly, W.S., Valle, D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York. Stabler, S.P., 2013. Vitamin B12 deficiency. N. Engl. J. Med. 368, 149!160. Whitehead, V.M., 2006. Acquired and inherited disorders of cobalamin and folate in children. Br. J. Haematol. 134 (2), 125!136. Lanzkowsky’s Manual of Pediatric Hematology and Oncology C H A P T E R 5 Lymphadenopathy and diseases of the spleen Philip Lanzkowsky1,2 1 Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States 2Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States Lymphadenopathy Enlarged lymph nodes are commonly found in children. Lymphadenopathy might be caused by proliferation of cells intrinsic to the node, such as lymphocytes, plasma cells, monocytes, or histiocytes, or by infiltration of cells extrinsic to the node, such as neutrophils and malignant cells. In most instances lymphadenopathy represents transient, self-limited proliferative responses to local or generalized infections. Reactive hyperplasia, defined as a polyclonal proliferation of one or more cell types, is the most frequent diagnosis found in lymph node biopsies in children. Lymphadenopathy, however, may be a presenting sign of malignancies such as leukemia, lymphoma, or neuroblastoma. It is important to be able to differentiate benign from malignant lymphadenopathy clinically. Lymphadenopathy in the head and neck region must be differentiated from several other nonlymp node masses due to congenital malformations (Table 5.1). Systematic palpation of the lymph nodes is important and should include examination of the occipital, posterior auricular, preauricular, tonsillar, submandibular, submental, upper anterior cervical, lower anterior cervical, posterior upper and lower cervical, supraclavicular, infraclavicular, axillary, epitrochlear, and popliteal lymph nodes. Many children have small palpable nodes in the cervical, axillary, and inguinal regions that are usually benign in nature. When a child presents with lymphadenopathy, management is based on the following factors. History This involves the duration of the lymphadenopathy; presence of fever; recent upper respiratory tract infection; sore throat; skin lesions or abrasions, or other infections in the lymphatic region drained by the enlarged lymph nodes; immunizations; medications; previous cat scratches, rodent bites, or tick bites; arthralgia; sexual history; transfusion history; travel history; and consumption of unpasteurized milk. Significant weight loss, night sweats, or other systemic symptoms should also be recorded as part of the patient’s history. Age Although in young children cervical lymphadenopathy, especially in the upper cervical region, is usually due to infection, more serious disorders may have to be considered. In children younger than 6 years the most common cancers of the head and neck are neuroblastoma, rhabdomyosarcoma, leukemia, and non-Hodgkin lymphoma. In children 7!13 years of age, non-Hodgkin lymphoma and Hodgkin lymphoma are equally common, followed by thyroid carcinoma and rhabdomyosarcoma; and for those older than 13 years, Hodgkin lymphoma is the more common cancer encountered. Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00023-4 81 © 2022 Elsevier Inc. All rights reserved. 82 5. Lymphadenopathy and diseases of the spleen TABLE 5.1 Differential diagnosis of nonlymph node masses in neck. Cystic hygroma Branchial cleft anomalies, branchial cysts Thyroglossal duct cysts Epidermoid cysts Neonatal torticollis Lateralprocess of lower cervical vertebra may be misdiagnosed as supraclavicular node Location Enlargement of tonsillar and inguinal lymph nodes is most likely secondary to localized infection; enlargement of supraclavicular and axillary lymph nodes is more likely to be of a serious nature. Enlargement of the left supraclavicular node, in particular, should suggest a malignant disease (e.g., lymphoma or rhabdomyosarcoma) arising in the abdomen and spreading via the thoracic duct to the left supraclavicular area. Enlargement of the right supraclavicular node indicates intrathoracic lesions because this node drains the superior areas of the lungs and mediastinum. Palpable supraclavicular nodes are an indication for a thorough search for intrathoracic or intraabdominal pathology. Localized or generalized Lymphadenopathy is either localized (one region affected) or generalized (two or more noncontiguous lymph node regions involved). Although localized lymphadenopathy is generally due to local infection in the region drained by the particular lymph nodes, it may also be due to malignant disease, such as Hodgkin lymphoma or neuroblastoma. Generalized lymphadenopathy is caused by many disease processes. Lymphadenopathy may initially be localized and subsequently become generalized. Size Nodes in excess of 2.5 cm should be regarded as pathologic. In addition, nodes that increase in size over time are significant. Character Malignant nodes are generally firm and rubbery. They are usually not tender or erythematous. Occasionally, a rapidly growing malignant node may be tender. Nodes due to infection or inflammation are generally warm, tender, and fluctuant. If infection is considered to be the cause of the adenopathy, it is reasonable to give a 2-week trial of antibiotics. If there is no reduction in the size of the lymph node within this period, careful observation of the lymph node is necessary. If the size, location, and character of the node suggest malignant disease, the node should be biopsied. Diagnosis of lymphadenopathy Table 5.2 outlines the differential diagnosis of lymphadenopathy. Fig. 5.1 provides a diagnostic algorithm for the evaluation of mononucleosis-like illness and Fig. 5.2 for diagnostic evaluation of cervical lymphadenitis. The following investigations should be carried out to elucidate the cause of either localized or generalized lymphadenopathy: • Thorough history of infection, contact with rodents or cats, and systemic complaints. • Careful examination of the lymphadenopathy, including size, consistency, mobility, warmth, tenderness, erythema, fluctuation, and location. All the lymph node!bearing areas as outlined earlier should be carefully examined. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Lymphadenopathy 83 TABLE 5.2 Differential diagnosis of lymphadenopathy. 1. Nonspecific reactive hyperplasia (polyclonal) 2. Infection a. Bacterial: Staphylococcus, Streptococcus, anaerobes, tuberculosis, atypical mycobacteria, Bartonella henselae (cat scratch disease), brucellosis, Salmonella typhi, diphtheria, Chlamydia trachomatis lymphogranuloma venereum, Calymmatobacterium granulomatis, and Francisella tularensis b. Viral: Epstein!Barr virus, cytomegalovirus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus, influenza, coxsackievirus, rubella, rubeola, varicella, HIV, herpes simplex virus, and HHV-6 c. Protozoal: toxoplasmosis, malaria, and trypanosomiasis d. Spirochetal: syphilis, Rickettsia typhi (murine typhus) e. Fungal: coccidioidomycosis (valley fever), histoplasmosis, Cryptococcus, and aspergillosis f. Postvaccination: smallpox, live attenuated measles, DPT, Salk vaccine, and typhoid fever 3. Connective tissue disorders a. Rheumatoid arthritis b. Systemic lupus erythematosus 4. Hypersensitivity states a. Serum sickness b. Drug reaction (e.g., Dilantin, mephenytoin, pyrimethamine, phenylbutazone, allopurinol, isoniazid, antileprosy, and antithyroid medications) 5. Lymphoproliferative disorders (Chapter 16: Lymphoproliferative Disorders) a. Angioimmunoblastic lymphadenopathy with dysproteinemia b. X-linked lymphoproliferative syndrome c. Lymphomatoid granulomatosis d. Sinus histiocytosis with massive lymphadenopathy (Rosai!Dorfman disease) e. Castleman disease benign (giant lymph node hyperplasia, angiofollicular lymph node hyperplasia) f. ALPS (Canale!Smith syndrome) g. PTLD 6. Neoplastic diseases a. Hodgkin and non-Hodgkin lymphomas b. Leukemia c. Metastatic disease from solid tumors: neuroblastoma, nasopharyngeal carcinoma, rhabdomyosarcoma, thyroid cancer d. Histiocytosis e. Langerhans cell histiocytosis f. Familial hemophagocytic lymphohistiocytosis g. Macrophage activation syndrome h. Malignant histiocytosis 7. Storage diseases a. Niemann!Pick disease b. Gaucher disease c. Cystinosis 8. Immunodeficiency states a. Chronic granulomatous disease b. Leukocyte adhesion deficiency c. Primary dysgammaglobulinemia with lymphadenopathy 9. Miscellaneous causes a. Kawasaki disease (mucocutaneous lymph node syndrome) b. Kikuchi!Fujimoto disease (self-limiting histiocytic necrotizing lymphadenitis) c. Sarcoidosis d. Beryllium exposure e. Hyperthyroidism f. PFAPA syndrome Abbreviations: ALPS, Autoimmune lymphoproliferative syndrome; DPT, Diptheriae-Pertussis-Tetanus vaccine; HHV-6, human herpesvirus 6; HIV, human immunodeficiency virus; PFAPA, periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis; PTLD, posttransplantation lymphoproliferative disorder. • Physical examination for evidence of hematologic disease, such as hepatosplenomegaly and petechiae. • Blood count and erythrocyte sedimentation rate (ESR). • Skin testing for tuberculosis. • Bacteriologic culture of regional lesions (e.g., throat). • Specific serologic tests for Epstein!Barr virus (EBV), Bartonella henselae (Immunofluorescence assay [IFA]), syphilis (venereal disease research laboratories) toxoplasmosis, cytomegalovirus (CMV), human immunodeficiency virus (HIV), tularemia, brucellosis, histoplasmosis, and coccidioidomycosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 84 5. Lymphadenopathy and diseases of the spleen FIGURE 5.1 Diagnostic algorithm Fever, pharyngitis, and LAD present? Pursue another diagnosis No Yes Heterophile test (“spot” test) Positive Negative Diagnosis of heterophile-positive IM* CBC with differential Of all WBCs: #50% lymphocytes and/or #10% atypical lymphocytes Of all WBCs: <50% lymphocytes and <10% atypical lymphocytes for the evaluation of MLI. Abbreviations: CBC, Complete blood count; CMV, cytomegalovirus; EBV, Epstein!Barr virus; HHV-6, human herpesvirus 6; HIV, human immunodeficiency virus; IM, infectious mononucleosis; LAD, lymphadenopathy; MLI, mononucleosis-like illness; VCA, viral capsid antigen; WBC, white blood cell. *Consider possibility of falsepositive heterophile test due to HIV-1 before finalizing diagnosis. Source: Adapted from Hurt, C., Tammaro, D., 2007. Diagnostic evaluation of mononucleosis-like illnesses. Am. J. Med. 120 (10), 911.e1!911.e8, with permission. EBV anti-VCA IgM and IgG EBV serologies positive Presumptive diagnosis of heterophile-negative EBV-induced IM EBV serologies negative Anti-CMV IgM and Anti-HHV-6 IgM Serologies positive Serologies negative Presumptive diagnosis of MLI caused by CMV or HHV-6 Reassess patient’s history and physical examination, with special attention to epidemiologic details and risk factors potentially missed initially Specific diagnostic testing guided by consideration of other diagnoses • Chest radiograph and computed tomography (CT) scan (if necessary); abdominal sonogram and CT, if indicated. • Ultrasonography is useful in an acute setting in assessing whether a swelling is nodal in origin, an infected cyst, or other soft tissue mass. It may detect an abscess requiring drainage. • EKG and echocardiogram if Kawasaki disease is suspected. • Lymph node aspiration and culture; helpful in isolating the causative organism and deciding on an appropriate antibiotic when infection is the cause of the lymphadenopathy. • Fine needle aspiration: may yield a definite or preliminary cytologic diagnosis and occasionally obviate the need for lymph node biopsy. It may provide limited material in the event that flow cytometry is required, so negative results cannot rule out malignancy because the sample may be inadequate. • Bone marrow examination if leukemia or lymphoma is suspected. • Lymph node biopsy is indicated if: • initial physical examination and history suggest malignancy, • lymph node size is greater than 2.5 cm in the absence of signs of infection, • lymph node persists or enlarges, • appropriate antibiotics fail to shrink node within 2 weeks, and • supraclavicular adenopathy. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 85 Lymphadenopathy Cervical lymphadenitis Asymptomatic Symptomatic Small node (<3 cm) Nonfluctuant Culture possible primary focus Large node (>3 cm) Small node (<3 cm) Fluctuant Observe Culture possible primary focus; needle aspirate with stains and cultures Culture possible primary focus; needle aspirate with stains and cultures; blood culture; draw acute serum Empiric antibiotic therapy for 2–3 weeks Node size persists or enlarges within 4–6 weeks Good clinical response but node remains large and tender Cultures negative, no change in size or larger Continue empiric treatment CBC, ESR, CXR, PPD skin test Serology (EBV, CMV, Toxoplasma gondii, VDRL, HIV ASO titer, tularemia, B. henselae, Brucella, fungi) Diagnosis in doubt; node persists, enlarges, is fixed to underlying tissues, or is hard Ultrasound to look for fluctuance, abscess formation Therapeutic needle aspiration/incision and drainage of abscess Excisional biopsy FIGURE 5.2 Diagnostic evaluation of cervical lymphadenitis. Abbreviations: ASO, Antistreptolysin titer; CBC, complete blood cell count; CMV, cytomegalovirus; CXR, chest radiography; EBV, Epstein!Barr virus; ESR, erythrocyte sedimentation rate; HIV, human immunodeficiency virus; PPD, purified protein derivative; VDRL, venereal disease research laboratories. Source: Adapted from Gosche, J.R., Vick, L., 2006. Acute, subacute and chronic cervical lymphadenitis in children. Semin. Pediatr. Surg. 15 (2), 99!106, with permission. Close communication between surgeon, oncologist, and pathologist is critical to maximize results from lymph node biopsy. In addition, the following precautions should be observed: • Upper cervical and inguinal areas should be avoided; lower cervical and axillary nodes are more likely to give reliable information. • The largest node should be biopsied, not the most accessible one. The oncologist should select the node to be biopsied in consultation with the surgeon. • The node should be removed intact with the capsule, not piecemeal. • The lymph node should be immediately submitted to the pathologist fresh or in sufficient tissue culture medium to prevent the tissue from drying out. The node must not be left in strong light, where it will be subject to heat and it should not be wrapped in dry gauze, which may produce a drying artifact. Fresh and frozen samples should be set aside for additional studies, as noted later. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 86 5. Lymphadenopathy and diseases of the spleen Intraoperative frozen section and cytologic smears should be performed. These findings, together with the clinical data, will determine which of the following additional studies may be required: • Gram stain and culture (bacterial, including mycobacterial, viral, and/or fungal) if clinically warranted or if intraoperative frozen section suggests an infection. • Tissue in tissue culture medium for cytogenetic analysis in the cases of suspected malignancy. Smears or touch preparations of the node on slides can be air-dried for fluorescent in situ hybridization studies to confirm certain malignancies. • Tissue frozen immediately for molecular studies. • Immunohistochemical stains to help differentiate and classify tumor types. • Flow cytometry for classifying and subtyping leukemias and lymphomas. • Gene rearrangement studies for the T-cell receptor and the immunoglobulin gene may be required to determine monoclonality in leukemia or lymphoma. (These can be performed on fresh frozen tissue or less optimally in formalin-fixed paraffin-embedded tissue.) • Formalin fixation for light microscopic analysis. Once the cause of the lymphadenopathy is ascertained, appropriate management can be undertaken. Diseases of the spleen The tip of the spleen is frequently palpable in otherwise normal infants and young children. It is usually palpable in premature infants and in about 30% of full-term infants. It may normally be felt in children up to 3 or 4 years of age. At an older age the spleen tip is generally not palpable below the costal margin and a palpable spleen usually indicates splenic enlargement two to three times its normal size. Asplenia Congenital asplenia is found in the rare Ivemark syndrome—trilobed lungs, centralized liver, and cardiac defects as well as a risk of infection. Diagnosis of asplenia (congenital or postsurgical) is made by the presence of Howell!Jolly bodies and the presence of intracellular vesicles (appearing as pits or pocks) in erythrocytes and no uptake by Tc99 colloid sulfur radionuclide. Congenital polysplenia This condition is characterized by the presence of several spleens of varying size and function, hepatobiliary abnormalities, and cardiac anomalies. Accessory spleen Accessory spleens occur in 15% of normal people and are usually present with no other abnormalities. The most frequent location is the splenic hilum or the tail of the pancreas or other locations in the abdomen or pelvis. Its identification is important when splenectomy is carried out for hematologic indications and the desired clinical effect is not obtained due to a functional accessory spleen. Splenosis Splenosis is autotransplantation of splenic tissue into the peritoneum or omentum and results from rupture of the spleen and spillage and subsequent implantation of splenocytes. Sequestration of spleen Sequestration of the spleen refers to splenic enlargement when blood enters the spleen but is unable to exit properly, for example, sickle cell anemia in young infants and congenital spherocytosis, and is characterized by a sudden severe drop in hemoglobin, occasionally hypovolemic shock, and abdominal pain in the left upper quadrant. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Diseases of the spleen 87 Splenoptosis (splenic visceroptosis) Splenoptosis occurs when the spleen is not fixed within the retroperitoneum, and a palpable spleen may be due to visceroptosis rather than true splenomegaly. This distinction is important to make so that extensive investigations for the cause of splenomegaly are not undertaken unnecessarily. Visceroptosis may result from congenital or acquired defects in the supporting mechanism responsible for maintaining the spleen in the correct position. The visceroptosed spleen may be felt anywhere from the upper abdomen to the pelvis and may undergo torsion. When the spleen is felt in the upper abdomen, it can easily be pushed under the left costal margin. This finding is helpful in diagnosing visceroptosis and differentiating it from true splenomegaly. In addition to this finding an abdominal radiograph in the upright position may reveal intestinal gas bubbles between the left dome of the diaphragm and the spleen. This sign may be helpful in differentiating true splenomegaly from visceroptosis of the spleen. Splenomegaly The significance of splenomegaly depends on the underlying disease. Splenomegaly can be caused by diseases that result in hyperplasia of the lymphoid and reticuloendothelial systems (e.g., infections, connective tissue disorders), infiltrative disorders (e.g., Gaucher disease, leukemia, lymphoma), hematologic disorders (e.g., thalassemia, hereditary spherocytosis), and conditions that cause the distention of the sinusoids whenever there is increased pressure in the portal or splenic veins (portal hypertension). Table 5.3 lists the various causes of splenomegaly. TABLE 5.3 Causes of splenomegaly. 1. Infectious (due to antigenic stimulation with hyperplasia of the reticuloendothelial and lymphoid systems) a. Bacterial: acute and chronic systemic infection, subacute bacterial endocarditis, abscesses, typhoid fever, miliary tuberculosis, tularemia, and plague b. Viral: infectious mononucleosis (Epstein!Barr virus), cytomegalovirus, HIV, hepatitis A, B, C c. Spirochetal: syphilis, Lyme disease, and leptospirosis d. Rickettsial: Rocky Mountain spotted fever, Q fever, and typhus e. Protozoal: malaria, babesiosis, toxoplasmosis, Toxocara canis, Toxocara catis, leishmaniasis, schistosomiasis, and trypanosomiasis f. Fungal: disseminated candidiasis, histoplasmosis, coccidioidomycosis, and South American blastomycosis 2. Hematologic a. Hemolytic anemias, such as thalassemia, splenic sequestration crisis in sickle cell disease, and hereditary spherocytosis b. Extramedullary hematopoiesis, as in osteopetrosis and myelofibrosis c. Myeloproliferative disorders (e.g., polycythemia vera, essential thrombocythemia) 3. Infiltrative a. Nonmalignant b. Langerhans cell histiocytosis c. Storage diseases such as Gaucher disease, Niemann!Pick disease, GM-1 gangliosidosis, glycogen storage disease type IV, Tangier disease, Wolman disease, mucopolysaccharidoses, hyperchylomicronemia types I and IV, amyloidosis, and sarcoidosis d. Malignant e. Leukemia f. Lymphoma: Hodgkin and non-Hodgkin 4. Congestive a. Intrahepatic (portal hypertension): Cirrhosis of the liver (e.g., neonatal hepatitis, α1-antitrypsin deficiency, Wilson disease, and cystic fibrosis) b. Prehepatosplenic or portal vein obstruction (e.g., thrombosis, vascular malformations) 5. Immunologic a. Serum sickness, GVHD b. Connective tissue disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis—Felty syndrome, mixed connective tissue disorder, Sjogren syndrome, macrophage activation syndrome, and systemic mastocytosis) c. Common variable immunodeficiency d. ALPS (Canale!Smith syndrome) 6. Primary splenic disorders a. Cysts b. Benign tumors (e.g., hemangioma, lymphangioma) c. Hemorrhage in spleen (e.g., subcapsular hematoma) d. Partial torsion of splenic pedicle leading to congestive splenomegaly, cyst, and abscess formation Abbreviations: ALPS, Autoimmune lymphoproliferative syndrome; GVHD, graft-versus-host disease; HIV, human immunodeficiency virus. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 88 5. Lymphadenopathy and diseases of the spleen Diagnostic approach to splenomegaly Detailed history 1. fever or rigors indicative of infection [e.g., subacute bacterial endocarditis (SBE), infectious mononucleosis, malaria] 2. history of neonatal omphalitis, umbilical venous catheterization leading to inferior vena cava, or portal vein thrombosis resulting in portal hypertension 3. jaundice (evidence of liver disease) leading to portal hypertension 4. abnormal bleeding or bruising (hematologic malignancy) 5. family history of hemolytic anemia (e.g., hereditary spherocytosis or thalassemia major) 6. travel to endemic areas (e.g., malaria) 7. trauma (splenic hematoma) Physical examination 1. Size of spleen (measured in centimeters below costal margin); consistency, tenderness, and audible rub. It is critical to differentiate splenoptosis from true enlargement of the spleen. 2. Hepatomegaly. 3. Lymphadenopathy. 4. Fever. 5. Ecchymoses, purpura, and petechiae. 6. Stigmata of liver disease, such as jaundice, spider angiomata, or caput medusa. 7. Stigmata of rheumatoid arthritis or systemic lupus erythematous. 8. Cardiac murmurs, Osler nodes, Janeway lesions, splinter hemorrhages, and fundal hemorrhages as evidence of SBE. Laboratory investigations The extent to which the following investigations are undertaken must be guided by clinical judgment. It is not necessary to perform all the evaluations. If the child appears well and the index of suspicion is low, it is reasonable to do no further investigations and reexamine the child in 1!2 weeks. If the splenomegaly persists, the following investigations should be done: • Blood count: red cell indices, reticulocyte count, platelet count, differential white blood cell count, and blood film (which may demonstrate evidence of hematologic malignancy, hemolytic disorders, and viral and protozoal infections). • Evaluation for infection: blood culture and viral studies (CMV, EBV panel, HIV, toxoplasmosis, smear for malaria, and tuberculin test). • Evaluation for evidence of hemolytic disease: blood count, reticulocyte count, blood smear, serum bilirubin, urinary urobilinogen, direct antiglobulin test (Coombs test), and red cell enzyme assays (if indicated). • Evaluation for liver disease: liver function tests, α1-antitrypsin deficiency, serum copper, ceruloplasmin (to exclude Wilson disease), and liver biopsy (if indicated). • Evaluation for portal hypertension: ultrasound and Doppler of portal venous system and endoscopy (if indicated to exclude esophageal varices). • Evaluation for connective tissue disease: ESR, C3, C4, CH50, antinuclear antibody, rheumatoid factor, urinalysis, blood urea nitrogen, and serum creatinine. • Evaluation for infiltrative disease (benign and malignant): bone marrow aspiration and biopsy, looking for blasts, Langerhans cell histiocytes, or storage cells. • Enzyme assay for Gaucher and other storage diseases. • Lymph node biopsy: if there is significant lymphadenopathy, lymph node biopsy may provide the diagnosis. • Imaging studies: abdominal CT scan, if indicated; magnetic resonance imaging, if indicated; and liver!spleen scans with 99mTc-sulfur colloid. • Splenectomy or partial splenectomy: if less invasive studies have failed to provide the diagnosis, it may be necessary to perform a splenectomy or a partial splenectomy on rare occasions to establish a diagnosis. Splenic tissue must be processed for cultures and Gram stain, as well as for histology, flow cytometry, histochemical stains, electron microscopy, and gene rearrangement studies. Once the etiology of the splenomegaly is ascertained, further management for the underlying disorder can be instituted. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 89 Surgery involving the spleen Splenectomy is usually done laparoscopically and partial splenectomy has become a therapeutic alternative to total splenectomy. Partial splenectomy leaving at least 20% splenic tissue is sufficient to preserve immune competence and is suitable when splenectomy is performed for indications other than for immune-mediated hematologic disorders such as autoimmune hemolytic anemia or immune thrombocytopenic purpura. The primary risk of splenectomy is overwhelming postsplenectomy infection (OPSI) and sepsis. Risk factors for OPSI are: 1. age of splenectomy—under 5 and especially infants under 2 years of age, 2. failure to receive presplenectomy immunization, and 3. noncompliance with prophylactic antibiotics. Reduction in incidence of OPSI can be achieved by: 1. presplenectomy immunization with protein-conjugated vaccines against Streptococcus pneumoniae and Haemophilus influenzae type b, 2. postsplenectomy prophylactic antibiotics, and 3. prompt, early, and effective medical treatment for fever. Further reading and references Behrman, R., Kliegman, R.M., Jenson, H.B., 2004. Nelson Textbook of Pediatrics, seventeenth ed. Saunders, Philadelphia, PA. Gosche, J.R., Vick, L., 2006. Acute, subacute and chronic cervical lymphadenitis in children. Semin. Pediatr. Surg. 15 (2), 99!106. Hoffman, R., Benz, E.J., Shattil, S.J., 2005. Hematology: Basic Principles and Practice, fourth ed. Churchill Livingstone. Hurt, C., Tammaro, D., 2007. Diagnostic evaluation of mononucleosis-like illnesses. Am. J. Med. 120 (10), 911.e1!911.e8. Kim, D.S., 2006. Kawasaki disease. Yonsei Med. J. 47 (6), 759!772. La Barge, D.V., Salzmam, K.L., Harnsberger, H.R., 2008. Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease): imaging manifestations in the head and neck. Am. J. Roentgenol. 191 (6), W299!W306. Leung, A.K.C., Robson, W.L.M., 2004. Childhood cervical lymphadenopathy. J. Pediatr. Health Care 18, 3!7. Nathan, D., Orkin, S., 2003. Nathan and Oski’s Hematology of Infancy and Childhood, sixth ed. Saunders, Philadelphia, PA. Paradela, S., Lorenzo, J., Martinez-Gomez, W., 2008. Interface dermatitis in skin lesions of Kikuchi-Fujimoto’s disease: a histopathological marker of evolution into systemic lupus erythematosus? Lupus 17 (12), 1127!1135. Tracy, T.F., Muratore, C.S., 2007. Management of common head and neck masses. Semin. Pediatr. Surg. 16 (1), 3!13. Twist, C.J., Link, M.P., 2002. Assessment of lymphadenopathy in children. Pediatr. Clin. N. Am. 49, 1009!1025. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 6 Bone marrow failure Adrianna Vlachos1,2,3, Michelle Nash1,2,3 and Jeffrey M. Lipton1,2,3 1 Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States 2Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States 3Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, NY, United States • Bone marrow failure may manifest as a single cytopenia (e.g., erythroid, myeloid, or megakaryocytic) or as pancytopenia. It may present with a hypoplastic or aplastic marrow or result from invasion of the bone marrow by neoplastic or nonneoplastic (e.g., storage cells) cells. • Bone marrow failure may be congenital (mostly inherited) or acquired (Table 6.1). Table 6.2 lists the inherited bone marrow failure syndromes (IBMFSs) with their causative genes. IBMFS can manifest with pancytopenia [e.g., Fanconi anemia (FA) and dyskeratosis congenita (DC)] or single cytopenias [e.g., Diamond Blackfan anemia (DBA), Shwachman Diamond syndrome (SDS), severe congenital neutropenia (SCN), Kostmann syndrome (KS), cyclic neutropenia, amegakaryocytic thrombocytopenia (AMT), and thrombocytopenia-absent radii (TAR) syndrome]. The “single cell-line cytopenias” may develop abnormalities in other hematopoietic cell lines (Table 6.1). • Congenital dyserythropoietic anemias (CDAs) result in moderate erythroid failure due to ineffective erythropoiesis with characteristic morphological abnormalities of erythroblasts. • Mitochondrial diseases may also present with bone marrow failure (Pearson syndrome, Wolfram syndrome, and various types of sideroblastic anemia). Fig. 6.1 shows the differential diagnosis of pancytopenia, and Table 6.3 lists the investigations to be carried out in a patient with pancytopenia. Aplastic anemia • Aplastic anemia is characterized by a marked decrease or absence of blood-forming elements with resulting pancytopenia and can be inherited or acquired. Various degrees of lymphopenia may be present. Splenomegaly, hepatomegaly, and lymphadenopathy do not generally occur in aplastic anemia. Acquired aplastic anemia Definition 1. Severe aplastic anemia (SAA) is defined by: a. bone marrow cellularity of less than 25% and Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00029-5 91 © 2022 Elsevier Inc. All rights reserved. 92 6. Bone marrow failure TABLE 6.1 Causes of single cytopenias and trilineage bone marrow failure. Single cytopenias 1. Red cells a. Inherited i. DBA ii. CDA iii. Pearson syndrome b. Acquired i. Idiopathic • TEC ii. Secondary • Drugs or toxins • Infection—parvovirus B19 infection in immunodeficiency patients (chronic bone marrow failure); SARS-CoV-2 • Malnutrition • Thymoma • Chronic hemolytic anemia with associated parvovirus B19 infection (transient bone marrow failure) • Connective tissue disease and autoimmune disease associated • Malignancy associated 2. White blood cells a. Inherited i. SCN ii. SDS iii. Reticular dysgenesis iv. Other rare genetic disorders b. Acquired i. Drugs ii. Autoimmune neutropenia of infancy iii. Other autoimmune associated (systemic lupus erythematosus, etc.) iv. Viral infections (e.g., SARS-CoV-2) 3. Platelets a. Inherited i. CAMT ii. TAR b. Acquired i. ITP ii. Other autoimmune associated (systemic lupus erythematosus, Crohn’s disease, etc.) iii. Viral infections (e.g., SARS-CoV-2) Trilineage bone marrow failure (generalized pancytopenia) 1. Inherited a. FA (associated with chromosomal breakages induced by clastogens, for example, DEB or MMC) b. DC (associated with short telomeres) c. Shwachman Diamond syndrome (predominantly neutropenia)a d. AMT (predominantly thrombocytopenia)a e. DBA (predominantly anemia)a f. Aplastic anemia with constitutional chromosomal abnormalities g. Dubowitz syndrome (congenital abnormalities, mental retardation, aplastic anemia) 2. Acquired a. Idiopathic (more than 70% of cases) b. Secondary i. Drugsb • Predictable, dose-dependent, rapidly reversible (affects rapidly dividing maturing hematopoietic cells rather than pluripotent stem cells) i. 6-Mercaptopurine ii. Methotrexate iii. Cyclophosphamide iv. Busulfan v. Chloramphenicol • Unpredictable to normal doses (defect or damage pluripotent stem cells) i. Antibiotics: chloramphenicol, sulfonamides ii. Anticonvulsants: mephenytoin, hydantoin iii. Antirheumatics: phenylbutazone, gold v. Antidiabetics: tolbutamide, chlorpropamide vi. Antimalarial: quinacrine (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 93 Aplastic anemia TABLE 6.1 (Continued) ii. Chemicals: insecticides (e.g., synthetic organochloride, pesticide, parathion, chlordane) iii. Toxins (e.g., benzene, carbon tetrachloride, glue, toluene) iv. Radiation v. Infections • Viral hepatitis (hepatitis A, B, and C and serotype-negative hepatitis) • Human immunodeficiency virus infection (HIV/AIDS) • Infectious mononucleosis (Epstein!Barr virus) • Rubellac • Influenzac • Parainfluenzac • Measlesc • Mumpsc • Venezuelan equine encephalitis • Rocky Mountain spotted feverc • Cytomegalovirus (in newborn) • Herpes virus (in newborn) • Chronic parvovirus • SARS-CoV-2 vi. Immunologic disorders • Graft-versus-host reaction in transfused immunologically incompetent subjects • X-linked lymphoproliferative syndrome (see Chapter 16: Lymphoproliferative Disorders) • Eosinophilic fasciitis • Hypogammaglobulinemia vii. Aplastic anemia preceding acute leukemia (hypoplastic preleukemia) viii. Myelodysplastic syndromes (see Chapter 17: Myelodysplastic Syndromes and Myeloproliferative Disorders) ix. Thymoma x. Paroxysmal nocturnal hemoglobinuria (see Chapter 7: General Considerations of Hemolytic Diseases, Red Cell Membrane and Enzyme Defects) xi. Malnutrition • Kwashiorkor • Marasmusc • Anorexia nervosac • Pregnancy a May have reduction in other cell lines. Partial listing. c Pancytopenia with temporary marrow hypoplasia. Abbreviations: AMT, Amegakaryocytic thrombocytopenia; CAMT, congenital amegakaryocytic thrombocytopenia; CDA, congenital dyserythropoietic anemia; DBA, Diamond Blackfan anemia; DEB, diepoxybutane; DC, dyskeratosis congenital; FA, Fanconi anemia; ITP, idiopathic/immune thrombocytopenic purpura; MMC, mitomycin C; SDS, Shwachman Diamond syndrome; SCN, severe congenital neutropenia; TAR, thrombocytopenia-absent radii syndrome; TEC, transient erythroblastopenia of childhood. b TABLE 6.2 Inherited bone marrow failure syndrome genes, known and presumed. Disorder Gene Locus Mode of inheritance FANCA 16q24.3 AR FANCB Xp22.31 XLR FANCC 9q22.3 AR FANCD1/BRCA2 13q12.3 AR FANCD2 3p25.3 AR FANCE 6p21.3 AR FANCF 11p15 AR FANCG/XRCC9 9p13 AR FANCI/KIAA1794 15q25!26 AR FANCJ/BRIP1/BACH1 17q22.3 AR FANCL/PHF9/POG 2p16.1 AR Fanconi anemia (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 94 6. Bone marrow failure TABLE 6.2 (Continued) Disorder Gene Locus Mode of inheritance FANCM/HEFa 14q21.3 AR FANCN/PALB2 16p12.1 AR FANCO/RAD51C 17q25.1 AR FANCP/SLX4 16p13.3 AR FANCQ/XPF/ERCC4 16p13.12 AR FANCR/RAD51 15q15.1 AD FANCS/BRCA1 17q21.31 AR FANCT/UBE2T 1q32.1 AR FANCU/XRCC2 7q36.1 AR FANCV/MAD2L2/REV7 1p36.22 AR FANCW/RFWD3 16q23.1 AR DKC1 Xq28 XLR TERC 3q26.2 AD TERT 5p15.33 AD/AR TINF2/TIN2 14q12 AD NHP2/NOLA2 5q35.3 AR NOP10/NOLA3 15q14 AR WRAP53/TCAB1 17p13.1 AR CTC1 17p13.1 AR ACD/TPP1 16q22.1 AD/AR PARN 16p13.12 AD/AR RTEL1 20q13.33 AD/AR NAF1 4q32.2 AD POT1 7q31.33 AR STN1 10q24.33 AR ZCCHC8 12q24.31 AD RPS7 2p25.3 AD RPS10 6p21.31 AD RPS15A 16p12.3 AD RPS17 15q25.2 AD RPS19 19q13.2 AD RPS24 10q22!23 AD RPS26 12q13.2 AD RPS27 1q21.3 AD RPS28 19p13.2 AD RPS29 14q21.3 AD RPL5 1p22.1 AD RPL11 1p36.11 AD Dyskeratosis congenita/telomere biology diseases Diamond Blackfan anemia (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 95 Aplastic anemia TABLE 6.2 (Continued) Disorder Gene Locus Mode of inheritance RPL15 3p24.2 AD RPL17 18q21.1 AD RPL18 19q13.33 AD RPL19 17q11 AD RPL26 17p13.1 AD RPL27 17q21.31 AD RPL31 2q11.2 AD RPL35 9q33.3 AD RPL35A 3q29 AD TSR2 Xp11.22 XLR GATA1 Xp11.23 XLR SBDS 7q11.21 AR DNAJC21 5p13.2 AR EFL1 15q25.2 AR SRP54 14q13.2 AD ELANE 19p13.3 AD HAX1 (Kostmann syndrome) 1q21.3 AR G6PC3 17q21.31 AR GFI1 1p22 AD WAS Xp11.4-p11.21 XLR JAGN1 3p25.2 AR CSF3R 1p34.3 AR TCIRG1 11q13.2 AD VPS45 1q21.2 AR GATA2 (MonoMac syndrome) 3q21.3 AD CXCR4 (WHIM syndrome) 2q22.1 AD MPL 1p34 AR HOXA11 7p15.2 AD THPO 3q27.1 AD MECOM 3q26.2 AD RBM8A 1q21.1 AR SAMD9 7q21.2 AD SAMD9L 7q21.2 AD Shwachman Diamond syndrome Severe congenital neutropenia Amegakaryocytic thrombocytopenia TAR syndrome Recently identified syndromes a FANCM is a member of the “core complex” but homozygosity has not yet been identified in patients with Fanconi anemia. Abbreviation: TAR, Thrombocytopenia-absent radii. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 96 6. Bone marrow failure Pancytopenia (low hemoglobin, hematocrit, white blood cell count, and platelet count) Splenomegaly No splenomegaly Bone marrow aspiration Bone marrow aspiration and biopsy Abnormal Normal Abnormal Hypocellular Leukemia/MDS Granulomata: sarcoid, TB Storage diseases Gaucher disease Niemann–Pick disease Other Lymphoma Hypersplenism Connective tissue disorder Portal hypertension Prehepatic Hepatic Primary splenic disease Cyst Leukemia Mild/moderate aplastic anemia Acellular (<25% cellularity) Aplastic anemia Inherited Acquired Fanconi anemia Dyskeratosis congenita Familial aplastic anemia Shwachman diamond syndrome Inherited thrombocytopenia Other rare inherited syndromes Idiopathic MDS PNH Secondary Drugs Toxins Radiation Immunologic Infections HIV FIGURE 6.1 Approach to the differential diagnosis of pancytopenia. Abbreviations: MDS, Myelodysplastic syndrome; PNH, paroxysmal nocturnal hemoglobinuria; TB, tuberculosis. Source: From Lanzkowsky, P., 1980. Pediatric Hematology-Oncology. McGraw-Hill. b. at least two of the following cytopenias: i. granulocyte count ,500/µL (,200 µL defines very SAA), ii. platelet count ,20,000/µL, and/or iii. reticulocyte count ,20,000/µL. Non-SAA occurs when the abovementioned criteria are not met. There is little consensus on distinguishing between mild and moderate aplastic anemia. Pathophysiology • Aplastic anemia results from an immunologically mediated, tissue-specific, organ-destructive mechanism. It is postulated that after exposure to an inciting antigen, cells and cytokines of the immune system destroy stem cells in the marrow resulting in pancytopenia. Treatment with immunosuppression can potentially lead to marrow recovery. Gamma-interferon (γ-IFN) plays a central role in the pathophysiology of aplastic anemia. In vitro studies show that the T cells from patients with aplastic anemia secrete γ-IFN and tumor necrosis factor (TNF). Long-term bone marrow cultures have shown that γ-IFN and TNF are potent inhibitors of both early and late hematopoietic progenitor cells. Both of these cytokines suppress hematopoiesis by their effects on the mitotic cycle and, more importantly, by the mechanism of cell killing. The mechanism of cell killing involves the pathway of apoptosis Lanzkowsky’s Manual of Pediatric Hematology and Oncology Aplastic anemia 97 TABLE 6.3 Investigations in patients with pancytopenia. 1. Detailed past history, medication history, toxin and radiation exposure, thorough physical examination for congenital anomalies, detailed family history of aplastic anemia, MDS or leukemia, and congenital anomalies in other family members 2. Blood count: Hb, Hct, MCV, absolute reticulocyte count, WBC, absolute neutrophil count, and platelet count 3. ANA and antidouble stranded DNA titers, DAT, rheumatoid factor, liver function tests, and tuberculin test 4. Viral serology: hepatitis A, B, C, EBV, parvovirus, varicella, CMV, and HIV. PCR for virus when indicated 5. Serum vitamin B12, red cell and serum folate levels 6. Bone marrow aspirate and trephine biopsy 7. Cytogenetic studies on bone marrow along with fluorescence in situ hybridization for chromosomes 5, 7, 8, 22, and perhaps others to evaluate for MDS 8. Chromosome breakage assay on blood lymphocytes or skin fibroblasts using clastogen stimulation (e.g., diepoxybutane or mitomycin C) to diagnose Fanconi anemia 9. Imaging: skeletal radiographs; renal, cardiac, and abdominal ultrasounds; chest radiograph to determine congenital anomalies 10. Telomere length determination for dyskeratosis congenita 11. Flow cytometric immunophenotypic analysis of erythrocytes for deficiency of GPI-linked surface protein (e.g., CD59) to exclude paroxysmal nocturnal hemoglobinuria 12. Skeletal radiograph, chest radiograph, pancreatic ultrasound or CT scan, 72-h fecal fat, serum trypsinogen and isoamylase, and fecal elastase for Shwachman Diamond syndrome 13. Mutation analysis for specific inherited bone marrow failure syndromes when suspected or for a comprehensive IBMFS genetic panel Abbreviations: ANA, Antinuclear antibody; CMV, cytomegalovirus; CT, computed tomography; DAT, direct antiglobulin test; EBV, Epstein!Barr virus; Hb, hemoglobin; Hct, hematocrit; HIV, human immunodeficiency virus; IBMFS, bone marrow failure syndrome; MCV, mean corpuscular volume; MDS, myelodysplastic syndrome; PCR, polymerase chain reaction; WBC, white blood cell; GPI, Glycosylphosphatidylinositol. (i.e., γ-IFN and TNF upregulate each other’s cellular receptors, as well as the Fas receptors in hematopoietic stem cells). Cytotoxic T cells also secrete interleukin-2 that causes polyclonal expansion of the T cells. Activation of the Fas receptor on the hematopoietic stem cell by the Fas ligand present on the lymphocytes leads to apoptosis of the targeted hematopoietic progenitor cells. Additionally, γ-IFN mediates its hematopoietic suppressive activity through IFN regulatory factor 1 that inhibits the transcription of cellular genes and their entry into the cell cycle. γ-IFN also induces the production of nitric oxide, the diffusion of which causes additional toxic effects on the hematopoietic progenitor cells. Direct cell!cell interactions between effective lymphocytes and targeted hematopoietic cells probably also occur. The oligoclonal expansion of CD41 and CD81 T cells that fluctuate with disease activity further supports an immune etiology. Table 6.1 lists the various causes of acquired aplastic anemia. Clinical manifestations • Acquired aplastic anemia may be idiopathic or secondary. At least 70% of cases are idiopathic. The incidence is approximately two cases per million per year in the West and higher in parts of Asia (B4!7.5 cases per million per year) and the male:female ratio is 1:1. The onset of acquired aplastic anemia is usually gradual and the symptoms are related to the pancytopenia. • Anemia results in pallor, easy fatigability, weakness, and loss of appetite. • Thrombocytopenia leads to petechiae, easy bruising, severe nosebleeds, gastrointestinal bleeding, and hematuria. • Leukopenia leads to increased susceptibility to infections and oral ulcerations and gingivitis that respond poorly to antibiotic therapy. • Hepatosplenomegaly and lymphadenopathy do not generally occur and their presence may suggest an underlying malignant, rheumatologic, or metabolic process. Laboratory investigations • Anemia: normocytic or macrocytic, normochromic • Reticulocytopenia: absolute count more reliable • Leukopenia: granulocytopenia often less than 1500/µL • Thrombocytopenia: platelets often less than 30,000/µL • Fetal hemoglobin: may slightly to moderately elevated • Bone marrow: • Marked depression or absence of hematopoietic cells and replacement by fatty tissue containing reticulum cells, lymphocytes, plasma cells, and usually tissue mast cells. • Megaloblastic changes and other features indicative of dyserythropoiesis frequently seen in the erythroid precursors. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 98 6. Bone marrow failure • Bone marrow biopsy essential to assess cellularity for diagnosis and to exclude the possibility of poor aspiration technique or poor bone marrow sampling; additionally, it will help to rule out granulomas, myelofibrosis, or leukemia. • Chromosomal analysis is normal and assists in excluding myelodysplastic syndromes (MDSs). Expert hematopathology assessment may help distinguish SAA from hypocellular MDS. • Bone marrow cultures, and molecular testing for infectious agents when indicated. • Chromosome breakage assay: performed on peripheral blood to screen for FA • Flow cytometry (CD59): performed on peripheral blood to diagnose paroxysmal nocturnal hemoglobinuria (PNH) • Telomere length: performed on peripheral blood to screen for DC • Liver function chemistries: to exclude hepatitis • Renal function chemistries: to exclude renal disease • Viral serology testing: hepatitis A, B, and C antibody panel (although posthepatitis aplastic anemia is generally associated with seronegative hepatitis); Epstein!Barr virus (EBV) antibody panel; parvovirus B19 IgG and IgM antibodies; varicella antibody titer; cytomegalovirus (CMV) antibody titer; human immunodeficiency virus antibody test • Quantitative immunoglobulins: to rule out immunodeficiency • Autoimmune disease evaluation: antinuclear antibody, total hemolytic complement (CH50), C3, C4, and direct antiglobulin test • Human leukocyte antigen (HLA) typing: patient and family, done at the diagnosis of SAA to identify a suitable donor and ensure a timely transplant Physical examination, appropriate laboratory screening assays and imaging studies, and, if warranted, mutation analysis should be performed to rule out other IBMFSs (FA, DC, DBA, SDS, AMT as well as rare genetic causes). Treatment Table 6.4 shows the recommendations for the treatment of moderate and SAA. Supportive care • Transfusion of red cells and platelets should be minimized but should not be withheld if clearly indicated. The risk of symptomatic anemia and serious bleeding must be balanced against transfusion sensitization and the risk of iron overload. TABLE 6.4 Recommendations for treatment of children with aplastic anemia. 1. Moderate aplastic anemia: observe with close follow-up and supportive care. Proceed to treatment if the patient develops: a. Severe aplastic anemia, and/or b. Severe thrombocytopenia with significant bleeding, and/or c. Chronic anemia requiring transfusion treatments, and/or d. Serious infections. 2. Severe aplastic anemia: allogeneic bone marrow transplantation when HLA-matched sibling donor available. In the absence of an HLAmatched sibling marrow donor: a. Treat the patient with ATG and CSA, with methylprednisolone for serum sickness prophylaxis. b. Eltrombopag has been approved as first-line treatment for SAA in combination with ATG and CSA. c. The use of growth factors such as G-CSF or GM-CCF is no longer routinely done. d. Complete response of IST is normalization of counts. e. Partial response is the absence of infections and transfusion dependency and sustained increase in all cell counts as follows: reticulocyte count $ 20,000/µL; platelet count $ 20,000/µL; and absolute neutrophil count $ 500/µL but not having achieved normal counts. Partial response and complete response are considered responses for the evaluation of the success of IST. f. If no response or partial response and recurrence of SAA, a second course of IST is controversial. g. In the case of no or partial response, or recurrence of SAA, HLA-matched unrelated bone marrow, peripheral blood or umbilical cord blood HSCT, if a suitable donor is available, is warranted. If a donor is not available, a second course of IST is acceptable. h. Newer thrombopoietin mimetic drugs such as eltrombopag have also shown response in patients with refractory severe aplastic anemia. i. HSCT clinical trials are ongoing and experience suggests that, particularly in light of late clonal evolution following IST, MUD transplants are likely to become a treatment of choice for newly diagnosed SAA. Abbreviations: ATG, Antithymocyte globulin; CSA, cyclosporine A; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colonystimulating factor; HLA, human leukocyte antigen; HSCT, hematopoietic stem cell transplantation; IST, immunosuppressive therapy; MUD, matched unrelated donor; SAA, severe aplastic anemia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Aplastic anemia 99 • Prior to any transfusion, perform extended blood group typing to minimize the risk of sensitization to minor blood group antigens and to permit the identification of antibodies should they subsequently develop. • Transfusions should be restricted, if possible, to unrelated blood product donors to decrease the likelihood of sensitization to donor antigens. • In all patients, blood products should be leukocyte-depleted to reduce the risk of sensitization and CMV infection. CMV-negative blood products are equivalent to CMV-safe blood products, even for CMVseronegative patients who may require future transplants. • Patients receiving chronic red cell transfusion should be followed for evidence of iron overload and receive appropriate iron chelation. • The use of single donor platelets, when available, is recommended. • Menses should be suppressed by the use of contraceptives. • Drugs that impair platelet function, such as aspirin, should be avoided. • Intramuscular injections should be given carefully, followed by ice pack application to injection sites. • The antifibrinolytic agent, ε-aminocaproic acid (100 mg/kg/dose every 6 hours, daily maximum 24 g) can be used to reduce mucosal bleeding in thrombocytopenic patients with good hepatic and renal function. Hematuria is a contraindication to its use. Teeth should be brushed with a cloth or soft toothbrush to avoid gum bleeding. • Avoid infection. Keep patients out of the hospital as much possible. Good dental care is important. Rectal temperatures should not be taken, and the rectal areas should be kept clean and free of fissures. If a patient is febrile: • Culture possible sources, including blood, sputum, urine, stool, skin, and sometimes spinal fluid and bone marrow, for aerobes, anaerobes, fungi, and tubercle bacilli. • Patients with fever and neutropenia should be treated with broad-spectrum antibiotic coverage (Chapter 32: Supportive Care of Patients With Cancer). The specific therapy depends upon the clinical status of the patient, the presence of an indwelling vascular access device, and knowledge of the local flora pending specific culture results and antibiotic sensitivities. Patients who remain febrile for 4!7 days, even with broad antibacterial coverage, should be started on antifungal therapy empirically. Therapy should be continued until the patient is afebrile and cultures are negative or a specific organism is identified. • Patients previously treated with immunosuppressive therapy (IST) should receive irradiated cellular blood products to prevent transfusion-acquired graft-versus-host disease (GVHD) (Chapter 35: Blood Banking Principles and Transfusion Medicine Practices). Patients receiving IST should also receive Pneumocystis jirovecii prophylaxis with trimethoprim/sulfamethoxazole or pentamidine. Patients with mild-to-moderate aplastic anemia should be observed for spontaneous improvement or complete resolution or progression to SAA. Hematopoietic stem cell transplantation • Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for SAA for patients who have an HLA-matched related donor. The role of matched unrelated donor transplants is being explored in clinical trials. HLA typing should be performed as soon as the diagnosis of SAA is suspected in children. Patients with related histocompatible donors should have an HSCT. FA, DC, PNH, or other IBMFSs should be ruled out prior to HSCT. Rapidly treating with HSCT is critical as prolonged neutropenia and multiple transfusions increase the risk of transplant-related morbidity and mortality. See Chapter 30, Hematopoietic Stem Cell Transplantation and Cellular Therapy, for preparatory regimens employed pretransplant. Immunosuppressive therapy • Patients unable to undergo matched related HSCT (because no suitable donor is present) should receive IST consisting of antithymocyte globulin (ATG) and cyclosporine A (CSA) (Table 6.5). Methylprednisolone or prednisone should be used to prevent serum sickness. The response rate using this regimen in children is 75!85% at 3!6 months. Contraindications to the use of immunosuppressive drugs include: • serum creatinine greater than 23 the upper limit of normal; • concurrent hepatic, renal, cardiac, or metabolic problems of such severity that death is likely to occur within 7!10 days; and • concurrent pregnancy. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 100 6. Bone marrow failure TABLE 6.5 Immunosuppressive therapy for severe aplastic anemia. 1. ATG: Atgam [ATG (equine) sterile solution] (Pharmacia & Upjohn Co, Pfizer, Inc.) is preferred at 40 mg/kg/day IV once daily, or thymoglobulin [ATG (rabbit)] (Genzyme Corp) 5-mg/kg/day IV once daily, on days 1!4 infused over 6!8 h. Premedication with diphenhydramine and acetaminophen is recommended. 2. Methylprednisolone, 2-mg/kg/day IV on days 1!4. Divide into 0.5-mg/kg/dose IV every 6 h. 3. Prednisone oral taper following the 4-day course of IV methylprednisolone. On days 5 through 14, start prednisone, 2 mg/kg/day PO to be divided into two equal daily doses. After day 14 institute a slow taper (e.g., on days 15 and 16, prednisone, 1-mg/kg/day PO to be divided into two equal daily doses). On days 17 and 18, prednisone, 0.5-mg/kg/day PO to be divided into two equal daily doses. On day 19, prednisone, 0.25-mg/kg/day PO to be given in one dose. 4. CSA (Sandimmune, Gengraf, Neoral)10-mg/kg/day PO initially starting on day 1, divided into two equal daily doses. Serum drug levels should be monitored as needed with the first level at 72 h postinitiation of therapy. CSA dose to be adjusted to keep serum trough levels between 200 and 400 ng/mL. CSA should be continued for 6 months to 1 year until and after a trilineage response is achieved. Decrease the dose by 2.0 mg/kg every 2 weeks once the taper is begun, watching for signs of recurrence of aplastic anemia. 5. G-CSF is no longer routinely used in most centers. Occasionally G-CSF is utilized at a dose of 5-µg/kg SC once daily to improve neutrophil counts in selected patients who are actively infected and persistently severely neutropenic (,200/µL), with reassessment and discontinuation after no more than a few days or weeks if there is no significant response. 6. Eltrombopag is dosed by age: 150-mg PO daily for patients 12 years of age and older, 75-mg PO daily for patients 6!11 years old, and 5 mg/kg/day for patients 2!5 years old. Patients of East and Southeast Asian ancestry must be dosed at 50% due to pharmacokinetic differences in this patient population. Clinical trial results have shown best results if used in combination with CSA and ATG. Abbreviations: ATG, Antithymocyte globulin; CSA, cyclosporine A; G-CSF, granulocyte colony-stimulating factor. Antithymocyte globulin The dose of ATG is shown in Table 6.5. Common adverse reactions to ATG: • Allergic reaction: ATG may cause allergic reactions, sometimes even anaphylaxis. Premedication with an antihistamine and often a corticosteroid is recommended. In some cases, skin testing may be done before the ATG infusion is given. An intradermal ATG skin test consists of 0.02 mL of a 1:1000 dilution in 0.9% sodium chloride solution for injection (5-µg equine IgG). The result is usually read at 10 minutes: a wheal at the ATG site 3 mm or larger in diameter suggests clinical sensitivity and increased possibility of a systemic allergic reaction. The ATG infusion may then require administration over a longer duration with increase in premedications. Most centers have eliminated the skin test in favor of premedication. • Thrombocytopenia: patients may require daily platelet transfusions to maintain a platelet count of more than 20,000/µL during administration of ATG. Only irradiated and leukocyte-filtered cellular blood products should be used. • Headache • Myalgia • Arthralgia • Chills and fever: treatment with an antipyretic, an antihistamine, and corticosteroid is indicated as premedication and may be required during the infusion as well. • Chemical phlebitis: a central line (high flow vein) for infusion of ATG should be used and peripheral veins should be avoided. • Itching and erythema: treatment with an antihistamine with or without corticosteroids is indicated. • Leukopenia. • Serum sickness: this may occur approximately 7!10 days following ATG administration. This should be treated by increasing the daily dose of methylprednisolone until the symptoms abate. Uncommon reactions may include dyspnea, chest, back and flank pain, diarrhea, nausea, vomiting, hypertension, herpes simplex infection, stomatitis, laryngospasm, anaphylaxis, tachycardia, edema, localized infection, malaise, seizures, gastrointestinal bleeding/perforation, thrombophlebitis, lymphadenopathy, hepatosplenomegaly, renal function impairment, liver function abnormalities, myocarditis, and congestive heart failure. Cyclosporine The starting dose of cyclosporine for patients with aplastic anemia is 10 mg/kg/day. CSA levels should be performed once a week for the first 2 weeks and then once every 2 weeks for the remainder of the treatment, or as necessary, to maintain a whole-blood CSA level between 200 and 400 ng/mL. An elevated serum creatinine level is the principal criterion for dose change. An increase in creatinine level of more than 30% above baseline Lanzkowsky’s Manual of Pediatric Hematology and Oncology Aplastic anemia 101 warrants a reduction in the dose of CSA by 2 mg/kg/day each week until the creatinine level has returned to normal. A serum CSA level of less than 100 ng/mL may be evidence of inadequate absorption and/or noncompliance; a CSA level above 500 ng/mL is considered an excessive dose and CSA should be held until the level is within the desired range. Levels should be repeated daily or every other day. When the level returns to 200 ng/mL or less, CSA should be resumed at a 20% reduced dose. In responders, CSA should be tapered very slowly, beginning at 6 months to a year from initiation of therapy although there is little to no evidence available for guidance regarding the target levels and tapering schedule. CSA should be administered on a consistent schedule with respect to time of day and meals. CSA is available in its original form (Sandimmune) and as a modified product (Gengraf and Neoral). Sandimmune is not bioequivalent to Neoral or Gengraf. The modified products are more absorbable and, therefore, the dose should be reduced from the usual Sandimmune dose. Principal side effects of CSA: renal dysfunction, tremor, hirsutism, hypomagnesemia, hyperkalemia, hypertension, and gingival hyperplasia. Uncommon side effects of CSA: hyperuricemia, hepatotoxicity, lipemia, central nervous system toxicity (including seizures), and gynecomastia. An increase of more than 100% in the bilirubin level or of liver enzymes is treated in the same way as an increase of more than 30% in creatinine and warrants a reduction in the dose of CSA by 2 mg/kg/day each week until the bilirubin and/or liver enzymes return to the normal range. Contraindications to the use of CSA: hypersensitivity to CSA. Pharmacokinetic interactions with CSA must be considered in addition to those listed: • carbamazepine, phenobarbital, phenytoin, rifampin—decrease half-life and blood levels of CSA; • sulfamethoxazole/trimethoprim IV—decreases serum levels of CSA; • erythromycin, fluconazole, ketoconazole, nifedipine—increase blood levels of CSA; • imipenem!cilastatin—increases blood levels of CSA and central nervous system toxicity; • methylprednisolone (high dose), prednisolone—increase plasma levels of CSA; • metoclopramide (Reglan)—increases absorption and increases plasma levels of CSA; • aminoglycosides, amphotericin B, nonsteroidal antiinflammatory drugs, trimethoprim/sulfamethoxazole— nephrotoxicity; • melphalan, quinolones—nephrotoxicity; • methylprednisolone—seizures; • azathioprine, corticosteroids—increase immunosuppression, infections, malignancy; • verapamil—increases immunosuppression; • digoxin—elevates digoxin level with toxicity; and • nondepolarizing muscle relaxants—prolong neuromuscular blockade. Hematopoietic growth factors • Granulocyte colony-stimulating factor (G-CSF) had been used to achieve a more rapid increment in the granulocyte count and theoretically to improve protection from infectious complications by stimulating granulopoiesis. G-CSF added to standard ATG and CSA reduces the rate of early infectious episodes and days of hospitalization in very SAA patients but has no effect on overall survival, event-free survival, remission, relapse rates, or mortality. • Eltrombopag (Promacta, Novartis), a thrombopoietin receptor agonist, has been FDA approved for use in combination with standard IST as first-line treatment for SAA in patients greater than 2 years of age (Tables 6.4 and 6.5). Patients who received IST with eltrombopag from day 1 to 6 months had higher overall and complete response rates at 6 months than historically observed with standard IST alone. Treatment choices and long-term follow-up • Although the short-term outcome with IST is comparable to that obtained with HLA-matched related HSCT, the decision to choose HSCT for younger patients with a histocompatible donor is based on the result of longterm follow-up. There are low rates of late mortality (due to chronic GVHD and therapy-related cancer) in patients undergoing HSCT, and the survival curves are relatively flat. Improved GVHD prophylaxis and safer preparative regimens have further improved these results. In contrast, there is a high risk of clonal hematopoietic disorders (MDS, AML, and PNH) in patients treated with IST compared to HSCT. Patients undergoing IST must be closely followed for the development of clonal disorders. Given the risk of clonal evolution and the fact that outcomes for unrelated transplantation are similar to those seen with Lanzkowsky’s Manual of Pediatric Hematology and Oncology 102 6. Bone marrow failure HLA-matched related donor transplants, there are active trials in children and adults to perform matched unrelated transplants at initial diagnosis with SAA. Salvage therapy • For patients who fail sibling donor HSCT, or have a partial response [absolute neutrophil count (ANC) $ 500/µL, but are red cell and platelet transfusion dependent], or relapse following IST, management choices include alternative donor HSCT or further IST. HSCT is preferred to IST if a suitable donor is available. Children and teenagers for whom a fully HLA-matched unrelated donor exists (as determined by high-resolution typing) are excellent candidates for an alternative donor HSCT. For patients without a good alternative donor, a second course of ATG/CSA is warranted although mismatched donors are being considered in some centers. Eltrombopag has been used for these patients in clinical trials. Long-term sequelae and outcomes for SAA • Outcomes for both IST and HSCT have improved considerably in recent years. The results of multiple cohorts report a slightly different response rate and incidence of the clonal evolution of SAA to MDS, AML, or PNH. These data vary based on length of follow-up, age of patient, as well as institution/consortium. • Complete or partial response rates in the range of 60!70%, largely from studies in adults, have been reported with IST. Horse ATG appears to be superior to rabbit ATG in these studies. Although the outcomes in children for IST are generally superior to those described for adults, disease-free survival for matched related HSCT is B95%. • IST improves hematopoiesis and achieves transfusion independence in the majority of patients, but the time to response is long. Hematopoietic response may be partial and relapses are relatively common. • The incidence of clonal hematopoietic disorders, including PNH, MDS, and AML in patients with SAA treated with IST, ranges from 10 to 40%. The European Bone Marrow Transplantation Working Party compared the rate of secondary malignancies following HSCT and IST. Forty-two malignancies developed in 860 patients receiving IST, compared to 9 in 748 patients who underwent HSCT. In this study, acute leukemia and MDS were seen exclusively in IST-treated patients while the incidence of solid tumors was similar in the two groups of patients. • From the aggregate data, there are a number of conclusions: • Matched sibling donor HSCT is always superior as primary therapy in young patients (,20 years of age) at any neutrophil count. • IST, due to transplant-related morbidity and mortality in older patients, is superior to HSCT in older patients (41!50 years). • For the 21- to 40-year-old age-group the differences are less clear. • In all age-groups, there are a higher percentage of late failures and clonal evolution in the IST-treated patients. • When considering the response rate (partial and complete) for IST, the low rate of transplant failure with alternative donor transplant, the incidence of GVHD, and the evolution of clonal disease after IST, the difference in survival between patients treated with matched unrelated donor HSCT and IST increases with time. Thus matched unrelated transplant (preferably within controlled clinical trials) is being considered primary therapy for SAA. Treatment of moderate aplastic anemia • The natural history of moderate aplastic anemia is uncertain and clinical experience varies widely. For this reason, it is generally thought that these patients should be treated initially with supportive therapy with very close follow-up. The majority of patients progress to SAA or develop significant and severe thrombocytopenia and bleeding, serious infections, or a chronic red blood transfusion requirement. These patients should be treated with the same treatment options as described for SAA. Inherited bone marrow failure syndromes • The key shared clinical manifestations of IBMFSs are as follows: • bone marrow failure • congenital anomalies Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes 103 • cancer predisposition • occasional presentation in adulthood The common pathophysiology is low apoptotic threshold of mutant cells. The advent of IBMFS genetic testing through large panels of IBMFS-associated genes has permitted recognition of less penetrant and differentially expressed phenotypes demonstrating significant overlap of phenotypes among these disorders. Fanconi anemia • FA is rare, with a heterozygote frequency in the general population of 1/181 in North America; 1/93 in Israel and less than 1/100 in Ashkenazi Jews (FANCC, BRCA2/FANCD1), South African Afrikaners (FANCA), Northern Europeans (FANCC), sub-Saharan Blacks (FANCG), and Spanish Gypsies (FANCA) due to the “founder effect.” It is a classic IBMFS associated with multiple congenital anomalies and a predisposition to cancer. The details of guidelines for the diagnosis and management of FA as reviewed in the text and tables are beyond the scope of this chapter but are available from the Fanconi Anemia Research Fund. Patients with FA should be registered with the International Fanconi Anemia Registry. This registry collects and maintains longterm outcome data as well as provides resources for physicians, patients, and families. Pathophysiology and genetics • 22 FA complementation groups have been thus far defined. All 22 FA genes have been cloned (Table 6.2). Complementation groups FANCA, C, and G represent B90% of the cases. The gene products of these genes have been shown to cooperate in a common pathway. After FANCM and the FA-associated protein FAAP24 detect DNA damage, eight of the FA proteins (FANCA, B, C, E, F, G, L, and M) assemble to form the FA core complex that is required to monoubiquitinate and activate FANCD2 and FANCI. Ubiquitinated FANCD2 and FANCI form a dimer that stabilizes the stalled replication fork and then in turn interacts in nuclear repair foci with the downstream FA gene products (FANCO, D1, N, and J) in the FA/BRCA DNA damage repair pathway. Damage repair is then achieved by the late FA proteins in cooperation with proteins from other DNA repair pathways. Of note, FANCD1 has been identified as BRCA2. Despite the identification of this pathway, the manner in which disruption in this cascade of events results in a faulty DNA damage response and genomic instability leading to hematopoietic failure, birth defects, and cancer predisposition is incompletely understood. FA cells are characterized by hypersensitivity to chromosomal breakage as well as hypersensitivity to G2/M cell cycle arrest induced by DNA crosslinking agents. In addition, there is sensitivity to oxygen-free radicals and to ionizing radiation. Clinical manifestations • FA is inherited as an autosomal recessive disorder ( . 99%), rarely as an X-linked recessive (FANCB, ,1%) or an autosomal dominant (FANCR/RAD51), and is the most frequently inherited aplastic anemia. FANCA is the most common complementation group, representing about 60!70% of cases. FANCC and FANCG are the next most common, representing B10% of cases each. The other complementation groups are quite rare, representing the remainder of cases (Table 6.2). • Genotype!phenotype correlations are complex and are emerging and relate to the complementation group as well as the specific allelic mutation (i.e., null versus hypomorphic gene product). In particular, certain associations relating genotype to specific congenital anomalies, early-onset aplastic anemia, leukemia, as well as Wilms’ tumor and medulloblastoma, have been confirmed. • All racial and ethnic groups are affected. • Pancytopenia is the usual finding. • The median age at hematologic presentation of patients with aplastic anemia is approximately 8!10 years. Leukemia tends to appear later in the teenage years and solid tumors appear in young adulthood and continue to occur as patients age. • Hematologic dysfunction usually presents with macrocytosis, followed by thrombocytopenia, often leading to progressive pancytopenia and SAA. FA frequently terminates in MDS and/or AML. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 104 6. Bone marrow failure • The diagnosis of FA should always be considered in any child with an isolated cytopenia even when the classical somatic anomalies are absent as a significant number of these cases are physically normal. • FA cells are hypersensitive to chromosomal breaks induced by DNA crosslinking agents. This observation is the basis for the commonly used chromosome breakage test for FA. The clastogens diepoxybutane (DEB) and mitomycin C (MMC) are the agents most frequently used in vitro to induce chromosome breaks, gaps, rearrangements, quadriradii, and other structural abnormalities. Clastogens also induce cell cycle arrest in G2/M. The hypersensitivity of FA lymphocytes to G2/M arrest, detected using cell cycle analysis by flow cytometry either de novo or clastogen induced, is being used by some as a screening tool for FA. • Bone marrow examination reveals hypocellularity and fatty replacement consistent with the degree of peripheral pancytopenia. Residual hematopoiesis may reveal dysplastic erythroid (megaloblastoid changes, multinuclearity) and myeloid (abnormal granulation) precursors and abnormal megakaryocytes. • Congenital anomalies include increased pigmentation of the skin along with café-au-lait spots and hypopigmented areas, short stature (impaired growth hormone secretion), skeletal anomalies (especially involving the thumb, radius, and long bones), male hypogenitalism, microcephaly, abnormalities of the eyes (microphthalmia, strabismus, ptosis, and nystagmus) and ears (deafness), hyperreflexia, developmental delay, and renal and cardiac anomalies. However, up to 40% of patients lack obvious physical abnormalities. There is great clinical heterogeneity even within a genotype (affected siblings may be phenotypically different). • There is a nearly 800-fold increased relative risk of developing AML and perhaps an even greater relative risk of nonhematologic solid tumors (e.g., squamous cell carcinoma of head and neck, cancer of the breast, kidney, lung, colon, bone, retinoblastoma, and female gynecologic) in patients with FA. In general, these occur at much younger ages than those seen in the general population. A relatively large number of patients only become aware that they have FA when they are diagnosed with cancer. Androgen-related liver neoplasia may also occur as adenoma or hepatoma and rarely carcinoma. The risk of solid tumors may become even higher as death from aplastic anemia is reduced, and as post-HSCT patients survive longer. These data must be considered in the context of HSCT, as the risk of nonhematologic malignancy is likely to increase as a result of HSCT conditioning regimens and chronic GVHD. Treatment for cancer is generally ineffective due to severe side effects of chemotherapy and radiation therapy experienced by these patients. • Prenatal diagnosis is possible by amniotic fluid cell cultures and chorionic villus biopsy. Diagnosis • Table 6.6 lists the indications for FA screening studies. • Table 6.7 lists the laboratory studies required to make the diagnosis of FA. • Table 6.8 lists the initial and follow-up investigations to be performed in a patient with an established diagnosis of FA. Differential diagnosis • The differential diagnosis of FA generally includes acquired aplastic anemia, AMT, TAR syndrome as well as VATER/VACTERL (vertebral anomalies, anal atresia, cardiac anomalies, tracheoesophageal fistula, renal anomalies, and limb anomalies) syndromes. FA is easily distinguished from TAR syndrome. There is an intercalary defect in TAR consisting of absent radii with normal thumbs, whereas in FA the defect is terminal, an abnormal radius always being associated with anomalies of the thumb. Table 6.9 lists the features differentiating FA from TAR syndrome. • FA testing is warranted in any child who presents with hematologic cytopenias, unexplained macrocytosis, aplastic anemia, or AML with congenital anomalies, as well as representative congenital abnormalities or solid tumors typical of FA such as head and neck, esophageal or gynecologic tumors presenting at an early age. • The critical investigations are aspiration and biopsy of the bone marrow and demonstration in peripheral blood of increased chromosomal fragility or G2/M arrest induced by clastogens (e.g., DEB, MMC). Complementation group analysis and/or mutation analysis are helpful after the demonstration of a positive screening test and should be obtained if possible. • FA somatic mosaics with DEB-positive and DEB-negative (double population) cells belong to distinct groups based upon the degree of mosaicism and may present diagnostic problems. Mosaicism leading to a “normal” T cell that is resistant to the less dose-intense HSCT conditioning used for FA may result in graft rejection. In cases where mosaicism is suspected, cultured skin fibroblasts should be studied. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes 105 TABLE 6.6 Indications for Fanconi anemia (FA) screening studies. All children with aplastic anemia or unexplained cytopenias Select children with solid tumors or acute myeloblastic leukemia (AML) (for example, patients with classic birth defects or those with severe prolonged cytopenias, and/or increased toxicity with chemotherapy) All children with MDS Patients with classic birth defects suggestive of FA • Patients with VATER/VACTERL (vertebral anomalies, anal atresia, cardiac anomalies, tracheoesophageal fistula, renal anomalies, and limb anomalies) • Patients with structural anomalies of the upper extremity and/or genitourinary system Siblings of known FA patients Patients with: • Excessive café-au-lait spots, hypo- or hyperpigmentation of skin (especially if increasing with age) • Microcephaly • Microphthalmia • Growth failure Patients with development of FA-associated cancers at a young age (e.g., squamous cell carcinoma in esophagus, head, and neck ,50 years of age, vulvar cancer ,40 years of age, and uterine cervical cancer ,30 years of age or liver tumors) Patients with karyotype testing with spontaneous chromosome breaks Patients with unexplained macrocytosis and an elevated HbF Patients with nonimmune thrombocytopenia Males with unexplained infertility Abbreviations: Hb, Hemoglobin; MDS, myelodysplastic syndrome. TABLE 6.7 Laboratory studies to establish the diagnosis of Fanconi anemia (FA). 1. Screening tests: a. Demonstration of the presence of increased chromosomal breakage in T lymphocytes cultured in the presence of DNA crosslinking agents such as DEB or MMC. DEB test is used more widely. Chromosome fragility includes breaks, gaps, rearrangements, radials, exchanges, and endoreduplication. Fibroblasts should be studied in patients for whom mosaicism is suspected.a b. A flow cytometric technique for the analysis of alkylating agent!treated cells can determine the percentage of cells arrested in G2/M and clearly distinguish FA cells from normal cells.b c. Western blot for D2-L (long protein formed by ubiquitination of FANCD2).b 2. Definitive tests: a. Complementation group analysis.b b. Targeted mutation analysis, copy number variant analysisb (see Table 6.2 for cloned FANC genes). 3. Prenatal diagnosis of FA: a. DEB test can be used in either chorionic villus- or amniocentesis-derived samples. 4. Detection of carrier state: a. In a FA family, if proband has been identified to have a defect in one of the cloned genes, molecular testing is available for the extended family members. Population-based screening is only done in at-risk populations. a Some patients with FA may have two populations of cells exhibiting either a normal or an FA phenotype. Such mosaicism may result in a false-negative chromosome breakage study if the percentage of normal cells is high. The study of fibroblasts is useful in this circumstance. b Done only in specialized laboratories. Abbreviations: DEB, Diepoxybutane; MMC, mitomycin C. Management • Serial assessments of the bone marrow should be performed to provide evidence of progression and the development, or evolution of cytogenetic abnormalities. • Bone marrow aspiration should be performed for cytology, cytogenetics with FISH analysis for cytogenetic abnormalities that may be predictive of leukemia (e.g., 13q, 11q, 27q, and/or monosomy 7) approximately yearly or more often if indicated by the emergence of specific clonal or morphological abnormalities. • Bone marrow biopsy should be done for cellularity. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 106 6. Bone marrow failure TABLE 6.8 Initial and follow-up investigations for a patient with Fanconi anemia (FA). 1. Endocrine studies for: a. Short stature (growth hormone deficiency) b. Glucose intolerance (diabetes mellitus) c. Hypothyroidism d. Pubertal delay e. Evaluation of undescended testes f. Reduced fertility 2. Imaging studiesa and evaluation of: a. Cardiac anomalies b. Orthopedic anomalies c. Genitourinary abnormalities 3. Hepatic ultrasound every 6 months if taking androgens 4. Serum chemistries for liver and kidney function 5. Hearing test 6. Monitoring for iron overload for patients on red cell transfusion therapy a. Liver enzymes b. MRI T2* for cardiac and hepatic liver iron quantification c. Liver biopsy, if needed 7. Survey of family members: a. Exclude diagnosis of FA in any other family members b. Type family members for the potential availability of an HLA-matched sibling for future consideration of stem cell transplant c. Provide genetic counseling to parents and patient 8. Prospective counseling and screening: a. Avoid exposure to potential mutagens or carcinogens (e.g., insecticides, organic solvents, hair dye, papillomavirus, sun exposure, radiographsa) b. Cancer surveillance: i. Examine bone marrow yearly with histologic, cytogenetic, and FISH studies for evidence of myelodysplasia or leukemia ii. Yearly head and neck examination over age 7 years iii. Yearly gynecologic examination beginning at age 16 years iv. Breast self-examination beginning at age 16 years v. Periodic oral cancer screening vi. Yearly dermatologic screening 9. Mutation analysis: These studies are performed in specialized laboratories only. Mutation analysis may help predict the phenotype as more data become available a Limit exposure to radiation by using appropriate restraint and nonradiologic imaging studies. The patient’s complete blood counts should be monitored. The degree of cytopenia guides management as follows: Mild Moderate Severe Hemoglobin level (g/dL) $ 8.0 ,8.0 ,8.0 ANC (/µL) ,1500 ,1000 ,500 Platelet count (/µL) 50,000!150,000 ,50,000 ,30,000 Abbreviation: ANC, Absolute neutrophil count. When cytopenias are in the mild-to-moderate range and in the absence of cytogenetic abnormalities, counts should be monitored every 3!4 months and bone marrow aspiration should be performed yearly. Monitoring of blood counts and bone marrow should be increased to every 1!2 months and every 1!6 months, respectively, for cytopenia in the presence of cytogenetic abnormalities or more significant dysplasia without frank MDS. With falling (or in some cases rising), counts surveillance must be increased. Treatment The majority of patients with FA will require treatment at some point for bone marrow failure. The only true treatment for the cytopenias seen in FA is HSCT. Androgen therapy has difficult side effects for children, and especially females, and is often not efficacious. HSCT, despite improved results over the recent years with overall survival advantage, has a known increase in nonhematologic malignancies, specifically head and neck squamous cell cancers. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes 107 TABLE 6.9 Features differentiating Fanconi anemia from thrombocytopenia-absent radii (TAR) syndrome. Feature Fanconi anemia TAR Age of onset of aplastic anemia symptoms Median of 8!10 years Birth to infancy (first year of life) Low birth weight B10% B10% Stature Short Short Skeletal deformities 66% 100% Absent radii with fingers and thumbs present 0% 100% Other hand deformities B40% B40% Lower extremity deformities B40% ,10% Cardiovascular anomalies 5!10% 5!10% Hyper- and hypopigmentation of skin 75%; increases with age 0% Hemangiomas 0% B10% Peripheral blood Pancytopenia, macrocytosis Thrombocytopenia, eosinophilia, leukemoid reactions, anemia Bone marrow Aplastic Absent or abnormal megakaryocytes, normal myeloid and erythroid precursors Marrow CFU-GM, CFU-E Decreased Normal (with decreased CFU-megakaryocytes) HbF Increased Normal Chromosomal breaks in leukocytes Present Absent Malignancy (solid tumors) Common No Associated leukemia Yes Rare Sex ratio (male/female) B1:1 B1:1 Inheritance pattern Autosomal recessive Autosomal recessive Prognosis Poor Good if a patient survives first year when platelet count improves Abbreviation: Hb, Hemoglobin. Modified from Hall, J.G., Levin, J., Kuhn, J.P., Ottenheimer, E.J., van Berkum, K.A., McKusick, V.A., 1969. Thrombocytopenia with absent radius (TAR). Medicine (Baltimore) 48 (6), 411!439. https://doi.org/10.1097/00005792-196948060-00001. PMID: 4951233. • Androgen therapy: oxymetholone 2!5 mg/kg/day and tapered to the lowest effective dose is effective in approximately 50% of patients. Recent anecdotal reports and small studies suggest that danazol may be an effective synthetic androgen with less virilizing effects. Doses in the range of B5 mg/kg have been effective. • Cytokines: G-CSF at a starting dose of 5 µg/kg/day, tapered to the lowest effective dose may be administered when moderate-to-severe neutropenia is present. • Transfusions: treatment with packed red blood cells and platelets should be minimized and reserved for patients who fail androgen therapy. Blood products should be irradiated, leukocyte-depleted, and of single donor origin, when possible. Blood relatives should not be used as blood donors. Iron status should be monitored at regular intervals to determine the degree of iron overload and the institution of chelation treatment in chronically transfused patients. • HSCT: HLA typing should be done at diagnosis to facilitate therapeutic planning. If an HLA-matched related donor is available, stem cell transplantation should be carried out. Indications for alternative donor HSCT are rapidly evolving and every patient, regardless of the availability of a matched related donor, should be considered for transplant as outcomes with unrelated transplant have improved. Patients should be evaluated for transplant at a center that has FA-transplant experience. Evidence of true MDS (as opposed to benign clonal abnormalities) or evolution to AML is a clear indication for transplant. The sensitivity of FA patients to traditional transplant conditioning regimens requires the use of lower dosages of chemotherapy and radiation therapy (Chapter 30: Hematopoietic Stem Cell Transplantation and Cellular Therapy). Before a family member is used as a donor, the donor must be evaluated to exclude a diagnosis of FA. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 108 6. Bone marrow failure Matched sibling donor Yes HLA-matched related SCT No* Anemia Thrombocytopenia Androgens Cytokines Transfusions Neutropenia Cytokines Androgens — oxymetholone: 2–5 mg/kg/day (may be able to taper) Cytokines — G-CSF 5 µg/kg every day or every other day *The use of matched unrelated and other alternative donor t ransplants is evolving. A center with extensive transplant experience should be consulted for all FA patients with significant bone marrow failure. FIGURE 6.2 Treatment of Fanconi anemia. • Human papillomavirus (HPV) vaccination: vaccination is recommended in patients with FA. • Growth hormone therapy: the majority of patients with FA have short stature. Up to 50% have deficient growth hormone. Given a theoretical association of growth hormone and leukemia, growth hormone should be used with that understanding in patients with FA. • Gene therapy: this approach is experimental and is being presently performed through two approved clinical trials in Spain and the United States. Both clinical trials are utilizing CD341 cells transduced with FANCA lentiviral vectors infused in an autologous transplant setting to provide correction of one copy of FANCA. Initial results have shown promise with a major limiting factor being low numbers of stem cells able to be collected in these patients. The treatment of FA is shown in Fig. 6.2. Prognosis • Current results of matched sibling transplantation prior to the development of overt leukemia show a longterm disease-free survival of 90%. However, the long-term risks of late sequelae from HSCT include an increase in cancer risk. Alternative donor transplant had historically been reserved for androgen-refractory patients and those with MDS or leukemia. However, this recommendation is rapidly evolving as improvements in HLA typing, conditioning regimens, and overall care as well as HSCT experience have improved outcomes considerably. Thus every patient should be evaluated for HSCT. Dyskeratosis congenita DC is characterized by the classic triad of ectodermal dysplasia consisting of: • abnormal skin pigmentation of the upper chest and neck, • dysplastic nails, and • leukoplakia of oral mucous membranes. Patients with DC are also known to have: • Predisposition to bone marrow failure. • Predisposition to cancer—hematologic (leukemia, MDS) and epithelial cancers. • Somatic findings in DC include epiphora (tearing due to obstructed tear ducts), blepharitis, developmental delay, pulmonary disease (fibrosis), short stature, esophageal webs, liver fibrosis, dental caries, tooth loss, premature gray hair and hair loss; ocular, dental, skeletal, cutaneous, genitourinary, gastrointestinal, neurologic abnormalities and immunodeficiency have been reported. • The classical clinical diagnosis of DC requires two of the three elements of the classic diagnostic triad and any other associated abnormality in patients with a known mutation or very short telomeres. The presence of short telomeres in a member of a pedigree with definitive DC is sufficient for the diagnosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 109 Inherited bone marrow failure syndromes • The median age at diagnosis is approximately 15 years. The median age for the onset of mucocutaneous abnormalities is 6!8 years. Nail changes occur first but hematologic abnormalities may precede mucocutaneous changes. The median age for the onset of pancytopenia is 10 years. Approximately 50% of patients develop SAA by age 50 years and greater than 90% develop at least a single cytopenia by 40 years of age. The anemia is associated with a high mean corpuscular volume (MCV) and elevated fetal hemoglobin. As with FA, it is the nonhematologic manifestations of DC that are of particular concern, especially when HSCT for bone marrow failure is considered. Pathophysiology • The DC phenotype results from deficient telomerase activity. Telomerase adds DNA sequence back to the ends of chromosomes that are eroded with each DNA replication. Telomerase activity is found in tissues with rapid turnover such as the basal layer of the epidermis, squamous epithelium of the oral cavity, hematopoietic stem cells and progenitors, and in other tissues affected in DC. The lack of telomerase activity may also give rise to chromosome instability resulting in the high rate of premature cancer. Table 6.10 shows the cells in various organs expressing telomerase and the defects that occur in telomerase failure. Epithelial malignancies develop at or beyond the third decade of life. Type II alveolar epithelial cells express telomerase, and as a consequence, about one in five patients develops progressive pulmonary disease characterized by fibrosis, resulting in diminished diffusion capacity and/or restrictive lung disease. It is likely that more pulmonary disease would be evident if patients did not succumb earlier to the complications of SAA and cancer. Genetics • Mutations in 14 genes in the telomerase maintenance pathway have been associated with DC. DC is most commonly inherited as an X-linked recessive but may also be autosomal dominant or recessive. The gene responsible for the X-linked form was mapped to Xq28 and subsequently identified as DKC1. DKC1 codes for dyskerin, a nucleolar protein associated with nucleolar RNAs. Dyskerin is associated with the telomerase complex. This latter function appears to be the one involved in the pathophysiology of DC, as most of the genes found to date in DC (Table 6.2) are involved in telomere biology. There are many features in common to all three genetic subtypes; however, the clinical phenotypes may vary widely in severity even within different mutations of the same allele. Affected members within the same family may exhibit wide variability in clinical presentation suggesting the influence of modifying genes and environmental factors. In addition, different allelic mutations in these genes may result in bone marrow failure in the absence of any physical anomalies. Thus in addition to FA, DC must be ruled out in all cases of aplastic anemia. Clinical manifestations The clinical manifestations are quite variable. Bone marrow failure: the incidence of bone marrow failure is 50% at 50 years of age. The majority of deaths (67%) are a result of bone marrow failure, followed by cancer and lung disease (pulmonary fibrosis) with or without HSCT. However, overall median survival has improved over the last decade. Malignancy: the causes of death are similar to those reported for FA with the exception of pulmonary fibrosis, which is unique to DC. MDS and AML are less frequent than in FA, as are solid tumors, including head and neck TABLE 6.10 Cells expressing telomerase and defects occurring in telomerase failure. Organ system Cells expressing telomerase Defect Hair Hair follicle Alopecia Oral cavity Squamous epithelium Leukoplakia Skin Epidermis, basal layer Abnormal pigmentation; dyskeratotic nails Lungs Type II alveolar cells Pulmonary fibrosis Liver Unknown cell type Cirrhosis Intestines Crypt cells Enteropathy Testes Spermatogonia Hypogonadism Bone marrow Progenitors Bone marrow failure Lanzkowsky’s Manual of Pediatric Hematology and Oncology 110 6. Bone marrow failure squamous cell carcinoma followed by anorectal, esophagus/stomach, brain, renal, and others. All these cancers occur at younger ages than these cancers occur in the population at large, but older than in patients with FA. Neurological: patients with the severe form of DC known as Hoyeraal!Hreidarsson (HH) syndrome have symptomatic cerebellar hypoplasia, microcephaly, and developmental delay. Revesz syndrome (RS) is associated with CNS calcification, occasionally cerebellar hypoplasia and exudative retinopathy. Multiple DC genes have been implicated in HH and mutations in the gene encoding TINF2 have been implicated in RS. Immunodeficiency: significant progressive immunodeficiency occurs in DC. Although DC is predominantly a cellular immune defect, humoral immunodeficiency as well as neutropenia probably plays a significant role in the infectious morbidity and mortality in DC. Outcome: the median survival is approximately 40!45 years for patients with DC. In HH, it is approximately 5 years of age and in RS the median has not yet been defined. Treatment • Supportive care: blood products, antibiotics, and antifibrinolytic agents are similar to those used for idiopathic aplastic anemia. • HSCT: transplantation should be considered for those patients with marrow failure who have an HLA-matched related donor or a matched alternative donor, and no DC-related contraindications. The results of HSCT have been poor predominantly due to preexisting nonhematologic organ damage. All DC patients are at a high risk of interstitial pulmonary disease when undergoing HSCT. A recent study demonstrated a 5-year overall survival of 59% and event-free survival of 51%. However, improvements in conditioning regimens and matching are necessary to decrease the 35% 3-year cumulative incidence of chronic GVHD. Also, overall and event-free survivals were found to continue to decrease with time due to infections early on, progressive pulmonary and other organ damage after infections, and later, due to secondary malignancies. An immunoablative rather than a myeloablative approach is currently being used to potentially reduce the incremental risk of pulmonary toxicity as well as the nonhematologic cancer risk. • Others: although responses to androgens, G-CSF as well as erythropoietin and rarely splenectomy have been documented, they have been transient. IST is ineffective. Congenital aplastic anemias of unknown inheritance Rare cases of aplastic anemia in the literature have been associated with Down syndrome, congenital trisomy 8 mosaicism, familial Robertsonian translocation (13;14), nonfamilial translocation in a male with t(1;20)(p22; q13.3), and increased spontaneous chromosomal breakage without further increase in breakage with MMC as well as other very rare cases with familial associations. Many of these cases were reported before the discovery of the genes associated with the IBMFSs and can now be categorized. However, a large number of cases are yet to be genetically diagnosed (20!40% in some series) and await the identification of new mutated genes while some represent known syndromes with atypical presentations that are missed with targeted mutation analysis. Diamond Blackfan anemia DBA is a rare, predominantly red cell aplasia presenting in 90% of the patients within the first year of life. The classic characteristics of DBA are macrocytic anemia and reticulocytopenia and a normocellular bone marrow with a selective paucity of erythroid precursors. Many countries have national patient registries. These registries collect and maintain long-term outcome data as well as provide resources for physicians, patients, and families. Patients in North America with DBA should be registered with the Diamond Blackfan Anemia Registry (DBAR). Pathophysiology • DBA results from defective ribosome biosynthesis. As a consequence, erythroid progenitors and precursors are highly sensitive to death by apoptosis. The majority of the genetically known cases are the consequence of either small or large subunit-associated ribosomal protein (RP) haploinsufficiency. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes 111 Genetics • Autosomal dominant inheritance is found in all cases of RP mutations. Two genes, GATA1, an erythroid transcription regulator, and TSR2, a chaperone for RPS26, have X-linked inheritance; cases with possible autosomal recessive inheritance are under investigation. • The first “DBA gene” was cloned in 1997 and identified as RPS19, a gene that codes for RPS19 and located at chromosome 19q13.2. RPS19 mutations account for 20!25% of both sporadic and familial cases. Since that time a total of 23 genes have been identified (Table 6.2) comprising approximately 80% of DBA cases analyzed. Along with RPS19, RPL5, RPL11, RPS10, and RPS26 make up the majority of the cases of DBA with the others being quite rare. The functions of the RP and how they lead to anemia are not fully understood. • Laboratory studies used for identification of dominant inheritance in family members of a proband with DBA include hemoglobin level, MCV, erythrocyte adenosine deaminase (eADA) activity (the absence of these markers clearly does not exclude dominant inheritance), and mutation analysis when available. By carefully evaluating families, it appears that at least 40!50% of cases of DBA may be dominantly inherited. • To provide genetic counseling, it is important to perform the previously mentioned laboratory studies to reduce the possibility of missing dominant inheritance in presumed sporadic cases. It is also important to perform these laboratory studies in potential family stem cell donors to increase the likelihood of detection of a silent phenotype. When there is a known mutation the parents should be evaluated, as well as extended family members as indicated. Clinical manifestations • The median age at presentation of anemia is 2 months and the median age at diagnosis of DBA is 3!4 months. Over 90% of the patients present during the first year of life. A small percentage of affected infants may be anemic at birth. • Platelet and white cell counts are usually normal; thrombocytosis occurs rarely, neutropenia and/or thrombocytopenia may occur. Instances of significant cytopenias, including aplastic anemia, have also been noted. • Physical anomalies, excluding short stature, are found in half of the patients. Of these, 50% are of the face and head (microcephaly, cleft palate, ear and eye anomalies), 38% upper limb and hand (thumb deformity, triphalangeal thumb, duplication of thumb, and bifid thumb), 39% genitourinary, and 30% cardiac. Over 20% of patients have more than one anomaly. • Low birth weight occurs in approximately 10% of all affected patients, with about half of this group being small for gestational age. Over 60% are below the 25th percentile for height. There appears to be a slight increase in the incidence of miscarriages, stillbirths, and complications of pregnancy among the mothers who have given birth to infants with this syndrome. • Karyotype is generally normal. • Hepatosplenomegaly is not a common feature. • DBA has been recognized as a cancer predisposition syndrome, with an incidence of cancer of almost 14% by 45 years of age. Colorectal and other gastrointestinal malignancies are the most common, with osteogenic sarcoma being the next most common. Cases of breast cancer and other solid tumors have been reported, all occurring at a younger age than expected for these malignancies. As patients age, MDS and AML become increasingly more likely. Diagnosis RP genes mutations have been identified in DBA and a number of genetically defined individuals have been identified who lack some or all of the classical clinical criteria. The following laboratory findings occur in DBA: Macrocytosis associated with reticulocytopenia. The white cell count and platelet counts are usually normal at presentation but neutropenia and thrombocytopenia are being more frequently recognized and trilineage marrow failure may become evident with increasing age. An elevated eADA activity is found in approximately 80!85% of patients. Elevated fetal hemoglobin. Bone marrow with the virtual absence of normoblasts, in some cases with relative increase in proerythroblasts or normal number of proerythroblasts, with a maturation arrest; normal myeloid and megakaryocytic series. Decrease in bone marrow cellularity is common. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 112 6. Bone marrow failure Importantly, these laboratory findings (macrocytosis, elevated fetal hemoglobin, and eADA activity) may be useful in avoiding potential matched related HSCT donors with genotypic DBA and have been helpful in distinguishing DBA from transient erythroblastopenia of childhood (TEC). Fetal hydrops secondary to fetal DBA has been reported. Patients presenting with hydrops fetalis should be evaluated for DBA. Table 6.11 lists the diagnostic criteria for DBA. Differential diagnosis DBA must be differentiated from: • TEC. Table 6.12 lists the differentiating features of TEC and DBA. • Congenital hypoplastic anemia due to transplacental infection with parvovirus B19: Performing reverse transcriptase polymerase chain reaction for parvovirus B19 on a bone marrow sample can identify this infection from DBA. Parvovirus may result in transient red cell failure in a patient with underlying hemolytic anemia or chronic red cell failure in a patient with an underlying immune deficiency. • Late hyporegenerative anemia due to severe Rh or ABO hemolytic disease of the newborn. This may rarely last for a few months and should be considered in the differential diagnosis of DBA. • Pearson syndrome: this very rare disorder is characterized by refractory aregenerative macrocytic sideroblastic anemia, neutropenia, vacuolization of bone marrow precursors with sideroblasts (usually ring sideroblasts), exocrine pancreatic dysfunction, and metabolic acidosis. The anemia presents at 1 month of age in 25% and at 6 months of age in 70% of affected individuals. A deletion in mitochondrial DNA has been found in Pearson syndrome. In many instances the anemia may resolve with age. However, many patients will develop neurodegenerative disease (Kearns!Sayre syndrome) later in childhood or adulthood. The natural history of Pearson syndrome is not well characterized. • Other IBMFSs as well as acquired marrow dysfunction due to viral infections or medications. Treatment • Packed red cell transfusions: when the blood product supply is safe and venous access is available, packed red cell transfusion should be used to support patients until approximately 1 year of age, if possible. This practice may avoid significant steroid-related problems encountered in infants, allow for adequate growth, and safe and effective immunizations. Leukocyte-depleted packed red cell transfusion should be used to reduce the incidence of nonhemolytic, febrile transfusion reactions, as well as the risk of transmission of CMV and the risk of HLA alloimmunization. Patients who are receiving or who have recently been treated with immunosuppressive drugs should receive irradiated blood products. Patients in whom stem cell transplantation is contemplated should receive CMV-safe blood products. Effective iron chelation must accompany a chronic transfusion protocol. TABLE 6.11 Diagnostic criteria for Diamond Blackfan anemia (DBA). Classical • Normochromic, usually macrocytic anemia, relative to patient’s age and occasionally normocytic anemia developing in early childhood with no other significant cytopenias • Reticulocytopenia • Normocellular marrow with selective paucity of erythroid precursors • Age less than 1 year Definitive but not essential • Presence of mutation described in classical DBAa Major • Positive family history Minor • Congenital abnormalities described in classical DBA • Macrocytosis • Elevated fetal hemoglobin • Elevated erythrocyte adenosine deaminase activity a These criteria are under constant analysis and may be modified as new DBA genes are identified. The diagnosis becomes less certain when there are fewer diagnostic criteria and the patient does not have a positive family history or a known mutation. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes TABLE 6.12 113 Features differentiating transient erythroblastopenia of childhood from Diamond Blackfan anemia. Feature Transient erythroblastopenia of childhood Diamond Blackfan anemia Frequency Common Rare (5!10 per 106 live births) Etiology Acquired (viral, idiopathic) Genetic Age at diagnosis 6 months to 4 years, occasionally older 50% by 3 months of age; 75% by 6 months and 90% by 1 year Familial No Yes (in up to 20% of cases) Antecedent history Viral illness None Congenital abnormalities Absent Present in B50% cases (heart, kidneys, musculoskeletal system) Course Spontaneous recovery in weeks to months Prolonged, 20% actuarial probability of remission Transfusion dependence Not dependent Transfusion or steroid dependent At diagnosis 20% 80% During recovery 90% 100% In remission 0% 100% At diagnosis 25% 100% During recovery 100% 100% In remission 0% 85% Erythrocyte adenosine deaminase activity Normal Elevated (B85% of cases) Treatment Packed cell transfusion, if required Packed red cell transfusion until 1 year of age; Prednisone 2 mg/kg/day and then weaned to ,0.5 mg/kg/day; stem cell transplantation MCV increased Hemoglobin F increased Abbreviation: MCV, Mean corpuscular volume. From Lanzkowsky, P., 1989. Manual of Pediatric Hematology and Oncology. Churchill Livingstone, New York. • Steroids: prednisone, or its equivalent, at a dose of 2 mg/kg/day in single or divided dosages is used to initiate therapy. Reticulocytosis usually occurs in 1!2 weeks but may take slightly longer. When the hemoglobin level reaches B10 g/dL, the prednisone dose should be tapered slowly to the minimum dose necessary to maintain a reasonable hemoglobin level on an alternate-day schedule. A dose equivalent of 1 mg/kg/every other day (50.5 mg/kg/day) is generally safe but the corticosteroid dose must be individualized. Any patient who experiences significant steroid-related side effects, including growth failure, should have steroid medication temporarily discontinued and should be placed on a chronic red cell transfusion regimen. Patients with DBA on low-dose alternate-day therapy of long duration, starting in early infancy, may manifest significant steroid toxicity. In the DBAR dataset, 40% of patients manifested cushingoid features, 12% had pathologic fractures, and 7% have cataracts. • HSCT: HLA-matched sibling donor transplantation should be considered for any patient with DBA, particularly those who are transfusion dependent. Consideration should be given to the fact that 20% of all patients attain spontaneous remission, balanced by the risk of hematologic malignancy, MDS, or SAA. A family marrow donor must be evaluated for the presence of a “silent phenotype.” Matched unrelated or mismatched related donor transplant outcomes have improved considerably over the last decade. The risk of developing nonhematologic malignancy is increased following preparative regimens used in HSCT and as the patients age. • Other therapies: a number of treatments, including erythropoietin, immunoglobulin, megadose corticosteroids, and androgens have been utilized in DBA patients with little success. Cyclosporine, IL-3, and metoclopramide have resulted in anecdotal responses in DBA and have been largely abandoned as therapeutic options. A recent DBAR clinical trial of L-leucine in transfusion-dependent patients demonstrated L-leucine to be safe, Lanzkowsky’s Manual of Pediatric Hematology and Oncology 114 6. Bone marrow failure resulted in an erythroid response in 16%, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable patients, respectively. Another L-leucine trial is planned in transfusion-dependent and in steroid-dependent patients, at higher doses. Two other trials are ongoing through the DBAR for adults with transfusion-dependent DBA—one with sotatercept and one with trifluoperazine. Prognosis • Approximately 80% of DBA patients initially respond to corticosteroid therapy; however, only half of the responders can remain on steroids at a dose required for an adequate red cell response due to significant side effects. The remaining patients will require transfusion therapy or stem cell transplantation. The major complication of chronic steroid therapy is poor growth especially when patients are maintained on higher doses than recommended. Some patients experience pathologic fractures on lower doses and should have the steroids discontinued. • The actuarial remission rate in DBA is approximately 20% by age 25, irrespective of their pattern of response to treatment, with the majority remitting during the first decade of life. • The major complication of chronic transfusion therapy is iron overload, the consequences of which include diabetes mellitus, cardiac and hepatic dysfunction, growth failure, as well as endocrine dysfunction. Iron chelation with either deferoxamine or deferasirox is, therefore, an essential component of a transfusion program. The oral chelator deferiprone (L1) has caused significant neutropenia in DBA and should only be considered with extreme caution in specific cases. Many patients find nearly daily subcutaneous and even oral chelation therapy onerous and compliance is often poor. The DBAR reports deaths due to iron overload complications as the leading cause of death in patients 20!30 years of age. • In summary, both chronic corticosteroid therapy and chronic transfusion therapy may lead to a number of significant short- and long-term complications, supporting a role for HSCT. Improvements in matched unrelated donor transplantation suggest that patients should be evaluated on an individual basis to determine the utility of that approach in a given patient. Survival of patients into adulthood is prolonged for patients in remission or sustainable on steroids. Only about 60% of transfusion-dependent patients currently survive to middle age. The overall actuarial survival for DBA at 40 years of age is 75.1 6 4.8%. • HLA-matched sibling stem cell transplant patients have long-term survival of over 90% if performed at age 9 years or younger. Well-matched unrelated donor transplants done over the last 10 years have resulted in an improved survival rate of 80%. Favorable transplantation outcomes are most likely if the patient is in good health at the time of HSCT without iron overload or allosensitization. Improvements in supportive care, GVHD prophylaxis, and infection control have resulted in a marked decrease in HLA-matched related HSCT transplant-related morbidity and mortality. Sibling HSCT is recommended for young DBA patients, prior to development of significant allosensitization or iron overload, when there is an available unaffected donor. • Death in DBA is primarily due to treatment-related causes (iron overload, infection, complications of stem cell transplant) in 67% of cases as opposed to disease-related causes (solid tumors, leukemia, and MDS, SAA). This will likely change as management of iron overload and HSCT improves and patients age. • Patients with DBA who become pregnant may develop either an increased requirement for steroid therapy or red cell transfusions due to worsening anemia and should be considered high risk. The worsening of anemia during pregnancy appears to be a hormonally induced problem because estrogen-containing oral contraceptives have the same effect. Contraceptives containing progesterone have been used safely in females with steroid-dependent DBA and those in remission. Transient erythroblastopenia of childhood TEC is much more common than DBA but must be differentiated from DBA (Table 6.12) in order to avoid unnecessary corticosteroid use. It is, however, not a bone marrow failure syndrome. TEC has the following features: Pathophysiology The following clinical and laboratory observations have shed light on the basic mechanisms of the pathogenesis of TEC: • Viral: there is usually a history of a preceding nonspecific viral illness 1!2 months prior to TEC. • Erythropoietin levels: serum erythropoietin levels are high, in keeping with the degree of anemia. • CFU-E and BFU-E: both are decreased in 30!50% of patients, suggesting that the defect might be at the CFU-E and BFU-E levels. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes 115 • Serum inhibitors of erythropoiesis: IgG inhibitors of normal progenitor cells have been found in 60!80% of patients with TEC. • Cellular inhibitors of erythropoiesis: inhibitory mononuclear cells have been observed in approximately 25% of patients with TEC. (On the basis of these observations, it has been speculated that a nonspecific virus is cleared as the host develops IgG antibody. This IgG antibody probably recognizes shared viral and erythroid progenitor epitopes.) • Age: usually between 6 months and 4 years of age. With more children attending daycare programs, younger patients with TEC are being identified. • Sex: equal frequency in boys and girls. • Hematologic values: • Hemoglobin falls to levels ranging from 3 to 8 g/dL. • Reticulocyte count is 0%. • White blood cell (WBC) and platelet count are usually normal. Approximately 10% of patients may have significant neutropenia (ANC, ,1000/µL) and 5% have thrombocytopenia (platelet count, ,100,000/µL). Table 6.12 lists the hematologic characteristics. An analysis of 50 patients presenting with TEC at a single institution revealed a high incidence of neutropenia (64% with an ANC of less than 1500/µL). • Bone marrow: the absence of red cell precursors, except when the diagnostic bone marrow is performed during early recovery (prior to a reticulocytosis) when variable degrees of erythroid maturation may be observed Prognosis Spontaneous recovery occurs within weeks to months with the vast majority of patients recovering within 1 month. TEC rarely recurs. Treatment Transfusion of packed red blood cells if there is impending cardiovascular compromise. As recovery is usually prompt, restraint should be exercised with regard to red cell transfusions. Other instances of transient red cell failure may occur secondary to: • Drugs—chloramphenicol, penicillin, phenobarbital, and diphenylhydantoin. • Infections—viral infections (e.g., mumps, EBV, parvovirus B19, atypical pneumonia) and bacterial sepsis. • Malnutrition—kwashiorkor and other disorders. • Chronic hemolytic anemia—hereditary spherocytosis, sickle cell anemia, ß-thalassemia, and other congenital or acquired hemolytic anemias. The etiologic agent is often human parvovirus B19. Congenital dyserythropoietic anemia • The CDAs are a group of conditions characterized by ineffective erythropoiesis (intramedullary red cell death, anemia with reticulocytopenia, and usually marrow erythroid hyperplasia) and by specific morphologic abnormalities in the bone marrow consisting of increased numbers of morphologically abnormal red cell precursors. There are three major types of CDA (I!III) for which the mutated genes have been identified (IA-CDAN1, IB-C15orf41, II-SEC23B, III-KIF23) and other variants (IV, V, and others) that have been described, with genes identified in two (IV-KLF1 and V-GATA1) recently. Type II is the most common of the CDAs. Patients with CDA in the United States should be registered in the Congenital Dyserythropoietic Anemia Registry so more information can be collected about these rare anemias. Clinical manifestations CDA has the following diagnostic/clinical manifestations: • chronic mild congenital anemia (red cells have nonspecific abnormalities; basophilic stippling, occasional normoblasts) usually presenting in childhood, • reticulocyte response insufficient for the degree of anemia in the context of erythroid hyperplasia in the marrow, • marrow with abnormal erythroid morphology that can usually distinguish the three types of CDA, • normal granulopoiesis and thrombopoiesis, • chronic or intermittent mild jaundice, Lanzkowsky’s Manual of Pediatric Hematology and Oncology 116 6. Bone marrow failure • splenomegaly, • high plasma iron turnover rate and low iron utilization by erythrocyte (ineffective erythropoiesis) resulting in progressive iron overload (hemosiderosis), and • shortened red cell survival time. Other manifestations of CDA include: • CDA associated with atypical hereditary ovalocytosis • CDA of neonatal onset (with severe anemia at birth, hepatosplenomegaly, jaundice, syndactyly, and small for gestational age) • CDA associated with hydrops fetalis and hypoproteinemia Table 6.13 lists the clinical and laboratory features of CDA types I!III. Table 6.14 represents CDA types IV and V and others for those variants that have yet to be classified. These types share the common features of a congenital, perhaps hereditary, anemia with an inappropriately low reticulocyte count for the degree of anemia and ineffective marrow dyserythropoiesis. Table 6.15 lists the diagnostic tests necessary when CDA is suspected. TABLE 6.13 Clinical and laboratory features of congenital dyserythropoietic anemia, types I!III. Feature Type IA/1B Type II (HEMPAS) Type III (familial); type III (sporadic) Gene CDAN1/C15orf41 SEC23B KIF23; Unknown Gene locus 15q15.2/15q14 20p11.23 15q23 Inheritance AR AR AD/AR Clinical Hepatosplenomegaly; jaundice Hepatosplenomegaly; variable jaundice; gallstones; hemochromatosis Familial: rare splenomegaly; intravascular hemolysis Sporadic: Hepatosplenomegaly Red cell size Macrocytic Normo- or macrocytic Macrocytic Anemia Moderate!severe (usually presenting in neonatal period) Mild!moderate Mild!moderate Hemoglobin 8!12 g/dL Hemoglobin 6!7 g/dL Hemoglobin 7!8.5 g/dL Reticulocytes 1.5% 6 2% 2!4% Smear Marked anisocytosis and poikilocytosis; basophilic stippling Anisocytosis and poikilocytosis; basophilic stippling; “tear drop” cells; irregular contracted cells; occasionally, normoblasts Anisocytosis; poikilocytosis; basophilic stippling Marrow normoblasts Megaloblastoid: binucleated, 3!7%; with internuclear chromatin bridges Normoblastic: binucleated, 10!30% Megaloblastic: multinucleared, 10!50% some with up to 12 nuclei Marrow iron Scantly increased Increased Mildly increased Elevated Elevated Serum bilirubin and urine Elevated urobilinogen Other Rare skeletal defects Treatment Interferon-α; HSCT Splenectomy; HSCT Familial: retinal angioid streaks; propensity for monoclonal gammopathy and multiple myeloma Sporadic: skeletal defects HSCT possibly Abbreviations: HEMPAS, hereditary erythroblastic multinuclearity with positive acidified serum lysis test; HSCT, Hematopoietic stem cell transplantation. Modified from Alter, B.P., 2003. Inherited bone marrow failure syndromes. In: Nathan, D.G., Orkin S.H., Ginsburg D., Look A.T. (Eds.), Nathan & Oski’s hematology of infancy and childhood, sixth ed. Saunders, Philadelphia, PA.; Iolascon, A., Heimpel, H., Wahlin, A., Tamary, H., 2013. Congenital dyserythropoietic anemias: molecular insights and diagnostic approach. Blood 122 (13), 2162!2166. https://doi.org/10.1182/blood-2013-05-468223. Epub 2013 Aug 12. PMID: 23940284; PMCID: PMC3785118. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 117 Inherited bone marrow failure syndromes TABLE 6.14 Clinical and laboratory features of congenital dyserythropoetic anemia, types IV, V, and others. Type IV Type V Others Gene KLF1 GATA1 Unknown Gene locus 19p13.13 Inheritance AD XLR Unknown Clinical Mild-severe splenomegaly Spleen palpable in few cases; unconjugated hyperbilirubinemia Spleen not palpable Anemia Severe, transfusion dependent Variable Mild MCV Normal or mildly elevated Normal or mildly elevated Very high (119!125) without vitamin B12, folic acid, or other causes of megaloblastic anemia Erythropoiesis Normoblastic or mildly to moderately megaloblastic Normoblastic Grossly megaloblastic Nonspecific erythroblast dysplasia Present Absent or little Present Other Cardiac anomalies Bleeding tendency Abbreviation: MCV, Mean corpuscular volume. Adapted from Wickramasinghe, S.N., 1997. Dyserythropoiesis and congenital dyserythropoietic anaemias. Br. J. Haematol. 98 (4), 785!797. https://doi.org/10.1046/j.13652141.1997.2513065.x. PMID: 9326170; Risinger, M., Emberesh, M., Kalfa, T.A., 2019. Rare hereditary hemolytic anemias: diagnostic approach and considerations in management. Hematol. Oncol. Clin. North Am. 33 (3), 373!392. https://doi.org/10.1016/j.hoc.2019.01.002. Epub 2019 Mar 29. PMID: 31030808. TABLE 6.15 Diagnostic tests for congenital dyserythropoietic anemia.a Complete blood count, including MCV, RDW, and blood smear examination Absolute reticulocyte count Quantitative light and, if needed, electron microscope analysis of the bone marrow Serum vitamin B12 and red cell folate measurements Parvovirus B19 Serum bilirubin levels Hb electrophoresis: Hb A2, HbF assays Red cell enzyme assays (pyruvate kinase, G6PD) Sodium dodecyl sulfate polyacrylamide gel electrophoresis of red cell membranes Test for urinary hemosiderin Cytogenetic studies of bone marrow cells Mutation analysis for known CDA genes Studies of globin chain synthesis Studies of globin gene analysis a This list is not exhaustive nor is it required in all patients. These may be required when there is a need to rule out ß-thalassemia, thiamine-responsive sideroblastic anemia, megaloblastic (B12, folate) anemia, iron deficiency, and other causes of ineffective erythropoiesis. Abbreviations: CDA, Congenital dyserythropoietic anemia; G6PD, glucose 6-phosphate dehydrogenase; Hb, hemoglobin; MCV, mean corpuscular volume; RDW, red cell distribution width. Differential diagnosis • The diagnosis of CDA can only be made after the exclusion of other causes of congenital hemolytic anemias associated with ineffective erythropoiesis, such as thalassemia syndromes and hereditary sideroblastic anemias, as well as metabolic abnormalities. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 118 6. Bone marrow failure Treatment • Splenectomy performed in severely affected patients with CDA type I may result in moderate to marked improvement. • A chronic transfusion program with the use of deferoxamine, deferasirox, or deferiprone to ameliorate the effects of iron overload may be required to maintain an acceptable hemoglobin level. • Folic acid 1 mg/week should be administered. Iron therapy is contraindicated. • Vitamin E has been used in the treatment of CDA type II with an apparent improvement in red cell survival and a reduction in serum bilirubin and reticulocyte count. • Recombinant interferon-α has been used in CDA type I, resulting in increased hemoglobin level, decrease in MCV and red cell distribution width, reduction in serum bilirubin and lactic dehydrogenase levels, improvement in morphology of erythroblasts, and reduction in ineffective erythropoiesis. • Successful stem cell transplant has been performed in patients with CDA types I and II. Sideroblastic anemias (mitochondrial diseases with bone marrow failure syndromes) • The sideroblastic anemias are a heterogeneous group of disorders characterized by iron deposition in erythroblast mitochondria. These anemias can arise from the primary or secondary defects of mitochondria. They are often secondary to defects in the enzymes of the heme biosynthetic pathway [such as δ-aminolevulinic acid synthase (δ-ALA-S) deficiency]. Impaired production of heme resulting from defects in these enzymes results in mitochondrial iron accumulation, damage to the mitochondrial machinery, and formation of ring sideroblasts. Porphyrias, however, do not display sideroblastic anemia because they are characterized by defects in the cytoplasmic steps of heme synthesis. Laboratory findings • Anemia that may be normocytic, normochromic or microcytic, and hypochromic except in Pearson syndrome, which is characterized by macrocytic anemia probably due to fetal-like erythropoiesis. • Reticulocytopenia. • Ineffective erythropoiesis (i.e., erythroid hyperplasia in bone marrow despite anemia). • The presence of iron-loaded normoblasts demonstrated as ring sideroblasts (greater than 10% of erythroid precursors) by Perls Prussian blue stain (this stain serves as a surrogate technique for electron microscopy or energy-dispersive X-ray analysis used for the demonstration of iron-loaded mitochondria in normoblasts). In congenital sideroblastic anemias, iron rings are predominantly seen in late normoblasts (i.e., orthochromatic and polychromatophilic normoblasts), whereas they are seen in earlier erythroid cells (i.e., basophilic normoblasts) in the acquired form. • Mild-to-moderate hemolysis due to peripheral red blood cell destruction of unknown etiology. Table 6.16 shows a classification of the sideroblastic anemias. Pathophysiology Heme biosynthesis involves eight enzymes, four of which are cytoplasmic and four that are localized in the mitochondria. • δ-ALA-S: there are two distinct types of ALA-S. ALA-S1 (housekeeping form) occurs in nonerythroid cells and its gene maps on 3p21.2, and ALA-S2 (erythroid-specific form) occurs in erythroid cells and its gene maps on Xp11.21. Distinct aspects of heme synthesis regulation in nonerythroid and erythroid cells are related to the differences between these two ALA-S enzymes. In nonerythroid cells the synthesis and activity of ALA-S1 is subject to feedback inhibition by heme, thus making ALA-S1 the rate-limiting enzyme for the heme pathway. In erythroid cells, heme does not inhibit either the activity or the synthesis of ALAS2 but it does inhibit cellular iron uptake from transferrin without affecting its utilization for heme synthesis. • There are distinct features of iron and heme metabolism in erythroid and nonerythroid cells. These differences explain the large amount of heme production by erythroid cells compared to the low amount produced by nonerythroid cells. They also explain the mitochondrial deposition of iron in iron-loaded erythroid precursors. • Injury to the mitochondria or defects attributed to the mitochondrial pathways of heme synthesis result in sideroblastic anemias. Mitochondrial injury can result from: Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes TABLE 6.16 119 Classification of the sideroblastic anemias (SAs) hereditary/congenital SA. Isolated heritable • XLSA • Glutaredoxin 5 deficiency • Associated with erythropoietic protoporphyria • Presumed autosomal • Suggested maternal • Sporadic congenital Associated with genetic syndromes • XLSA/A • TRMA • MLASA • Mitochondrial cytopathy (Pearson syndrome) Acquired clonal SA • RARS/PSA • RARS-T • RCMD-RS Acquired reversible SA Associated with: • Alcoholism • Certain drugs (isoniazid, chloramphenicol) • Copper deficiency (idiopathic, zinc ingestion, copper chelation, nutritional, malabsorption) • Hypothermia Abbreviations: MLASA, Myopathy, lactic acidosis, and sideroblastic anemia; PSA, pure sideroblastic anemia; RARS, refractory anemia with ring sideroblasts; RARS-T, refractory anemia with ring sideroblasts and thrombocytosis; RCMD-RS, refractory cytopenia with multilineage dysplasia and ring sideroblasts; TRMA, thiamine-responsive megaloblastic anemia; XLSA, X-linked sideroblastic anemia; XLSA/A, X-linked sideroblastic anemia with ataxia. Modified from Bottomley, S.S., 2009. Sideroblastic anemia. In: Wintrobe’s Clinical Hematology, twelfth ed. Lippincott Williams & Wilkins, Philadelphia, PA. A Erythroid cell normoblast B Non-erythroid cell Damaged mitochondria, e.g., as a result of mitochondrial DNA damage, ALAS deficiency, antibiotic toxins Damaged mitochondria due to mitochondrial DNA mutation ↓ Increased accumulation of non-utilized iron ↓ Formation of hydroxyl radical through Fenton reaction ↓ Decreased ATP production ↓ Damage to cellular organelles and cell membranes ↓ Cross-linking of OH radical to DNA, proteins, and lipids ↓ Damage to cellular organelles and cell membranes FIGURE 6.3 Simplified view of pathophysiologic consequences of mitochondrial diseases. • Defective heme synthesis and the accumulation of iron, especially in erythroid precursors. This iron accumulation causes oxidative damage to the mitochondrial machinery through a Fenton reaction (i.e., the formation of a hydroxyl radical catalyzed by iron and reactive oxygen species damaging mitochondrial DNA by crosslinking DNA strands or by promoting the formation of DNA protein crosslinks). • Congenital deletions of mitochondrial DNA. As a result of mitochondrial damage, there is increased deposition of iron in heme-containing cells (e.g., erythroid cells). Additionally, there is decreased oxidative phosphorylation and decreased adenosine triphosphate synthesis in many organs as observed in Pearson syndrome. Fig. 6.3 shows a simplified view of the Lanzkowsky’s Manual of Pediatric Hematology and Oncology 120 6. Bone marrow failure pathophysiologic relationship of various mitochondrial diseases in the context of sideroblastic anemias, bone marrow failure, and/or mitochondrial cytopathies. Treatment • Oral pyridoxine is used in some patients with either congenital or acquired sideroblastic anemia with partial response. • The removal of the toxin/drug responsible for causing sideroblastic anemia may be effective. • Stem cell transplantation is employed for the treatment of sideroblastic anemia secondary to MDS. Treatment of Pearson syndrome is largely palliative and consists of the following: • Correction of the metabolic acidosis (e.g., avoidance of fasting, administration of thiamine, riboflavin, carnitine, and coenzyme Q to bypass deleted respiratory enzymes). • The removal of reactive oxygen radical by the use of ascorbate, vitamin E, or lipoic acid. The efficacy of these therapies is not clear at this time. • Anemia is treated with red cell transfusions. G-CSF may be used to support clinically significant neutropenia. If patients do not succumb to metabolic acidosis and organ failure, the bone marrow will often improve within the first decade of life. • HSCT has been performed and although engraftment occurred, the patient succumbed to nonhematopoietic manifestations of the disease. Severe congenital neutropenia and Kostmann syndrome Severe chronic neutropenias include a heterogeneous group of disorders with different patterns of inheritance. SCN is a rare form of autosomal dominant chronic neutropenia presenting with severe bacterial infections within the first few months after birth. KS is reserved for a form of SCN with an autosomal recessive pattern of inheritance. Patients with severe chronic neutropenia should be registered with the Severe Chronic Neutropenia International Registry. This registry collects and maintains long-term outcome data as well as provides resources for physicians, patients, and families. Epidemiology • In this group of patients with SCN, B60% have diverse mutations in the neutrophil elastase gene (ELANE). These mutations affect only one allele. The majority of patients present with a sporadic pattern, as, in the past, autosomal dominant inheritance was relatively more lethal and the patients did not live on to adulthood. In some patients, there may be germline mosaicism. Incidence • The incidence of SCN is two per million population. Pathogenesis and genetics • In vitro bone marrow studies show a reduced number of granulocyte!macrophage colonies in SCN patients. There is also a reduced number of CD341 /Kit1 /G-CSFR1 myeloid progenitor cells in the bone marrow. • It has been hypothesized that mutations of ELANE in SCN result in a high rate of premature apoptosis in neutrophil precursors, which results in decreased myelopoiesis. Neutrophil elastase is a serine protease localized in the granules of neutrophils and monocytes. A mutant enzyme has a dominant-negative effect on the normal wild-type elastase. This explains the defective proteolysis in the SCN neutrophils even though half of the normal amount of the elastase is present in the neutrophils of these patients. • The gene responsible for the autosomal recessive form of SCN in the original cases described by Kostmann has been identified as the HAX1 gene located on chromosome 1. HAX1 is an inhibitor of apoptosis. In its absence, apoptosis proceeds unchecked. G-CSF can overcome this deficiency by activating other antiapoptotic pathways. The mutated genes for other rare SCN syndromes are listed in Table 6.2. Some of these syndromes have unique features: • Glucose 6-phosphatase, catalytic subunit (G6PC3): cardiac and urogenital malformations, neurologic findings Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited bone marrow failure syndromes 121 • GFI1: may be milder but clinically resembles SCN seen with ELANE mutations as GFI1 is a repressor of ELANE • WAS mutation: X-linked • JAGN1: autosomal recessive • MonoMac syndrome (GATA2): is inherited as an autosomal dominant with severe persistent monocytopenia • WHIM syndrome (CXCR4): Warts, hypogammaglobulinemia, infections, myelokathexis with extensive HPV infection is inherited as an autosomal dominant Clinical manifestations and laboratory Investigations • During the first year of life, severe infections with omphalitis, pneumonia, pneumonitis, skin abscesses, and liver abscesses occur commonly with positive cultures for staphylococci, streptococci, Pseudomonas, Peptostreptococcus, and fungi. Splenomegaly may be present. Other manifestations include: • Blood counts reveal a normal WBC with an ANC less than 200/µL and a compensatory eosinophilia and monocytosis. Mild anemia and thrombocytosis may be present. • Bone marrow examination shows a maturation arrest of myelopoiesis at the promyelocyte or myelocyte stage with marked paucity of mature neutrophils. There is an increase in monocytes, eosinophils, macrophages, and plasma cells. Treatment • G-CSF: prior to G-CSF, the patients succumbed to infections before 2 years of age. With the use of G-CSF, patients have been able to survive well into adulthood. The initial dose of G-CSF employed is 5 µg/kg/day. The response occurs 7!14 days from the start of treatment. The goal of therapy is to achieve an ANC of approximately 1000!1500/µL and maintain the patient free of infections. More than 95% of patients with SCN will respond to G-CSF. After beginning G-CSF therapy the dose should be adjusted up or down at 1- to 2-week intervals until the lowest effective dose is reached. • Complications associated with the use of G-CSF include bone pain, splenomegaly, hepatomegaly, thrombocytopenia, osteopenia/osteoporosis, Henoch!Schonlein purpura type of immune complex!induced vasculitis of the skin, and/or glomerulonephritis. • Baseline bone marrow cytogenetics should be obtained prior to G-CSF therapy. Initial cytogenetic studies at diagnosis are usually normal. However, during the course of the disease, clonal abnormalities may emerge, 50% of which are monosomy 7. Since 12% of patients with SCN develop MDS and/or AML, it is important to perform periodic bone marrow examinations for morphology and cytogenetic studies in the follow-up of these patients. Patients who require higher doses of G-CSF (more than 8 µg/kg/day) are at higher risk to develop MDS/AML than those who are more G-CSF responsive (40 vs 11% after 10 years of therapy). • The G-CSF receptor is normal in patients with SCN. However, patients with SCN are predisposed to develop acquired (somatic) mutations of the cytoplasmic domain of the G-CSF receptor. There is a good correlation between the development of AML/MDS and the acquisition of G-CSF receptor mutations in these patients. The time interval between these two events varies considerably. • HSCT: the following are the indications for HSCT: • Patients who require greater than B8 µg/kg/day of G-CSF are statistically much more likely to succumb to infection or leukemia. • Refractoriness to G-CSF treatment. • Emergence of MDS/AML. • HSCT can be considered treatment for all patients with SCN who have an HLA-matched sibling donor available, with matched unrelated transplant reserved for high-risk patients. Prognosis The leading causes of death in SCN are infection and MDS/AML. SCN and cyclic neutropenia are discussed in Chapter 11, Disorders of White Blood Cells. Reticular dysgenesis Reticular dysgenesis is a disorder of stem cells in which maturation of both myeloid and lymphoid lineages is defective. Platelet and red cell production are normal. Affected individuals have severe neutropenia and Lanzkowsky’s Manual of Pediatric Hematology and Oncology 122 6. Bone marrow failure moderate-to-severe lymphopenia. In addition, there is the absence of peripheral lymphoid tissues, Peyer’s patches, tonsils, and splenic follicles. The mortality rate is high from infection at an early age. Treatment HSCT can be curative. Shwachman Diamond syndrome SDS is an IBMFS presenting with neutropenia, in the majority of patients, and pancreatic insufficiency. Patients occasionally may develop macrocytic anemia as well. The majority of these patients (B90%) have biallelic mutations in SBDS. Patients in North America with SDS should be registered with the Shwachman Diamond Syndrome Registry. This registry collects and maintains long-term outcome data as well as provides resources for physicians, patients, and families. SDS is discussed in more detail in Chapter 11, Disorders of White Blood Cells. SAMD9/9L-related syndromes Pancytopenia with potential progression to MDS/AML has been seen in various syndromes. Not all the symptoms are evident in each patient however, and mutations have been identified in SAMD9 and SAMD9L in patients with bone marrow failure without the remainder of the syndromic findings. • In 2015 gene mutations in SAMD9L were found to be the cause of ataxia!pancytopenia syndrome, with autosomal dominant inheritance. These patients are noted to have transient cytopenia, occasionally pancytopenia, with a predisposition to developing MDS/AML with monosomy 7, along with neurologic symptoms, including ataxia, nystagmus, and/or cerebellar atrophy. They may also have bacterial infections and pulmonary alveolar proteinosis. The diagnosis of MDS/AML is often in very young patients. Treatment is variable as reversion of the SAMD9L mutation has been noted in some patients with improvement in cytopenias by a copy neutral loss of heterozygosity of chromosome 7q leading to loss of the mutant allele. • SAMD9 is a gene contiguous to SAMD9L and was found in 2016 to be mutated in patients with MIRAGE syndrome—MDS, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes, Enteropathy. This is a heterozygous mutation but most patients have been found with de novo mutations, possibly due to the early death seen in these children. These patients may also present with anemia, thrombocytopenia, and progress to MDS/AML with monosomy 7. Genetic reversion has also been seen in these patients. • Genetic testing for SAMD9 and SAMD9L may need to be done in patients from blood and nonhematopoietic tissue DNA due to the decreased variant allele frequency possible from reversion in the hematopoietic tissue DNA. • The only definitive treatment for the cytopenia/pancytopenia in these cases is stem cell transplantation but the timing is controversial due to the possibility of genetic reversion. Once MDS/AML has been diagnosed, HSCT must be done but the comorbidities due to organ involvement may make success difficult. Further reading and references Albers, C.A., Newbury-Ecob, R., Ouwehand, W.H., Ghevaert, C., 2013. New insights into the genetic basis of TAR (thrombocytopenia-absent radii) syndrome. Curr. Opin. Genet. Dev. 23 (3), 316!323. Available from: https://doi.org/10.1016/j.gde.2013.02.015. Epub 2013 Apr 17. PMID: 23602329. Alter, B.P., 2003. Inherited bone marrow failure syndromes. In: Nathan, D.G., Orkin, S.H., Ginsburg, D., Look, A.T. (Eds.), Nathan & Oski’s Hematology of Infancy and Childhood, sixth ed. Saunders, Philadelphia, PA. Alter, B.P., Giri, N., Savage, S.A., Rosenberg, P.S., 2018. Cancer in the National Cancer Institute inherited bone marrow failure syndrome cohort after fifteen years of follow-up. Haematologica. 103 (1), 30!39. Available from: https://doi.org/10.3324/haematol.2017.178111. Epub 2017 Oct 19. PMID: 29051281; PMCID: PMC5777188. Bacigalupo, A., Socié, G., Hamladji, R.M., et al., 2015. Aplastic anemia working party of the European Group for blood marrow transplantation. Current outcome of HLA identical sibling versus unrelated donor transplants in severe aplastic anemia: an EBMT analysis. Haematologica 100 (5), 696!702. Available from: https://doi.org/10.3324/haematol.2014.115345. Epub 2015 Jan 23. PMID: 25616576; PMCID: PMC4420220. Bluteau, O., Sebert, M., Leblanc, T., et al., 2018. A landscape of germ line mutations in a cohort of inherited bone marrow failure patients. Blood. 131 (7), 717!732. Available from: https://doi.org/10.1182/blood-2017-09-806489. Epub 2017 Nov 16. PMID: 29146883. Bottomley, S.S., 2009. Sideroblastic anemia, Wintrobe’s Clinical Hematology, twelfth ed. Lippincott Williams & Wilkins, Philadelphia, PA. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 123 Bottomley, S.S., Fleming, M.D., 2014. Sideroblastic anemia: diagnosis and management. Hematol. Oncol. Clin. North Am. 28 (4), 653!670, v. Available from: https://doi.org/10.1016/j.hoc.2014.04.008. Epub 2014 Jun 2. PMID: 25064706. Cherrick, I., Karayalcin, G., Lanzkowsky, P., 1994. Transient erythroblastopenia of childhood. Prospective study of fifty patients. Am. J. Pediatr. Hematol. Oncol. 16 (4), 320!324. PMID: 7978049. Davidsson, J., Puschmann, A., Tedgård, U., Bryder, D., Nilsson, L., Cammenga, J., 2018. SAMD9 and SAMD9L in inherited predisposition to ataxia, pancytopenia, and myeloid malignancies. Leukemia 32 (5), 1106!1115. Available from: https://doi.org/10.1038/s41375-018-0074-4. Epub 2018 Feb 25. PMID: 29535429; PMCID: PMC5940635. Desmond, R., Townsley, D.M., Dumitriu, B., et al., 2014. Eltrombopag restores trilineage hematopoiesis in refractory severe aplastic anemia that can be sustained on discontinuation of drug. Blood 123 (12), 1818!1825. Available from: https://doi.org/10.1182/blood-2013-10534743. Epub 2013 Dec 17. PMID: 24345753; PMCID: PMC3962161. Dufour, C., 2017. How I manage patients with Fanconi anaemia. Br. J. Haematol. 178 (1), 32!47. Available from: https://doi.org/10.1111/ bjh.14615. Epub 2017 May 5. PMID: 28474441. Frohnmayer, D., Frohnmayer, L., Guinan, E., Kennedy, T., Larsen, K. (Eds.), 2014. Fanconi Anemia: Guidelines for Diagnosis and Management. fourth ed. Fanconi Anemia Research Fund, Inc., Eugene, OR. Fioredda, F., Iacobelli, S., Korthof, E.T., et al., 2018. Outcome of haematopoietic stem cell transplantation in dyskeratosis congenita. Br. J. Haematol. 183 (1), 110!118. Available from: https://doi.org/10.1111/bjh.15495. Epub 2018 Jul 9. PMID: 29984823. Fujiwara, T., Harigae, H., 2013. Pathophysiology and genetic mutations in congenital sideroblastic anemia. Pediatr. Int. 55 (6), 675!679. Available from: https://doi.org/10.1111/ped.12217. PMID: 24003969. Germeshausen, M., Ancliff, P., Estrada, J., et al., 2018. MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia. Blood Adv. 2 (6), 586!596. Available from: https://doi.org/10.1182/bloodadvances.2018016501. PMID: 29540340; PMCID: PMC5873238. Hall, J.G., Levin, J., Kuhn, J.P., Ottenheimer, E.J., van Berkum, K.A., McKusick, V.A., 1969. Thrombocytopenia with absent radius (TAR). Medicine (Baltimore) 48 (6), 411!439. Available from: https://doi.org/10.1097/00005792-196948060-00001. PMID: 4951233. Iolascon, A., Heimpel, H., Wahlin, A., Tamary, H., 2013. Congenital dyserythropoietic anemias: molecular insights and diagnostic approach. Blood 122 (13), 2162!2166. Available from: https://doi.org/10.1182/blood-2013-05-468223. Epub 2013 Aug 12. PMID: 23940284; PMCID: PMC3785118. Niewisch, M.R., Savage, S.A., 2019. An update on the biology and management of dyskeratosis congenita and related telomere biology disorders. Expert Rev. Hematol. 12 (12), 1037!1052. Available from: https://doi.org/10.1080/17474086.2019.1662720. Epub 2019 Sep 10. PMID: 31478401. Rı́o, P., Navarro, S., Wang, W., et al., 2019. Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia. Nat. Med. 25 (9), 1396!1401. Available from: https://doi.org/10.1038/s41591-019-0550-z. Epub 2019 Sep 9. PMID: 31501599. Risinger, M., Emberesh, M., Kalfa, T.A., 2019. Rare hereditary hemolytic anemias: diagnostic approach and considerations in management. Hematol. Oncol. Clin. North. Am. 33 (3), 373!392. Available from: https://doi.org/10.1016/j.hoc.2019.01.002. Epub 2019 Mar 29. PMID: 31030808. Savage, S.A., Walsh, M.F., 2018. Myelodysplastic syndrome, acute myeloid leukemia, and cancer surveillance in Fanconi anemia. Hematol. Oncol. Clin. North Am. 32 (4), 657!668. Available from: https://doi.org/10.1016/j.hoc.2018.04.002. PMID: 30047418; PMCID: PMC6071325. Scheinberg, P., 2020. Novel therapeutic choices in immune aplastic anemia. F1000Res 9: F1000: Faculty Rev-1118. PMID: 32953089; PMCID: PMC7484718. https://doi.org/10.12688/f1000research.22214.1. Townsley, D.M., Scheinberg, P., Winkler, T., et al., 2017. Eltrombopag added to standard immunosuppression for aplastic anemia. N. Engl. J. Med. 376 (16), 1540!1550. Available from: https://doi.org/10.1056/NEJMoa1613878. PMID: 28423296; PMCID: PMC5548296. Vlachos, A., Muir, E., 2010. How I treat Diamond-Blackfan anemia. Blood 116 (19), 3715!3723. Available from: https://doi.org/10.1182/ blood-2010-02-251090. Epub 2010 Jul 22. PMID: 20651069; PMCID: PMC2981532. Vlachos, A., Rosenberg, P.S., Atsidaftos, E., et al., 2018. Increased risk of colon cancer and osteogenic sarcoma in Diamond-Blackfan anemia. Blood 132 (20), 2205!2208. Available from: https://doi.org/10.1182/blood-2018-05-848937. Epub 2018 Sep 28. PMID: 30266775; PMCID: PMC6238192. Walkovich, K., Connelly, J.A., 2019. Congenital neutropenia and rare functional phagocyte disorders in children. Hematol. Oncol. Clin. North Am. 33 (3), 533!551. Available from: https://doi.org/10.1016/j.hoc.2019.01.004. Epub 2019 Apr 1. PMID: 31030818. Wickramasinghe, S.N., 1997. Dyserythropoiesis and congenital dyserythropoietic anaemias. Br. J. Haematol. 98 (4), 785!797. Available from: https://doi.org/10.1046/j.1365-2141.1997.2513065.x. PMID: 9326170. Williams, D.A., Bennett, C., Bertuch, A., et al., 2014. Diagnosis and treatment of pediatric acquired aplastic anemia (AAA): an initial survey of the North American Pediatric Aplastic Anemia Consortium (NAPAAC). Pediatr. Blood Cancer. 61 (5), 869!874. Available from: https:// doi.org/10.1002/pbc.24875. Epub 2013 Nov 27. PMID: 24285674; PMCID: PMC4280184. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 7 General considerations of hemolytic diseases, red cell membrane, and enzyme defects Lionel Blanc1,2,3 and Lawrence C. Wolfe1,2 1 Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States 2Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States 3Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, NY, United States Hemolysis is a reduction in the normal red cell survival of 120 days. A general approach to diagnosing hemolytic anemia in the context of other causes of anemia is described in Chapter 3, Classification and Diagnosis of Anemia in Children and Neonates. It may result from corpuscular abnormalities such as membrane, cytoskeleton, enzyme, or hemoglobin (Hb) defects, or from extracorpuscular abnormalities involving immune or nonimmune mechanisms (see Chapter 8: Extracorpuscular Hemolytic Anemia). Clinical features of hemolytic disease The following clinical features suggest a hemolytic process in a child with anemia: 1. history of anemia, jaundice, or gallstones in family 2. persistent or recurrent anemia associated with reticulocytosis 3. anemia unresponsive to hematinics 4. intermittent bouts or persistent indirect hyperbilirubinemia/jaundice 5. splenomegaly 6. hemoglobinuria 7. presence of multiple gallstones 8. chronic leg ulcers 9. development of anemia or hemoglobinuria after exposure to certain drugs 10. cyanosis without cardiorespiratory distress 11. polycythemia (2,3-diphosphoglycerate mutase deficiency) 12. dark urine due to dipyroluria (unstable hemoglobins, thalassemia, and ineffective erythropoiesis) 13. ethnic factors: a. Incidence of sickle gene carrier in the African-American population (8%) b. High incidence of thalassemia trait in people of Mediterranean ancestry c. High incidence of glucose-6-phosphate dehydrogenase (G6PD) deficiency among those with ethnic origins arising in territories near the Mediterranean Sea, Africa, and Southeast Asia d. High incidence of hereditary ovalocytosis in Southeast Asian populations 14. age factors: anemia and jaundice in a Rh-positive infant born to a mother who is Rh negative or a group A or group B infant born to a group O mother (setting for a hemolytic anemia). Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00030-1 125 © 2022 Elsevier Inc. All rights reserved. 126 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects Laboratory findings Laboratory findings of hemolytic anemia consist of: • evidence of accelerated hemoglobin catabolism due to reduced red cell survival and • evidence of increased erythropoiesis. Accelerated hemoglobin catabolism Accelerated hemoglobin catabolism varies with the type of hemolysis as follows: • extravascular hemoglobin catabolism (see Fig. 7.1) • intravascular hemoglobin catabolism (see Fig. 7.2). The two may not be easily distinguished if the cause for hemolysis is not obvious, hence the long lists of markers of testing indicated next. The presence of hemoglobinuria and hemosiderinuria and the absence of haptoglobin are the major markers of intravascular hemolysis in practice. Markers of extravascular hemolysis 1. Increased unconjugated bilirubin 2. Increased lactic acid dehydrogenase in serum 3. Decreased plasma haptoglobin (normal level, 36!195 mg/dL) 4. Increased fecal and urinary urobilinogen 5. Increased rate of carbon monoxide production. Reticuloendothelial cell (RE cell) Phagocytosis of RBC by RE cell and disruption of its membrane resulting in release of hemoglobin RBC Breakdown of hemoglobin by lysosymal enzymes to heme and globin Hemoglobin Globin Heme Heme oxygenase Amino acids Protein pool Biliverdin Biliverdin reductase Bilirubin Carbon monoxide Lungs Fe++ Bilirubin glucuronide (conjugated bilirubin) Transferrin Unconjugated bilirubin in circulating blood Enterohepatic circulation of bilirubin Deconjugation of bilirubin Urobilinogen in blood: to kidney and enterohepatic circulation Urobilinogen Urinary urobilinogen FIGURE 7.1 Extravascular hemoglobin catabolism following extravascular hemolysis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 127 Laboratory findings Intravascular disruption of red blood cell (RBC) membrane and release of hemoglobin in circulating blood Hemoglobin–haptoglobin Hemopexin–heme Haptoglobin Hemopexin Free hemoglobin Heme O2 Methemoglobin Albumin Methemalbumin Hemoglobinuria Oxyhemoglobin Methemoglobin Urobilinogen FIGURE 7.2 Intravascular hemoglobin catabolism following intravascular hemolysis. Hemoglobin!haptoglobin, hemopexin!heme, and methemalbumin are cleared by hepatocytes. Heme is converted to iron and bilirubin. The common pathway for both extravascular and intravascular hemolysis is the conjugation of bilirubin (bilirubin glucuronide) by the hepatocytes, its excretion in bile and ultimately formation of urobilinogen by the bacteria in the gut. Part of urobilinogen enters the enterohepatic circulation and part is excreted by the kidney in urine, and the remainder of urobilinogen is excreted in stool. Markers of intravascular hemolysis 1. Increased unconjugated bilirubin (although often less than extravascular hemolysis as urinary losses leave less hemoglobin to be scavenged and processed to bilirubin) 2. Increased lactic acid dehydrogenase in serum 3. Hemoglobinuria (Fig. 7.3 lists the causes of hemoglobinuria) 4. Low or absent plasma haptoglobin 5. Hemosiderinuria (due to sloughing of iron-laden tubular cells into urine) 6. Raised plasma hemoglobin level (normal value ,1-mg hemoglobin/dL plasma, visibly red plasma contains greater than 50-mg hemoglobin/dL plasma) 7. Raised plasma methemalbumin (albumin bound to heme; unlike haptoglobin, albumin does not bind intact hemoglobin) 8. Raised plasma met hemoglobin (oxidized free plasma hemoglobin) and raised levels of hemopexin!heme complex in plasma Increased erythropoiesis Erythropoiesis increases in response to a reduction in hemoglobin and is manifested by: 1. Reticulocytosis: frequently up to 10!20%; rarely, as high as 80% 2. Increased mean corpuscular volume (MCV) due to the presence of reticulocytosis Lanzkowsky’s Manual of Pediatric Hematology and Oncology 128 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects I. Acute A. Mismatched blood transfusions B. Warm antibody-induced autoimmune hemolytic anemia C. Drugs and chemicals C.1. Regularly causing hemolytic anemia C.1.1. Drugs: phenylhydrazine, sulfones (dapsone), phenacetin, acetanilid (large doses) C.1.2. Chemicals: nitrobenzene, lead, inadvertent infusion of water C.1.3. Toxins: snake and spider bites II. Chronic A. Paroxysmal cold hemoglobinuria; syphilis; idiopathic B. Paroxysmal nocturnal hemoglobinuria C. March hemoglobinuria D. Cold agglutinin hemolysis C.2. Occasionally causing hemolytic anemia C.2.1. Associated with G6PD deficiency: antimalarials (primaquine, chloroquine), antipyretics (aspirin, phenacetin), sulfonamides (Gantrisin, lederkyn), nitrofurans (Furadantin, Furacin), miscellaneous [naphthalene, vitamin K, British anti-Lewisite (BAL), favism] C.2.2. Associated with Hb Zürich: sulfonamides C.2.3. Hypersensitivity: quinine, quinidine, paraaminosalicylic acid (PAS), phenacetin D. Infections D.1. Bacterial: Clostridium perfringens, Bartonella bacilliformis (Oroya fever) D.2. Parasitic: Malaria E. Burns F. Mechanical (e.g., prosthetic valves) FIGURE 7.3 Causes of hemoglobinuria. Abbreviation: G-6-P, Glucose-6-phosphate. 3. Increased red cell distribution width (RDW) as the hemoglobin level falls 4. Normoblasts in the peripheral blood 5. Specific morphologic abnormalities: sickle cells, target cells, basophilic stippling, irregularly contracted cells or fragments (schistocytes), elliptocytes, acanthocytes, and spherocytes 6. Erythroid hyperplasia of the bone marrow: erythroid:myeloid ratio in the marrow increasing from 1:5 to 1:1 7. Expansion of marrow space in chronic hemolysis resulting in: a. Prominence of the frontal bones and broadened cheekbones b. Widened intratrabecular spaces with hair-on-end appearance of skull radiographs c. Biconcave vertebrae with fish-mouth intervertebral spaces 8. Decreased red cell survival demonstrated by 51Cr red cell labeling 9. Red cell creatine levels increased Fig. 7.4 lists the tests used to establish the cause of the hemolytic anemia. Membrane defects Fig. 7.5 lists causes of hemolytic anemia due to corpuscular defects. Structure of the red cell membrane Spectrin, the major red cell membrane protein, is largely responsible for maintaining the normal red cell shape and overall morphology. It is composed of two large subunits, α- and β-spectrin, which are encoded by separate genes and are structurally distinct. Spectrin is integrated vertically into the lipid bilayer of the red cell membrane through the intercession of smaller proteins (ankyrin, 4.2) to integral membrane-spanning proteins (band 3, Rh antigen, and glycophorin A). These vertical interactions maintain red cell membrane cohesion. Spectrin associates with itself head to head, while the tail associates with actin and other members Lanzkowsky’s Manual of Pediatric Hematology and Oncology 129 Membrane defects I. Corpuscular defects A. Membrane defects A.1. Blood smear a spherocytes, ovalocytes, pyknocytes, stomatocytes A.2. Osmotic fragility a (fresh and incubated) A.3. Eosin-5-maleimide dye staining with flow cytometry b A.4. Ektacytometry A.5. Autohemolysis a A.6. Cation permeability studies A.7. Membrane phospholipid composition A.8. Scanning electron microscopy B. Hemoglobin defects B.1. Blood smear: sickle cells, target cells (HbC) a B.2. Sickling test a B.3. Hemoglobin electrophoresis a B.4. Quantitative fetal hemoglobin determination a B.5. Kleihauer–Betke smear a B.6. Heat stability test for unstable hemoglobin B.7. Oxygen dissociation curves B.8. Rates of synthesis of polypeptide chain production B.9. Fingerprinting of hemoglobin II. Extracorpuscular defects Direct antiglobulin test: IgG-γ, C3 (complement), broad-spectrum (both IgG-γ and C3) a III. Serological testing for unusual immune defects IgA-induced hemolysis, DAT negative hemolytic anemia Donath–Landsteiner test a Flow cytometric analysis of red cells with monoclonal antibodies to GP1linked surface antigens (for PNH) a C. Enzyme defects C.1. Heinz-body preparation a C.2. Osmotic fragility a C.3. Autohemolysis test a C.4. Screening test for enzyme deficiencies a C.5. Specific enzyme assays a FIGURE 7.4 Tests used to establish a specific cause of hemolytic anemia. aTests commonly employed and most useful in establishing a diagnosis; bTest available in reference laboratories. Test of choice for hereditary spherocytosis. Abbreviations: DAT, Direct antiglobulin test; PNH, paroxysmal nocturnal hemoglobinuria. of the junctional complex (4.1R, adducin). These horizontal interactions maintain membrane stability. Fig. 7.6 summarizes the structure of the normal red cell membrane, highlighting the vertical and horizontal interactions. The red cell membrane is semipermeable and must maintain its volume in order for the erythrocyte to negotiate the narrower spaces in the circulatory system. Red cell volume is maintained by a number of passive, gradient-driven cation and anion channels as well as active transporters. Red cell membrane disorders Hereditary spherocytosis (HS), elliptocytosis, stomatocytosis, acanthocytosis, xerocytosis, and pyropoikilocytosis can be diagnosed on the basis of their characteristic morphologic abnormalities. Alterations in the quality and/or quantity of the proteins involved in the maintenance of the unique properties of the red cell membrane (deformability and stability) lead to the red cell membrane disorders: • HS: perturbations in the vertical linkage • Hereditary elliptocytosis (HE): perturbations in the horizontal linkage • Stomatocytosis: perturbations in the function of the ion transporters. Note: Enzyme defects and many hemoglobinopathies have nonspecific morphologic abnormalities related to secondary effects on red cell membrane proteins and pumps [e.g., adenosine triphosphate (ATP) depletion]. Hereditary spherocytosis Genetics • Autosomal dominant inheritance (75% of cases). The severity of anemia and the degree of spherocytosis may not be uniform within an affected family Lanzkowsky’s Manual of Pediatric Hematology and Oncology 130 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects Primary membrane defects with specific morphologic abnormalities I. Membrane defects Secondary membrane defects Hereditary Hereditary stomatocytosis elliptocytosis / pyropoikilocytosis Hereditary spherocytosis Abetalipoproteinemia / neuroacanthocytosis Over-hydrated stomatocytosis (increased RBC volume) Dehydrated stomatocytosis/xerocytosis/desicytosis (decreased RBC volume) Rh null Normal osmotic fragility/normal volume Phosphofructokinase Pyruvate kinase Hexokinase Glucose II. Enzyme defects phosphate isomerase Energy potential defects Triose phosphate isomerase 2,3-Diphosphoglyceromutase Phosphoglycerate Reduction potential defects kinase (polycythemia, no hemolysis) 6-Phosphogluconate dehydrogenase (6PGD) Glutathione synthetase Glutathione reductase G6PD Congenital erythropoietic porphyria Globin Adenosine Adenylate triphosphatas kinase e deficiency deficiency Adenosine deaminase Pyrimidine 5’excess nucleotidase (P5N) deficiency 2,3-Glutamylcysteine synthetase IV. Congenital dyserythropoietic anemias (see Chapter 10) III. Hemoglobin defects (see Chapter 10) Heme Abnormalities of erythrocyte nucleotide metabolism Qualitative Hemoglobinopathies (e.g., HbS, C, M) Type I Type II Type III Type IV Quantitative (α- and βthalassemia) Ver!cal linkage FIGURE 7.5 Causes of hemolytic anemia due to corpuscular defects. WHO classification of G6PD variant: Class I variant: Chronic hemolysis due to severe G6PD deficiency, for example, G6PD deficiency Harilaou. Class II variant: intermittent hemolysis in spite of severe G6PD deficiency, for example, G6PD Mediterranean. Class III variant: intermittent hemolysis associated usually with drugs/infections and moderate G6PD deficiency, for example, G6PDA variant. Class IV variant: no hemolysis, no G6PD deficiency, for example, normal G6PD (B variant). Abbreviations: ATPase, Adenosine triphosphatase; G6PD, glucose-6-phosphate dehydrogenase; RBC, red blood cell; WHO, World Health Organization. Horizontal linkage FIGURE 7.6 Schematic representation of the normal red cell membrane. The vertical and horizontal interactions leading, respectively, to HS and HE are highlighted. Abbreviations: HS, Hereditary spherocytosis; HE, hereditary elliptocytosis. • No family history in 25% of cases. Some show minor laboratory abnormalities suggesting a carrier (recessive) state. Others are due to a de novo mutation • Most common in people of northern European heritage, with an incidence of 1 in 5000. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Membrane defects 131 Pathogenesis The primary defect is membrane instability due to dysfunction or deficiency of a red cell skeletal or membrane protein, including: • Ankyrin mutations account for 50!60% of HS. In many patients, both spectrin and ankyrin proteins are deficient. Mutations of ankyrin occur in both dominant and recessive forms of HS. The clinical course varies from mild to severe. Red cells are typically spherocytes • α-Spectrin mutations occur in recessive HS and account for less than 5% of HS. The clinical course is severe. Contracted cells, poikilocytes, and spherocytes are seen • β-Spectrin mutations occur in dominant HS and account for 15!20% of HS. The clinical course is mild to moderate. Acanthocytes, spherocytic elliptocytes, and spherocytes are seen • Protein 4.2 mutations occur in the recessive form of HS and account for less than 5% of HS. The clinical course is mild to moderate. Spherocytes, acanthocytes, and ovalocytes are seen • Band 3 mutations occur in the dominant form of HS and account for 15!20% of HS. The clinical course is mild to moderate. Spherocytes are occasionally mushroom-shaped or pincered cells. The deficiency of these proteins in HS results in a vertical defect, which causes progressive loss of membrane lipid and surface area. The loss of surface area results in characteristic microspherocytic morphology of HS red cells. The sequelae are as follows: • Depletion of membrane lipid • Decrease in membrane surface area relative to volume, resulting in a decrease in surface-area-to-volume ratio • Tendency to spherocytosis • Influx and efflux of sodium increased; cell dehydration • Sequestration of red cells in the spleen due to reduced erythrocyte deformability • Rapid ATP utilization and increased glycolysis leading to increased loss of surface area under ATP-depleted conditions. This leads to the observation of splenic conditioning where the changes in glucose utilization, as well as cell volume control, are dramatically exacerbated with each circulatory passage through the spleen. • Premature red cell destruction. Hematologic features 1. Anemia: mild to moderate when there is a compensated hemolytic anemia. In erythroblastopenic (aplastic or hypoplastic) crisis, hemoglobin may drop to 2!3 g/dL 2. MCV usually decreased, mean corpuscular hemoglobin concentration (MCHC) raised, and RDW elevated. a. The MCHC is raised in HS, hereditary xerocytosis, hereditary pyropoikilocytosis (HPP), pyruvate kinase (PK) deficiency (which has acquired xerocytosis), and cold agglutinin disease. The presence of elevated RDW and MCHC (performed by aperture impedance instruments, e.g., Coulter) makes the likelihood of HS very high, because these two tests used together are very specific for HS 3. Reticulocytosis (3!15%) 4. Blood smear: spherocytes, microspherocytes (vary in number); hyperdense cells with or without polychromasia. The percentage of microspherocytes is the best indicator of the severity of the disease but not a good discriminator of the HS genotype. Hyperdense cells are seen in HbSC disease, HbCC disease, and xerocytosis. In HS, hyperdense cells are a poor indicator of disease severity but an effective discriminating feature of the HS phenotype 5. Direct antiglobulin test (DAT) negative 6. Increased red cell osmotic fragility (spherocytes lyse in higher concentrations of saline than normal red cells) occasionally only demonstrated after incubation of blood sample at 37" C for 24 h (therefore always do this test incubated). In spite of normal osmotic fragility, increased MCHC or an increase in hyperdense red cells is highly suggestive of HS 7. Autohemolysis at 24 and 48 h increased, corrected by the addition of glucose 8. Survival of 51Cr-labeled cells reduced with increased splenic sequestration 9. Marrow: normoblastic hyperplasia; increased iron 10. Eosin-5-maleimide dye staining of red cells and analysis by flow cytometry is the test of choice to diagnose HS but is only available in special reference laboratories 11. Genetic analysis for the α- and β-spectrin, ankyrin, and band 3 mutations is available, but rarely necessary to be performed for diagnosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 132 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects Biochemical features 1. Indirect hyperbilirubinemia 2. Obstructive jaundice with increased direct bilirubin; may develop due to gallstones, a consequence of increased pigment excretion. Clinical features 1. Anemia and jaundice: severity depends on rate of hemolysis, degree of compensation of anemia by reticulocytosis, and ability of liver to conjugate and excrete indirect hyperbilirubinemia 2. Splenomegaly 3. Presents in newborn period in 50% of cases with hyperbilirubinemia, reticulocytosis, normoblastosis, spherocytosis, negative DAT, and splenomegaly. Patients may present with a requirement for transfusion in the first 8 weeks of life that may not be reflective of their ultimate clinical severity 4. Presents before puberty in most patients 5. Diagnosis sometimes made much later in life, often after the birth of an infant with neonatal jaundice caused by HS 6. Coinheritance of HS with HbSC disease may increase the risk of splenic sequestration crisis 7. Coinheritance of α- or β-thalassemia trait and HS has been reported to have variable effects on hemolysis 8. Iron deficiency may correct the laboratory values (artificially reducing the MCHC, etc.) but not the red cell life span in HS patients 9. HS with other system involvement: a. interstitial deletion of chromosome 8p11.1!8p21.1 causes ankyrin deficiency, psychomotor retardation, and hypogonadism, b. HS may be associated with neurologic abnormalities such as cerebellar disturbances, muscle atrophy, and a tabes-like syndrome, c. in patients presenting with common bile duct obstruction associated with gallstones, the increased cholesterol and triglyceride load from the induced dyslipidemia can correct the membrane defect and the resulting spherocyte morphology, MCHC, and osmotic fragility results, hence masking the diagnostic features of the disease. The removal of bile duct obstruction leads to a reappearance of the disease phenotype. Classification Table 7.1 lists a classification of HS in accordance with clinical severity and indications for splenectomy. Diagnosis • Clinical features and family history • Hematologic features. Complications 1. Hemolytic crisis: with more pronounced jaundice due to accelerated hemolysis (may be precipitated by viral infection) 2. Erythroblastopenic crisis (hypoplastic crisis): dramatic fall in hemoglobin level (and reticulocyte count); usually due to maturation arrest and often associated with giant pronormoblasts in the recovery phase; often associated with parvovirus B19 infection. Parvovirus B19 infects developing normoblasts, causing a transient cessation of production. The virus specifically infects Colony-Forming Unit-Erythroid and prevents their maturation. Giant pronormoblasts are seen in bone marrow. Diagnosis is made by increased IgM antibody titer against parvovirus and polymerase chain reaction for parvovirus on bone marrow 3. Folate deficiency: caused by increased red cell turnover; may lead to superimposed megaloblastic anemia. Megaloblastic anemia may mask HS morphology as well as its diagnosis by osmotic fragility 4. Gallstones (in approximately one-half of untreated patients): increased incidence with age, can occur as early as 4!5 years of age. Occasionally, HS may be masked or improved in obstructive jaundice due to increase in surface area of red cells and the coinheritance of Gilbert syndrome markedly increases the incidence of gallstones 5. Complications of chronic anemia: patients with more severe HS (see Table 7.1) may suffer growth retardation, anemic heart failure, and failure to thrive, necessitating intermittent or chronic transfusion Lanzkowsky’s Manual of Pediatric Hematology and Oncology 133 Membrane defects TABLE 7.1 Classification of spherocytosis and indications for splenectomy. Classification Trait Mild spherocytosis Moderate spherocytosis Severe spherocytosisa Hemoglobin (g/dL) Normal 11!15 8!12 6!8 Reticulocyte count (%) #3 3.1!6 $6 $10 Bilirubin (mg/dL) #1.0 1.0!2.0 $2.0 $3.0 Reticulocyte production index ,1.8 1.8!3 .3 Spectrin per erythrocyteb (percentage of normal) 100 80!100 50!80 40!60 Fresh blood Normal Normal to slightly increased Distinctly increased Distinctly increased Incubated blood Slightly increased Distinctly increased Distinctly increased Distinctly increased Without glucose (%) .60 .60 0!80 50 With glucose (%) ,10 $10 $10 $10 Splenectomy Not necessary Usually not necessary during childhood and adolescence Decision based on quality of life Necessary, not before 5 years of age Symptoms None None Pallor, erythroblastopenic crises, splenomegaly, gallstones Osmotic fragility Autohemolysis Pallor, erythroblastopenic crises, splenomegaly, gallstones a Value before transfusion. Normal (mean 6 SD): 226 6 54 3 103 molecules per cell. Adapted from Eber, S.W., Armburst, R., Schröter, W.J., 1990. Variable clinical severity of Hereditary spherocytosis: relation to erythrocytic spectrin concentration, osmotic fragility, and autohemolysis. J. Pediatr. 117 (1990), 409. b 6. Hemochromatosis: rarely, this may occur more frequently when a restricted or partial splenectomy is carried out (see next) 7. Splenic rupture: the risk of splenic rupture in HS is similar to that of the normal population. Nonetheless, a patient with a large spleen below the costal margin should be cautioned against contact sports or other activities known to lead to blunt trauma to the abdomen Treatment 1. Folic acid supplement (1 mg/day). 2. Leukocyte-depleted packed red cell transfusion for severe erythroblastopenic crisis. 3. Splenectomy for moderate-to-severe cases (see Table 7.1). Most patients with less than 80% of normal spectrin content require splenectomy. Splenectomy should be carried out early in severe cases but not before 5 years of age, if possible. The management of the splenectomized patient is detailed in Chapter 5, Lymphadenopathy and Diseases of the Spleen. Although spherocytosis persists postsplenectomy, the red cell life span normalizes and complications are prevented, especially transient erythroblastopenia and hyperbilirubinemia. There may, however, be an increased risk of arterial and venous thrombosis in later life as well as an increased risk for idiopathic pulmonary hypertension. Patients are at risk of sepsis after splenectomy, especially for those under 5 years of age. In partial splenectomy, up to 90% of the splenic mass is removed with small studies suggesting that the technique leaves enough splenic tissue to protect against infection. The technique is not widely utilized but its use should certainly be entertained in transfusion-dependent patients who are under 5 years of age. It is being offered more frequently as an elective alternative to full splenectomy in patients in the moderate-to-severe category. There may be an increased risk for iron loading in patients with HS who have not undergone splenectomy. Since patients in this situation are unlikely to tolerate phlebotomy, iron overload may make the decision for splenectomy or even partial splenectomy even more complex. Laparoscopic splenectomy is safe in children. Although it requires more operative time than open splenectomy, it is superior with regard to postoperative analgesia, smaller abdominal wall scars, duration of hospital stay, and more rapid return to a regular diet and daily activities. It is not known whether accessory spleens are readily Lanzkowsky’s Manual of Pediatric Hematology and Oncology 134 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects identified with the laparoscope although the magnification afforded by the laparoscope might be advantageous in some cases. 4. Ultrasound should be carried out before splenectomy to exclude the presence of gallstones. If present, cholecystectomy is also indicated. Hereditary elliptocytosis HE is clinically and genetically a heterogeneous disorder. Genetics HE is characterized by an autosomal dominant or codominant mode of inheritance with variable penetrance, affecting about 1 in 25,000 of the population. The prevalence of HE is much higher in regions where malaria is endemic. This could be explained by the resistance of elliptocytes to malarial invasion. Occasionally, patients who are severely affected appear to be the offspring of a family with only a single affected parent. In this case a “silent carrier”!like mutation in an α-spectrin gene of the unaffected parent may be the cause. Pathogenesis HE is due to various defects not only in the skeletal proteins, spectrin, and protein 4.1, but also in the integral protein glycophorin C. The basic membrane defects consist of • defects of spectrin self-association involving the α-chains (65%) • defects of spectrin self-association involving the β-chains (30%) • deficiency of protein 4.1 • deficiency of glycophorin C • “silent carrier” effect: α-spectrin mutant genes which produce less α-spectrin when paired with an α-spectrin structural mutant. They lead to more severe disease (see next). The mechanically unstable membrane of HE leads to shape change from discocyte to elliptocyte as the membrane is buffeted by shear stress in the circulation. Patients who are heterozygotes for these defects have milder disease while double heterozygotes and homozygotes for these mutants have progressively more severe syndromes. Hematologic features • Blood smear: 25!90% of cells have elongated oval elliptocytes • Osmotic fragility is normal or increased • Autohemolysis is usually normal but may be increased and usually corrected by the addition of glucose or ATP. Clinical features • Varies from patients who are symptom-free to severe anemia requiring blood transfusions. The percentage of microcytes best reflects the severity of the disease • About 12% have symptoms indistinguishable from HS • The percentage of elliptocytes varies from 50% to 90%. No correlation has been established between the degree of elliptocytosis and the severity of the anemia. Classification HE has been classified into the following clinical subtypes: 1. Common HE, which is divided into several groups: a. silent carrier state b. mild HE c. HE with infantile pyknocytosis 2. Common HE with chronic hemolysis, which is divided into two groups: a. HE with dyserythropoiesis b. homozygous common HE, which is clinically indistinguishable from HPP (see later discussion) 3. Spherocytic HE, which clinically resembles HS; however, a family member usually has evidence of HE Lanzkowsky’s Manual of Pediatric Hematology and Oncology Membrane defects 135 4. Southeast Asian ovalocytosis, in which the majority of cells are oval. Some red cells contain either a longitudinal or transverse ridge 5. Infantile hemolytic elliptocytosis of infancy: these patients present with hemolytic elliptocytosis (mimicking HPP) that changes over the first 2 years of life to a clinical picture of mild HE as fetal hemoglobin changes to adult hemoglobin. Usually, there is a single affected parent with HE. Treatment The indications and considerations for transfusion, splenectomy, and prophylactic folic acid are the same as for HS. Hereditary pyropoikilocytosis Genetics Homozygous or doubly heterozygous for spectrin chain mutants (e.g., Sp-a1/74 and Sp-a1/76). The spectrin chain defects found in HPP are similar to those found in HE. Pathogenesis HPP is a congenital hemolytic anemia associated with in vivo red cell fragmentation and marked in vitro fragmentation of red cells at 45" C. Because of the similarities in the membrane defect in this condition and HE, it is viewed as a subtype of HE. Biochemical and biophysical features 1. Increased ratio of cholesterol to membrane protein 2. Decreased cell deformability. Clinical features 1. Anemia characterized by extreme anisocytosis and poikilocytosis: a. red cell fragments, spherocytes, and budding red cells (the red cells are exquisitely sensitive to temperature and fragment after 10 min of incubation time at 45" C!46" C in vitro; heating for 6 h at 37" C explains in vivo formation of fragmented red cells and chronic hemolysis) b. hemoglobin level reduced to 7!9 g/dL c. Marked reduction in MCV and elevated MCHC 2. Jaundice 3. Splenomegaly 4. Osmotic fragility and autohemolysis increased 5. Mild HE present in a parent or sibling. Differential diagnosis Similar cells are seen in microangiopathic hemolytic anemias, after severe burns or oxidant stress and in PK deficiency. Treatment In infancy, these patients require intermittent transfusion for hypoplastic crises. Patients respond well to splenectomy with a rise in hemoglobin to 12 g/dL. Following splenectomy, hemolysis is decreased but not totally eliminated. Hereditary stomatocytosis Definition and genetics The stomatocyte has a linear slit-like area of central pallor rather than a circular area. When suspended in plasma, the cells assume a bowl-shaped form. This hereditary hemolytic anemia of variable severity is characterized by an autosomal dominant mode of inheritance. There are two forms of this inherited disorder related to failure to maintain normal red cell volume: 1. Overhydrated stomatocytosis (previously referred to as “hereditary stomatocytosis”). 2. Dehydrated stomatocytosis (previously referred to as “hereditary desicytosis or xerocytosis”). This is characterized by a relative paucity of stomatocytes with cells that appear very hyperchromic. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 136 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects Etiology The cells contain high Na1 and low K1 concentrations. The disorder is probably due to a membrane and protein defect. Although both forms share the relative increase in red cell sodium, overhydrated stomatocytosis is associated with an increase in red cell volume as the total cation content increases from unbridled sodium entry, while dehydrated stomatocytosis has a reduced red cell volume as the potassium cation loss is not matched by sodium accumulation. The cells are abnormally rigid and poorly deformable, contributing to their rapid rate of destruction. There are many biochemical variants. The properties of the stomatocytosis syndromes are listed in Table 7.2. Red cells from most patients with overhydrated stomatocytosis lack the membrane protein stomatin (B and 7.2b). Mutations in PIEZO1 (a mechanotransduction protein) or in the Gardos channel (a calcium channel) lead to dehydrated stomatocytosis. Clinical features Overhydrated stomatocytosis 1. Jaundice at birth 2. Pallor: marked variability depending on severity of anemia 3. Splenomegaly 4. Hematology a. Variable degrees of anemia b. Smear, 10!50% stomatocytes c. Reticulocytosis d. Increased MCV e. Decreased MCHC f. Increased osmotic fragility and autohemolysis. Dehydrated stomatocytosis 1. Mild anemia 2. Variable neonatal presentation 3. Splenomegaly and gallstones 4. Mild increase of MCV 5. Increased MCHC 6. Decreased osmotic fragility (i.e., increased osmotic resistance) 7. Increased heat stability (46" C and 49" C for 60 min). TABLE 7.2 Properties of the stomatocytosis syndromes. Severe stomatocytosis Mild stomatocytosis Cryohydrocytosis Xerocytosis Hemolysis Severe Mild!moderate Mild!moderate Moderate Smear Stomatocytes Stomatocytes Stomatocytes Target cells MCV fl. 110!150 95!130 90!105 85!125 MCHC (%) 24!30 26!29 34!38 34!38 Osmotic fragility Very increased Increased Normal/slightly increased 1 60!100 RBC Na 1 20!55 RBC K a " Very decreased 30!60 6!25 at 20 C 10!30 40!85 " 60!90 55!90 at 20 C " Cation leak 10!50 B3!10 2!10 at 20 C 2!4 Cold lysis No No Yes No Pseudohyperkalemia ? Yes ? Yes Yes Occasionally Perinatal ascites No No No Occasionally Genetics AD AD AD AD a Times normal value. Abbreviations: AD, Autosomal dominant; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBC, red blood cell. Provided by Dr. Samuel Lux, personal communication, 2009. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Membrane defects 137 Differential diagnosis Stomatocytosis morphology may occur with thalassemia, some red cell enzyme defects (glutathione peroxidase deficiency, glucose phosphate isomerase deficiency), Rhnull red cells, viral infections, lead poisoning, some drugs (e.g., quinidine and chlorpromazine), some malignancies, liver disease, and alcoholism. Dehydrated stomatocytosis syndrome resembles PK deficiency and infantile pyknocytosis on blood smear. Treatment Most patients have mild-to-moderate hemolysis that occasionally requires transfusion. Splenectomy should be avoided in these syndromes as there seems to be a consistent finding of significant venous thromboembolic complications postsplenectomy in these disorders. Hereditary acanthocytosis Definition Acanthocytes have thorn-like projections that vary in length and width and are irregularly distributed over the surface of red cells. There are apparently a number of genetic syndromes associated with acanthocytosis and their molecular basis is not yet well defined. Genetics The mode of inheritance is autosomal recessive. Clinical features 1. Steatorrhea: in cases when acanthocytosis is associated with severe fat malabsorption 2. Neurologic symptoms: weakness, ataxia and nystagmus, atypical retinitis pigmentosa with macular atrophy, blindness 3. Anemia: mild hemolytic anemia; 10!80% acanthocytes; slight reticulocytosis. Diagnosis 1. Inherited acanthocytosis is associated with the following clinical syndromes: a. Abetalipoproteinemia (absent beta-lipoprotein in blood) b. Chorea-acanthocytosis c. Huntington-like disease 2 d. Pantothenate kinase!associated neurodegeneration e. Hypobetalipoproteinemia, acanthocytosis, retinitis pigmentosa, and pallidal syndrome degeneration f. McLeod syndrome (X-linked anomaly of Kell blood group syndrome) 2. Acquired acanthocytosis is associated with the following clinical conditions: a. Anorexia nervosa b. Renal failure c. Microangiopathic hemolytic anemia d. Subgroup of HS e. Thyroid disease f. Liver disease: when associated with liver disease, the acanthocytosis is due to an imbalanced loading of cholesterol and phospholipid on to the red cell membrane. Hemolysis may be more brisk in this situation g. Zieve’s syndrome: a syndrome of hemolytic anemia with acanthocytosis, severe hyperlipoproteinemia, liver disease with jaundice, and abdominal pain in the setting of alcohol abuse. Obviously, a syndrome is more commonly seen in adults but can occur in any patient with a prolonged history of alcohol abuse. Differential diagnosis During the neonatal period, hereditary acanthocytosis may have to be distinguished from the benign nonhereditary disorder of infantile pyknocytosis. Later, acquired causes of acanthocytosis must be considered. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 138 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects Paroxysmal nocturnal hemoglobinuria Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a nonmalignant clonal expansion of hematopoietic stem cells that are mutated at phosphatidylinositol glycan complementation group A (PIG-A). PIG-A encodes the glycosylphosphatidylinositol (GPI) anchor, the mutation of which results in a deficiency of GPI anchor proteins. Many of these are complement regulatory surface proteins, a deficiency of which results in hemolytic anemia by increasing sensitivity to complement-induced hemolysis. Pathogenesis Patients with PNH have a somatic mutation in the PIG-A gene (phosphatidylinositol glycan complementation group A). This mutation occurs in hematopoietic stem cells. A protein product (probably α-1, 6N-acetylglucosamine transferase) of the PIG-A gene is normally responsible for the transfer of N-acetylglucosamine to phosphatidylinositol. In patients with PNH, there is a mutation in the PIG-A gene, which results in a decrease in its protein product and leads to a metabolic block in the biosynthesis of the glycolipid (i.e., GPI) anchor. This anchoring molecule is required for several surface proteins of the hematopoietic cells. Table 7.3 lists the surface proteins missing on PNH blood cells as a result of a deficiency in the GPI anchor. Thus the primary defect in PNH resides in the deficient assembly of the GPI anchor and, as a result, all GPIlinked antigens are absent on the surface of PNH cells. Mechanism of hemolysis and hemoglobinuria in PNH The absence of the surface complement regulatory proteins CD55 and CD59 allows deposition of complement factors and C3 convertase complexes. This leads to chronic complement-mediated intravascular hemolysis, resulting in hemoglobinuria. Mechanism of hypercoagulable state The mechanism of a hypercoagulable state in PNH is not well understood. A theory is that complement deposition on platelets results in vesiculations of their plasma membranes, which leads to increased procoagulant activity of the platelets. The monocytes and granulocytes of PNH cells lack the receptor for the GPI-linked urokinase plasminogen activator and this deficiency may lead to impaired fibrinolysis. The antithrombin, protein C, and protein S levels are normal in PNH patients. Mechanism of defective hematopoiesis The mechanism of defective hematopoiesis (macrocytosis with bone marrow erythroid dysplasia) evolving to severe aplastic anemia (AA) in some patients is not well understood. Possible explanations include: • The initial step is the development of the PIG-A mutation. This is followed by a bone marrow insult. • Resistance of PNH clones to injury compared with the susceptibility of normal hematopoietic stem cells. • Intrinsic proliferation advantage of PNH stem cells compared with normal hematopoietic stem cells. • Suppression of normal hematopoietic stem cells by PNH cells and evolution to Myelodysplastic Syndrome or Acute Myeloid Leukemia (AML). In the preceding explanation, it is assumed that two populations of stem cells normally reside in bone marrow: (1) a large population of normal stem cells and (2) a minor population of PNH stem cells. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Paroxysmal nocturnal hemoglobinuria 139 TABLE 7.3 Surface proteins missing on paroxysmal nocturnal hemoglobinuria blood cells. Protein Expression pattern Enzymes AChE Red blood cells 50 -Ectonucleotidase (CD73) Some B and T lymphocytes Leukocyte alkaline phosphatase Neutrophils Adhesion molecules Blast-1/CD48 Lymphocytes LFA-3 (or CD58) All blood cellsa Complement regulating surface proteins DAF (or CD55) All blood cellsb HRF (or C8bp) All blood cellsc MIRL (or CD59) All blood cells Receptors Fcγ III (or CD16) Neutrophils, NK cells,d macrophages,d some T lymphocytesd Endotoxin binding protein (CD14) Monocytes, macrophages, granulocytes Urokinase-type plasminogen activator receptor (CD87) Monocytes, granulocytes Blood group antigens Comer antigens (DAF) Red blood cells Yt antigens (AChE) Red blood cells Holley Gregory antigen Red blood cells JMH-bearing protein (CD108) Red blood cells, lymphocytes Dombrock residue Red blood cells Neutrophil antigens NA1/NA2 (CD16) Neutrophils NB1/NB2 Neutrophils Other surface proteins Various CD52 (CAMPATH) CD109 CD24 CD157 CD48 GP500 CD66c GP175 CD67 Folate receptor CD90 a On lymphocytes expressed in GPI-linked and transmembrane form. Level of expression on T lymphocytes varies. Expression of C8bp on human blood cells is controversial (personal communication, Taroh Kinoshita). d Expressed in a transmembrane form. Abbreviations: AChE, Acetylcholinesterase; DAF, decay accelerating factor; Fcγ III, Fcγ receptor III; HRF, homologous restriction factor; JMH, John Milton Hagen; LFA-3, lymphocyte function!associated antigen-3; MIRL, membrane inhibitor of reactive lysis. Adapted from Young, N.S., Bressler, M., Casper, J.T., Liu, J., Schacter, G.P., McArthur, T.R., 1996. Biology and therapy of aplastic anemia. In: Schacter, G.P., McArthur, T.R. (Eds.), Hematology. American Society of Hematology, Washington, DC; Ware, R.E., Orkin, S.H., Fisher, D.E., Ginsburg, D., Look, T.A., Lux, S.E., et al., 2014. Autoimmune hemolytic anemia. In: Orkin, S.H., Fisher, D.E., Ginsburg, D., Look, T.A., Lux, S.E., Nathan, D.G. (Eds.), Nathan and Oski’s Hematology of Infancy and Childhood. Elsevier Saunders, Philadelphia, PA. b c Lanzkowsky’s Manual of Pediatric Hematology and Oncology 140 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects Clinical manifestations The three main clinical features of PNH are as follows: • Paroxysmal intravascular hemolysis more frequent at night associated with hemoglobinuria and abdominal and back pain. In most cases hemolytic episodes occur every few weeks although some patients have chronic unrelenting hemolysis with severe anemia. • Bone marrow failure (macrocytosis, pancytopenia to severe AA). • Tendency to venous thrombosis. PNH can present as a primary “classic” intravascular hemolysis or it may arise during the course of AA as AA!PNH syndrome. The nature of the pathogenetic link between the two conditions remains unknown. They may be differentiated from each other by the clinical findings shown in Table 7.4. Many patients have an overlap of the aforementioned findings and do not fit precisely into one of these two groups. Course of the disease The onset of PNH is insidious. There is no familial tendency. Venous thrombosis is more often responsible for death than bone marrow failure in patients with PNH. Spontaneous long-term remission or leukemia transformation or AA may occur in some patients. Anemia is the most common finding and AA is found in approximately 10% of patients. Patients with classic PNH may have cytopenia of one or all blood cell lineages and the degree of bone marrow failure may vary from mild to severe. About 15% of patients with AA develop overt PNH; however, 35!50% of AA patients may have flow cytometric evidence of deficiency of GPI-linked molecules at some stage of their disease as evidence of subclinical PNH. Complications Intravascular hemolysis (DAT negative) • Hemoglobinuria (dark urine) • Iron deficiency • Acute renal failure. Venous thrombosis • Peripheral veins • Dermal veins • Portal venous thrombosis • Superior and inferior vena cava • Hepatic veins (Budd!Chiari syndrome)a • Mesenteric veins • Sagittal sinusa • Splenic vein TABLE 7.4 Clinical findings in classic paroxysmal nocturnal hemoglobinuria (PNH) syndrome and in aplastic anemia!PNH syndrome. Findings Classic PNH syndrome Aplastic anemia!PNH syndrome Hemolysis Chronic with acute exacerbation Hemolysis clinically subtle Thrombotic complications More often present Occurs less frequently Acute hemolysis may be preceded by abdominal pain, thought to be due to temporary occlusion of the gastrointestinal veins. Thrombosis of larger abdominal veins may be present Bone marrow failure predominant clinical finding Abnormal erythrocyte or Positive from the time of diagnosis granulocyte CD55/CD59 Positive in 20!50% of patients with SAA. May evolve postimmunosuppressive therapy Abbreviation: SAA, Severe aplastic anemia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Paroxysmal nocturnal hemoglobinuria 141 • Abdominal wall veins • Intrathoracic veins a Potential cause of death. Defective hematopoiesis • Aplastic Anemia • Macrocytosis • Evolution to myelodysplastic or AML. Infectious • Sinopulmonary • Bloodborne. Other • Dysphagia Table 7.5 lists the laboratory findings in PNH. Diagnosis PNH is definitively diagnosed through flow cytometric analysis of blood cells with the use of monoclonal antibodies to GPI-linked surface antigens. All blood cell lineages (i.e., red blood cells, lymphocytes, monocytes, granulocytes) can be analyzed by the flow cytometric technique, and heterogeneous patterns of the phenotypic expressions of various blood cells can be identified. For example, red blood cell phenotypes can be identified by their CD59 expression: • PNH type I: normal expression of CD59 • PNH type II: partially deficient or residual expression of CD59 • PNH type III: complete absence of expression of CD59. The proportion of the three different phenotypes may vary from patient to patient. Because other blood cell lineages can be analyzed, the transfusion of red blood cells to a patient does not interfere with the diagnosis of PNH. TABLE 7.5 Laboratory findings in paroxysmal nocturnal hemoglobinuria. Nonspecific findings Cytopenia involving one or more cell lineages Macrocytosis, anisocytosis, polychromasia Reticulocytosis Decreased neutrophil alkaline phosphatase Increased level of lactate dehydrogenase Decreased haptoglobin Hemoglobinuria, hemosiderinuria Iron deficiency, folate deficiency Bone marrow findings Varies from hyperplastic with predominant erythropoiesis to hypoplastic with little or patchy hematopoiesis Hypoplasia or aplasia of one or more hematopoietic lineages Increased number of mast cells Cytogenesis Usually normal Specific test for PNH Flow cytometric analysis for GPI-linked cell surface proteins (e.g., CD59) on peripheral blood or bone marrow cells Abbreviation: GPI, Glycosylphosphatidylinositol. Adapted from Young, N.S., Bressler, M., Casper, J.T., Liu, J., Schacter, G.P., McArthur, T.R., 1996. Biology and therapy of aplastic anemia. In: Schacter, G.P., McArthur, T.R. (Eds.), Hematology. American Society of Hematology, Washington, DC. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 142 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects The percentage of granulocytes with a PNH phenotype is usually higher than the percentage of red cells lacking CD59. Thus flow cytometric analysis of the granulocytes increases sensitivity in the diagnosis of PNH. Management Hematopoietic stem cell transplantation Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for PNH. If a fully matched family donor is available, then HSCT is the treatment of choice, especially for patients who develop bone marrow failure. In the absence of a matched unrelated donor, alternative donor transplantations can be considered based on the quality of the available alternative donor and the severity of the PNH (see Chapter 30: Hematopoietic Stem Cell Transplant and Cellular Therapy). Eculizumab Eculizumab, a humanized monoclonal antibody that blocks complement activation at C5 preventing the formation of C5a, is the standard of care for PNH. It reduces hemolysis and thromboembolism and dramatically improves the patient’s quality of life. Due to the importance of complement in immunity against Neisseria meningitidis, patients receiving eculizumab must be vaccinated. Immunosuppressive therapy Therapy with cyclosporine and antithymocyte globulin is indicated in the setting of PNH-associated AA. This treatment may lead to improvement in AA but not in the hemolysis of PNH. Prednisone 1!2 mg/kg daily can ameliorate hemolysis and can be used for 24!72 h around the time of a hemolytic episode. Use of hematopoietic growth factor The use of Granulocyte-Colony Stimulating Factor may be attempted in the setting of a clinically significant neutropenia. Supportive therapy • Long-term anticoagulant therapy (e.g., with warfarin or low-molecular-weight heparin) is indicated for patients with venous thrombosis. Women with PNH should be discouraged from using birth control pills • Iron and folate supplements are indicated due to chronic hemoglobinuria accompanied by iron loss and chronic hemolysis with increased erythroid marrow activity requiring supplementation of additional folate • Sildenafil may be effective in treating dysphagia and intestinal spasm and impotence, which are the consequence of decreased nitric oxide secondary to consumption by plasma-free hemoglobin • Red blood cell transfusion as needed for symptomatic anemic patients. Enzyme defects There are two major biochemical pathways in the red cell: the Embden!Meyerhof anaerobic pathway (energy potential of the cell) and the hexose monophosphate shunt (reduction potential of the cell). Fig. 7.7 illustrates the enzyme reactions in the red cell. PK deficiency PK is an enzyme active in the penultimate conversion in the Embden!Meyerhof pathway. Although deficiency is rare, it is the most common enzyme abnormality in the Embden!Meyerhof pathway. Genetics • Autosomal recessive inheritance • Significant hemolysis seen in homozygotes • Found predominantly in people of northern European origin • Deficiency not simply quantitative; probably often reflects the production of PK variants with abnormal characteristics. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 143 Enzyme defects Glucose Hexokinase ATP AOP %-Glut-Cyst + Gly Glucose-6phosphate dehydrogenase Mg$$ G-6-P Glucose phosphate isomerase NADP ATP Mg$$ AOP 6-PG Glutathione synthetase GSH NADP NADPH H 2O 2 F-6-P Phosphofructokinase ATP AOP Phosphogluconate dehydrogenase Mg$$ F-1,6-P CO2 Fructose diphosphate aldolase Glutathione reductase NADPH GSSG Glutathione peroxidase H2O R-5-P DHAP Triosephosphate isomerase G-3-P Pi Glyceraldehyde-3-phosphate dehydrogenase NAD NADH 1.3-DPG Phosphoglycerate kinase ADP 3-PG $$ Mg ATP 3-PG 2.3-DPG Phosphoglyceromutase Diphosphoglyceromutase Pi 2.3-DPG Diphosphoglycerate phosphatase 2-PG Mg$$ Phosphopyruvate hydratase PEP Pyruvate kinase ADP ATP Mg$$ K$ Pyruvate NADH Lactate dehydrogenase NAD Lactate FIGURE 7.7 Enzyme reactions of Embden!Meyerhof and hexose monophosphate pathways of metabolism. Documented hereditary deficiency diseases are indicated by enclosing dotted lines. Abbreviations: ATP, Adenosine triphosphate; G-6-P, glucose-6-phosphate; ADP, adenosine di-phosphate; DHAP, dihydroxyacetone phosphate; DPG, diphosphoglycerate; GSH, reduced glutathione; GSSG, oxidized glutathione; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of Nicotinamide adenine dinucleotide phosphate; PEP, phosphoenolpyruvate. Pathogenesis • Defective red cell glycolysis with reduced ATP formation • Red cells rigid, deformed and metabolically and physically vulnerable (reticulocytes less vulnerable because of the ability to generate ATP by oxidative phosphorylation). Hematology • Features of nonspherocytic hemolytic anemia: macrocytes, oval forms, polychromatophilia, anisocytosis, occasional spherocytes, contracted red cells with multiple projecting spicules, rather like echinocytes or pyknocytes Lanzkowsky’s Manual of Pediatric Hematology and Oncology 144 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects • Erythrocyte PK activity decreased to 5!20% of normal; 2,3-bisphosphoglycerate (2,3-BPG) and other glycolytic intermediary metabolites increased (because of two- to three-fold increase in 2,3-BPG, there is a shift to the right in P50)1 • Autohemolysis markedly increased, showing marked correction with ATP but not with glucose. Clinical features • Variable severity; can cause moderately severe anemia (not drug-induced). Patients may tolerate their anemia better because of the increase in 2,3-BPG shifting the hemoglobin oxygen dissociation curve to the right. This leads to superior off-loading of oxygen to the tissues and may mitigate the anemia. • Usually presents with neonatal jaundice. • Splenomegaly common but not invariable. • Late: gallstones, bone changes of chronic hemolytic anemia, cardiomegaly secondary to severe anemia. • Erythroblastopenic crisis due to parvovirus B19 infection. • Hemochromatosis. These patients seem to have a risk of hemochromatosis beyond the number of transfusions they received. Careful attention should be paid to their iron loading. Treatment • Folic acid supplementation • Transfusions as required • Splenectomy (if transfusion requirements increase); splenectomy does not arrest hemolysis but decreases transfusion requirements. Note that there is a paradoxical increase in reticulocytosis after splenectomy even as transfusion requirement and hemolytic rate abate • Surveillance for iron overload • Experimental studies with a small molecule oral activator of PK are still underway. Other enzyme deficiencies 1. Hexokinase deficiency, with many variants 2. Glucose phosphate isomerase deficiency 3. Phosphofructokinase deficiency, with variants 4. Aldolase 5. Triosephosphate isomerase deficiency 6. Phosphoglycerate kinase deficiency 7. 2,3-BPG deficiency due to deficiency of diphosphoglycerate mutase 8. Adenosine triphosphatase deficiency 9. Enolase deficiency 10. Pyrimidine 50 -nucleotidase deficiency 11. Adenosine deaminase overexpression 12. Adenylate kinase deficiency. These enzyme deficiencies have the following features: 1. General hematologic features: a. Autosomal recessive disorders except phosphoglycerate kinase deficiency, which is sex linked and enolase deficiency that presents as an autosomal dominant b. Chronic nonspherocytic hemolytic anemias (CNSHAs) of variable severity c. Osmotic fragility and autohemolysis normal or increased d. Improvement in anemia after splenectomy e. Diagnosed by specific red cell assays with increased availability of genetic testing. 2. Specific nonhematologic features: a. Phosphofructokinase deficiency associated with type VII glycogen storage disease. Hematologic symptoms are mild compared to the significant myopathy b. Triosephosphate isomerase deficiency associated with progressive debilitating neuromuscular disease with generalized spasticity and recurrent infections (some patients have died of sudden cardiac arrest) 1 Because of the right shift of P50, patients do not exhibit fatigue and exercise intolerance proportionate to the degree of anemia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 145 Enzyme defects c. Phosphoglycerate kinase deficiency associated with mental retardation, myopathy, and a behavioral disorder. Note the three exceptions to the general hematologic features listed earlier: • Adenosine deaminase excess (i.e., not an enzyme deficiency) is an autosomal dominant disorder. • Pyrimidine 50 -nucleotidase deficiency is characterized by marked basophilic stippling, although the other CNSHAs lack any specific morphologic abnormalities. • Deficiency of diphosphoglycerate mutase results in polycythemia. Glucose-6-phosphate dehydrogenase deficiency G6PD is the first enzyme in the pentose phosphate pathway of glucose metabolism. Deficiency diminishes the reductive energy of the red cell and may result in hemolysis, the severity of which depends on the quantity and type of G6PD and the nature of the hemolytic agent (usually an oxidation mediator that can oxidize NADPH, generated in the pentose phosphate pathway in red cells). Genetics • Sex-linked recessive mode of inheritance by a gene located on the X chromosome (similar to hemophilia) • Disease is fully expressed in hemizygous males and homozygous females • Variable intermediate expression is shown by heterozygous females (due to random deletion of X chromosome, according to Lyon hypothesis) • As much as 3% of the world’s population is affected; most frequent among African-American, Asian, and Mediterranean peoples. The molecular basis of G6PD deficiency and its clinical implications follow: • Deletions of G6PD genes are incompatible with life because it is a housekeeping gene and complete absence of G6PD activity, called hydeletions, will result in death of the embryo • Point mutations are responsible for G6PD deficiencies. They result in: ! Sporadic mutations: they are not specific to any geographic areas. The same mutation may be encountered in different parts of the world that have no causal (e.g., encountering G6PD Guadalajara in Belfast) relationship with malarial selection. These patients manifest with CNSHA World Health Organization (WHO) Class I ! Polymorphic mutations: these mutations have resulted from malaria selection; hence, they correlate with specific geographic areas. They are usually WHO Class II or III and not Class I. The WHO classifies G6PD variants on the basis of magnitude of the enzyme deficiency and the severity of hemolysis (Table 7.6). Pathogenesis • Red cell G6PD activity falls rapidly and prematurely as red cells age • Decreased glucose metabolism • Diminished NADPH/NADP and Reduced Glutathione/GSSG ratios • Impaired elimination of oxidants (e.g., H2O2) • Oxidation of hemoglobin and of sulfhydryl groups in the membrane TABLE 7.6 World Health Organization (WHO) classification of glucose-6-phosphate dehydrogenase variants. WHO class Variant Magnitude of enzyme deficiency Severity of hemolysis I Harilaou, Tokyo, Guadalajara, Stony Brook, Minnesota 2% of normal activity Chronic nonspherocytic hemolytic anemia II Mediterranean 3% of normal activity Intermittent hemolysis 2 III A 10!60% of normal activity Intermittent hemolysis usually associated with infections or drugs IV B (Normal) 100% normal activity No hemolysis Lanzkowsky’s Manual of Pediatric Hematology and Oncology 146 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects • Red cell integrity impaired, especially on exposure to oxidant drugs, oxidant response to infection and chemicals • Oxidized hemoglobin precipitates to form Heinz bodies that are plucked out of the red cell leading to hemolysis and “bite cell” and “blister cell” morphology. Clinical features Episodes of hemolysis may be produced by: • Drugs. Table 7.7 lists the agents capable of inducing hemolysis in G6PD-deficient subjects • Fava bean (broad bean, Vicia faba): ingestion or exposure to pollen from the bean’s flower (hence favism) • Infection. Drug-induced hemolysis 1. Typically in African-Americans but also in Mediterranean and Canton types 2. List of drugs (see Table 7.7); occasionally need additional stress of infection or the neonatal state 3. Acute self-limiting hemolytic anemia with hemoglobinuria 4. Heinz bodies in circulating red cells 5. Blister cells, fragmented cells, and spherocytes 6. Reticulocytosis 7. Hemoglobin normal between episodes. Favism 1. Acute life-threatening hemolysis, often leading to acute renal failure, caused by ingestion of fava beans 2. Associated with Mediterranean and Canton varieties 3. Blood transfusion required. Neonatal jaundice 1. Usually associated with Mediterranean and Canton varieties but can occur with all variants 2. Infants may present with pallor, jaundice (can be severe and produce kernicterus), and dark urine a. The excessive jaundice resulting in kernicterus is not only due to hemolysis but also may be due to reduced glucuronidation of bilirubin caused by defective G6PD activity in the hepatocytes. Often no exposure to drugs, occasionally exposure to naphthalene (mothballs), aniline dye, marking ink, or a drug. In a majority of neonates the jaundice is not hemolytic but hepatic in origin. Chronic nonspherocytic hemolytic anemia 1. Occurs mainly with sporadic inheritance. Clinical picture: 1. Variable but can be severe with transfusion dependence 2. Reticulocytosis 3. Intense neonatal presentation 4. Shortened red cell survival 5. Increased autohemolysis with only partial correction by glucose 6. Slight jaundice 7. Mild splenomegaly. Treatment 1. Avoidance of agents that are deleterious in G6PD deficiency. (For a consistent and up-to-date list of drug susceptibilities, visit www.favism.org) 2. Education of families and patients in recognition of food prohibition (fava beans), drug avoidance, heightened vigilance during infection and the symptoms and signs of hemolytic crisis (orange/dark urine, lethargy, fatigue, jaundice) 3. Indication for transfusion of packed red blood cell in children presenting with acute hemolytic anemia: a. Hb level below 7 g/dL b. persistent hemoglobinuria and Hb below 9 g/dL Lanzkowsky’s Manual of Pediatric Hematology and Oncology 147 Enzyme defects TABLE 7.7 Agents capable of inducing hemolysis in glucose-6-phosphate dehydrogenase-deficient subjects. Clinically significant hemolysis Usually not clinically significant hemolysis Analgesics and antipyretics Acetanilid Acetophenetidin (phenacetin) Acetylsalicylic acid (large doses) Antipyrinea,b Aminopyrineb p-Aminosalicylic acid Antimalarial agents Pentaquine Quinacrine (Atabrine) Pamaquine Quinineb Primaquine Chloroquinec Quinocide Pyrimethamine (Daraprim) Plasmoquine Flouroquinones Ciprofloxacin Sulfonamides Sulfanilamide Sulfadiazine N-Acetylsulfanilamide Sulfamerazine Sulfapyridine Sulfisoxazole (Gantrisin)c Sulfamethoxypyridazine (Kynex) Sulfathiazole Salicylazosulfapyridine (Azulfidine) Sulfacetamide Nitrofurans Nitrofurazone (Furacin) Nitrofurantoin (Furadantin) Furaltadone (Altafur) Furazolidone (Furoxone) Sulfones Thiazolsulfone (Promizole) DDS (dapsone) Sulfoxone sodium (Diasone) Miscellaneous Naphthalene Phenylhydrazine Menadione Acetylphenylhydrazine Dimercaprol (BAL) Toluidine blue Methylene blue Nalidixic acid (NegGram) Chloramphenicolb Neoarsphenamine (Neosalvarsan) Probenecid (Benemid) Infections Quinidineb Diabetic ketoacidosis Fava beansb (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 148 7. General considerations of hemolytic diseases, red cell membrane, and enzyme defects TABLE 7.7 (Continued) Clinically significant hemolysis Usually not clinically significant hemolysis Doxorubicin Urate oxidase (Rasburicase) Foods a Many other compounds have been tested but are free of hemolytic activity. Penicillin, the tetracyclines, and erythromycin, for example, will not cause hemolysis and the incidence of allergic reactions in G6PD-deficient persons is not any greater than that observed in others. Hemolysis in Whites only. c Mild hemolysis in African-Americans, if given in large doses. Note: Drugs associated with hemolysis in any WHO class are listed as clinically significant. DSS, Diaminodiphenylsulfone; GPI, glycosylphosphatidylinositol. b 4. CNSHA: a. In patients with severe chronic anemia: transfuse red blood cells to maintain Hb level 8!10 g/dL and iron chelation, when needed. b. Splenectomy has only occasionally ameliorated severe anemia in this disease. Indications for splenectomy are as follows: i. hypersplenism ii. severe chronic anemia iii. splenomegaly causing physical impediment 5. Genetic counseling and prenatal diagnosis for severe CNSHA if the mother is a heterozygote. Other defects of glutathione metabolism Glutathione reductase In this autosomal dominant disorder, hemolytic anemia is precipitated by drugs having an oxidant action. Thrombocytopenia has occasionally been reported. Neurologic symptoms occur in some patients. The disease is mimicked by riboflavin deficiency. Glutamylcysteine synthetase In this autosomal recessive disorder, there is a well-compensated hemolytic anemia. This very rare disease has been associated with spinocerebellar degeneration in one patient. Glutathione synthetase In this autosomal recessive disorder, there is a well-compensated hemolytic anemia, exacerbated by drugs having an oxidant action. This is the most common disorder of the group and can also present as a systemic metabolic disorder with acidosis, hemolysis, and susceptibility to infection. Glutathione peroxidase In this autosomal recessive disorder, acute hemolytic episodes occur after exposure to drugs having an oxidant action. Further reading and references Becker, P., Lux, S., Nathan, D., Oski, F., 1993. Disorders of the red cell membrane. In: Nathan, D., Oski, F. (Eds.), Hematology of Infancy and Childhood. WB Saunders, Philadelphia, PA. Bolton-Maggs, P.H., Stevens, R.F., 2004. On behalf of the General Haematology Task Force of the British Committee for the Standards in Haematology, Guidelines for the diagnosis and management of hereditary spherocytosis. Br. J. Haematol. 126 (2004), 455!474. Da Costa, L., Gallimand, J., Fenneteau, O., Mohandas, N., 2013. Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders. Blood Rev. 27 (4), 167!178. Dacie, J., 1992. The Haemolytic Anaemias 3. The Auto-Immune Haemolytic Anaemias, third ed. Churchill Livingstone, Edinburgh. Eber, S.W., Armburst, R., Schröter, W.J., 1990. Variable clinical severity of Hereditary Spherocytosis: relation to erythrocytic spectrin concentration, osmotic fragility, and autohemolysis. J. Pediatr. 117 (1990), 409. Gallagher, P.G., 2013. Abnormalities of the erythrocyte membrane. Pediatr. Clin. North Am. 60 (6), 1349!1362. Grace, R.F., Rose, C., Layton, D.M., Galactéros, F., Barcellini, W., Morton, D.H., et al., 2019. Safety and efficacy of mitapivat in pyruvate kinase deficiency. N. Engl. J. Med. 381 (2019), 933!944. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 149 Hillmen, P., Lewis, S.M., Bressler, M., 1995. Natural history of paroxysmal nocturnal hemoglobinuria. N. Engl. J. Med. 333 (1995), 1253!1258. Inoue, N., Izui-Sarumaru, T., 2006. Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH). Blood 108 (13), 4232!4236. King, M.-J., Behrens, J., 2000. Rapid flow cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anemia. Br. J. Haematol. 111 (2000), 924!933. Lanzkowsky, P., 1980. Hemolytic Anemia Pediatric Hematology Oncology: A Treatise for the Clinician. McGraw-Hill, New York. Mohandas, N., 2018. Inherited hemolytic anemia: a possessive beginner’s guide. Hematol. Am. Soc. Hematol. Educ. Program. 2018 (1), 377!381. Narla, J., Mohandas, N., 2017. Red cell membrane disorders. Int. J. Lab. Hematol. 39 (2017), 47!52. Schilling, R.F., Gangnon, R.E., 2007. Delayed diverse vascular events after splenectomy in hereditary spherocytosis. J. Thromb. Haemost. 6 (2007), 1289!1295. Tracy, E., Rice, H., 2008. Partial splenectomy for hereditary spherocytosis. Pediatr. Clin. North Am. 55 (2008), 503!519. Ware, R.E., Orkin, S.H., Fisher, D.E., Ginsburg, D., Look, T.A., Lux, S.E., et al., 2014. Autoimmune hemolytic anemia. Nathan and Oski’’s Hematology of Infancy and Childhood. In: Orkin, S.H., Fisher, D.E., Ginsburg, D., Look, T.A., Lux, S.E., Nathan, D.G. (Eds.), Nathan and Oski’’s Hematology of Infancy and Childhood. Elsevier Saunders, Philadelphia. Wolfe, L., Manley, P., Arceci, R., Hann, I., Smith, O., 2006. Disorders of erythrocyte metabolism including porphyria. In: Arceci, R., Hann, I., Smith, O. (Eds.), Pediatric Hematology. Blackwell Publishing Ltd., Boston, MA. Young, N.S., Bressler, M., Casper, J.T., Liu, J., Schacter, G.P., McArthur, T.R., 1996. Biology and therapy of aplastic anemia. In: Schacter, G.P., McArthur, T.R. (Eds.), Hematology. American Society of Hematology, Washington, DC. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 8 Extracorpuscular hemolytic anemia Anshul Vagrecha1,2 and Lawrence C. Wolfe1,2 1 Division of Pediatric Hematology/Oncology and Stem Cell Transplantation, Cohen Children’s Medical Center of New York, New Hyde Park, NY, United States 2Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, United States The causes of extracorpuscular hemolytic anemia are listed in Table 8.1; they may be immune or nonimmune. Immune hemolytic anemia Immune hemolytic anemia can be isoimmune or autoimmune. Isoimmune hemolytic anemia results from a mismatched blood transfusion or from hemolytic disease of the newborn. In autoimmune hemolytic anemia (AIHA), shortened red cell survival is caused by the action of autoantibodies with or without the participation of complement on the red cell membrane. The red cell autoantibodies may be of the warm type [usually immunoglobulin G (IgG)], the cold type [usually complement-mediated hemolysis by immunoglobulin M (IgM)], mixed with both the types present, or the cold Donath!Landsteiner type (cold IgG). Immune hemolytic anemia is confirmed by a positive direct antiglobulin test (DAT) (previously referred to as a Coombs’ test) that demonstrates an immunoglobulin and/or complement on the red blood cell (RBC) surface. A DAT is considered positive in the presence of agglutination of antibody (Ab)-coated red cells. Most commercial laboratories use a polyspecific AHG reagent that will be positive in both IgG and C3. Later, monospecific IgG and complement reagents are used. Most laboratories do not test for other autoantibodies leading to a falsenegative result in about 3!11% of AIHA cases. If AIHA is strongly suspected despite a negative DAT, consider testing for Donath!Landsteiner Ab or further testing in reference laboratories for unusual autoantibodies that may not appear in conventional testing, either due to low numbers of attached antibodies or failure of the reagent to detect [e.g., immunoglobulin A (IgA) because the reagent is anti-IgG]. Complement participation is usually confined to the IgM type of antibody; only rarely is it associated with IgG (see Section 8.1.4). AIHA may be idiopathic or secondary to several conditions listed in Table 8.1. Warm AIHA Antibodies of the IgG class are most commonly responsible for AIHA in children (responsible for about 48!70% of cases). The IgG autoantibody is directed against one of the Rh erythrocyte antigens in over 70% of cases. This antibody usually has its maximal activity at 37" C, and the resultant hemolysis is called warm antibody-induced hemolytic anemia. Rarely, warm reacting IgA and IgM antibodies may be responsible for hemolytic anemia. As in all patients with AIHA, erythrocyte survival is proportional to the amount of antibody on the erythrocyte surface, although rarely hemolysis can occur in patients with too few antibodies on the surface of the red cell to cause a positive DAT (DAT-negative hemolytic anemia). Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00007-6 151 © 2022 Elsevier Inc. All rights reserved. 152 8. Extracorpuscular hemolytic anemia TABLE 8.1 Causes of hemolytic anemia due to extracorpuscular defects. Immune Isoimmune • Hemolytic disease of the newborn • Incompatible blood transfusion Autoimmune • IgG only • Complement only • Mixed IgG and complement • Other antibody-mediated mechanisms Idiopathic • Warm antibody • Cold antibody • Cold!warm hemolysis (Donath!Landsteiner antibody) Secondary • Viral infections with EBV, CMV, hepatitis, herpes simplex, measles, varicella, influenza A, coxsackie virus B, HIV, and the novel SARS-CoV-2 • Bacterial infections: streptococcal, typhoid fever, Escherichia coli septicemia, Mycoplasma pneumoniae (atypical pneumonia) • Drugs and chemicals: quinine, quinidine, phenacetin, p-aminosalicylic acid, sodium cephalothin (Keflin), ceftriaxone, penicillin, tetracycline, rifampin, sulfonamides, chlorpromazine, pyridone, dipyrone, insulin, and lead • Hematologic disorders: leukemias, lymphomas, lymphoproliferative syndrome, paroxysmal cold hemoglobinuria, PNH • Immunopathic disorders: systemic lupus erythematosus, periarteritis nodosa, scleroderma, dermatomyositis, rheumatoid arthritis, ulcerative colitis, agammaglobulinemia, Wiskott!Aldrich syndrome, dysgammaglobulinemia, IgA deficiency, thyroid disorders, giant cell hepatitis, Evans syndrome (immunemediated anemia associated with immune thrombocytopenia), ALPS, and common variable immune deficiency • Tumors: ovarian teratomata, dermoids, thymoma, and lymphomas Nonimmune Idiopathic Infection, viral: infectious mononucleosis, viral hepatitis; bacterial: streptococcal, E. coli septicemia, Clostridium perfringens, Bartonella bacilliformis; parasites: malaria, histoplasmosis Drugs and chemicals: phenylhydrazine, vitamin K, benzene, nitrobenzene, sulfones, phenacetin, acetanilide, and lead Hematologic disorders: leukemia, aplastic anemia, megaloblastic anemia, hypersplenism, and pyknocytosis Microangiopathic hemolytic anemia: TTP, HUS, chronic relapsing schistocytic hemolytic anemia, burns, postcardiac surgery, and march hemoglobinuria Miscellaneous: Wilson disease, erythropoietic porphyria, osteopetrosis, hypersplenism, posthematopoietic stem cell transplantation Abbreviations: ALPS, autoimmune lymphoproliferative syndrome; CMV, cytomegalovirus; EBV, Epstein!Barr virus; HIV, human immunodeficiency virus; HUS, hemolytic!uremic syndrome; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. Clinical features • Severe, life-threatening condition • Sudden onset of pallor, jaundice, and dark urine • Splenomegaly Laboratory findings • A hemoglobin level can be very low in fulminant disease or normal in indolent disease. • Reticulocytosis is common, although often the reticulocytes are destroyed by the antibody as well and reticulocytopenia may occur. • Mean corpuscular hemoglobin concentration may be elevated. • Smear shows prominent spherocytes (due to antibody-mediated membrane loss), polychromasia, macrocytes, autoagglutination (IgM), nucleated RBCs, and erythrophagocytosis. • Neutropenia and thrombocytopenia (occasionally). Lanzkowsky’s Manual of Pediatric Hematology and Oncology Immune hemolytic anemia 153 • Increased osmotic fragility and autohemolysis proportional to spherocytes. • DAT positivity establishes the diagnosis of AIHA. • Hyperbilirubinemia and increased serum lactate dehydrogenase. • Haptoglobin level is markedly decreased. • Hemoglobinuria, usually only at first presentation, increased urinary urobilinogen. Initial management Warm AIHA is potentially life-threatening and the following must be monitored carefully: • Hemoglobin level (every 4 hours) • Reticulocyte count (daily) • Spleen size (daily) • Hemoglobinuria (daily) • Haptoglobin level (weekly) • DAT (weekly) Close attention should always be paid to supportive care issues such as folic acid supplementation, hydration status, urine output, and cardiac status. Blood transfusion Transfusion should be avoided when possible, because there will be no truly compatible blood available and the survival of transfused cells in this situation is quite limited and may fail to elevate the hemoglobin level significantly. Nonetheless, using the “least incompatible” blood by crossmatching may be required in properly selected situations in order to avoid cardiopulmonary compromise. The guidelines listed below should be followed: • If a specific antibody is identified, a compatible donor may be selected. The antibody usually behaves as a pan agglutinin, and totally compatible blood cannot be found. • Washed packed red cells should be used from donors whose erythrocytes show the least agglutination in the patient’s serum. Despite the autoantibody the absence of an alloantibody should be confirmed prior to transfusion. • The volume of transfused blood should only be of sufficient quantity to relieve any cardiopulmonary embarrassment from the anemia. Aliquots of 5 mL/kg are taken from a single unit and transfused at a rate of 2 mL/kg/h. • The use of such incompletely matched blood is made relatively safe by biologic crossmatching, transfusing of relatively small volumes of blood, and concomitant use of high-dose corticosteroid therapy. Corticosteroid therapy • Prednisone 2!10 mg/kg/day orally or methylprednisolone 4!8 mg/kg/day IV (both given over four doses each day) for 3 days followed by oral prednisone. • High-dose corticosteroid therapy should be maintained for several days. Thereafter, corticosteroid therapy in the form of prednisone should be slowly tapered over 3!4 weeks. • The dose of prednisone should be tailored to maintain the hemoglobin at a reasonable level; when the hemoglobin stabilizes, the corticosteroids should be discontinued. The presence of a continued positive DAT does not prevent continuing to taper steroids as long as the hemoglobin is stable or rising, and reticulocytosis continues to decrease or remain normal. 70!85% of patients respond within 4!7 days to corticosteroids, but some patients experience ongoing profound hemolysis for the first week after initiation. For these patients and patients who appear dependent on steroids, alternative treatment should be considered. Rituximab In patients with a severe disease not responding early on or in patients exhibiting steroid dependence, rituximab (a manufactured monoclonal antibody targeting CD20) should be used in doses of 375 mg/m2 once a week for 4 weeks. It has a very high rate of remission induction in AIHA in children. Immunoglobulin levels should be measured prior to the use of rituximab to rule out common variable immunodeficiency and also to assess for the possibility of autoimmune lymphoproliferative syndrome (ALPS) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 154 8. Extracorpuscular hemolytic anemia (see Chapter 16: Lymphoproliferative Disorders) or other immunological diseases prior to starting rituximab therapy when possible. The short-term side effects include: • Itching • Hives • Hypotension • Chest pain These can be prevented through premedication with acetaminophen 15 mg/kg, diphenhydramine 1 mg/kg, and, if necessary, corticosteroids. Patients should be monitored carefully during each infusion. Although rituximab eliminates the B-cell compartment, there have not been increased rates of infection, and intravenous gamma globulin has been administered to offset the loss of B-cell function. If steroids and rituximab fail to induce a remission, alternative therapies listed later can be used. If remission is induced followed by a relapse, the patient can be treated with another course of rituximab or one of the following therapies. Intravenous gamma globulin Doses between 1 and 5 g/kg have been employed but the response in children is poor (about 54%). It should be considered in patients with severe hemolysis who are requiring transfusion and are having poor responses to transfusion. Plasmapheresis In IgG warm immune hemolytic anemia, plasmapheresis should always be combined with moderate immunosuppression (e.g., rituximab). This ensures that both antibody production and antibody titer reduction are employed concomitantly. Plasmapheresis has been successful in slowing the rate of hemolysis in patients with severe IgG-induced immune hemolytic anemia. The effect is short-lived if antibody production is ongoing and success is limited. The limited efficacy of plasmapheresis is likely because: • More than half of the IgG is extravascular • Most of the antibodies are on the red cell surface with little remaining free in the plasma Chronic management There are several agents available for chronic management of warm AIHA after the initial treatment with steroids and/or rituximab. These agents often require 4!12 weeks to exert their effect and are usually started as steroids are being tapered. Immunomodulating agents Mycophenolate mofetil (MMF) This drug is showing promise in the treatment of several autoimmune diseases, including AIHA. It is also effective in Evans syndrome [autoimmune bicytopenia (immune thrombocytopenia with AIHA)]. • Dose: 15 mg/kg twice daily (max 2000 mg/day) given in combination with rituximab. Sirolimus Sirolimus is the appropriate agent for AIHA associated with the ALPS and should also be considered in patients with autoimmune bi- or pancytopenia or a background of autoimmune serology or disease due to the high success rate in such cases. • Dose: 2!2.5mg/m2/day PO once daily OR divided BID (max initial dose 4 mg/m2/day). Drug levels are monitored every 2 weeks until target level between 4.6 and 20 ng/mL, then switched to every 3 months. Bortezomib Bortezomib has been used in adults with ongoing refractory AIHA either alone or with solid organ transplant. Patients with warm AIHA refractory to multiple therapies received bortezomib combined with dexamethasone. • Dose: In adult patients, IV bortezomib was initiated at 1.3mg/m2 on Day 1,4,8, and 11 in a 21 day cycle. One pediatric case report indicated dosing of 1!1.3 mg/m2 given every 3 days. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Immune hemolytic anemia 155 Cyclosporine This drug has been frequently used in immune cytopenias, and in Evans syndrome for patients who respond poorly to steroids. Rituximab, MMF, and sirolimus are preferred over cyclosporine due to the sideeffect profile. • Dose: 5!6 mg/kg/day PO divided BID (dose range 1.4!10 mg/kg/day). Higher doses ( . 5 mg/kg/day) has been shown to be more nephrotoxic. Cyclosporine (modified) (Gengraf, Neoral) and cyclosporine (nonmodified) (Sandimmune) are not bioequivalent and cannot be used interchangeably. • Doses should be adjusted to achieve desired target cyclosporine concentration. ! Goal plasma levels: 150!300 ng/mL ! Will require slow taper when coming off therapy ! Goal residual level: .100 ng/mL Danazol There has been some success with this synthetic androgen. Its early effect is likely due to decreased macrophage Fc-receptor activity. The virilizing effect makes it less desirable. • Dose: ! There is limited dosing for pediatric patients. Dosing of 600!800 mg/day has been used in adult patients initially in conjunction with corticosteroids. ! Once at remission, a dose of danazol can be reduced to 200!400 mg/day. ! Recommend to limit use to patients $ 16 years old. Azathioprine and 6-mercaptopurine (Antimetabolites) As with the immunomodulators, they may take 4!12 weeks to provide a steroid-sparing effect. • Doses: ! Azathioprine: 2!5 mg/kg/day PO once daily. Therapy with azathioprine has been generally used in combination with corticosteroids. ! Mercaptopurine: 50!75 mg/m2/day PO once daily. Children with BSA , 1 m2; dose at 2.5 mg/kg/day PO once daily. Thiopurine methyltransferase gene status should be evaluated prior to initiation. Mercaptopurine/metabolite levels are recommended to be assessed for efficacy and toxicity. Cyclophosphamide (alkylating agent) This drug is used in patients with severe disease that is unresponsive to steroids, rituximab, or immunomodulators. It is rarely used. • Dose: 50 mg/kg/day for 4 days. Vincristine and vinblastine (mitotic inhibitors) These drugs are rarely used, but when given, are used as a bridge to suppress hemolysis while waiting for an immunomodulator or cytotoxic agent to take effect. • Doses: ! Vincristine: 1.5 mg/m2 (max dose 2 mg) IV weekly. ! Vinblastine: 6 mg/m2 (max dose 10 mg) IV weekly. (Limited data on dosing. Some literature refers to vinblastine-loaded platelets, but no specific dosing is provided. An adult case report vinblastine every 14 days for an adult patient. Dosing is extrapolated from pediatric ITP dosing.) Splenectomy Splenectomy is indicated if the hemolytic process is brisk despite the use of high-dose corticosteroid therapy, rituximab, and transfusions, and the patient cannot maintain a reasonable hemoglobin level safely, or if chronic hemolysis develops. Splenectomy is beneficial in 60!75% of patients. Whenever possible, children should be older than 5 years of age and the disease should be present for at least 6!12 months with no significant response to medical treatment prior to undertaking splenectomy. Presplenectomy immunization should be instituted. This approach is rarely necessary in the age of immunomodulatory therapy. Recombinant erythropoietin (rEPO) Although most cases with AIHA have reticulocytosis, a few children have a low reticulocyte count. This is mostly due to the autoantibody removal of reticulocytes. Rarely, there may be no evidence of overt hemolysis and a hypoplastic response. Only in these cases should erythropoietin be considered. A retrospective study showed Lanzkowsky’s Manual of Pediatric Hematology and Oncology 156 8. Extracorpuscular hemolytic anemia that high doses of rEPO have been effective in increasing Hb and reticulocyte count in about 70% of children with severe, refractory AIHA with reticulocytopenia. However, prospective data are lacking and rEPO is not without potential toxicity (see FDA black box warnings before prescribing for this indication). • Dose: Epoetin alpha 20,000!40,000 IU per week. Hematopoietic stem cell transplant (HSCT) Rarely, HSCT is indicated in patients with severe, life-threatening AIHA who have failed multiple therapies. Data for autologous and allogeneic HSCT are sparse. Giant cell hepatitis and DAT-positive AIHA This is a specific rare entity of unknown etiology, although an autoimmune component has been suggested because of the association of DAT-positive AIHA and response to immunosuppression. The prognosis is poor. Clinical findings • Age: 6!24 months, occasionally older age • Fever • Pallor • Jaundice (progressing to cirrhosis and liver failure) • Firm hepatomegaly and splenomegaly • Associated convulsions Laboratory findings • DAT: mixed (IgG and complement); no evidence of other autoimmunity • Hemolytic anemia • Liver function abnormality: high direct bilirubin, transaminase, and serum globulin values; prolonged prothrombin time • Liver histology: marked lobular fibrosis, extensive necrosis with central!portal bridging, and giant cell transformation Treatment Corticosteroids in combination with immunosuppressive agents are the primary therapy as steroids alone cannot typically control the hemolysis. Cytoxan, rituximab, cyclosporine, splenectomy, and azathioprine have all been used. Survival has been achieved with more intensive immunosuppression. Cold AIHA IgM antibody-associated hemolysis occurs less often in children than in adults. Most IgM autoantibodies causing immune hemolytic anemia are cold agglutinins, and cold agglutinin disease is almost always caused by an IgM antibody. The destruction of RBCs is triggered by cold exposure. Cold hemagglutinin disease typically occurs during Mycoplasma pneumoniae infection, although it may also occur with infectious mononucleosis, cytomegalovirus, mumps, and rarely other infections. Cold hemagglutinin disease or IgM-induced hemolysis is usually due to the production of antibodies targeting the I/i system (red cell surface antigens). Anti-I is characteristic of M. pneumoniae!associated hemolysis, and anti-i cold agglutinins are found in infectious mononucleosis. M. pneumoniae adherence to the red cell membrane appears to be mediated by sialic acid!containing receptors, associated with terminal galactose residues of the I antigen. The association of the infecting organism with the RBC may alter the antigenic structure of RBC membrane antigen, rendering it immunogenic. In children, the IgM antibody is usually polyclonal and immunologically heterogeneous. Clinical features This disease may be idiopathic but is more frequently seen with infections such as M. pneumoniae (atypical pneumonia) and less commonly with lymphoproliferative disorders. The features are: • Hemoglobin is usually normal or mildly decreased, and the reticulocyte count may be elevated. • The blood smear may show agglutination and polychromatophilia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Immune hemolytic anemia 157 • Spherocytosis is usually absent. • The DAT is positive for complement (polyspecific and anti-C3-agents) only and is negative for anti-IgG. • Most blood banks do cold agglutinin testing only when the DAT is positive for complement. It is important to keep the specimen tubes for cold agglutinin testing warm or immediate delivery to the blood bank to prevent binding of antibody to red cells (and exclusion from serum), while tube is cooling toward room temperature. Treatment • Control of the underlying disorder. • Transfusions may be necessary for patients with significant hemolysis who may be symptomatic. Identification of compatible blood may prove difficult, and the blood bank may have to release a “least incompatible” unit of blood. Warming the blood to 37" C during administration by means of a heating coil or water bath is indicated to avoid further temperature activation of the antibody. Efficient in-line blood warmers (McGaw Water Bath; Fenwall Dry Heat Warmer) are designed to deliver blood at 37" C to the patient. Unmonitored or uncontrolled heating of blood is extremely dangerous and should not be attempted. Red cells heated too long are rapidly destroyed in vivo and can be lethal to the patient. • Warm the patient’s room. Keeping a patient warm will help diminish hemolysis and peripheral agglutination. • Plasmapheresis is very efficient for the treatment of IgM disease, as IgM is largely intravascular. Patients with severe hemolysis should undergo plasmapheresis. • Autotransfusion of RBCs can be performed if the blood is obtained at 37" C, with the patient’s arm warmed by hot pads. The warm unit can be separated quickly by centrifugation and the red cells returned to the patient through an efficient in-line blood warmer. • Drug therapy: If the anemia is severe, a drug trial is appropriate. Rituximab and cyclophosphamide have been used with plasmapheresis. Steroids are of marginal value in cold agglutinin disease. Other therapies like ibrutinib, eculizumab, bortezomib, and sutimlimab are being evaluated in clinical trials for Cold AIHA. Paroxysmal cold hemoglobinuria due to Donath!Landsteiner cold hemolysin Paroxysmal cold hemoglobinuria (PCH) is caused by an unusual IgG antibody with anti-P specificity and a cold thermal amplitude, originally described in cases of syphilis. This antibody, although uncommon, is most frequently found in young children with viral infections. Hemolysis is most commonly intravascular as a result of the unusual complement-activating efficiency of this IgG antibody. Clinical features The most common clinical finding is a sudden bout of hemolysis with a drop in hemoglobin and hemoglobinuria. The hemoglobin drop is often serious enough to require transfusion (and sudden death from this disease has been reported). Children usually have a short-lived, explosive illness where the antibody is only produced for a short time. Although blood for transfusion will appear compatible, all red cells carry the P blood group specificity against which the antibody is directed. Laboratory findings A positive complement test is present on antiglobulin testing, and this should lead to testing for the Donath!Landsteiner antibody (the IgG cold binding antibody) in the absence of an obvious IgM cold agglutinin. Differential diagnosis Acute intravascular hemolysis with hemoglobinuria is a relatively rare event in childhood. It may occur due to: 1. An acute transfusion reaction 2. Drug-induced acute hemolysis (e.g., ceftriaxone) 3. PCH 4. An oxidant hemolysis event (e.g., as would occur in G6PD deficiency) Treatment Keeping a patient warm is the mainstay of treatment, and warming blood in a blood warmer prior to transfusion is important. Most patients will stabilize in a warm environment and not require more transfusion or Lanzkowsky’s Manual of Pediatric Hematology and Oncology 158 8. Extracorpuscular hemolytic anemia treatment with full recovery in 6!8 weeks. Patients with ongoing severe hemolysis may benefit from plasmapheresis, although the disease is caused by an IgG antibody. Steroid use is controversial. If the disease is severe enough to consider plasmapheresis, or if it appears to be chronic or relapsing (which is rare), rituximab has been shown to be effective. DAT-negative immune hemolytic anemia Sometimes, a patient can have a negative DAT despite having an immune hemolytic anemia. The causes can include: • Non-IgG and IgM AIHA (IgA) • Autoantibody threshold below that of the DAT reagent • Low-affinity autoantibodies • NK cell!mediated hemolysis Nonimmune hemolytic anemia This group of conditions is due to extracorpuscular causes of hemolytic anemia in which the DAT is negative. The various causes are listed in Table 8.1. Conditions caused by various infections, drugs, and underlying hematologic disease respond to the treatment of the underlying condition, as well as the necessary acute supportive care, including red cell transfusions. Microangiopathic hemolytic anemia Microangiopathic hemolytic anemia (MAHA) is a result of diverse causes that lead to a uniform hematologic picture and is generally caused by a common pathogenesis. Table 8.2 lists the various causes of MAHA. TABLE 8.2 Causes of microangiopathic hemolytic anemia. Renal disease Hemolytic!uremic syndrome Renal vein thrombosis Renal transplantation rejection Radiation nephritis Chronic renal failure Cardiac conditions Malignant hypertension Coarctation of aorta Severe valvular heart disease Subacute bacterial endocarditis of aortic valve Intracardiac prosthesis Liver disease Severe hepatocellular disease Infections Disseminated herpes infection Meningococcal septicemia Cerebral falciparum malaria Hematologic Thrombotic thrombocytopenic purpura (hereditary or secondary) (see Chapter 12: Disorders of Platelets) Miscellaneous Severe burns Giant hemangioma (Kasabach!Merritt syndrome) Disseminated intravascular coagulation of any cause, sometimes accompanied by the consumption of circulating coagulation factors (consumption coagulopathy) Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 159 Diagnosis The blood smear is characterized by the presence of burr erythrocytes, schistocytes, helmet cells, and microspherocytes. This occurs in association with the evidence of hemolysis and there may be associated thrombocytopenia depending on the initial etiology of the microangiopathy [e.g., disseminated intravascular coagulation and hemolytic!uremic syndrome (HUS), see Table 8.2]. The severity of anemia varies and depends on the underlying pathophysiology. Intravascular hemolysis occurs in all forms; plasma hemoglobin levels may be elevated, haptoglobin absent, hemosiderinuria present, and urinary iron excretion increased in the more chronic forms. Treatment involves improving the underlying condition and transfusion as needed. Patients can become iron deficient due to the ongoing hemoglobinuria. Special attention should be paid to the simultaneous occurrence of microangiopathic hemolysis with thrombocytopenia or other serious systemic issues (like a renal injury). Diseases requiring very specific early treatment, such as HUS or hemophagocytic lymphohistiocytosis (see Chapter 15: Histiocytic Disorders) may present this way and should be considered when microangiopathy is seen. Hypersplenism Shortened red cell survival with excessive sequestration can be demonstrated in many patients with clinical splenomegaly. This can occur whether splenic enlargement is caused by infection or is secondary to such diseases as thalassemia, portal hypertension, or storage diseases. Typically, hypersplenism is accompanied by moderate neutropenia and thrombocytopenia with active erythropoiesis, myelopoiesis, and thrombopoiesis in the marrow. There may also be mild spherocytosis. The blood values return to normal following splenectomy. Wilson disease Wilson disease is a rare inherited disease of copper metabolism that leads to copper deposition most prominently in the liver and central nervous system (CNS). It has an autosomal recessive inheritance pattern and usually presents with liver or CNS symptoms. Wilson disease rarely presents with anemia, which is normochromic and normocytic with reticulocytosis or indirect hyperbilirubinemia. Because of the lethal nature of the disease without treatment and the potential successful treatment, if the disease is detected early, Wilson disease should be considered in any patient with unexplained hemolytic anemia that has no abnormal morphology and a negative DAT. Further reading and references Bride, K.L., Vincent, T., Smith-Whitley, K., Lambert, M.P., Bleesing, J.J., Seif, A.E., et al., 2016. Sirolimus is effective in relapsed/refractory autoimmune cytopenias: results of a prospective multi-institutional trial. Blood 127 (1), 17!28. Dacie, J., 1992. The Haemolytic Anaemias: The Auto-Immune Haemolytic Anaemias, third ed Churchill Livingstone, Edinburgh. Gertz, M., 2007. Management of cold haemolytic syndrome. Br. J. Haematol. 138, 422!429. Giulino, L.B., Bussel, J.B., Neufeld, E., 2007. Treatment with rituximab in benign and malignant hematologic disorders in children. J. Pediatr. 150, 338!344. Glader, Arceci, R., Hann, I., Smith, O., 2006. Autoimmune Hemolytic Anemia. Pediatric Hematology. Blackwell Publishing Ltd, Boston, MA. Hoffman, P.C., 2009. Immune Hemolytic Anemia-Selected Topics. ASH Education Book. Koppel, A., Lim, S., Osby, M., Garratty, G., Goldfinger, D., 2007. Rituximab as successful therapy in a patient with refractory paroxysmal cold hemoglobinuria. Transfusion 47 (10), 1902!1904. Ladogana, S., Maruzzi, M., Samperi, P., et al., 2017. Diagnosis and management of newly diagnosed childhood autoimmune haemolytic anaemia. Recommendations from the Red Cell Study Group of the Paediatric Haemato-Oncology Italian Association. Blood Transfus. 15 (3), 259!267. Ladogana, S., Maruzzi, M., Samperi, P., et al., 2018. Second-line therapy in paediatric warm autoimmune haemolytic anaemia. Guidelines from the Associazione Italiana Onco-Ematologia Pediatrica (AIEOP). Blood Transfus. 16 (4), 352!357. Lewis, S.M., Bain, B.J., Bates, I., 2006. Acquired Haemolytic Anemias. Dacie and Lewis Practical Hematology. Elsevier, Ltd, Philadelphia, PA. Maggiore, G., Sciveres, M., Fabre, M., Gori, L., Pacifico, L., Resti, M., 2011. Giant cell hepatitis with autoimmune hemolytic anemia in early childhood: long-term outcome in 16 children. J. Pediatr. 159 (1), 127!132.e1. Sève, P., Philippe, P., Dufour, J.F., Broussolle, C., Michel, M., 2008. Autoimmune hemolytic anemia: classification and therapeutic approaches. Expert. Rev. Hematol. 1 (2), 189!204. Zanella, A., Barcellini, W., 2014. Treatment of autoimmune hemolytic anemias. Haematologica 99 (10), 1547!1554. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 9 Hemoglobinopathies Eugene Khandros1,2 and Janet L. Kwiatkowski1,2 1 Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States 2Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States Sickle cell disease Pathophysiology HbS arises as a result of a point mutation (A-T) in the sixth codon of the β-globin gene on chromosome 11, which causes a single amino acid substitution (glutamic acid to valine at position 6 of the β-globin chain). HbS is more positively charged than HbA and hence has a different electrophoretic mobility. Deoxygenated HbS polymerizes, leading to cellular alterations that distort the red cell into a rigid, sickled form. Vaso-occlusion with ischemia-reperfusion injury is the central event, but the underlying pathophysiology is complex, involving a number of factors, including hemolysis-associated reduction in nitric oxide bioavailability, chronic inflammation, oxidative stress, altered red cell adhesive properties, activated white blood cells and platelets, altered hemostasis, including platelet activation, thrombin activation, lowered levels of anticoagulants, impaired fibrinolysis, and increased viscosity. Fetal hemoglobin (HbF, γ-globin) affects HbS by decreasing polymer content in cells. The effect of HbF on HbS may have direct and indirect effects on other red blood cell (RBC) characteristics [i.e., percentage of HbF affects the RBC adhesive properties in patients with sickle cell disease (SCD)]. Elevated HbF concentration is associated with a reduction in certain complications of SCD. Incidence Approximately 300,000 children with SCD are born every year worldwide. Sickle hemoglobin is the most common abnormal hemoglobin found in the United States. Among African-Americans the prevalence of all types of SCD at birth is approximately one in 365 and the prevalence of SCD-type SS is about one in 700. Taking into account early mortality in individuals with SCD, the most recent population estimate of individuals in the United States is 72,000!98,000. Genetics 1. SCD is transmitted as an autosomal codominant trait. 2. Homozygotes for HbS have SCD-type SS, also referred to as sickle cell anemia. 3. Compound heterozygotes for HbS and HbC have a form of SCD, SCD-type SC. 4. Compound heterozygotes for HbS and β-thalassemia trait (β0 or β1) have a form of SCD (Sβ0-thalassemia or Sβ1-thalassemia). 5. Heterozygotes (AS), sickle cell trait, have red cells containing 35!45% HbS. 6. Sickle cell trait provides selective advantage against Plasmodium falciparum malaria (balanced polymorphism). 7. α-Thalassemia may be commonly coinherited with sickle cell trait or disease (one or two α-globin gene deletions have a 35% frequency in African-Americans). Individuals who have both α-thalassemia and SCD-SS Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00013-1 161 © 2022 Elsevier Inc. All rights reserved. 162 9. Hemoglobinopathies tend to be less anemic than those who have SCD-SS alone, have a reduced risk of some complications such as stroke, but no reduction in frequency or severity of vaso-occlusive pain episodes. Prognosis 1. In utero: SCD can be diagnosed accurately in utero by mutation analysis of DNA prepared from chorionic villus biopsy (10- to 14-week gestation) or fetal fibroblasts obtained by amniocentesis (15- to 20-week gestation). Noninvasive prenatal testing with cell-free DNA analysis is currently being studied. 2. During the newborn period: The diagnosis of SCD can be established by electrophoresis of hemoglobins recovered from dried blood specimens blotted on filter paper (Guthrie cards) using a. isoelectric focusing (most commonly used in screening programs); b. high-performance liquid chromatography; c. citrate agar with a pH of 6.2, a system that provides distinct separation of HbS, HbA, and HbF; and d. DNA-based mutation analysis. 3. In older children: Table 9.1 lists the diagnosis and differential diagnosis of various sickle cell syndromes. Clinical features Hematology 1. Anemia with reticulocytosis—moderate to severe in SS and Sβ0-thalassemia, milder with SC or Sβ1-thalassemia. 2. Mean corpuscular volume (MCV) is normal with SS and microcytic with concomitant α-thalassemia; MCV is also microcytic with Sβ-thalassemia and SC genotypes. 3. Neutrophilia is common. 4. Platelet count often increased. 5. Blood smear—sickle cells (not infants or others with high HbF), increased polychromasia, nucleated red cells, and target cells (Howell!Jolly bodies may indicate hyposplenism). 6. Erythrocyte sedimentation rate—usually low despite inflammation (sickle cells fail to form rouleaux). 7. Hemoglobin electrophoresis—HbS migrates slower than hemoglobin A. Newborn screening shows FS, FSC, or FSA pattern depending on genotype. Acute complications 1. Vaso-occlusive pain event (VOE) a. Episodic microvascular occlusion at one or more sites resulting in pain and inflammation. Common locations and manifestations of VOE are shown in Table 9.2. Symptoms of fever, erythema, swelling, and focal bone pain may accompany VOE, making it difficult to distinguish from osteomyelitis. Unfortunately, no test clearly distinguishes these two entities, but Table 9.3 describes clinical, laboratory, and radiographic features that may be helpful in differentiating bone infarction from osteomyelitis. b. The average rate of VOE prompting medical evaluation in SCD-type SS is 0.8 events/year. Approximately 40% of patients never seek medical attention for pain, while about 5% of patients account for a third of all VOE. These numbers underestimate the true incidence of VOE because many episodes are managed at home. c. Risk factors for pain include high baseline hemoglobin level, low hemoglobin F levels, nocturnal hypoxemia, and asthma. d. Evidence-based guidelines for the management of VOE are lacking. The typical approach to pain management involves a stepwise progression, beginning with a nonsteroidal anti-inflammatory pain medication for mild-to-moderate pain and adding an opioid pain medication rapidly if pain is not resolving or for moderate-to-severe pain. Rapid alleviation of VOE pain is a basic tenet of SCD management (Table 9.4). e. Additional therapies under investigation for the treatment of acute VOE include recombinant ADAMTS13, intravenous (IV) immunoglobulin, and inhaled nitrous oxide. f. Treatments to reduce VOE rates: i. Hydroxyurea (HU). ii. Prophylactic red cell transfusions. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Mild/moderate SC S/β0-Thalassemia S/β1-Thalassemia SS/α-Thalassemia-1 Mild/moderate S/HPFH (1) YC (1) OC (2) 14 10 10 8.5 11 7.5 Normal 40 27 32 28 33 22 Normal 85 70 72 65 80 85 Normal Mean Mean corpuscular hematocrit (%) volume (fL) 1!3 5!10 2!6 3!20 2!6 5!30 Normal Occasional target cells, no ICSs Mild hypochromia and microcytosis; few ISCs (21) Mild microcytosis and hypochromia; many target cells few ISCs (11) Marked hypochromia and microcytosis; many target cells, ISCs (31) and NRBCs Many target cells, few ISCs (11) Many target cells, ISCs (41) and NRBCs Few target cells Red cell Reticulocytes (%) morphology b All syndromes have positive sickle preparations. Hemoglobin F distribution; heterogeneous. c Hemoglobin F distribution; homogeneous. (2) Absent and (1) present. Abbreviations: HPFH, Hereditary persistence of fetal hemoglobin; ISC, irreversibly sickled cell; NRBC, nucleated red blood cell; OC, older child; YC, young child. a (2) Mild/moderate SS Asymptomatic (1) Severe Moderate/severe (1) (1) Asymptomatic AS (2) Clinical severity Syndromea Mean hemoglobin Splenomegaly (g/dL) TABLE 9.1 Differential diagnosis in sickle cell syndromes. 60!80% S; 15!35% Fc 80!100% S; 0!20% Fb 50!80% S; 10!30% A; 0!20% Fb; ,3.5% A2 50!85% S; 2!30% Fb; .3.5% A2 50!55% S; 45!50% C; Fb 80!96% S; 2!20% Fb 35!45% S; 55!60% A; Fb Electrophoresis 164 9. Hemoglobinopathies iii. Crizanlizumab was approved by the FDA in 2019 for the prevention of VOE in SCD patients 16 years and older. It is a humanized anti-P-selectin antibody given at a dose of 5 mg/kg intravenously every 4 weeks (following an initial loading period). Crizanlizumab reduced annual VOE rates by approximately 50%, and patients concurrently on HU were more likely to be VOE-free over the study period. 2. Acute chest syndrome (ACS) a. ACS is the most common cause of death and the second most common cause of hospitalization in children with SCD. It is generally defined as the development of a new pulmonary infiltrate accompanied by symptoms, including fever, chest pain, tachypnea, cough, hypoxemia, and wheezing. b. ACS is caused by infection, infarction, and/or fat embolization and iatrogenically by overhydration. About 50% of ACS events are associated with infections, including viruses, atypical bacteria, including Mycoplasma and Chlamydia, and less frequently with Streptococcus pneumoniae. Parvovirus B19 infection can also result in ACS. In about half of cases, ACS develops during hospitalization, often for vaso-occlusive pain, where fat embolization, hypoventilation, and iatrogenic overhydration contribute to the pathophysiology. c. The incidence of ACS in SCD-SS is about 13 events per 100 patients in children with a peak of about 25 events/100 patients in 2- to 5-year-old patients. The incidence in other sickle cell genotypes is lower (SS . Sβ0-thalassemia . SC . Sβ1-thalassemia), and concomitant α-thalassemia does not appear to affect ACS rates. d. The risk of ACS is directly proportional to the hemoglobin level and white blood cell count; increased levels of cytokines and/or white cell adhesion to the endothelium may play a role. Rates of ACS are also higher in children with asthma. Higher HbF levels appear to be protective. TABLE 9.2 Common location of vaso-occlusive pain. Site Manifestations Hands/feet (dactylitis) Most common in children younger than 3 years old. Painful swelling of the hands and/or feet. Often can be managed with acetaminophen or nonsteroidal antiinflammatory medication. Unusual in older children because as the child ages, the sites of hematopoiesis move from peripheral locations such as the fingers and toes to more central locations such as arms, legs, ribs, and sternum. Bone More common after age 3 years. Often involves long bones, sternum, ribs, spine, and pelvis. May involve more than one site during a single episode. Swelling and erythema may be present. May be difficult to differentiate from osteomyelitis because clinical symptoms, laboratory studies, and radiological imaging may be similar. Features that may aid in distinguishing these two diagnoses are shown in Table 9.3. Abdomen Caused by microvascular occlusion of mesenteric blood supply and infarction in the liver, spleen, or lymph nodes that result in capsular stretching. Symptoms of abdominal pain and distension mimic acute abdomen. TABLE 9.3 Differentiation between bone infarction and osteomyelitis. Features Favoring osteomyelitis Favoring vaso-occlusion History No previous history Preceding painful crisis Pain, tenderness, erythema, swelling Single site Multiple sites Fever Present Present Leukocytosis Elevated band count ( . 1000/mm3) Present Erythrocyte sedimentation rate Elevated Normal to low Magnetic resonance imaging Abnormal Abnormal Bone scana Abnormal 99mTc-diphosphonate Abnormal 99mTc-diphosphonate Normal 99m Tc-colloid marrow uptake Decreased 99mTc-colloid marrow uptake Blood culture Positive (Salmonella, Staphylococcus) Negative Recovery Only with appropriate antibiotic therapy Spontaneous a Obtained within 3 days of symptom onset. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 165 TABLE 9.4 Management of vaso-occlusive pain episodes. Setting Management recommendations Home Ibuprofen and/or acetaminophen If continued pain, add oral opioid: • mild pain—codeine • moderate pain—oxycodone, hydrocodone, morphine Supportive measures: • heating pad • fluids • stool softeners and/or laxative if taking opioids for .1!2 days If pain persists or worsens, patient should be evaluated and treated in an acute care setting Emergency department or acute Rapid triage and administration of pain medication care unit If no pain medications were taken prior to arrival and pain not severe, may use ibuprofen and oral opioid If prior pain medications were taken or pain is severe • ketorolac tromethamine (NSAID) • IV opioid Fluids to maintain euvolemia. IV normal saline bolus should only be used if evidence of decreased oral intake/dehydration Inpatient Continue nonsteroidal antiinflammatory agent Continue IV opioids. Should be given as scheduled medication rather than “as needed” Consider patient-controlled analgesia pump if pain not adequately controlled Consider addition of long-acting opioid (e.g., sustained-release morphine) Ongoing evaluation of adequacy of pain control is essential—utilize pain scales Supportive care Fluids (oral 1 IV) to maintain euvolemia Incentive spirometry Heating pad—must be used carefully to avoid burns Bowel regimen to prevent/treat constipation secondary to opioid use Stool softeners (e.g., docusate) Laxative (e.g., senna) Antihistamines (e.g., diphenhydramine, hydroxyzine) for pruritis Venous thromboembolism prophylaxis if mobility limited (age 12 years and older, serial compression devices, low molecular weight heparin if indicated) Transition to oral nonsteroidal and oral opioid as pain level improves • • • • • • • • Abbreviations: IV, Intravenous; NSAID, nonsteroidal antiinflammatory drug. e. Laboratory findings i. White blood cell count is often elevated. ii. Hemoglobin level often falls to 1.5 g/dL below baseline values. iii. Thrombocytosis may be present and often follows an episode of ACS. f. The management of ACS is described in Table 9.5. g. Prevention of ACS: Patients with a history of recurrent ACS are candidates for preventative/curative therapies, including: i. HU. ii. Prophylactic red cell transfusions. Optimal target HbS level is not known, but usually a goal of 30!50% is used. iii. Hematopoietic stem cell transplantation (HSCT). 3. Overt stroke a. Acute symptomatic stroke is usually ischemic in children, although hemorrhagic stroke may occur, particularly in older children and adults. b. The most common underlying lesion is intracranial arterial stenosis or occlusion, usually involving the large arteries of the circle of Willis, particularly the distal internal carotid artery (ICA), the middle (MCA), and anterior cerebral arteries (ACAs). c. Chronic injury to the endothelium of vessels by sickled RBCs results in changes in the intima with the proliferation of fibroblasts and smooth muscle; the lumen is narrowed or completely obliterated. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 166 9. Hemoglobinopathies TABLE 9.5 Management of acute chest syndrome in children. Evaluation • • • • • • • Chest radiograph Complete blood count and reticulocyte count Blood type and screen Blood culture Viral studies—influenza, respiratory panel, and SARS-COV-2 Pulse oximetry Consider arterial blood gas in room air Treatment • Antibiotics: Broad-spectrum intravenous antibiotic such as ampicillin plus an oral macrolide (erythromycin or azithromycin) to cover atypical bacteria • Supplemental oxygen if hypoxemic • Fluids: Intravenous 1 oral fluids should be kept at maintenance. Avoid overhydration • Pain control: Must be carefully monitored. Goal is to relieve pain to reduce splinting/poor aeration but avoid oversedation with hypoventilation • Transfusion: • Simple transfusion (10!15 cm3/kg)—do not exceed posttransfusion hemoglobin level of B10 g/dL • Exchange transfusion—if no improvement with simple transfusion or with severe hypoxemia/respiratory distress • Bronchodilators—particularly if history of reactive airways disease or if wheezing present • Steroids may be beneficial for severe acute chest syndrome or if reactive airways disease component. There is a risk of rebound pain with discontinuation of the steroids • Incentive spirometry to reduce atelectasis • Mechanical ventilation as needed • Consider thoracentesis if significant pleural effusion Small friable collateral blood vessels known as moyamoya may develop. Infarction of brain tissue occurs acutely as a result of in situ occlusion of the damaged vessel or distal embolization of a thrombus. Perfusional and/or oxygen delivery deficits related to changes in blood pressure or other factors also may contribute to infarction, particularly in watershed zones. d. Stroke is most common in SCD-SS. Prior to transcranial Doppler (TCD) ultrasound screening with transfusions for high-risk children, stroke prevalence in children with SCD-SS was estimated at 11%, with the highest incidence rates occurring in the first decade of life (1.02 per 100 patient-years in 2- to 5-year olds and 0.79 per 100 patient-years in 6- to 9-year olds). The incidence of first ischemic stroke in the postSTOP era has been reduced to 0.09!0.24 per 100 patient-years. e. A number of clinical, laboratory, and radiological risk factors for stroke have been identified (Table 9.6). f. Symptoms of stroke include: i. focal motor deficits (e.g., hemiparesis and gait dysfunction), ii. speech defects, iii. altered mental status, iv. seizures, and v. headache. g. Gross neurological recovery occurs in approximately two-thirds of children, but neurocognitive deficits are common. h. In untreated patients, about 70% of patients experience a recurrence within 3 years. Outcome after recurrent stroke is worse. i. Any child with SCD who develops acute neurological symptoms requires immediate medical evaluation. The acute management involves prompt diagnosis and treatment and should involve consultation by neurology or a dedicated stroke team. i. Diagnosis: • Physical examination with detailed neurological examination. Treatment in the setting of clinical suspicion must not await imaging confirmation. Head CT scan is useful for detecting intracranial hemorrhage and often more readily available than magnetic resonance imaging (MRI). CT scan may not be positive for acute infarction within the first 6 hours. • Brain MRI with diffusion-weighted imaging is more sensitive to early ischemic changes and may be abnormal within 1 hour. It should be performed as soon as possible in a child with SCD presenting with acute neurological symptoms but should not delay empiric treatment. • Magnetic resonance arterial angiography (MRA)—demonstrates large-vessel disease. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 167 TABLE 9.6 Factors associated with increased risk of overt infarctive stroke in sickle cell disease. Clinical History of transient ischemic attacks History of bacterial meningitis Sibling with SCD-SS and stroke Recent episode of acute chest syndrome (within 2 weeks) Frequent acute chest syndrome Systolic hypertension Nocturnal hypoxemia Laboratory Low steady-state hemoglobin level No α-gene deletion Certain HLA haplotypes Radiological Abnormal transcranial Doppler ultrasound Silent infarct Abbreviations: HLA, Histocompatibility locus antigen; SCD, sickle cell disease. ii. Treatment: • Transfusion. Exchange transfusion, either automated or manual, should be performed as soon as possible. The goal is to reduce the amount of HbS to , 15!20% and to raise the hemoglobin level to approximately 10 g/dL. If exchange transfusion is not readily available, a simple transfusion to raise the hemoglobin level to no greater than 10 g/dL may be used while awaiting exchange. Exchange transfusion may be associated with a decreased risk of stroke recurrence compared with simple transfusion. • Supportive therapy, including avoiding hypotension and maintaining adequate oxygenation and euthermia, should be initiated as adjunctive therapy. j. Long-term management. i. Secondary prophylaxis of recurrent stroke • A chronic red cell transfusion program should be instituted, with the goal of maintaining the pretransfusion HbS level at , 30%. Transfusions must be continued indefinitely due to the high risk of stroke recurrence after discontinuation of therapy. After a period of 3!4 years after the initial stroke, it may be possible to allow the pretransfusion HbS level to rise to , 50% in low-risk patients, without increased risk of stroke recurrence. This approach is associated with decreased transfusional iron loading. • HSCT. • The uses of revascularization procedures such as encephaloduroarteriosynangiosis or a newer modification, pial synangiosis, may be beneficial in children with significant vasculopathy, particularly if symptomatic (transient ischemic attacks, recurrent stroke). • Prophylactic aspirin may also be useful in children with progressive vasculopathy, but the risks of hemorrhage must be weighed against the potential benefit. • HU generally is not recommended for secondary stroke prevention. A multicenter phase 3 trial of HU and phlebotomy compared with transfusions and chelation therapy was terminated due to a higher rate of recurrent stroke (10%) in the HU arm compared with continued transfusions (0%) without any difference in iron reduction between the two treatment arms. The stroke recurrence rate on HU still was lower than historical rates in untreated patients; thus HU may be considered in patients who are unable to be transfused, particularly if there is no significant stenosis or occlusion of cerebral blood vessels on MRA. ii. Rehabilitation • Physical and occupational therapy as needed. • Neuropsychological testing should be performed with educational interventions if indicated. iii. Screening for stroke risk—primary stroke prevention • TCD ultrasonography is a noninvasive study used to measure the blood flow velocity in the large intracranial vessels of the circle of Willis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 168 9. Hemoglobinopathies • The highest time-averaged mean velocity (TAMMvel) in the distal ICA, its bifurcation, and the MCA are used to categorize studies into risk groups: ◦ Normal (velocity , 170 cm/s), low risk. ◦ Conditional (170!199 cm/s), moderate risk. ◦ Abnormal ( $ 200 cm/s), high risk. ◦ Inadequate—unable to obtain velocity in the ICA or MCA on either side, in the absence of a clearly abnormal value in another vessel. Inadequate TCD may be due to technique, skull thickness, or severely stenosed vessel. ◦ Very low velocity (ICA/MCA velocity , 70 cm/s) may indicate vessel stenosis and increased risk of stroke. • Elevated velocity in the ACA ( . 170 cm/s) is associated with increased stroke risk. Treatment of children with isolated high ACA velocities has not been established. Brain MRI/MRA should be obtained. Chronic transfusion should be instituted for children with ACA velocity $ 200 cm/s, and with elevated ACA velocity if there is evidence of significant cerebral blood vessel stenosis on MRA. • TCD screening is recommended for children with SCD-SS or SCD-Sβ0-thalassemia ages 2!16 years. Screening is performed annually, but more frequently if the prior study was not normal. An approach to screening is shown in Table 9.7. In addition, more frequent screening should be considered if other known stroke risk factors are present (such as sibling with SCD-SS and stroke or abnormal TCD). • Brain MRI/MRA should be obtained in children with abnormal TCD and should be considered for children with conditional TCD. • Brain MRA is helpful to evaluate cerebral vasculature in children with repeatedly inadequate TCD, with very low velocity, and with isolated elevated velocity in the ACA or posterior cerebral artery. iv. Treatment • Chronic transfusion to maintain the HbS level , 30% reduces the risk of stroke by . 90% in children with abnormal TCD. • Discontinuation of transfusion therapy without alternative therapy after $ 30 months of transfusion in children whose TCD normalized is not recommended as this was associated with a high risk of reversion to abnormal TCD and stroke. • In children with a history of abnormal TCD who have received $ 1 year of red cell transfusions and have no MRA evidence of severe vasculopathy, a switch to HU therapy can be considered. ◦ The TWITCH trial showed that HU was not inferior to continued transfusions for the control of TCD velocities in that patient population. ◦ A gradual switch to HU at maximum tolerated dose, with a period of transfusion overlap, typically 6!9 months, is recommended. ◦ Ongoing monitoring and support of adherence with HU are essential. • HSCT with a histocompatibility locus antigen (HLA)-identical sibling donor should be considered. 4. Priapism a. Priapism is a sustained, painful erection of the penis. Priapism may be prolonged (lasts . 3 hours), or stuttering (lasts , 3 hours). Stuttering episodes often recur or may develop into a prolonged episode. TABLE 9.7 Transcranial Doppler ultrasonography screening protocol. Last TCD result (TAMMvel in ICA/MCA) Screening interval Normal (,170 cm/s) Annual Low conditional (170!184 cm/s) 3!6 monthsa High conditional (185!199 cm/s) 6 weeks!3 monthsa Abnormal (200!219 cm/s) 1 week High abnormal (220 cm/s or higher) No confirmation needed—recommend treatment a Use the shorter time interval for children ,10 years old. Abbreviations: ICA, Internal carotid artery; MCA, middle cerebral artery; TCD, transcranial Doppler. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 169 b. Occurs in 30!45% of patients with SCD, most commonly in the SS type. The prevalence is likely underestimated due to underreporting by patients. c. Mean age at the first episode of priapism in patients with SCD is about 12!15 years; 75% have their first episode before age 20 years. d. Priapism often occurs in the early morning hours, when normal erections occur, and is probably related to nocturnal acidosis and dehydration. The normal slow blood flow pattern in the penis is similar to the blood flow in the spleen and renal medulla. Failure of detumescence is due to venous outflow obstruction or to prolonged smooth muscle relaxation, either singly or in combination. e. A history of priapism in childhood is associated with later sexual dysfunction, with 10!50% of adults with SCD and a history of priapism reporting impotence. f. Treatment i. At home, patients may try warm baths, oral analgesics, increased oral hydration, exercise, and pseudoephedrine. ii. Patients should be evaluated in an emergency room for episodes lasting over 2 hours. iii. Initial treatment includes IV hydration and parenteral analgesia. iv. Episodes lasting $ 4 hours are associated with an increased risk of irreversible ischemic injury and thus warrant more aggressive management. Urological consultation should be obtained. Treatment involves aspiration of the corpus cavernosum followed by irrigation with or without intracavernous administration of a dilute (1:1,000,000) epinephrine solution. Although published data in SCD are lacking, a dilute solution of phenylephrine, an alpha-adrenergic agent, rather than epinephrine, has also been utilized in some centers. v. Inhaled nitrous oxide (maximum 60%) was associated with detumescence within 4!15 minutes in a case report of two children with priapism; further study is needed. g. The role of transfusion for the management of priapism is controversial and the clinical response is variable. Furthermore, exchange transfusion for acute priapism has been associated with the development of acute neurological events. h. Surgical shunting procedures (corpus cavernosum—corpus spongiosum or cavernosaphenous) may be considered if the previous treatments fail, although shunt occlusion is a common complication. i. Prevention of priapism i. Pseudoephedrine, 30!60 mg orally at bedtime. ii. HU therapy has been employed, although this treatment has not been studied for this indication. iii. Leuprolide injections, a gonadotropin-releasing hormone analog that suppresses the hypothalamic!pituitary access, reducing testosterone production. iv. Phosphodiesterase type 5 inhibitors may have some benefit, but studies of sildenafil and tadalafil have been limited; further research is needed. v. Transfusion protocol for 6!12 months following an episode of priapism requiring irrigation and injection. 5. Splenic sequestration a. Highest prevalence between 5 and 24 months of age in SCD-SS; may occur at older ages in patients taking HU or receiving regular transfusions and with other genotypes (Sβ-thalassemia, SC). b. May occur in association with fever or infection, including parvovirus B19. c. Splenomegaly due to pooling of large amounts of blood in the spleen. d. Rapid onset of pallor and fatigue. Abdominal pain is often present. e. Hemoglobin level may drop precipitously, followed by hypovolemic shock and death. f. Reticulocytosis and nucleated RBCs often present. g. Platelet and white blood cell count also usually fall from baseline. h. Treatment of splenic sequestration is shown in Table 9.8. 6. Transient pure red cell aplasia a. Cessation of red cell production that may persist for 7!14 days with profound drop in hemoglobin. b. Reticulocyte count and the number of nucleated red cells in the marrow sharply decrease; platelet and white blood cell counts are generally unaffected. c. May occur in several members of a family and can occur at any age. d. Almost invariably associated with parvovirus B19 infection. e. Terminates spontaneously usually after about 10 days (recovery occurs with reticulocytosis and nucleated red cells in the blood). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 170 9. Hemoglobinopathies TABLE 9.8 Management of splenic sequestration. Treatment of acute splenic sequestration episode Monitor cardiovascular status, spleen size, and hemoglobin level closely. Normal saline bolus of 10!20 cm3/kg. Red cell transfusion. Administer in small aliquots because transfusion often results in reduction in spleen size with “autotransfusion” of previously trapped red cells. Rapid infusion used for cardiovascular instability. Pain management. Prevention of recurrent splenic sequestration Splenectomy if history of one major or two minor acute splenic sequestration episodes. For children ,2 years old, chronic transfusion therapy can be considered to postpone splenectomy, though this may not prevent recurrent episodes. f. Vaso-occlusive pain and/or splenic sequestration may occur in association with parvovirus B19/transient pure red cell aplasia. g. Treatment i. close monitoring of complete blood count (CBC) and reticulocyte count, ii. red cell transfusion to raise hemoglobin level to no greater than 9!10 g/dL, and iii. monitor siblings with SCD closely [CBC, reticulocyte count, parvovirus polymerase chain reaction, and/or titers]. Chronic complications and end-organ damage 1. Central nervous system (CNS) a. Silent cerebral infarction i. defined as one or more focal T2-weighted signal hyperintensities demonstrated on brain MRI, in the absence of a focal neurological deficit corresponding to the anatomical distribution of the brain lesion; ii. present in approximately 39% of children with SCD-SS and occurs less commonly in other sickle cell genotypes; iii. associated with neuropsychological deficits and impaired school performance; and iv. silent infarcts may progress in size and number over time and are associated with an increased risk of overt stroke. b. Management of children with silent infarcts includes neuropsychological testing and monitoring of academic performance. c. Chronic transfusion therapy to maintain the hemoglobin level above 9 g/dL and the HbS below 30% for children with silent cerebral infarcts is associated with a reduction in infarct recurrence. In a large multicenter trial, new or enlarged silent infarcts or overt stroke occurred in 6% of children receiving transfusions compared with 14% of children in the observation group. d. HU has not been studied for this indication. 2. Cardiovascular system a. Abnormal cardiac findings are present in most patients as a result of chronic anemia and the compensatory increased cardiac output. b. Cardiomegaly is found in most patients and left ventricular hypertrophy occurs in about 50%. c. Prolonged QTc . 440 ms occurs in 9!38% of children with SCD, most commonly the SS type. Prolonged QTc is associated with increased risk of mortality in adults with SCD. d. A moderate-intensity systolic flow murmur is often present. e. Echocardiogram may show left and right ventricular dilatation, increased stroke volume, and abnormal septal motion. Diastolic dysfunction occurs in 11!77% of patients with SCD and is associated with myocardial fibrosis, exercise impairment, and increased risk of mortality. f. Pulmonary hypertension i. Defined as a resting pulmonary artery systolic pressure $ 25 mmHg. Right-heart catheterization is required to make a definitive diagnosis. Noninvasive echocardiography often is used to screen for the possible presence of pulmonary hypertension. Tricuspid regurgitant jet velocity (TRV) of $ 2.5 m/s is an indicator of possible pulmonary hypertension. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 171 ii. Prevalence of pulmonary hypertension documented by right-heart catheterization in adults is estimated at 6!11%, with 10% of these adults having moderate-to-severe pulmonary hypertension (pressure above 45 mmHg). An elevated TRV is found in approximately 30% of adults with SCD. The prevalence of elevated TRV in children appears to be about 11% and is most common with the SS genotype. Diagnosis of pulmonary hypertension by TRV alone has been questioned. Children with elevated TRV should be managed along with a cardiologist. iii. In adults, pulmonary hypertension by right-heart catheterization, elevated TRV, and increased serum N-terminal probrain natriuretic peptide are independent risk factors for mortality; the significance of these findings in children is unclear. iv. A central role for hemolysis and altered NO bioavailability has been postulated. v. The optimal treatment is unknown, but HU or red cell transfusions have been used. Treatment with sildenafil, an agent used to treat pulmonary hypertension in other patient groups, is associated with an increased risk of vaso-occlusive pain episodes. 3. Pulmonary a. Reduced PaO2. b. Reduced O2 saturation. Pulse oximetry may not correlate with PaO2 in steady state. Changes in pulse oximetry are useful for monitoring children with ACS. Daytime and/or nocturnal hypoxemia may be present. c. Chronic lung disease—pulmonary fibrosis: This is a prime contributor to mortality in young adults with SCD. i. Early identification of progressive lung disease using pulmonary function testing is imperative. Aggressive treatment has little benefit in end-stage lung disease and this should be avoided by prophylactic transfusions. ii. The staging system for chronic lung disease is based on clinical, physiological, and radiographic criteria, with progression from stage to stage every 2!3 years. • Stage 1 is characterized by a mild reduction in lung volumes (vital capacity and total lung capacity) and forced expiratory volume in 1 s /forced vital capacity (FVC) ratio (defines airflow obstruction). • Stage 2 is characterized by moderate reduction in these measurements. • Stage 3 is where hypoxemia is first observed during stable periods, and a severe reduction in lung volumes and flows is seen with associated borderline pulmonary hypertension and fibrosis on chest radiograph. • Stage 4 is characterized by severe pulmonary fibrosis and pulmonary hypertension d. Asthma—prevalence appears to be higher than in general population in children with SCD. Asthma is associated with complications of SCD, including pain, ACS, stroke, and pulmonary hypertension. Aggressive management is warranted. Using steroid for asthma exacerbations may lead to rebound VOE. 4. Renal a. Increased renal flow and glomerular filtration rate. b. Enlargement of kidneys; distortion of collecting system. c. Hyposthenuria (urine concentration defect): Hyposthenuria is the first manifestation of sickle cell!induced obliteration of the vasa recta of the renal medulla. Edema in the medullary vasculature is followed by focal scarring, interstitial fibrosis, and destruction of the countercurrent mechanism. Hyposthenuria results in a concentration capacity of . 400!450 mOsmol/kg and an obligatory urinary output as high as 2000 mL/m2 per day, causing the patient to be particularly susceptible to dehydration. The increased urine output is associated with nocturia, often manifesting as enuresis. The treatment of nocturnal enuresis includes behavioral modifications and 1-deamino-8-D-arginine vasopressin at bedtime. d. Hematuria: Papillary necrosis is usually the underlying anatomic defect. The treatment of papillary necrosis is IV hydration and rest. Frank hematuria usually resolves, although bleeding can be prolonged. Antifibrinolytic agents such as epsilon-aminocaproic acid have been used for recalcitrant bleeding with variable success. However, caution must be taken when using this drug because of the risk of thrombosis and urinary obstruction. Evaluation for other causes of hematuria (e.g., renal medullary carcinoma) is indicated for the first episode of hematuria. e. Renal tubular acidification defect. f. Increased urinary sodium loss (may result in hyponatremia). g. Hyporeninemic hypoaldosteronism and impaired potassium excretion are results of renal vasodilating prostaglandin increase in patients with SCD. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 172 9. Hemoglobinopathies h. Proteinuria: Persistent increasing proteinuria is an indication of glomerular insufficiency, perihilar focal segmental sclerosis, and renal failure. Intraglomerular hypertension with sustained elevations of pressure and flow is the prime etiology of the hemodynamic changes and subsequent proteinuria. If proteinuria persists for . 4!8 weeks, angiotensin-converting enzyme inhibitors (i.e., enalapril) are recommended. i. Nephrotic syndrome: A 24-hour urine protein of . 2 g/day, edema, hypoalbuminemia, and hyperlipidemia may indicate progressive renal insufficiency. The efficacy of steroid therapy in the management of nephrotic syndrome in SCD is not clear. Carefully monitored use of diuretics is indicated to control edema. j. Chronic renal failure and uremia. 5. Liver and biliary system a. Chronic hepatomegaly. b. Liver function tests: Increased serum aspartate transaminase and serum alanine transaminase. c. Cholelithiasis. i. Chronic hemolysis with increased bilirubin turnover causes pigmented stones ii. Occurs as early as 2 years old and affects $ 30% by age 18 years iii. Sonographic examinations of the gallbladder should be performed in children with symptoms iv. The treatment for symptomatic cholelithiasis is laparoscopic cholecystectomy. The role of screening and treatment of asymptomatic patients is unclear d. Transfusion-related hepatitis. Hepatitis C is more common in older patients who received red cell transfusions prior to the availability of screening of blood products. e. Intrahepatic crisis: Intrahepatic sickling can result in massive hyperbilirubinemia, elevated liver enzyme values, and a painful syndrome mimicking acute cholecystitis or viral hepatitis. Progression to multiorgan system failure may occur. Early exchange transfusion is indicated. f. Hepatic necrosis, portal fibrosis, regenerative nodules, and cirrhosis are common postmortem findings that may be a consequence of recurrent vascular obstruction and repair. g. Transfusional iron overload, secondary to repeated intermittent or chronic transfusions, may cause hepatic fibrosis. 6. Bones a. Skeletal changes in SCD are common because of expansion of the marrow cavity, bone infarcts, or both. b. Widening of medullary cavity and cortical thinning: Hair-on-end appearance of skull on radiograph. c. Fish-mouth vertebra sign on radiograph. d. Avascular necrosis (AVN): i. SCD is the most common cause of AVN of the femoral head in childhood. AVN of the humeral head is less common. ii. The cumulative incidence rate for AVN of the femoral head in SCD is estimated at 22%. iii. The incidence is higher with coexistent α-thalassemia, in patients who have frequent painful events, history of ACS, and in those with the highest hematocrits. iv. The pathophysiology is due to repeated ischemia!reperfusion injury of vulnerable articular surfaces. v. About 50% of patients are asymptomatic. Symptomatic patients have significant chronic pain and limited joint mobility. vi. The diagnosis is made radiographically and shows subepiphyseal lucency and widened joint space, flattening or fragmentation and scarring of the epiphysis. AVN of femoral head can be detected by MRI before deformities are apparent on radiograph. vii. AVN of the hip may have its onset in childhood, so thorough musculoskeletal examination with concentration on the hips should be performed at least yearly in children with SCD. viii. Progression of early-stage AVN of the femoral hip to collapse is extremely common. • Spontaneous regression of AVN in some typically younger children has been reported. ix. Treatment: • Therapy for AVN is largely supportive, with bed rest, nonsteroidal antiinflammatory drugs, and limitation of movement during the acute painful episode. • Transfusion therapy and HU do not seem to delay the progression of AVN. • Physical therapy is helpful and may reduce the risk of progression. • Core decompression of the affected hip has been reported to reduce pain and stop progression of the disease. In this procedure, avascularized bone is removed to decompress the area with the Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 173 potential for subsequent new bone formation. This procedure seems to be beneficial only in the early stages of AVN and before loss of the integrity of the femoral head. • Total hip replacement: Recent data show favorable outcomes and improved quality of life using cementless grafts. Careful perioperative management, including appropriate hydration, transfusion, antibiotics, and pain management, are needed. 7. Eyes a. Retinopathy: Sickle retinopathy is common in all forms of SCD, but particularly in those patients with SCD, type SC. i. Nonproliferative: Occlusion of small blood vessels of the eye detected on the dilated ophthalmological examination and usually not associated with defects in visual acuity. Treatment is usually not needed. ii. Proliferative: Occlusion of small blood vessels in the peripheral retina may be followed by enlargement of existing capillaries or the development of new vessels. Clusters of neovascular tissue “sea fans” grow into the vitreous and along the surface of the retina. Sea fans may cause vitreous hemorrhage, which results in transient or prolonged loss of vision. Small hemorrhages resorb but repeated leaks cause the formation of fibrous strands. Shrinkage of these strands can cause retinal detachment. iii. Treatment: In proliferative retinopathy, neovascularization may not progress or may regress spontaneously. Indications for treatment include bilateral proliferative disease, rapid growth of neovascularization, and large elevated neovascular fronds. Laser photocoagulation and other methods are used to induce regression of neovascularization. iv. Screening: With proper screening and new methods such as laser surgery, most of the complications of retinopathy can be avoided. Annual ophthalmologic examinations, including inspection of the retina, are indicated for children from the age of 5 years for children with SCD-SC and 8 years for children with SCD-SS. v. Patients should be instructed to seek medical attention for new vision changes, flashing lights, or new visual floaters. b. Angioid streaks: These are pigmented striae in the fundus caused by abnormalities in the Baruch membrane due to iron or calcium deposits or both. They usually produce no problems for the patient, but occasionally they can lead to neovascularization that can bleed into the macula and decrease vision. c. Hyphema: Blood in the anterior chamber (hyphema) rarely occurs secondary to sickling in the aqueous humor, because of its low pH and pO2. Traumatic hyphema may occur as in any individual. Anterior chamber paracentesis should be performed if pressure is increased. d. Conjunctivae: Comma-shaped blood vessels, seemingly disconnected from other vasculature, can be seen in the bulbar conjunctiva of patients with SCD and variants (SS . SC . Sβ-thalassemia). These produce no clinical disability. Their frequency may be related to the number of irreversibly sickled cells in the blood. This abnormality can be identified by using the 1 40 lens of an ophthalmoscope. 8. Ear, nose, and throat a. Sensorineural hearing loss. Up to 12% of pediatric patients have high-frequency sensorineural hearing loss, and as high as 30% of adults in some studies. The pathophysiology may involve sickling in the cochlear vasculature with destruction of hair cells. b. Adenotonsillar hypertrophy giving rise to upper airway obstruction can become a problem from the age of 18 months. The marked hypertrophy is postulated to be compensation for the loss of lymphoid tissue in the spleen. It occurs in $ 18% of patients and up to 50% in some studies. In severe cases, this can cause hypoxemia at night with consequent sickling and should be evaluated with a sleep study. Early tonsillectomy and adenoidectomy may be indicated in these patients. 9. Skin a. Cutaneous ulcers of the legs occur over the external or internal malleoli. Leg ulcers occur less commonly in children, and rarely before age 10 years. Ulcers are most common in homozygous SCD. Ulceration may result from increased venous pressure in the legs caused by the expanded blood volume in the hypertrophied bone marrow. b. Treatment includes rest and elevation, physical protection with soft sponge-rubber doughnut, debridement and scrupulous hygiene, elastic stockings to improve venous circulation, and oral administration of zinc sulfate. If ulcers persist despite optimal care, consider transfusion therapy for 3!6 months. More severe cases can require split-thickness skin grafts. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 174 9. Hemoglobinopathies 10. Growth and development a. Growth delay: i. Birth weight is normal. ii. By 2!6 years of age the height and weight are significantly delayed. The weight is more affected than the height, and patients with SCD-SS and Sβ0-thalassemia experience more delay in growth than patients with SCD-SC and Sβ1-thalassemia. iii. By the end of adolescence, patients with SCD have caught up with controls in height but not weight. The poor weight gain is likely to represent increased caloric requirements in anemic patients with increased bone marrow activity and cardiovascular compensation. iv. Growth hormone levels and growth hormone stimulation studies appear to be normal in most children who have impaired growth. b. Delayed sexual maturation: Tanner 5 is not achieved until the median ages of 17.3 and 17.6 years for girls and boys, respectively. In males, decreased fertility with abnormal sperm motility, morphology, and numbers is prominent. c. Zinc deficiency may contribute to poor growth and delays in sexual maturity; supplementation with elemental zinc, 10 mg daily, was associated with improved linear growth and weight gain in a pilot study of children ages 4!10 with SCD-SS. 11. Functional hyposplenism a. By 6 months of age, mild splenomegaly may be apparent and persists during early childhood, after which the spleen undergoes progressive fibrosis (autosplenectomy). b. Functional reduction of splenic activity occurs in early life due to altered intrasplenic circulation caused by intrasplenic sickling. It can be temporarily reversed by transfusion of normal red cells. c. Children with functional hyposplenia are 300!600 times more likely to develop overwhelming pneumococcal and Haemophilus influenzae sepsis and meningitis than are healthy children; other organisms involved are Gram-negative enteric organisms and Salmonella. The period of greatest risk of death from severe infection occurs during the first 5 years of life. d. Functional hyposplenism may be demonstrated by the following: i. the presence of Howell!Jolly bodies on blood smear, ii. 99mTc-gelatin sulfur colloid spleen scan—no uptake of the radioactive colloid by enlarged spleen, and iii. pitted RBC count . 3.5%. Prognosis 1. The survival time is unpredictable and is related in part to the severity of the disease and its complications. The median age of death is around 43 years. Survival data with early HU use are not yet available. 2. The adolescent and young adult period and transition from pediatric to adult care is a period of high mortality. 3. Causes of death include: a. infection (sepsis, meningitis) with a peak incidence between 1 and 3 years of age; b. ACS/respiratory failure; c. stroke (especially hemorrhagic); and d. organ failure, including heart, liver, and renal failure. Management 1. Comprehensive care: Prevention of complications is as important as treatment. Optimal care is best provided in a comprehensive setting. Recommended screening studies are shown in Table 9.9. 2. Infection: Because of a marked incidence of bacterial sepsis and meningitis and fatal outcome under 5 years of age, the following management is recommended: a. Prophylactic antibiotics: i. All children with SCD should receive oral penicillin prophylaxis starting by 3!4 months of age, with 125 mg bid under 3 years old and 250 mg bid for 3 years and older. ii. In patients allergic to penicillin erythromycin ethyl succinate 10 mg/kg orally twice a day should be prescribed. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 175 TABLE 9.9 Routine health maintenance!related laboratory and special studies in patients with sickle cell disease. Laboratory studies Starting age Frequency Complete blood count/ reticulocyte count At diagnosis Quarterly to yearly; with differential monthly if receiving HU Hemoglobin quantitation At diagnosis Yearly; 2!4 times a year if receiving HU Red cell antigen typing and Rh genotyping At diagnosis ! Liver and renal functions At diagnosis Yearly; monthly if receiving deferasirox Urinalysis 1 year Yearly; monthly if receiving deferasirox HIV, hepatitis B, C Yearly if receiving transfusions Ferritin Every 1!3 months if receiving transfusions Special studies Pulse oximetry At diagnosis Quarterly Pulmonary function 5 years Every 3 years Sleep study If symptoms present Eye examinations 10 years Yearly Transcranial Doppler 2 years At least annually, more frequently if indicated based on prior results Brain MRI/MRA If school difficulties, abnormal or repeatedly conditional TCD, neurological symptoms; consider baseline at 5!6 years for SCD-SS Abdominal ultrasound If symptoms of cholelithiasis Hip radiograph/MRI If symptoms of AVN Echocardiogram 10 years Every 3 years or more frequent if abnormal Abbreviations: AVN, Avascular necrosis; HU, hydroxyurea; MRI, magnetic resonance imaging; SCD, sickle cell disease; TCD, transcranial Doppler. iii. Penicillin prophylaxis should be continued at least through the age of 5 years. As the incidence of invasive bacterial infections declines with age, it may be reasonable to discontinue penicillin in older children. However, given that the rate of infection remains higher than the rate in individuals with spleens, some centers advocate continuing penicillin indefinitely. b. Vaccination: i. All children with SCD should receive routine childhood immunizations, including conjugate H. influenzae and Hepatitis B. ii. The 23-valent pneumococcal vaccine (PPV-23) should be administered at 2 years of age with a booster administered 5 years later. iii. The conjugate 13-valent pneumococcal vaccine (PCV-13) should be administered according to the routine childhood schedule. Children aged 6!18 years who have not previously received PCV-13 should receive a single dose of the vaccine. iv. Adults who have received PPV-23 should receive a single dose of PCV-13 $ 1 year after receiving PPV-23. v. Meningococcal vaccination should also be administered. vi. Influenza virus vaccine should be given yearly, each fall. c. Early diagnosis and treatment of infections: i. Families should be instructed to call their physician immediately if their child develops a single temperature .38.5" C (by mouth) or three elevations between 38" C and 38.4" C. The child should be seen immediately by a physician. ii. Evaluation should include a careful examination, CBC with differential and reticulocyte count, and blood culture. Chest radiograph is obtained in children under 3 years of age and in older children with respiratory symptoms. Urinalysis and culture are indicated in children ,3 years or in older children with symptoms. Lumbar puncture is performed in young infants (,2!3 months) and in older infants Lanzkowsky’s Manual of Pediatric Hematology and Oncology 176 9. Hemoglobinopathies and children with symptoms of meningitis. Other studies such as viral studies, stool cultures, and sputum cultures are performed based on symptoms. iii. Prompt antibiotic treatment with a broad-spectrum IV antibiotic that covers encapsulated organisms, such as ampicillin or a third-generation cephalosporin should be given. iv. Many centers recommend inpatient hospitalization for all children younger than 5 years because this group is at highest risk of infection. In addition, all children, regardless of age, with the following highrisk features should be admitted: • Ill appearance, • ACS, • meningeal signs, • enlarging spleen, • elevated leukocyte count (. 30,000/mm3), and • falling blood counts or low reticulocyte count. v. A subset of lower risk children, over the age of 12 months and without the previous high-risk features, may be considered for discharge after a shorter period of observation (4!18 hours) after having received a long-acting antibiotic such as ceftriaxone. This option should only be considered if the family can be contacted readily, follow-up is ensured, and continuous blood culture monitoring is available. 3. Treatment of specific complications of SCD are provided earlier in the acute and chronic complication sections: 4. Transfusion therapy: a. Indications for transfusions in SCD are shown in Table 9.10. b. Risks of transfusion include infection (hepatitis B virus, hepatitis C virus, HIV, and bacterial), alloimmunization, and iron overload. c. The incidence of alloimmunization is 17.6%: Mostly Kell (26%) and Rh [E (24%) and C (16%), respectively] antibodies. Other antibodies also occur in the following order of frequency: Jkb (10%), Fya (6%), M (4%), Lea (4%), S (3%), Fyb (3%), e (2%), and Jka (2%). d. All children with SCD should have a red cell phenotype when available identified at diagnosis. This allows the determination of the child’s red cell antigen phenotype before any transfusion. e. Patients should receive blood that is phenotypically matched to the patient for the Rh and Kell antigens. However, a high rate of Rh alloimmunization may still be seen with such an approach, likely due to the high prevalence of Rh variants that are not detected by routine phenotyping in the African-American population. Whether RH genotyping of patients and donors may further reduce the rate of alloimmunization due to variant RH alleles needs further study. TABLE 9.10 Generally accepted indications for transfusions in sickle cell disease. Episodic transfusion Overt stroke Transient pure red cell aplastic episode Splenic sequestration Acute chest syndrome Preoperatively for surgical procedure with general anesthesiaa Acute multiorgan failure Retinal artery occlusion Chronic transfusion Stroke Abnormal transcranial Doppler ultrasound Silent cerebral infarcts Recurrent acute chest syndrome Pulmonary hypertension Recurrent severe pain a Moderate- to high-risk surgical procedures. Controversial for low-risk procedures. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Sickle cell disease 177 f. Blood should be leukoreduced, and sickle negative blood should be administered to children receiving chronic transfusion therapy to allow accurate monitoring of HbS levels. g. Chronic red cell transfusion therapy or repeated intermittent transfusions lead to iron overload. Complications of iron overload include hepatic fibrosis, endocrinopathies, and cardiac disease and are best defined for thalassemia. The prevalence of certain complications such as heart disease is lower in SCD than in thalassemia. The treatment is similar to the approach used for thalassemia described later in this chapter. h. In SCD, exchange transfusion limits or prevents iron loading and should be utilized when possible for chronic transfusion therapy. 5. Induction of HbF: a. Sustained elevations in HbF ($20%) are associated with reduced clinical severity in SCD, as HbF interferes with HbS polymerization and RBC sickling. b. HU is the only approved drug for HbF modulatory therapy. Other effects of HU include increased red cell hydration and decreased expression of red cell adhesion molecules, increased NO production, and lowering of white blood cell count, reticulocytes, and platelets. c. Numerous studies in adults and children have shown the beneficial effects of HU in SCD-SS and Sβ0-thalassemia, including reduced number of VOEs, reduced incidence of ACS, and reduced mortality. d. In infants of ages 9!18 months, treatment with HU significantly reduces the number of episodes of dactylitis, pain, and ACS. Early use of HU is not associated with a reduction in the development of splenic dysfunction or glomerular hyperfiltration in these children. HU is FDA approved for children over 2 years of age with SCD, although recommendations are to offer treatment at 9 months of age. There are no large studies on the use of HU for patients with SCD-SC and Sβ1-thalassemia. e. Dose: The starting dose of HU is 15!20 mg/kg per day. It is increased every 8 weeks by 5 mg/kg per day until a total dose of 35 mg/kg per day is reached or until a favorable response is obtained or until signs of toxicity appear. Evidence of toxicity includes: i. neutrophil count ,1000/mm3; ii. platelet count ,80,000/mm3; iii. hemoglobin drop of 2 g/dL; and iv. absolute reticulocyte count ,80,000/mm3 f. Response is indicated by clinical improvement (reduction in VOE, ACS, etc.) and by laboratory response, including rise in HbF (typically 10!20%), a rise in hemoglobin level of 1!2 g/dL, and increased MCV. g. Follow-up: When HU is started, the patient should be monitored with a CBC every 2!4 weeks and HbF level at least quarterly. Once a stable and maximum tolerated dose is obtained, the patient can be monitored with CBCs monthly to quarterly. h. Indications. Given the clinical benefits of HU, treatment with this drug should be discussed with the families of all children, 9 months of age and older, with SCD-SS or Sβ0-thalassemia. Frequent VOE and/ or ACS are indications for treatment. i. Side effects. i. myelosuppression, ii. rarely hair loss; skin and nail pigment changes, iii. headache, iv. gastrointestinal (GI) disturbance, v potential birth defects (HU should not be taken during pregnancy and birth control should be discussed with patients on HU), and vi. reduced sperm count and motility. j. Another drug, IMR-687, a selective phosphodiesterase 9 inhibitor that raises HbF levels is being studied in phase 2 trials for patients with SCD. 6. Newly approved therapies: a. L-Glutamine was approved by the FDA in 2017 for the prevention of acute SCD complications in adults and children of 5 years and older. The proposed mechanism of action is to alter the redox state of RBCs and decrease RBC adhesion to endothelium. It is administered orally as a powder reconstituted in liquid at a dose of 5!15 g twice daily, based on weight. In a randomized placebo-controlled phase 3 trial, over 48 weeks of treatment, patients taking L-glutamine had a 25% decrease in pain episodes and a 33% Lanzkowsky’s Manual of Pediatric Hematology and Oncology 178 9. Hemoglobinopathies decrease in emergency department visits and hospitalizations. The medication is well tolerated and the primary side effects are constipation, nausea, headache, abdominal pain, cough, and pain. b. Voxelotor received accelerated FDA approval in 2019 for SCD in adults and children 12 years and older. It is a small molecule inhibitor of HbS polymerization and is administered orally at 1500 mg/day (tablet form only). In a randomized placebo-controlled phase 3 trial (HOPE), the 1500-mg voxelotor dose resulted in a greater proportion of patients with a hemoglobin increase of .1 g/dL (51% vs 7%), with a mean Hb change of 1.1 g/dL. Although there was a decrease in markers of hemolysis, data have not yet shown a significant change in VOEs or other acute SCD complications. The medication is well tolerated with primary adverse effects of headache, diarrhea, abdominal pain, nausea, rash, fatigue, and pyrexia. c. Crizanlizumab—see VOE pain section. 7. HSCT: a. Currently HSCT [including umbilical cord blood (UCB)] is the only curative therapy. b. The results of transplantation are best when performed in children with a sibling donor who is HLA identical. Only about 15% of patients with SCD are likely to have an HLA-identical sibling donor. Unrelated donor stem cell transplantation, haploidentical stem cell transplantation, and reduced intensity conditioning protocols are under investigation and should be considered only in the context of a clinical trial in an experienced center. c. Eligibility criteria for HSCT for SCD-SS or SCD-Sβ0-thalassemia include one or more of the following complications: i. stroke or CNS event lasting longer than 24 hours; ii. impaired neuropsychological performance with abnormal brain MRI; iii. recurrent ACS (at least two episodes in the last 2 years) or stage 1 or 2 sickle lung disease; iv. recurrent severe, debilitating VOE (three or more severe pain events per year for the past 2 years); vi. recurrent priapism; vii. sickle nephropathy; viii. bilateral proliferative retinopathy with major visual impairment in at least one eye; ix. AVN of multiple joints; and x. significant red cell alloimmunization during long-term transfusion therapy. d. Outcomes: With HLA-matched sibling donor HSCT, the survival rate is .90%. Over 85% survive free from SCD after HLA-matched sibling HSCT. Patients who have stable engraftment of donor cells experience no subsequent sickle cell!related events and stabilization of preexisting organ damage. The majority of patients have the stabilization or improvement of cerebrovascular disease after transplantation. Similarly, other organ toxicity (such as lung disease) related to SCD tends to stabilize posttransplantation. Linear growth is normal or accelerated after transplantation in the majority of patients. About 5% of the patients develop clinical grade III acute or extensive graft-versus-host disease (GVHD) (see Chapter 30: Hematopoietic Stem Cell Transplant and Cellular Therapy). The risk of secondary cancers is estimated to be ,5%. e. Recommendations: i. Children with SCD who experience significant sickle cell complications should be considered for HSCT. ii. HLA typing should be performed on all siblings. iii. Families should be counseled about the collection of UCB from prospective siblings/donors. iv. For severely affected children who have HLA-identical sibling donors, families should be informed about the benefits, risks, and treatment alternatives regarding HSCT. 8. Gene therapy approaches: Several different approaches are currently under study. The general method involves the collection of autologous CD34 1 hematopoietic stem cells, ex vivo manipulation by gene addition or gene editing, followed by conditioning and reinfusion of the modified stem cells. Several trials with promising early data in patients with SCD are highlighted: a. Gene addition: Adding an antisickling β-globin variant or γ-globin. i. Lentiglobin BB305: Utilizes a beta globin gene that contains a single amino acid substitution (βT87Q) that confers antisickling properties. Myeloablative conditioning is utilized. Preliminary results of a phase 1/2 study showed reduction in HbS to B50% in adolescent and adult patients with a substantial decrease in VOE and ACS. A phase 3 study is underway. ii. ARU-1801: Utilizes a lentiviral vector containing a gamma globin gene. Preliminary results of a phase 1/2 study with reduced intensity conditioning showed sustained HbF expression and some reduction in symptoms in the first two adult patients treated. iii. Other active studies that utilize lentiviral vectors containing antisickling β-globin genes are underway. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Hemoglobin C 179 b. Manipulation of HbF repressor BCL11A to induce HbF. i. Lentiviral vector expressing a short-hairpin RNA against BCL11A. Preliminary results of a phase 1/2 trial showed B20% HbF induction in three analyzed patients. ii. Gene editing of the erythrocyte-specific enhancer of the BCL11a gene: Preliminary results of a phase 1/2 trial utilizing CRISPR Cas9 editing and myeloablative conditioning showed that the first adult patient treated had a hemoglobin level of 11.8 g/dL with 46.1% HbF at 9 months post infusion. A second trial utilizing zinc finger nuclease gene editing is recruiting adult subjects. c. Manipulation of regulatory elements of the γ-globin genes to induce HbF. d. Gene editing approaches for the correction of the HbS mutation are in early preclinical trials. 9. Psychological support. As for any chronic disease, patients require psychological support. Major problems that occur are: a. coping with chronic pain, b. inability to keep up with peers, c. fears of premature death, d. delayed sexual maturity, and e. increased doubts about self-worth. Sickle cell trait (heterozygous form, AS) Pathophysiology The concentration of HbS in red cells is low, and sickling does not occur under normal conditions. Hematology 1. Indices—usually normal 2. Blood smears—normal with few target cells 3. Sickle cell preparation—reducing agents (e.g., sodium metabisulfite) to induce sickling in vitro 4. Hemoglobin electrophoresis—AS pattern (HbA 55!60%; HbS, 35!45%) Clinical features 1. Usually asymptomatic. 2. Hematuria rarely. 3. Increased propensity for renal medullary cancer. 4. Exertional rhabdomyolysis-/exercise-related sudden death. Ensure adequate hydration with sports activities. 5. Complicated hyphema—with secondary hemorrhage, increased intraocular pressure, and central retinal artery occlusion. This requires evaluation/treatment by an ophthalmologist. 6. Infarction rare, occurring during flights in unpressurized aircraft. Significance The genetic implications mandate counseling. Table 9.1 lists the differential diagnosis of sickle cell syndromes. Hemoglobin C Basic features and pathology 1. Carrier state—2% in African-Americans. 2. Amino acid substitution (the same codon in the β-chain as in HbS)—lysine for glutamic acid. 3. HbC tendency to form rhomboidal crystals with increases in osmolality—red cell deformability impaired and splenic sequestration increased. 4. HbC trait (heterozygous form, AC) is asymptomatic, with only genetic implications. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 180 9. Hemoglobinopathies HbC disease (homozygous CC) 1. Anemia—usually mild, microcytic, and hemolytic. 2. Blood smear—numerous target cells, as well as some spherocytes (the result of membrane loss in the spleen); a bar of crystalline hemoglobin across cell due to alteration in intracellular hemoglobin is a frequent finding. 3. Hemoglobin electrophoresis—CC pattern. 4. Clinical manifestations—mild: a. Splenomegaly. b. Dehydration, leading to marked hemolysis and microcirculatory problems. c. Retinopathy has been observed in individuals with HbC disease. Hemoglobin E 1. Mutation in β-globin gene that creates an alternate splice site which leads to decreased production of an abnormal globin chain. 2. Heterozygotes (HbE trait) and homozygotes (HbE disease) are clinically asymptomatic. The MCV is reduced and target cells are seen on peripheral blood smear. Mild anemia is seen with HbE disease and less commonly with HbE trait. Important to distinguish HbE disease from HbE/β-thalassemia as the latter is clinically significant. 3. HbE/β-thalassemia—causes nontransfusion-dependent or transfusion-dependent thalassemia (NTDT or TDT) phenotype (see later in chapter). 4. HbE/HbS—coinheritance of HbE and HbS leads to a form of SCD, type SE. Unstable hemoglobins 1. Unlike the amino acid substitutions in HbS and HbC, which affect the polarity of the external surface of the hemoglobin molecule, resulting in polymerization (HbS) or crystallization (HbC), the substitutions in unstable hemoglobins are more likely to occur within the heme cavity or pocket of the α- or β-polypeptide chain. Substitution in the region of heme attachment causes gross molecular instability. 2. Changes in the oxygen affinity have also been found in some of the unstable hemoglobins and some of the M hemoglobins. An increase in oxygen affinity results in greater tissue anoxia and greater erythropoietin stimulation for a given level of anemia. In at least one hemoglobinopathy, hemoglobin Chesapeake, the only clinical manifestation is mild polycythemia. 3. Table 9.11 lists the various clinical manifestations that suggest unstable hemoglobinopathies. Table 9.12 presents laboratory data that suggest unstable hemoglobinopathies. 4. The hereditary methemoglobinopathies are closely related to the unstable hemoglobins. The substitution in these cases is also in the region of heme attachment, but it results in increased susceptibility to oxidation of heme Fe21 to Fe31 with consequent methemoglobin accumulation and cyanosis rather than hemolysis. There is some overlap between these two disorders, insofar as there is an increase in methemoglobin formation in most types of unstable hemoglobinopathies. Thalassemias Basic features 1. Thalassemia syndromes are characterized by varying degrees of ineffective hematopoiesis and increased hemolysis. 2. Clinical syndromes are divided into α- and β-thalassemias, each with varying numbers of their respective globin genes mutated or deleted. There is a wide array of genetic defects and a corresponding diversity of clinical syndromes. Severity usually correlates with the degree of chain imbalance, and excess α-globin chains are more toxic than β-globin chains. 3. Most β-thalassemias are due to point mutations, usually in both of the two β-globin genes (chromosome 11), which can affect every step in the pathway of β-globin expression from the initiation of transcription to Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thalassemias TABLE 9.11 181 Clinical manifestations of unstable hemoglobins. Chronic nonspherocytic hemolytic anemia, varying from mild to severe Intraerythrocyte inclusions (Heinz bodies) demonstrable by the incubation of the cells with brilliant cresyl blue or methyl violet Urinary dipyrrolic pigment excretion Drug-induced hemolytic anemia Methemoglobinemia Cyanosis Polycythemia Chronic hemolytic anemia with normal hemoglobin electrophoresis Variable response of hemolytic anemia to splenectomy TABLE 9.12 Laboratory data in unstable hemoglobinopathies. Chronic hemolytic anemia with normal red cell morphology, red cell enzymes, and hemoglobin electrophoresis Abnormal heat stability test; tendency to precipitate on heating at 50" C The presence of Heinz bodies Raised methemoglobin levels messenger RNA (mRNA) synthesis to translation and posttranslation modification. A mutation in a single β-globin gene inherited along with triplicated α-genes also may cause a β-thalassemia syndrome. Autosomal dominant forms of β-thalassemia also occur rarely. Fig. 9.1 shows the organization of the genes (i.e., ε and γ, which are active in embryonic and fetal life, respectively) and activation of the genes by the locus control region, an essential regulatory element. 4. Mutations of β-globin genes occur predominantly in children of Mediterranean, African, Asian, and Southeast Asian ancestry. 5. There are four genes for α-globin synthesis (two on each chromosome 16). Most α-thalassemia syndromes are due to deletions of the α-globin genes rather than to point mutations. 6. Deletions of α-globin genes are most common in those of Southeast Asian and African ancestry. Main genetic variants 1. β-Thalassemia a. β0-Thalassemia: No detectable β-chain synthesis due to absent β-chain mRNA. b. β1-Thalassemia: Reduced β-chain synthesis due to reduced or nonfunctional β-chain mRNA. c. δβ-Thalassemia: δ- and β-chain genes deleted. d. Eβ-Thalassemia: HbE (lysine-glutamic acid at 26) combined with β-thalassemia mutation. May be β0 or β1. e. Hb Lepore: A fusion globin due to unequal crossover of the β- and δ-globin genes (the globin is produced at a low level because it is under δ-globin regulation). 2. α-Thalassemia a. Silent carrier α-thalassemia: Deletion of one α-globin gene. b. α-Thalassemia trait: Deletion of two α-globin genes. c. Hb Constant Spring: Abnormal α-chain variant produced in very small amounts, thereby mimicking deficiency of the gene. d. HbH disease: Deletion of three α-globin genes resulting in significant reduction of α-chain synthesis. e. Hydrops fetalis: Deletion of all four α-globin genes; no normal adult or HbF production. f. In many populations, α- and β-thalassemia and structural hemoglobin variants (hemoglobinopathies) exist together, resulting in a wide spectrum of clinical disorders. g. Tables 9.13 and 9.14 list some features of the heterozygous and homozygous states of β-thalassemia and its variants. Table 9.15 lists the α-thalassemia syndromes. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 182 9. Hemoglobinopathies Chromosome 11 β-globin locus ε 5 4 3 2 1 Locus Control Embryonic Region (LCR) γ G A γ δ β Fetal Adult HbF (α2γ2) HbA2 HbA (α2δ2) (α2β2) FIGURE 9.1 The structure of the human β-globin locus in chromosome 11. The LCR regulates the transcription of the different genes at each indicated developmental stage. Abbreviation: LCR, Locus control region. TABLE 9.13 Heterozygous states of β-thalassemia and variants. Type HbA2 HbF β -Thalassemia Increased Normal to slightly increased β -Thalassemia Increased Normal to slightly increased δβ-Thalassemia Normal Increased (5!15%) HPFH Normal Increased (15!30%) 1 0 Abbreviations: HbF, Fetal hemoglobin; HPFH, hereditary persistence of fetal hemoglobin. β-Thalassemia: homozygous or compound heterozygous forms Pathogenesis 1. Variable reduction of β-chain synthesis (β0, β1, and variants). 2. Relative α-globin chain excess resulting in intracellular precipitation of insoluble α-chains. 3. Increased but ineffective erythropoiesis with many red cell precursors prematurely destroyed; related to α-chain excess. 4. Shortened red cell life span; variable splenic trapping. Sequelae 1. Hyperplastic marrow (bone marrow expansion with cortical thinning and bony abnormalities). 2. Increased iron absorption and iron overload (especially with repeated blood transfusion), resulting in: a. fibrosis/cirrhosis of the liver, b. endocrine disturbances (e.g., diabetes mellitus, hypothyroidism, hypogonadism, hypoparathyroidism, and hypopituitarism), c. skin hyperpigmentation, and d. cardiac hemochromatosis causing arrhythmias and cardiac failure. 3. Hypersplenism: a. shortened red cell life (of autologous and donor cells) b. leukopenia c. thrombocytopenia Hematology 1. Anemia—hypochromic, microcytic. 2. Mild reticulocytosis. 3. Leukopenia and thrombocytopenia (may develop with hypersplenism). 4. Blood smear—target cells, hypochromia, extreme anisocytosis, poikilocytosis, polychromasia, punctate basophilia, and nucleated RBCs. 5. Increased HbF and HbA2. 6. Bone marrow—erythroid hyperplasia, may be megaloblastic (due to folate depletion). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 183 Thalassemias TABLE 9.14 Homozygous or compound heterozygous states of β-thalassemia and variants. Type Anemia δ-Globin chain β-Globin chain β-Globin mRNA β-Globin gene mutation 1 β -Thalassemia Severe Present Decreased Decreased Point mutations or deletions β -Thalassemia Severe Present Absent Absent or abnormal Point mutations or deletions δβ-Thalassemia Mild Absent Absent Absent Deletion mutation HPFH None Absent Absent Absent Point mutations or deletions 0 Abbreviations: HPFH, Hereditary persistence of fetal hemoglobin; mRNA, messenger RNA. TABLE 9.15 α-Thalassemia syndromes. Number of α-genes deleted Newborn Hb Barts (γ4) (%) α/β Synthesis ratio 1 1!2 0.8!0.9 No anemia; no microcytosis; detectable by genetic interaction (i.e., two silent carriers can produce a child with α-thalassemia trait; a silent carrier and a person with α-thalassemia trait can produce a child with HbH disease); also detectable by molecular studies 2 Heterozygous α-thalassemia trait OR Homozygous silent carrier OR Homozygous Hb Constant spring Hemoglobin H Deletional: Heterozygous 3 disease α-thalassemia trait/silent carrier OR α-Thalassemia trait/ Constant Spring or other point mutation Hydrops Homozygous 4 fetalis α-thalassemia trait Hb Barts (γ4) 3!10 0.7!0.8 Microcytosis; hypochromia; mild anemia 25 0.3!0.6 Hemolytic anemia of variable severity; relatively little ineffective erythropoiesis; no transfusion requirement; intermittent transfusions may be needed (especially with illness or stressors); HbH (β4) present 80!100 0 Death in utero or shortly after birth Syndrome Genetics Silent carrier of Heterozygous silent α-thalassemia carrier α-Thalassemia trait Comments Biochemistry 1. Raised bilirubin (chiefly indirect). 2. Evidence of liver dysfunction (late, as cirrhosis develops). 3. Evidence of endocrine abnormalities [e.g., diabetes (typically late), hypogonadism (low estrogen and testosterone), hypothyroidism (elevated thyroid stimulating hormone), growth hormone deficiency]. 4. Elevated transferrin saturation and ferritin levels. Clinical features Because of the variability in the severity of the fundamental defect, there is a spectrum of clinical severity, which considerably influences management. Homozygous or compound heterozygous thalassemia that does not require regular transfusions for management is known as NTDT (also known as β-thalassemia intermedia) and if regular transfusions are required, TDT (also known as β-thalassemia major). If untreated, 80% of patients die in the first decade of life. With current management, including monitoring of iron burden with MRI and chelation therapy, life expectancy has dramatically increased. Patients now reach the seventh decade of life and are expected to live even longer. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 184 9. Hemoglobinopathies Complications Complications develop as a result of ineffective erythropoiesis and hemolysis (in patients who are undertransfused or in untransfused NTDT) as well as from iron overload. These complications include: 1. Anemia. 2. Failure to thrive in early childhood. 3. Growth retardation, delayed puberty, primary amenorrhea in females, and other endocrine disturbances secondary to chronic anemia and iron overload. 4. Jaundice, usually mild; cholelithiasis. 5. Hepatosplenomegaly, which may be massive; hypersplenism. 6. Bone abnormalities. a. Abnormal facies, prominence of malar eminences, frontal bossing, depression of bridge of the nose, and exposure of upper central teeth. b. Skull radiographs showing hair-on-end appearance due to widening of diploic spaces. c. Osteopenia and osteoporosis are common. The risk is directly proportional to age (the prevalence of osteoporosis is about 60% in patients 20 years and older). Multifactorial causes include medullary expansion, deficiency of estrogen and testosterone, nutritional deficiency (including calcium, vitamin D, and zinc), chelator effects and genetic factors. d. Fractures may occur due to marrow expansion, abnormal bone structure, and osteopenia. 7. Pulmonary hypertension (suggested by TRV of $ 2.5 cm/s) occurs in both TDT and NTDT. Splenectomy may exacerbate this risk, particularly in patients who are not regularly transfused. 8. Thrombosis that is exacerbated by splenectomy. 9. Leg ulcers. Causes of death 1. Congestive heart failure. 2. Arrhythmia. 3. Infection. 4. Multiple organ failure due to iron overload. 5. Complication of HSCT. 6. Malignancy. Management Transfusion therapy 1. Indications for the initiation of regular red cell transfusions include hemoglobin level ,7 g/dL (on at least two measurements; some patients with hemoglobin E β-thalassemia may tolerate hemoglobin levels below 7 g/dL) OR: a. poor growth, b. facial bone changes, c. fractures, and d. the development of other complications (pulmonary hypertension, extramedullary hematopoiesis, etc.). 2. Transfusion regimen: a. Goal is to maintain the pretransfusion hemoglobin .9.5 g/dL. Higher trough levels may be indicated in the setting of heart failure or clinical symptoms. b. Typical programs involve transfusion of 10!15 cm3/kg of packed leukodepleted red cells every 3!5 weeks. c. Blood should be matched for ABO, Cc, Ee, and Kell antigens to reduce the risk of alloimmunization (some centers perform extended red cell antigen matching). 3. Goals of successful transfusion regimen: a. maximizing growth and development, b. minimizing extramedullary hematopoiesis and decreasing facial and skeletal abnormalities, c. reducing excessive GI iron absorption, d. retarding the development of splenomegaly and hypersplenism by reducing the number of red cells containing α-chain precipitates that reach the spleen, and e. reducing and/or delaying the onset of complications (e.g., cardiac). Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thalassemias 185 4. Causes of iron overload: a. repeated red cell transfusions (regular in TDT and intermittent in NTDT) and b. increased absorption of dietary iron, especially NTDT. 5. Complications of iron overload include: a. endocrine disturbances (e.g., growth retardation, pituitary failure with impaired gonadotropins, hypogonadism, insulin-dependent diabetes mellitus, adrenal insufficiency, hypothyroidism, hypoparathyroidism, osteopenia, and osteoporosis), b. cirrhosis of the liver and liver failure (exacerbated if concomitant hepatitis B or C infection), c. cardiac failure and arrhythmias due to myocardial iron overload, and d. skin bronzing. Monitoring iron overload A number of tests are available to monitor iron loading, including: 1. Serum ferritin is particularly useful to follow trends but a measurement of ferritin in a chronically transfused patient may not accurately reflect the iron burden. Value may be altered by infection, inflammation, and vitamin C deficiency. 2. Liver iron concentration (LIC): Target range on iron chelation typically is 2!5 mg Fe/g dw (dry weight). LIC of $15 mg/g dw of liver is associated with an increased risk of cardiac disease and death. Methods to measure LIC include: a. MRI: Using R2 or R2* methodologies. MRI is noninvasive and has become the most frequently utilized modality to assess LIC. b. Superconducting quantum interference device: Highly specialized equipment is required and is available in only a few centers worldwide. c. Liver biopsy: The gold standard, but invasive. This is the method of choice if histopathological examination is needed. 3. Cardiac iron measurement by T2* MRI. Cardiac iron may be high even if the LIC is low, particularly in patients with a history of poorly controlled iron overload in the past with recent intensification of chelation. a. T2* $ 20 ms indicates minimal cardiac iron loading and is the goal level. b. T2* of 10!19 ms indicates mild-to-moderate cardiac iron loading. This result should prompt a discussion with patient/family about adherence with chelation. Intensification of chelation may be warranted. c. T2* , 10 ms indicates severe iron loading and is associated with a high risk of cardiac disease (arrhythmias, congestive heart failure). Improved adherence and/or intensification of chelation therapy (with consideration for dual therapy) is indicated. Chelation therapy 1. The objectives of chelation therapy are: a. bind and detoxify free (nontransferrin bound) extracellular iron b. remove excess intracellular iron c. maintain a safe level of body iron burden i. reduce previous iron loading ii. reverse organ dysfunction iii. prevent new iron loading 2. Chelation therapy is generally initiated after 1!2 years of regular transfusions. Chelation typically is not used in children younger than 2 years old, though earlier initiation of deferiprone was safe and effective in a pilot study. Indications for chelation therapy in patients receiving chronic transfusions include: a. received $ 10!20 transfusions b. serum ferritin level .1000 ng/mL on two occasions when well c. LIC . 5 mg/g dw 3. Transfusion requirements and iron burden should be monitored closely and doses of chelation adjusted to maintain LIC at 2!5 mg/g dw. If LIC determination is not available, a serum ferritin level between 500 and 1500 ng/mL is a reasonable goal. Some centers advocate more aggressive chelation, often using deferiprone, to maintain “normal” body iron burden, which has been associated with reversal of some endocrine complications. The safety of this approach has not been studied in children. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 186 TABLE 9.16 9. Hemoglobinopathies Properties of common chelators. Property Deferoxamine Deferiprone Deferasirox Chelator:iron binding 1:1 3:1 2:1 Route of administration Subcutaneous or intravenous Oral tablet or solution Oral dispersible tablet Oral film-coated tablet or granules Usual dosage 25!50 mg/kg per day 75!99 mg/kg per day 20!40 mg/kg per day 14!28 mg/kg per day Schedule Administered over 8!24 h, 5!7 days/week Divided three times a day; new twice daily tablet formulation available Daily Route of excretion Urine/feces Urine Feces Adverse effects Local reactions—swelling, rash Ophthalmologic—cataracts, reduction of visual fields and visual acuity and night vision Hearing impairment Bone abnormalities Pulmonary Neurologic Allergic reactions Gastrointestinal disturbances Transaminase elevations Agranulocytosis/ neutropenia Arthralgia Gastrointestinal disturbancesa Transaminase elevations Hepatic failure Gastrointestinal bleeding Rise in serum creatinine Proteinuria Renal tubular dysfunction Rash Weekly complete blood count with differential Monthly blood urea nitrogen, creatinine, hepatic transaminases (also obtain 2 weeks after starting the medication), and urinalysis Special monitoring considerations a May be reduced with film-coated tablet and granule forms. Adapted from Kwiatkowski, J.L., 2013. Evaluation and treatment of transfusional iron overload in children. Pediatr. Clin. North. Am. 60 (6), 1393!1406, with permission. 4. Currently available options for chelation therapy in the United States include parenterally administered deferoxamine and two oral chelators, deferasirox and deferiprone (approved as a second-line agent). The properties of the common chelators are summarized in Table 9.16. a. Deferoxamine was the first available chelator, in clinical use for about 50 years. Due to its poor oral bioavailability, this drug must be administered parenterally, usually as a subcutaneous infusion over 8!24 hours. Potential complications of deferoxamine are listed in Table 11.16. Audiological and ophthalmological toxicities are more common when the iron burden is low relative to the chelator dose. Similarly, bone changes, including metaphyseal dysplasia, are more common in young children with lower iron burden. Thus it is important to avoid “overchelation” in all patients, and lower doses of chelation therapy should be used in young children to avoid toxicity. To avoid overchelation, it is recommended to keep the ratio of the mean daily deferoxamine dose (mg/kg per day) to serum ferritin level (ng/mL) below 0.025. Nightly subcutaneous administration of deferoxamine is time-consuming and painful and interferes with the lifestyle of the patient. For this reason, treatment adherence is often suboptimal with poorly controlled iron burden. b. Deferasirox. The drug initially was supplied as orally dispersible tablets (Exjade), which are dissolved in a glass of water or apple juice and administered 1.5 hours before meals. Subsequently, film-coated tablet and granule forms (Jadenu) became available; dosing for these forms is 30% lower than the dispersible tablet form. Studies have shown efficacy similar to that of deferoxamine. GI disturbances, including abdominal pain, nausea, vomiting, and diarrhea, are common and may improve with continued administration of the drug. The GI effects may be related to lactose intolerance as lactose is present in the dispersible tablet drug preparation and may be less with the film-coated tablet and granule formulations. Elevations in hepatic transaminases to more than five times above normal can occur, and fulminant hepatic failure has been reported rarely. Liver function tests should be measured every 2 weeks for the first month after starting the medication and tested monthly thereafter. Gastric and duodenal ulcers also have been reported and complaints of abdominal pain should be investigated. Elevations in serum creatinine are also common, although renal insufficiency is rare. Renal function Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thalassemias 187 should be monitored monthly. Proteinuria also can develop and urinalysis with protein:creatinine should be followed. Less commonly, renal Fanconi syndrome can develop and may be more common with low iron burden. c. Deferiprone is a second-line agent for patients with TDT. The most significant side effect is agranulocytosis that occurs in 1!2% of patients; milder forms of neutropenia also may occur. Therefore weekly monitoring of the CBC with differential is required. The drug should be held during all febrile illnesses, and a CBC with differential should be checked. Other adverse effects include arthropathy and elevated hepatic transaminases. Deferiprone appears to be particularly useful in reducing cardiac iron overload either as a single agent, or in combination with deferoxamine or deferasirox. Splenectomy 1. Splenectomy reduces the transfusion requirements in patients with hypersplenism. It is used in patients with severe leukopenia and/or thrombocytopenia due to hypersplenism and for patients with very high annual packed RBC requirements ( . 250 mL/kg per year) and uncontrolled iron overload. 2. More recently, splenectomy is utilized less frequently due to the increased risk of pulmonary hypertension, thromboembolism, and infection after splenectomy. 3. At least 2 weeks prior to splenectomy, a polyvalent pneumococcal and meningococcal vaccine should be given. If the patient has not received a H. influenzae vaccine, this should also be given. Following splenectomy, prophylactic penicillin 250 mg bid is given to reduce the risk of overwhelming postsplenectomy infection. Management of the febrile splenectomized patient is detailed in Chapter 5, Lymphadenopathy and Diseases of the Spleen. Supportive care Follow-up recommendations for patients with thalassemia major are shown in Table 9.17: 1. Folic acid, 1 mg daily orally, is given to patients who are not receiving regular red cell transfusions. 2. Hepatitis A and B vaccination should be given to all patients. 3. Cardiology consultation and administration of appropriate inotropic, antihypertensive, and antiarrhythmic drugs when indicated for cardiac dysfunction. 4. Endocrine intervention (i.e., thyroxine, growth hormone, estrogen, and testosterone) should be implemented when indicated. 5. Cholecystectomy should be performed if symptomatic gallstones are present. 6. Referral to gastroenterology for the management of chronic hepatitis B or C infection. 7. HIV-positive patients should be treated with the appropriate antiviral medications. 8. Genetic counseling and prenatal diagnosis (when indicated) should be carried out using chorionic villus sampling or amniocentesis. 9. Management of osteoporosis includes: a. Periodic screening, prevention, and treatment. b. Yearly bone densitometry and gonadal hormone evaluation should be performed starting by age 10 years. c. Calcium and vitamin D intake should be monitored; encourage the recommended daily allowance for calcium in the diet and administer vitamin D supplements if low vitamin D levels. d. Zinc supplementation can be considered. In a pilot study of patients aged 10!30 years with thalassemia major and low bone mass, supplementation with zinc, 25 mg daily, for 18 months resulted in a greater rise in whole-body bone mineral content compared to placebo. Assess copper levels and supplement if low. e. Hormonal replacement therapy (estrogen/progesterone; testosterone) should be administered to those with gonadal insufficiency. f. Encourage physical activity. Discourage smoking. g. Bisphosphonates, which inhibit osteoclast-mediated bone resorption, have been used to treat osteoporosis in thalassemia, with some efficacy. Pharmacologic enhancement of HbF synthesis High levels of HbF ameliorate the symptoms of β-thalassemia by increasing the hemoglobin concentration of the thalassemic red cells and decreasing the accumulation of unmatched α-chains, which cause ineffective erythropoiesis. HU has been demonstrated to increase HbF production and mean hemoglobin levels in patients with NTDT, including hemoglobin E β-thalassemia. Additionally, there are reports of a few TDT patients who became Lanzkowsky’s Manual of Pediatric Hematology and Oncology 188 TABLE 9.17 Baseline 9. Hemoglobinopathies Routine monitoring of β-thalassemia patients. α- and β-Globin genotype Transplant evaluation/HLA typing Red blood cell antigen profile Weekly Complete blood count with differential if taking deferiprone Monthly Complete blood count Complete blood chemistry (including liver function tests, BUN, creatinine, phosphate) if taking deferasirox Record transfusion volume Monthly to bimonthly Measure ferritin (trends in ferritin used to adjust chelation) Every 3 months Measure height and weight Complete blood chemistry, including liver function tests Every 6 months Complete physical examination, including Tanner staging, monitor growth and development, dental examination Every year Cardiac function—echocardiogram, ECG, Holter monitor (as indicated) Endocrine function (TFTs, PTH, and FSH/LH), vitamin D levels starting at age 6 years; fasting glucose, fructosamine, testosterone/estradiol, FSH, LH, and IGF-1, starting at the age of 10 years Vitamin C level, zinc level Ophthalmological examination and audiogram Viral serologies [HAV, HBV panel, HCV (or if HCV1 , quantitative HCV RNA PCR), HIV] Bone densitometry (from the age of 10 years) Ongoing psychosocial support Every 1!2 years Evaluation of tissue iron burden: Liver iron measurement—MRI, SQUID, or biopsy T2* MRI measurement of cardiac iron (beginning at the age of 10 years; earlier if chelation history is unknown such as with international adoption, or if there is a history of elevated liver iron .15 mg/g dw) Abbreviations: BUN, Blood urea nitrogen; HLA, histocompatibility locus antigen; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; SQUID, superconducting quantum interference device, TFT, thyroid function testing. transfusion independent using HU. However, neutropenia may limit adequate dose escalation. Typically, a starting dose of 10 mg/kg per day is utilized and dose escalation beyond 20 mg/kg per day is usually not tolerated. Erythroid maturation agents 1. Luspatercept is a recombinant fusion protein that binds TGF-β family ligands, blocking a signaling pathway involved in ineffective erythropoiesis, and improving erythroid maturation. 2. A phase 3 trial in adults with transfusion-dependent β-thalassemia or hemoglobin E β-thalassemia showed at least a 33% reduction from baseline in transfusion requirements and a reduction of at least two red cell units between weeks 13 and 24 in 21.4% of subjects who received luspatercept compared with 4.5% of controls. 3. Adverse effects include bone pain, arthralgia, dizziness, hypertension, and hyperuricemia. Thromboembolic complications occurred in 3.6% of patients in the luspatercept group compared with 0.9% in the placebo arm. a. Patients should be monitored for thrombosis. b. Aspirin or prophylactic anticoagulation may be considered in patients with additional risk factors such as splenectomy or prior history of thrombosis. 4. Luspatercept received FDA approval in November 2019 for the treatment of individuals $ 18 years old with TDT. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thalassemias 189 5. The drug is given as a subcutaneous injection, at a dose of 1!1.25 mg/kg every 3 weeks; if there is no evidence of effect after 9 weeks at the higher dose, the drug should be discontinued. 6. In a phase 2 trial of luspatercept in NTDT, 58% of patients receiving doses of luspatercept of 0.6!1.25 mg/kg achieved a rise in hemoglobin level of 1.5 g/dL or higher. A phase 3 trial in NTDT is ongoing. Hematopoietic stem cell transplantation 1. Stem cell transplantation is a curative mode of therapy. 2. Outcome is best for children ,14 years with an HLA-identical sibling donor. Overall survival is .90%. 3. The presence of hepatomegaly, liver fibrosis, and/or history of poor adherence with chelation therapy has been associated with worse outcome; however, with the use of modified conditioning regimens for those with two or more of these risk factors, outcome is similar. 4. Although limited data are available, the outcome for matched unrelated donor transplantation with highresolution molecular testing at HLA Class 1 and 2 loci appears to be comparable to matched sibling donor transplantation. Chronic GVHD is seen in 18%. 5. Reduced intensity conditioning regimens and haploidentical transplantation are under study. Gene therapy The general method, as for SCD therapy, involves the collection of autologous CD341 hematopoietic stem cells, ex vivo manipulation by gene addition or gene editing, followed by conditioning and reinfusion of the modified stem cells, with the primary goal of increasing β-like globins. 1. Gene addition: Adding β-globin or variant a. The Lentiglobin BB305 vector includes a β-globin gene with a single amino acid substitution (T87Q) as in the SCD trials. In two phase 1/2 trials of Lentiglobin BB305 in TDT, 12 of 13 patients with non-β0/β0 genotypes were able to discontinue red cell transfusions, but many had persistent mild-to-moderate anemia. Patients with more severe β0/β0 or IVS1!110 (severe β1) genotypes did not fare as well, with a significant reduction in transfusion requirements in most patients but only three of nine being able to discontinue transfusions. Adverse events were typical of myeloablative conditioning regimens and no clonal dominance or replication competent lentivirus was observed. Preliminary results from an ongoing phase 3 trial of Lentiglobin BB305 (NCT03655678) in severe β-thalassemia using an improved transduction method showed improved results with three of four patients who had $ 6 months of follow-up were able to discontinue transfusions maintaining hemoglobin levels of 10.5!13.6 g/dL. b. An ongoing phase 1/2 trial in patients with severe β-thalassemia using a different lentiviral vector encoding the β-globin gene, Globe, showed better outcomes in children than adults: three of four children discontinued transfusion, while the three adult patients had a reduction in transfusion requirements. 2. Manipulation of HbF repressor BCL11A to induce HbF a. Early results of a phase 1/2 trial utilizing CRISPR Cas9 editing and myeloablative conditioning appear promising. The first two adult patients with TDT treated have discontinued transfusions, with HbF production of 13.5 g/dL at 15 months in one patient and 12.2 g/dL at 5 months in a second patient. Safety was consistent with myeloablative conditioning. b. A phase 1/2 trial using genome editing with a zinc finger nuclease is ongoing. Management of the acutely ill patient with thalassemia 1. Acute illness requiring urgent treatment occurs secondary to: a. Sepsis, usually with encapsulated organisms. Iron overload and chelation with deferoxamine also increase the risk of infection with Yersinia enterocolitica. b. Cardiomyopathy secondary to myocardial iron overload. c. Endocrine crises such as diabetic ketoacidosis. 2. Prevention of these complications should be the primary treatment. Preventive measures include: a. management of the splenectomized patient as outlined in Chapter 5, Lymphadenopathy and Diseases of the Spleen. b. adequate chelation to prevent secondary hemochromatosis, and c. routine monitoring of cardiac and endocrine function. 3. If a patient presents with signs of shock, the following measures should be instituted: a. Determine hemoglobin, electrolyte, calcium, and glucose levels; perform urinalysis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 190 9. Hemoglobinopathies b. Obtain blood cultures. c. Distinguish between cardiogenic shock and septic shock because the management of each differs. To distinguish between the two, obtain: i. ECG; ii. echocardiogram, looking at left ventricular contractility; and iii. central venous pressure (CVP). 4. If the patient is in cardiogenic shock, management includes: a. continuous electrocardiographic and hemodynamic monitoring; b. immediately initiate intensive chelation, with deferoxamine as a continuous IV infusion at a dose of 50!60 mg/kg per day administered over 24 hours; add deferiprone, 75!99 mg/kg per day divided tid as soon as possible; c. diuretics—use carefully because baseline preload is high due to chronic anemia and overdiuresis can precipitate acute renal failure; d. presumptive administration of hydrocortisone given the high rate of adrenal insufficiency; e. meticulous glucose control; and f. obtain cardiac T2* as soon as practical. 5. If the patient is in septic shock, management consists of: a. blood cultures, at least two peripheral sites; b. broad-spectrum antibiotics IV (e.g., third-generation cephalosporin and an aminoglycoside); c. hold chelation until infection is under control; d. fluid boluses of 10 cm3/kg normal saline to restore blood pressure; e. pressors such as dopamine, as indicated; f. presumptive administration of hydrocortisone given the high rate of adrenal insufficiency; g. coagulation studies to evaluate for disseminated intravascular coagulation; h. CVP monitoring to guide fluid management; and i. arterial blood gas and chest radiograph. 6. If the patient is in diabetic ketoacidosis, manage the ketoacidosis in the usual manner with careful monitoring of cardiac function, when the patient is being vigorously hydrated. Nontransfusion-dependent β-thalassemia (β-thalassemia intermedia) Although patients are homozygous or compound heterozygous, the resultant anemia is milder than in thalassemia major. Clinical features 1. Patients generally do not require transfusions and maintain a hemoglobin level between 7 and 10 g/dL. 2. Medullary expansion may result in nerve compression, extramedullary hematopoiesis, hepatosplenomegaly, growth retardation, and facial anomalies. 3. Pulmonary hypertension and increased risk of thrombosis, particularly in splenectomized patients. 4. Patients are most healthy if management is as vigorous as that for TDT. Management 1. Folic acid 1 mg/day PO should be administered. 2. Iron-fortified foods should be avoided. A cup of tea with every meal will reduce the absorption of nonheme iron. 3. Chelation therapy is required at an older age than in thalassemia major because patients have received fewer transfusions. Iron overload develops secondary to increased absorption of dietary iron. Ferritin levels may not correlate well with total iron burden (usually lower than expected for the degree of iron loading). Indications for chelation include elevated transferrin saturation of 70%, ferritin of 800 ng/mL or higher, or LIC of 5 mg/g dw or higher. Liver iron quantitation may also be used to guide treatment. 4. Transfusions generally are not required except during periods of erythroblastopenia (erythroid aplastia due to parvovirus B19) or during acute infection. If hemoglobin falls below 7 g/dL, growth is poor, or other complications develop, chronic transfusion therapy should be initiated. Children should be monitored for facial bone changes, which can be prevented, but not reversed, by chronic transfusions. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 191 5. Splenectomy may improve hemoglobin level. However, the risk of infection with encapsulated organisms, pulmonary hypertension, and hypercoagulability are increased following splenectomy; therefore splenectomy is often avoided. 6. Cardiac (including evaluation for pulmonary hypertension) and endocrine evaluation and bone densitometry should be performed as in TDT. β-Thalassemia minor or trait (heterozygous β0 or β1) 1. Asymptomatic (physical examination is normal). a. Discovered on routine blood test—slightly reduced hemoglobin, basophilic stippling, low MCV, normal red cell distribution width. b. Discovered in family investigation or family history of heterozygous or homozygous β-thalassemia. c. Confirmed with hemoglobin electrophoresis, demonstrating slightly decreased HbA (90!95% typically) increased HbA2 ( . 3.5%); hemoglobin F mildly elevated in 50% of cases. 2. Thalassemia trait of unusual severity. There are cases of β-thalassemia trait of unusual severity secondary to the coinheritance of α-gene duplication with increased α-globin synthesis, thereby increasing α- and β-chain imbalance, causing a β-thalassemia intermedia (NTDT) phenotype. α-Thalassemias The syndromes resulting from decreased α-chain synthesis are listed in Table 9.15. α-Thalassemia may present as a silent carrier, thalassemia trait, HbH disease, or hydrops fetalis. Deletional HbH disease is clinically milder than homozygous β-thalassemia and usually does not require regular red cell transfusions. HbH Constant Spring or other point mutations generally results in a more severe phenotype, with more severe anemia. Acute worsening of anemia with infections that requires treatment with acute red cell transfusion may occur in HbH Constant Spring, and less commonly with deletional HbH disease. Hydrops fetalis is not compatible with life and presents with intrauterine or neonatal death, though some babies have survived with fetal packed RBC transfusions when antenatal diagnosis was made. These patients should continue on hypertransfusion regimens and be treated like β-thalassemia major, or treated with allogeneic hematopoietic stem cell transplant. Further reading and references Adams, R.J., Brambilla, D., 2005. Discontinuing prophylactic transfusions used to prevent stroke in sickle cell disease. N. Engl. J. Med. 353, 2769!2778. Adams, R.J., McKie, V.C., Hsu, L., Files, B., Vichinsky, E., Pegelow, C., 1998. Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography. N. Engl. J. Med. 339, 5!11. Bhatia, M., Walters, M.C., 2008. Hematopoietic cell transplantation for thalassemia and sickle cell disease: past, present and future. Bone Marrow Transplant 41, 109!117. Borgna-Pignatti, C., 2007. Modern treatment of thalassaemia intermedia. Br. J. Haematol. 138, 291!304. Cappellini, M.D., Viprakasit, V., Taher, A., Georgiev, P., Kuo, K., 2020. A Phase 3 Trial of Luspatercept in Patients with TransfusionDependent Beta-thalassemia. New England Journal of Medicine 382, 1219!1231. Chou, S.T., Jackson, T., Vege, S., Smith-Whitley, K., Friedman, D.F., Westhoff, C.M., 2013. High prevalence of red blood cell alloimmunization in sickle cell disease despite transfusion from Rh-matched minority donors. Blood 122, 1062!1071. Cunningham, M.J., Macklin, E.A., Neufeld, E.J., Cohen, A.R., 2004. Complications of beta-thalassemia major in North America. Blood 104, 34!39. DeBaun, M.R., Gordon, M., McKinstry, R.C., Noetzel, M.J., White, D.A., Sarnaik, E.R., 2014. Controlled trial of transfusions for silent cerebral infarcts in sickle cell anemia. N. Engl. J. Med. 371, 699!710. Frangoul, H., Altshuler, D., Cappellini, D., Chen, Y.-S., Jennifer, D., Brenda, E., et al., 2021. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and Beta Thalassemia. New England Journal of Medicine 384, 252!260. Howard, J., Llwelyn, C., Choo, L., Hodge, R., Johnson, T., Purohit, S., 2013. The Transfusion Alternatives Preoperatively in Sickle Cell Disease (TAPS) study: a randomised, controlled, multicentre clinical trial. Lancet 381, 930!938. Koshy, M., Weiner, S.J., Miller, S.T., Sleeper, L.A., Vichinsky, E., Brown, A.K., 1995. Surgery and anesthesia in sickle cell disease. Cooperative study of sickle cell diseases. Blood 86, 3676!3684. Kwiatkowski, J.L., 2013. Evaluation and treatment of transfusional iron overload in children. Pediatr. Clin. North. Am. 60 (6), 1393!1406. Lanzkron, S., Carroll, C.P., Haywood, C., 2013. Mortality rates and age at death from sickle cell disease: US, 1979!2005. Public Health Rep. 128, 110!116. Marktel, S., Scaramuzza, S., Cicalese, M., Giglio, F., Galimberti, S., Lidonnici, M., et al., 2019. Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia. Nat. Med. 25, 234!241. Ohene-Frempong, K., Weiner, S.J., Sleeper, L.A., Miller, S.T., Embury, S., Moohr, J.W., 1998. Cerebrovascular accidents in sickle cell disease: rates and risk factors. Blood 91, 288!294. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 192 9. Hemoglobinopathies Paulukonis, S., Eckman, J., Snyder, A., Hagar, W., Feuchtbaum, L., Zhou, M., et al., 2016. Defining sickle cell disease mortality using a population-based surveillance system, 2004 through 2008. Public Health Rep. 131, 367!375. Pegelow, C.H., Macklin, E.A., Moser, F.G., Wang, W.C., Bello, J.A., Miller, S.T., et al., 2002. Longitudinal changes in brain magnetic resonance imaging findings in children with sickle cell disease. Blood 99, 3014!3018. Pennell, D.J., Udelson, J.E., Arai, A.E., Bozkurt, B., Cohen, A.R., Galaello, R., 2013. Cardiovascular function and treatment in β-thalassemia major: a consensus statement from the American Heart Association. Circulation 128, 281!308. Piel, F., Steinberg, M., Rees, D., 2017. Sickle cell disease. N. Engl. J. Med. 376, 1561!1573. Platt, O.S., Thorington, B.D., Brambilla, D.J., Milner, P.F., Rosse, W.F., Vichinsky, E., 1991. Pain in sickle cell disease. Rates and risk factors. N. Engl. J. Med. 325, 11!16. Rosse, W.F., Gallagher, D., Kinney, T.R., Castro, O., Dosik, H., Moohr, J., 1990. Transfusion and alloimmunization in sickle cell disease. The Cooperative Study of Sickle Cell Disease. Blood 76, 1431!1437. Rund, D., Rachmilewitz, E., 2005. Beta-thalassemia. N. Engl. J. Med. 353 (2005), 1135!1146. St Pierre, T.G., Clark, P.R., Chua-anusorn, W., Fleming, A.J., Jeffrey, G.P., Olynyk, J.K., 2005. Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance. Blood 105 (2005), 855!861. Thompson, A.A., Walters, M.C., Kwiatkowski, J., Rasko, J.E.J., Ribeil, J.-A., Hongeng, S., et al., 2018. Gene therapy in patients with transfusiondependent B-thalassemia. N. Engl. J. Med. 378 (2018), 1479!1493. Vichinsky, E.P., Haberkern, C.M., Neumayr, L., Earles, A.N., Black, D., Koshy, M., 1995. A comparison of conservative and aggressive transfusion regimens in the perioperative management of sickle cell disease. The Preoperative Transfusion in Sickle Cell Disease Study Group. N. Engl. J. Med. 333, 206!213. Vichinsky, E.P., Styles, L.A., Colangelo, L.H., Wright, E.C., Castro, O., Nickerson, B., 1997. Acute chest syndrome in sickle cell disease: clinical presentation and course. Cooperative Study of Sickle Cell Disease. Blood 89, 1787!1792. Wang, W.C., Ware, R.E., Miller, S.T., Iyer, R.V., Casella, J.F., MinnitiBABY HUG Investigators, C.P., 2011. Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG). Lancet 377, 1663!1672. Ware, R.E., Zimmerman, S.A., Sylvestre, P.B., Mortier, N.A., Davis, J.S., Treem, W.R., 2004. Prevention of a secondary stroke and resolution of transfusional iron overload in children with sickle cell anemia using hydroxyurea and phlebotomy. J. Pediatr. 145, 346!352. Ware, R.E., Helms, R.W., For the SWiTCH Investigators, 2012. Stroke with transfusions changing to hydroxyurea (SWiTCH). Blood 119, 3925!3932. Ware, R., Davis, B., Schultz, W., Brown, R., Aygun, B., Sarnaik, S., et al., 2015. Hydroxycarbamide versus chronic transfusion for maintenance of transcranial Doppler flow velocities in children with sickle cell anaemia—TCD With Transfusions Changing to Hydroxyurea (TWiTCH): a multicentre, open-label, phase 3, non-inferiority trial. Lancet 387, 661!670. Lanzkowsky’s Manual of Pediatric Hematology and Oncology C H A P T E R 10 Primary and secondary erythrocytosis Tsewang Tashi1,2 and Josef T. Prchal1,2,3 1 2 Division of Hematology and Hematologic Malignancies, University of Utah, Salt Lake City, UT, United States Division of Hematology/Oncology, VA Medical Center, Salt Lake City, UT, United States 3Huntsman Cancer Center, University of Utah, Salt Lake City, UT, United States The term “polycythemia,” particularly as it pertains to newborns and children, should be more accurately termed erythrocytosis because it generally refers to conditions in which only erythrocytes are increased in number. It is usually a response to tissue hypoxia from any disorder causing inadequate oxygen, the presence of high-oxygen-affinity hemoglobins or increased production of erythropoietin (EPO) or other circulating erythropoietic stimulating factors, or mutations making erythroid progenitors intrinsically hyperproliferative. Erythrocytosis can be primary or secondary. In primary erythrocytosis the erythroid progenitors are either independent or hypersensitive to EPO and, thus, have very low EPO levels. Secondary erythrocytoses, on the other hand, have normal responsive erythroid progenitors but excessive production of EPO. Elevated hematocrit can also be associated with normal red cell mass and decreased plasma volume, so-called spurious, stress, or relative polycythemia. Erythrocytosis or polycythemia Increased red cell mass, mostly accompanied by elevated hemoglobin concentration and hematocrit, is variably denoted as polycythemia or erythrocytosis. However, there is no consensus on the use of either term. The term “polycythemia,” particularly as it pertains to newborns and children, should be more accurately termed erythrocytosis because it generally refers to conditions in which only erythrocytes are increased in number. Some founders of modern hematology have reasoned that the proper meaning of polycythemia is too many cells in blood, while others have reasoned that polycythemia implies that several lineages are increased along with erythrocytosis, that is, increased neutrophils, and/or platelet counts. With this definition, the only form of polycythemia is polycythemia vera (PV). As the acceptance of this definition is now increasing, this chapter will reserve term the “polycythemia” only for PV, while other conditions with elevated red cell mass will be referred to as “erythrocytosis.” The term “erythrocytosis” applies to an increase in circulating red cell mass to above the normal upper limits of 30 mL/kg body weight (excluding hemoconcentration due to dehydration). However, the measurement of red cell mass and plasma volumes that uses radioactive isotopes is no longer available in the United States, and estimates of the red cell mass can be made using measured hemoglobin and hematocrit. A hemoglobin level greater than the 99th percentile of the method-specific reference range for age and sex, and adjusted for normal range at the altitude of residence should be applied. It should be noted that a patient may have decreased plasma volume and elevated hemoglobin and hematocrit, so-called spurious erythrocytosis, while high altitude dwellers such as Tibetans and Sherpas may have a normal hemoglobin levels but increased both red cell mass and plasma volumes, and, thus, have true erythrocytosis. Erythrocytosis can be primary or secondary. Both can be congenital or acquired. Primary erythrocytosis, both congenital and acquired, has lower than normal serum EPO levels because the underlying genetic defect in the hematopoietic progenitors makes them hypersensitive to, or even independent of EPO. Congenital primary Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00001-5 193 © 2022 Elsevier Inc. All rights reserved. 194 10. Primary and secondary erythrocytosis erythrocytosis, such as primary familial and congenital erythrocytosis/polycythemia (PFCP), has mutations in the EPO receptors (EPORs) and is associated with polyclonal hematopoiesis, while acquired primary erythrocytosis such as PV has clonal hematopoiesis due to mutations in hematopoietic progenitors, mainly JAK2 mutation that confers EPO independence. Secondary erythrocytosis, on the other hand, has normally responsive erythroid progenitors but excessive production of erythropoietic stimulating factors such as EPO. From a functional perspective, the increased red cell mass in secondary erythrocytosis can be physiologically appropriate or inappropriate. Appropriate responses are due to tissue hypoxia that increases red cell mass to assure adequate tissue oxygen delivery that can be either from congenital causes (high-oxygen-affinity hemoglobins) or acquired causes (Eisenmenger complex, exposure to low oxygen environment such as high altitude, or lung disease). Inappropriate responses can also be congenital (mutations of normal hypoxia sensing pathways) or acquired (EPO producing tumors, postrenal transplant erythrocytosis, and EPO doping). See Table 10.1 for various causes and classification of erythrocytosis. TABLE 10.1 Classification of erythrocytosis. 1. Relative erythrocytosis (hemoconcentration, dehydration) 2. Primary erythrocytosis (resulting from somatic or germline mutations of erythroid progenitor cells that make them exquisitely sensitive to erythropoietin or other cytokines) a. Congenital: EPOR mutation resulting from germline mutation b. Acquired: polycythemia vera resulting from somatic mutation 3. Secondary erythrocytosis a. Inadequate oxygen delivery (appropriate erythrocytosis due to physiologic compensation) i. Physiologic a. Fetal life b. Low environmental O2 (high altitude) ii. Pathologic a. Impaired ventilation: cardiopulmonary disease, obesity b. Pulmonary arteriovenous fistula c. Congenital heart disease with left-to-right shunt (e.g., tetralogy of Fallot, Eisenmenger syndrome) d. Abnormal hemoglobins (reduced P50 in whole blood) i. Methemoglobinemia (congenital and acquired) ii. Carboxyhemoglobin iii. Sulfhemoglobinemia iv. High-oxygen-affinity hemoglobinopathies (hemoglobin Chesapeake, Ranier, Yakima, Osler, Tsurumai, Kempsey, and Ypsilanti) v. 2,3-BPG deficiency b. Increase in erythropoietin (inappropriate erythrocytosis from an aberrant production of erythropoietin or other growth factors) i. Endogenous a. Renal: Wilms’ tumor, hypernephroma, renal ischemia, for example, renal vascular disorder, congenital polycystic kidney, benign renal lesions (cysts, hydronephrosis), renal cell carcinoma. Postrenal transplantation erythrocytosis (occurs in 10!15% of renal graft recipients). Contributing factors include persistence of erythropoietin secretion from the recipients’ diseased and ischemic kidney and secretion of angiotensin II androgen and insulin-like growth factor b. Endocrine: pheochromocytoma, Cushing’s syndrome, congenital adrenal hyperplasia, and adrenal adenoma with primary aldosteronism c. Liver: hepatoma, focal nodular hyperplasia, hepatocellular carcinoma, hepatic hemangioma, Budd!Chiari syndrome (some of these patients may have overt or latent myeloproliferative disorder) d. Cerebellum: hemangioblastoma, hemangioma, and meningioma e. Uterus: leiomyoma, leiomyosarcoma f. Ovaries: dermoid cysts g. TEMPI syndrome ii. Exogenous a. Administration of testosterone and related steroids b. Administration of growth hormone c. Heavy metal toxicity (cobalt, manganese) c. Erythrocytosis with characteristics of both primary and secondary erythrocytosis i. Chuvash erythrocytosis and other VHL gene mutations ii. EPAS1 and EGLN1 mutations 4. Neonatal erythrocytosis (erythrocytosis) Abbreviations: BPG, Bisphosphoglycerate; EPOR, primary familial congenital erythrocytosis; TEMPI, Telangiectasias, Erythrocytosis, Monoclonal gammopathy, Perinephric fluid, Intrapulmonary shunt; VHL, von Hippel!Lindau. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Primary erythrocytosis 195 Primary erythrocytosis Polycythemia vera PV is an acquired primary erythrocytosis—a clonal disorder arising from a pluripotent hematopoietic stem cell manifesting by the excess production of erythrocytes with low EPO levels and variable overproduction of leukocytes and platelets. It is one of the Philadelphia chromosome-negative myeloproliferative neoplasms and can be differentiated from other myeloproliferative disorders by the predominance of erythrocyte production. This is a well-characterized disease in middle- to older age adults, but it is extremely rare in childhood and adolescence, and, thus, published literature on clinical presentation, treatment, and long-term prognosis in children is very limited. However, overall clinical course, disease biology, and management do not differ significantly from the adults and much of the information available is extrapolated from the adult literature. Pathophysiology The biology of PV is characterized by clonality and EPO independence. In PV a single clonal population of erythrocytes, granulocytes, and platelets arises when a hematopoietic stem cell that preferentially differentiates to myelopoiesis gains a proliferative advantage over other nonmutated stem cells. Genome-wide scanning that compared clonal PV and nonclonal cells from the same individuals revealed a loss of heterozygosity in chromosome 9p, found in approximately 50% of patients with PV. This is not a classical chromosomal deletion, but, rather, duplication of a portion of the chromosome and loss of the corresponding parental region, a process referred to as uniparental disomy. The 9p region contains a gene encoding for JAK2 tyrosine kinase, which transmits an activating signal in the EPOR-signaling pathway. A point mutation involving valine-to-phenylalanine substitution at codon 617 in the pseudokinase JAK2 domain on exon 14, known as JAK2V617F, leads to constitutive gain-of-function of the kinase, which at least partly explains EPO hypersensitivity/independence. Over 98% of adult patients with PV carry the JAK2V617F mutation, as well as approximately 50% of adults with essential thrombocythemia and idiopathic myelofibrosis. However, in children, the frequency of the JAK2V617F mutation is reported much lower, in the range of 25!40% by several studies, although the number of studied patients was small due to the rarity of the disease in the children. It is very likely that many of these children did not actually have PV and had some other, possibly inherited, polycythemic disorder. In about 2% of JAK2V617F-negative PV patients, other JAK2 mutations have been found in exon 12 and these mutations are heterogeneous, consisting of insertions, deletions, or stop codons. These patients may have marked erythrocytosis without affecting other cell lines. However, the risk of thrombosis and transformation to myelofibrosis are similar to JAK2V617F-positive adult PV patients. There is compelling evidence against JAK2V617F and JAK2 exon 12 mutations being a disease-initiating mutation, but rather that these mutations play a major role in the behavior of the PV clone. Leukemic transformation from PV that is seen in adults, however, can sometimes arise from JAK2V617F-negative PV progenitor cells. Clinical features PV in children is extremely rare. The incidence in adults is approximately 10!20 per 100,000, of which 1% present before the age of 25 and 0.1% present before the age of 20. Patients usually present with elevated hemoglobin and hematocrit found on routine testing. Some patients may initially present with isolated elevated platelet count, often diagnosed as essential thrombocythemia, but later develop erythrocytosis, thus, transforming to PV. Some patients are asymptomatic, while others may have various nonspecific symptoms recognized retrospectively to be consistent with PV. Overall, children tend to be less symptomatic than adults. In adults, about one-third of patients present with thrombosis or hemorrhage. Thrombosis is about equally distributed between arterial and venous thrombosis. Less frequent, but more specific for PV, is Budd!Chiari syndrome (hepatic vein thrombosis). In younger adults, about 20!30% may present with Budd!Chiari syndrome or mesenteric thrombosis. The presence of leukocytosis at presentation has been shown to be an independent risk factor for thrombosis. A few studies have shown that the rate of thrombosis is much lower in children with PV, at about 5%, and the thrombosis invariably occurs in the setting of leukocytosis associated with infections. It has been suggested that children may have much better vascular integrity than adults, which may negate some prothrombotic factors associated with PV. Transformation to more advanced myelofibrosis and secondary acute leukemia has been reported to be extremely rare in the pediatric population. Less than 5% of patients will have erythromelalgia, that is, erythema and warmth of the distal extremities, especially the hands and feet, with a painful burning sensation that can progress to digital ischemia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 196 10. Primary and secondary erythrocytosis Erythromelalgia is associated with augmented platelet aggregation and frequently responds within hours to lowor regular-dose aspirin therapy. Less commonly, PV may present with elevated uric acid, with or without associated gout, due to increased cell turnover. Hemorrhagic presentations are usually mild, with gum bleeding and easy bruising, although serious gastrointestinal hemorrhage can occur, typically when the platelet count is .1 million which can be associated with acquired von Willebrand disease. About 40% of adult patients present with pruritus, which typically gets worse after a warm bath or shower, known as aquagenic pruritus. This has been attributed to increased numbers of mast cells and elevated histamine levels, and these patients may have plethora and ruddiness of the face. Diagnosis The WHO criteria which were updated in 2016, listed in Table 10.2, are used for diagnosis. While the presence of EPO-independent erythroid colonies is specific for PV, this test is difficult and not widely available and is now removed from the criteria. The hemoglobin level for the criteria has been lowered to 16.5 g/dL for men (from 18.5 g/dL) and 16.0 g/dL for women (from 18 g/dL). This was done to increase the sensitivity to detect “masked” PV where many had PV but did not fulfill the older WHO criteria. Treatment Unlike for adult patients, there are no consensus on treatment guidelines for pediatric patients. Therefore the treatment approach in pediatric population is extrapolated from the guidelines for adults. Thromboembolism is the major cause of morbidity in PV and the goal of the treatment is primarily directed at reducing vascular events by restraining clonal proliferation with cytoreductive therapy. In adults, treatment is initiated for those with “high-risk” disease, that is, those who have a prior history of thrombosis, those who are .60 years old and with multiple cardiovascular risk. However, in children the rate of thrombosis is significantly lower than adults, and typically children do not have significant cardiovascular risks; therefore some experts have suggested adopting a conservative initial approach. • Phlebotomy is performed by many to maintain hematocrits ,45%. As more blood is removed and the patient becomes iron deficient, the hematocrit becomes easier to control and the phlebotomy schedule should be adjusted accordingly. However, in some patients, iron deficiency can become symptomatic and can cause neurocognitive impairment and decreased exercise tolerance. Although phlebotomy is effective for controlling erythrocytosis, it does not affect variable leukocytosis, thrombocytosis, risk of thromboembolic events, or overall natural course of the disease. In fact, there have been reports that the risk of thrombosis is slightly higher during the immediate postphlebotomy period. • Low-dose aspirin is employed to reduce the risk of thromboembolic events and results in a minor, but statistically significant, decreased risk of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, pulmonary embolism, and major venous thrombosis without a significant increase in rates of hemorrhage. • Chemotherapeutic cytoreductive therapy: Cytoreductive therapy can be achieved either with hydroxyurea or interferon-alpha (IFN-α). Indications are: • Prior history of thrombosis or transient ischemic attacks. • A platelet count .1.5 million/mm3. Platelet counts at this level are a risk factor for bleeding. The following cytoreductive therapy is used: • Hydroxyurea. Initial dose of 20!30 mg/kg daily. This dose is adjusted depending on the hematological response or signs of toxicity. Hydroxyurea reduces the risk of thrombosis compared to phlebotomy or TABLE 10.2 Revised 2016 WHO criteria. Diagnosis requires the presence of all three major criteria or the presence of the first two major criteria together with one minor criteriona Major criteria 1. Hemoglobin. 16.5 g/dL in men, 16.0 g/dL in women, or other evidence of increased red cell mass 2. Bone marrow biopsy showing hypercellularity for age with panmyelosis with prominent erythroid, granulocytic and megakaryocytic proliferation 3. The presence of JAK2V617F or other functionally similar mutation such as JAK2 exon 12 mutation Minor criteria 1. Serum erythropoietin level below the reference range for normal a Bone marrow biopsy (criterion 2) may be not be required if there is sustained erythrocytosis, JAK2 mutation positive and subnormal serum erythropoietin level. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Secondary erythrocytosis 197 phlebotomy and aspirin. The safety and efficacy are unclear in pediatric patients. Leukemogenic risk of hydroxyurea has long been debated, but unlike alkylating agents and radioactive phosphorus that lead to an increase in fatal PV leukemic transformation, such an association with hydroxyurea has not been proven. • IFN-α achieves a complete hematological response in a high percentage of cases, and some patients achieve durable reduction in their JAK2V617F mutant allele. However, many patients do not tolerate IFN-α well because of a high rate of side effects and inconvenience of frequent intravenous administration. Newer pegylated formulations are much better tolerated, with less side effects and better efficacy, and some consider pegylatedIFNα as the first-line therapy in children, mainly due to its ability to achieve molecular response. However, safety and efficacy are unclear in patients younger than 18 years old. • JAK2 inhibitor—ruxolitinib is FDA approved for PV if they are intolerant or resistant to first-line hydroxyurea. It is effective in controlling the hematocrit level and achieving spleen volume reduction if the patient has concomitant splenomegaly. It can also be very effective for symptoms of aquagenic pruritus. However, there are no conclusive evidence on the prevention of thromboses or slowing disease transformation to myelofibrosis or acute leukemia in adults. • Anagrelide is useful to decrease platelet counts in patients presenting with thrombocytosis. The induction dose of anagrelide in children is 0.5 mg twice daily, followed by a maintenance dose of 0.5!1.0 mg twice a day, adjusted to the lowest effective dosage required to maintain platelet counts below 600,000/mm3 and ideally to maintain it in the normal range. Primary familial and congenital erythrocytosis PFCP is a primary erythrocytosis that is an autosomal dominant condition where the defect exists in erythroid progenitor cells. In contrast to PV, PFCP is characterized by a polyclonal hematopoiesis and is not associated leukocytosis, thrombocytosis, or splenomegaly and does not progress to myelofibrosis or leukemia. Although PFCP is a rare disease, it is frequently misdiagnosed as PV. To date, .20 pathologic EPOR mutations associated with PFCP have been described so far. Most of the mutations are single-nucleotide variants or frameshift mutations located in the exon 8 of EPOR gene which encodes the negative regulatory domain. Therefore these EPOR mutations cause a gain-of-function involving deletion of the cytoplasmic negative regulatory subunit of EPOR. These patients invariably have low EPO levels because EPO production is appropriately suppressed by the absence of tissue hypoxia and additionally the erythroid progenitor cells are hypersensitive to EPO. PFCP patients tend to have erythrocytosis quite early in their life and majority of them are asymptomatic. However, some patients may have symptoms of hyperviscosity such as headaches and dizziness, while others may develop cardiovascular diseases such as hypertension and coronary artery diseases. There are no established guidelines for treatment, although phlebotomy is the mainstay of therapy, it should only be used only in patients who manifest hyperviscosity symptoms. These patients may need to avoid any activities that may increase the blood viscosity such as dehydration and have regular cardiology surveillance. Low-dose aspirin may be used for thromboprophylaxis but there are no evidence to prove its efficacy. Given the autosomal dominant inheritance, referral to genetic counseling may be appropriate. Secondary erythrocytosis Erythrocytosis in the newborn Hypoxia is the major regulator and determinant of red cell mass and EPO transcription. Neonatal erythrocytosis is an appropriate physiological response to intrauterine hypoxia and is also contributed to by fetal hemoglobin, which has increased oxygen affinity. Humans have the highest hematocrit at the time of birth, which then dramatically decreases during the first 2 weeks of life. This decrease is consistent with preferential destruction of young red blood cells, a process called neocytolysis. However, a venous hematocrit reading of .65% or venous hemoglobin concentration in excess of 22 g/dL at any time during the first week of life should be considered an evidence of erythrocytosis. Capillary blood samples should not be relied on for the diagnosis of erythrocytosis because their measured hemoglobin and hematocrit are significantly higher than venous hemoglobin or venous hematocrit and these measurements also vary with the temperature of the extremity from which the sample is obtained. Hematocrit values determined on a microcentrifuge include a small amount of trapped plasma and have a higher value than hematocrit values determined from automated analyzers. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 198 TABLE 10.3 10. Primary and secondary erythrocytosis Causes of neonatal erythrocytosis. 1. Intrauterine hypoxia a. Placental insufficiency i. SGA (intrauterine growth factor) ii. Dysmaturity iii. Postmaturity iv. Placenta previa v. Maternal hypertension syndromes (toxemia of pregnancy) b. Severe maternal cyanotic heart disease c. Maternal smoking 2. Hypertransfusion a. Twin-to-twin transfusion b. Maternal-to-fetal transfusion c. Placental cord transfusion (delayed cord clamping, cord stripping, third stage of labor underwater at body temperature, holding baby below mother with cord attached) 3. Endocrine causes a. Congenital adrenal hyperplasia b. Neonatal thyrotoxicosis c. Congenital hypothyroidism d. Maternal diabetes mellitus 4. Miscellaneous a. Chromosomal abnormalities i. Trisomy 13 (Patau syndrome) ii. Trisomy 18 (Edwards syndrome) iii. Trisomy 21 (Down syndrome) b. Beckwith!Wiedemann syndrome (hyperplastic visceromegaly) c. Oligohydramnios d. Maternal use of propranolol e. High altitude conditions f. High oxygen affinity hemoglobinopathies Abbreviation: SGA, Small-for-gestational age. The incidence of neonatal erythrocytosis is 0.4!4.0% of all births and is higher at high altitudes than at sea level. However, the normal range of hematocrit progressively increases with altitude, and appropriate adjustment needs to be made. The causes of neonatal erythrocytosis are listed in Table 10.3. Symptoms The majority of the time, there are no clinical symptoms from erythrocytosis but when symptoms manifest, they are mainly due to an increase in blood viscosity. Hematocrit up to 65% has a linear correlation with viscosity and beyond 65% has an exponential relationship. Viscosity depends on a number of factors, as listed in Table 10.4. However, optimal oxygen delivery to the tissues is also markedly influenced by total blood volume. In physiological-appropriate secondary erythrocytosis, both hematocrit and total blood volume may be increased for optimal tissue oxygenation. Since this is a normal physiologic response, decreasing the hematocrit may be detrimental in such patients. Therefore it is important to take the total blood volume into consideration while assessing the symptoms. Dehydration should be considered in neonates if erythrocytosis persists beyond the first 48 hours of life. Laboratory findings Hyperbilirubinemia can be found in about 20!30% of neonates with erythrocytosis. This is thought to be from the increased breakdown of erythrocytes but also compounded by inadequate processing of bilirubin by the immature liver. Furthermore, increased circulating erythrocytes results in increased glucose utilization, leading to hypoglycemia. This is seen in about 40% of the cases. When erythrocytosis is due to maternofetal transfusion, the following laboratory findings may be present: • Increased quantities of immunoglobulin (IgA and IgM) in the infant’s serum • Reduction in fetal hemoglobin to ,60% • The presence of red cells bearing maternal blood group antigens in the infant’s circulation • The presence of cells of maternal origin (bearing XX chromosomes) in the infant’s circulation if the infant is male Lanzkowsky’s Manual of Pediatric Hematology and Oncology Secondary erythrocytosis TABLE 10.4 199 Factors increasing viscosity. 1. Hematocrit .60% 2. Larger MCV 3. Decreased deformability of erythrocytes 4. High plasma protein levels, especially high fibrinogen 5. Decreased flow rate—vessel diameter and endothelial integrity, for example, increased levels of erythropoietin, in addition to inducing erythrocytosis, may induce other effects such as: a. hematocrit-independent, vasoconstriction-dependent hypertension b. upregulation of tissue renin c. increased endothelin production d. stimulation of endothelial and vascular smooth muscle proliferation e. change in vascular tissue prostaglandin production f. stimulation of angiogenesis Abbreviation: MCV, Mean cell volume. If erythrocytosis is due to intrauterine hypoxia, it is usually accompanied by an increase in nucleated red blood cells (nRBCs) in peripheral blood during the early neonatal period. The mean value of nRBC in the first few hours of life in a healthy full-term neonate is 500 nRBC/mm3 or 0!10 nRBC/100 WBC. A value of .1000 nRBC/mm3 or 10!20 nRBC/100 WBC is considered abnormal. Other hematologic indices of fetal hypoxia include higher absolute lymphocyte count and lower platelet count in comparison with normal full-term neonates without hypoxia during fetal life. Treatment Treatment should be reserved for infants who have a venous hematocrit of .65%, with respiratory, cardiac, or central nervous system symptoms, or an asymptomatic infant with a venous hematocrit .70%. All polycythemic infants, however, should be carefully monitored for evidence of hypoglycemia, hypocalcemia, and hyperbilirubinemia. Treatment should be designed to reduce the venous hematocrit to approximately 50!55%. This can be accomplished by a partial exchange transfusion using 5% human albumin, Ringer’s lactate, or normal saline. It is better to avoid the use of fresh frozen plasma because it may potentially transmit infectious agents. Normal saline or Ringer’s lactate solutions have the advantage that they are easily available and equally effective. Serum sodium level and renal function should be carefully monitored during the exchange transfusion procedure to avoid sodium overload. The following formula is employed to approximate the volume of exchange required to reduce the hematocrit reading to the desired level: Volume of exchange ðmLÞ 5 ½ðobserved Hct 2 desired HctÞ 3 blood volumeðmLÞ& observed Hct Partial exchange transfusion has been shown to increase capillary perfusion, cerebral blood flow, and cardiac function and reduces the risk of tissue ischemia in various organs resulting from severe microcirculation slowing due to high hematocrit and high shear rates. However, there is little evidence that the long-term outcome of infants is improved by this procedure. A few cases of necrotizing enterocolitis after partial exchange transfusions have been reported; however, a causative association has not been conclusively established. Congenital erythrocytosis due to altered hypoxia sensing The response to hypoxia is controlled by transcriptional factors termed hypoxia-inducible factors (HIFs). These adaptive physiologic responses to hypoxia serve to increase O2 delivery to cells and allow cells to survive under reduced O2 and by activating glycolysis. HIFs are heterodimeric transcription factors composed of a highly regulated α subunit and a constitutively expressed β subunit. Of the three HIF homologues, HIF-2 has more limited tissue expression and is the principal regulator of EPO expression. In normoxia, HIF-1 and HIF-2α subunits are prolyl hydroxylated and then rapidly degraded by the von Hippel!Lindau (VHL) protein!ubiquitin!proteasome pathway, and, thus have extremely short half-life, measured in seconds. The targeting and subsequent polyubiquitination of HIFα subunits and proline hydroxylase enzymes requires iron, O2 and alpha-ketoglutarate, and VHL; this complex constitutes the oxygen sensor. In hypoxia, HIF-1 and HIF-2α subunits have much longer half-life as they are not prolyl hydroxylated and, thus, are not degraded. Thereafter, the HIFs enter the nucleus Lanzkowsky’s Manual of Pediatric Hematology and Oncology 200 10. Primary and secondary erythrocytosis and initiate the transcription of myriad of HIF responsive genes that forms the basis of physiologic response to hypoxia, including erythrocytosis. Mutations in any component of the oxygen sensor complex, including HIF, prolyl hydroxylases, and VHL, can lead to aberrant hypoxia signaling, resulting in erythrocytosis among other symptoms. Chuvash erythrocytosis and other von Hippel!Lindau mutations Chuvash erythrocytosis (in most publications referred to as Chuvash polycythemia) is an endemic erythrocytosis found with high frequency on the west bank of the Volga River in the Chuvash Autonomous Republic in western Russia, the Italian island of Ischia, and sporadically worldwide in all ethnic and racial groups. It is an autosomal recessive disorder characterized by a loss-of-function mutation of the VHL gene, thus, impairing degradation of alpha subunits of HIFs and resulting in their accumulation, leading to upregulation of transcription in a number of target genes, including EPO and vascular-endothelial growth factor. Because EPO can be high normal or increased, Chuvash erythrocytosis can be grouped with the secondary erythrocytosis. However, because the erythroid progenitors are hypersensitive to EPO, Chuvash erythrocytosis also has features of primary erythrocytosis. Patients with Chuvash erythrocytosis have normal arterial blood gases and normal P50. (P50 is defined as the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen, and reference range is 22!28 mmHg.) They often have a relatively low blood pressure, varicose veins, benign vascular abnormalities, and increased risk of pulmonary hypertension. There is increased risk for arterial and venous thrombotic complications and strokes that is increased by those undergoing therapeutic phlebotomies, but no predisposition for developing malignancies which is typical of VHL-tumor-predisposition syndrome. Thromboses occur from childhood as documented in case!control prospective registry study. Other congenital VHL mutations have been described in which there are compound heterozygous and even homozygous genotypes. These patients may present with isolated erythrocytosis, elevated EPO level and a normal P50. Table 10.5 compares the clinical manifestations of PV, PFCP, and Chuvash erythrocytosis. TABLE 10.5 Clinical manifestations of polycythemia vera (PV), primary familial and congenital erythrocytosis (PFCP), and Chuvash erythrocytosis/polycythemia (CP). Clinical entities PV PFCP CP Inheritance None Dominant Recessive Underlying cause Acquired somatic mutation Erythropoietin receptor mutation Hypomorphic mutations of VHL Symptoms of erythrocytosis (e.g., headache, dizziness lethargy, and blurred vision) Present Variable; majority are asymptomatic, some are symptomatic Present Signs Plethora, may have splenomegaly Plethora, no splenomegaly Plethora, no splenomegaly varicosities of peripheral veins Erythropoietin level Undetectable or subnormal Normal or low Increased but high or normal in sporadic nonCP Course Thrombosis or hemorrhage Benign Thrombosis or hemorrhage Diagnosis JAK2V617F or JAK2 exon 12 mutation, endogenous erythroid colonies Molecular analysis for the truncation of cytosolic portion of ER and in vitro hypersensitivity to EPO Molecular analysis of VHL, EPAS1, and EGLN1 mutations Treatment Phlebotomy, Peg-IFN-α, ASA, Phlebotomy if symptomatic hydroxyurea, anagrelide Phlebotomy Abbreviations: ASA, Aspirin; EGLN1, Egl-9 family hypoxia-inducible factor 1; EPAS1, endothelial PAS domain-containing protein 1; EPO, erythropoietin; ER, endoplasmic reticulum; IFN-α, interferon-alpha; VHL, von Hippel!Lindau. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Secondary erythrocytosis 201 Hypoxia-inducible factor 2α mutations HIF-2 is the main regulator of EPO transcription encoded by the EPAS1 gene. Several missense gain-offunction mutations of the EPAS1 gene associated with isolated erythrocytosis and elevated EPO levels have been reported. It is typically inherited as an autosomal dominant disorder. In one large pedigree the affected relatives had increased thromboses from young age regardless of their hematocrit being kept below 45% by phlebotomies. A unique syndrome of congenital erythrocytosis and mosaicism of EPAS1 gene associated (Hopfeld-Fogel et al., 2020) with recurrent paraganglioma/pheochromocytoma and somatostatinomas in later life has been reported. The predisposing EPAS1 mutation can be found in adrenals, pancreas, retina, and other nonhematopoietic organs but only in very low proportion, or even absent in blood cells. Therefore the erythrocytosiscausing EPAS1 mutation escapes detection in the peripheral blood. This suggests that the mutation is most likely acquired postzygotic leading to somatic mosaicism. While this syndrome is generally not seen in other family members, rarely some cases of familial involvement have been reported. Prolyl hydroxylase domain-2 mutations Prolyl hydroxylase domain (PHD)-containing enzymes hydroxylate alpha subunits of HIF, which facilitates binding to VHL, thus, leading to ubiquitin-mediated proteasome degradation of HIFα subunits. Several mutations of the EGLN1 gene, which encodes for PHD2, have been reported. These mutations lead to the loss-offunction of PHD2, thus, decreasing hydroxylation of HIFα subunits and its subsequent proteosomal degradation, leading to increased HIF stability and levels. These patients present with congenital autosomal dominant erythrocytosis but generally have normal EPO levels. Phlebotomy in these patients does not alter the risk of thrombotic complications or natural course of the disease. Similar to other congenital disorders of hypoxia sensing, several HIF inhibitors have been identified; however, no data from preclinical trials yet exist. High-affinity hemoglobinopathies High-affinity hemoglobinopathies are autosomal dominant conditions. Most of these mutations occur within the beta-globin chain where α1 and β2 chains come in contact. This change impairs intramolecular rotation or 2,3-bisphosphoglycerate (BPG) binding, making hemoglobin unable to transition from high-oxygen-affinity to low-oxygen-affinity states, thus, causing tissue hypoxia and compensatory erythrocytosis. Hemoglobin electrophoresis is insufficient to identify hemoglobin structural defects, and some hemoglobin mutants will be missed. The only reliable screening test is P50 measurement either by Hemox-Analyzer or calculated by using pH, PO2, and O2% saturation obtained from venous blood gas. (See the formula at the end of the chapter.) Identification of these mutations can only be determined by sequencing of globin genes. Since the erythrocytosis in patients with high-affinity hemoglobinopathies is a compensatory mechanism for tissue hypoxia, phlebotomy in these patients is not beneficial, but in fact detrimental as it only further augments tissue hypoxia, as reflected by increased EPO after phlebotomies. 2,3-Bisphosphoglycerate deficiency 2,3-BPG, also known as 2,3-diphosphoglycerate, promotes hemoglobin transition from a high-oxygenaffinity state to a low-oxygen-affinity state. 2,3-BPG binds to the central compartment of the hemoglobin tetramer, changing its conformation and shifting the oxygen disassociation curve to the right. The deficiency is created by ineffective bisphosphoglyceromutase (BPGM), a red cell enzyme of the early glycolytic pathway that converts 1,3-BPG to 2,3-BPG. Mutations of BPGM are extremely rare and not clear if autosomal recessive as some heterozygotes also had elevated hemoglobin. Diagnosis is confirmed by establishing a decreased P50 and excluding hemoglobin mutants, and by establishing a decreased 2,3-BPG level and BPGM enzyme activity. Methemoglobinemia Methemoglobinemia is usually suspected when a patient is cyanotic with low oxygen saturation by pulse oximetry, but with normal PaO2 level. Methemoglobin levels are often included in blood gas measurements. Methemoglobin is generated when oxygen-carrying ferrous iron (Fe21) has been oxidized to ferric iron (Fe31). The ferric hemes of methemoglobin are unable to bind oxygen and, additionally, if a ferriheme subunit is part of a hemoglobin tetramer, the oxygen affinity of the accompanying ferrous hemes in the hemoglobin tetramer is Lanzkowsky’s Manual of Pediatric Hematology and Oncology 202 10. Primary and secondary erythrocytosis increased. As a result, the oxygen dissociation curve is left-shifted and oxygen tissue delivery is impaired and in the congenital forms is often associated with erythrocytosis. In normal physiological conditions, methemoglobin is converted to hemoglobin by enzyme cytochrome b5 reductase (also known as methemoglobin reductase or b5R). Congenital methemoglobinemia from b5R-inherited mutations is an autosomal recessive disorder. There are two types of b5R deficiency, in type 1 the enzyme deficiency is restricted to erythrocytes and the mutations generate unstable protein leading to deficiency in the erythrocytes lacking the protein-synthesis capacity. Patients with this type chronic methemoglobinemia can be asymptomatic to minimally symptomatic and often have erythrocytosis. The less common type 2 b5R deficiency, wherein the enzyme deficiency is generalized in all cells, is associated with developmental and neurological abnormalities; most infant die within first year of life. Methemoglobinemia can also be caused by various mutations of globin genes, known as hemoglobins M, inherited as an autosomal dominant phenotype. Acquired methemoglobinemia is usually caused by exposure to oxidizing substances or drugs, including nitrates and sulfa-containing antibiotics. Acute methemoglobinemia is a medical emergency, and early recognition is critical because it can be life-threatening. It usually does not cause erythrocytosis unless it has been chronic for weeks. Other causes of erythrocytosis Physiologically inappropriate erythrocytosis is often due to exogenous sources of EPO. Several malignancies, for example, hepatocellular carcinoma, renal cell carcinoma, uterine myomas, and cerebellar hemangiomas have been shown to produce EPO. Large, bulky tumors produce erythrocytosis by mechanical interference with blood supply to the kidneys, resulting in false sensing of hypoxia and EPO production. Renal erythrocytosis is due to EPO produced by renal cysts, polycystic kidney disease, or hydronephrosis. Doping of EPO by athletes can cause iatrogenic erythrocytosis. Endocrine disorders, such as pheochromocytomas, aldosterone-producing adenomas, Barter syndrome, and dermoid cysts of the ovary, can result in inappropriate EPO production through mechanical interference with renal blood supply or hypertensive damage to renal parenchyma resulting in a false sensing of hypoxia by the kidneys. Excess androgens can also cause an increase in hematocrit, mainly by two mechanisms—stimulation of EPO production or an independent hyperproliferative effect on erythrocyte precursors. It is often seen in patients taking exogenous testosterone supplements. Postrenal transplantation erythrocytosis occurs following kidney transplantation in adult patients and is associated with dysregulation of angiotensin receptor. These patients respond to drugs that cause inactivation of the renin!angiotensin system, such as angiotensin-converting enzyme inhibitors (ACE inhibitors). Patients unable to tolerate ACE inhibitors can be treated with an angiotensin II AT1 receptor antagonist. Cobalt toxicity has been associated with erythrocytosis. Excessive cobalt exposure leading to erythrocytosis was noted in miners in Peru, and also in patients with hip prosthesis. With time the prosthesis head wears down, exposing the chrome cobalt leading to increased blood concentration of cobalt and systemic manifestation of toxicity, including cardiomyopathy, cognitive decline, and neuropathy, in addition to erythrocytosis. Elevated level of manganese has also been shown to be associated with erythrocytosis. A congenital disorder with manganese transport leading to elevated blood level of manganese, liver cirrhosis, neurological deficits, and erythrocytosis was reported in a child born to consanguineous parents in 1978. Several similar symptoms have been described since then. Severe lung diseases affecting gas exchange and causing hypoxia, such as cystic fibrosis and α-1 antitrypsin deficiency!related lung diseases, may cause secondary compensatory erythrocytosis. However, quite often, erythrocytosis may not be clinically apparent because of concomitant anemia due to chronic inflammation, as well as increase in plasma volume. Smoking causes carboxyhemoglobinemia (HbCO), which can be measured with standard arterial blood gas measurements. Carbon monoxide has a 200-fold higher affinity for hemoglobin than oxygen and HbCO is fairly stable. In Eisenmenger complex, patients have right-to-left shunting of blood, resulting in increased pulmonary vascular resistance. Because of the admixture of desaturated venous blood with arterial circulation, they develop compensatory erythrocytosis due to tissue hypoxia. Clinically, they may manifest symptoms of hyperviscosity. Phlebotomy may alleviate symptoms of hyperviscosity; however, excessive phlebotomies may cause iron deficiency leading to an increase in HIF, which may further complicate pulmonary vasoconstriction. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Diagnostic approach to erythrocytosis 203 A rare cause of erythrocytosis has been described lately, known as TEMPI syndrome. It comprises Telangiectasias, Elevated EPO and erythrocytosis, Monoclonal gammopathy, Perinephric fluid collection and Intrapulmonary shunting. Telangiectasias often occur on the face, arms, and trunk. Elevated EPO and erythrocytosis is reported to precede the hypoxia from intrapulmonary shunting. The symptoms are slowly progressive and the cause is still largely unknown, but successful treatment has been reported using therapy directed against the monoclonal gammopathy, such as proteasome inhibitors. High altitude erythrocytosis is a physiologic response to hypoxia. As altitude increases, a decrease in atmospheric pressure reduces the partial pressure of inspired oxygen, even though the percentage of oxygen in the air is constant. At an altitude of 5000 m, the atmospheric pressure and partial pressure of oxygen decreases by 50% compared to sea level. This fall in pressure reduces the driving force for gas exchange in the lungs, leading to arterial hypoxemia and tissue hypoxia, which causes secondary erythrocytosis as a compensatory response. Acute and chronic exposure to high altitude hypoxia leads to many physiologic changes that have overall detrimental effects, such as acute mountain sickness and chronic mountain sickness. However, in native highaltitude dwellers such as Tibetans highlanders, distinct evolutionary adaptations have been observed which protect them from erythrocytosis and many of the detrimental effects of chronic exposure to hypoxia, unlike the Andean and Ethiopian highlanders in whom erythrocytosis is more common. With recent advances in genomics, we are beginning to understand more about the molecular and genetic mechanisms underlying these high-altitude human adaptations. Diagnostic approach to erythrocytosis Fig. 10.1 shows a diagnostic algorithm for the diagnosis of erythrocytosis in children. The initial step is to apply the appropriate age-specific reference range for confirmation and then repeat laboratory studies, as m FIGURE 10.1 Diagnostic algorithm for erythrocytosis. Abbreviations: 2,3-BPG, 2,3-Bisphosphoglycerate; EPO, erythropoietin; EPOR, erythropoietin receptor; HbM, hemoglobin M; HIF2α, hypoxia-inducible factor 2α; PHD2, prolyl hydroxylase-2; TEMPI, Telangiectasias, Elevated erythropoietin and erythrocytosis, Monoclonal gammopathy, Perinephric fluid collection and Intrapulmonary shunting; VHL, von Hippel!Lindau. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 204 10. Primary and secondary erythrocytosis hemoglobin concentration may reflect a transient decrease in plasma volume due to dehydration, causing hemoconcentration (relative erythrocytosis). Next, a determination has to be made as to whether the increased hemoglobin is acquired or congenital and, if congenital, whether it is familial; obtaining a thorough family history is crucial. If the hemoglobin is persistently elevated, hypoxia should be considered the most common cause. An arterial oxygen saturation level of ,92% suggests cardiac or pulmonary etiologies. A complete blood count (CBC), serum EPO level, arterial blood gas, and P50 value (either by Hemox-Analyzer or calculated from venous blood gasses) should be done, as well as red cell and plasma volume studies to rule out spurious erythrocytosis due to chronic contraction of plasma volume. Arterial blood gas studies are helpful in determining the presence of arterial hypoxemia due to inadequate oxygenation, HbCO due to smoking or carbon monoxide poisoning, or methemoglobinemia. The CBC may reveal increased leukocytes, platelets, and erythrocytes, which may often coexist in PV. JAK2V617F mutation testing is now a widely available test, and 98% of PV patients are positive for the mutation. JAK2 exon 12 mutations can be done for those negative for JAK2V617F. If JAK2 mutations are negative, it is highly unlikely that the patient has PV. However, testing for endogenous erythroid colony formation can be considered in these JAK2-negative patients who harbor a strong suspicion for PV, but this test is not standardized or available widely. Bone marrow biopsy is suggested by some in determining marrow hyperproliferation, but in our experience it has not been very helpful in determining a specific cause of erythrocytosis. • Primary familial congenital erythrocytosis is another primary erythrocytosis that has low serum EPO level and presents with isolated erythrocytosis without leukocytosis or thrombocytosis; it has autosomal dominant pattern of inheritance (although de novo cases have been known to occur). • EPO levels will be low in primary erythrocytoses, while in secondary erythrocytosis the EPO level will be inappropriately elevated or high normal that is inappropriate to the high hemoglobin level. Disorders resulting from high hemoglobin oxygen affinity such as high-affinity hemoglobin mutants or low 2,3-BPG concentrations are diagnosed with a decreased P50 from a Hemox-Analyzer, an instrument that records blood oxygen equilibrium curves. If a Hemox-Analyzer is not available, the P50 value can be calculated from freshly obtained venous blood gasses by applying the formula: P50 std 5 antilog logð1=kÞ ; n where 1/k 5 [antilog (nlogPO2(7.4))] ' ð100 2 SO2 Þ=SO2 ; n 5 Hill’s constant. The PO2 in venous blood at 37" C can be converted to PO2 at pH 7.4 with the formula: # ! "$ log PO2ð7:4Þ 5 log PO2 2 0:5 7:40 ! pH where pH is measured from the antecubital venous blood. P50 can be calculated using this formula in excel sheet which can be found on the Internet at http://www. medsci.org/v04/p0232/ijmsv04p0232s1.xls. Further reading and references Alsafadi, T.R., Hashmi, S.M., et al., 2014. Polycythemia in neonatal intensive care unit, risk factors, symptoms, pattern, and management controversy. J. Clin. Neonatol. 3 (2), 93!98. Cario, H., McMullin, M.F., et al., 2013. Erythrocytosis in children and adolescents-classification, characterization, and consensus recommendations for the diagnostic approach. Pediatr. Blood Cancer 60 (11), 1734!1738. Goina, F., Teofili, L., et al., 2012. Thrombocythemia and polycythemia in patients younger than 20 years at diagnosis: clinical and biologic features, treatment, and long-term outcome. Blood 119 (10), 2219!2227. Gordeuk, V.R., Key, N.S., Prchal, J.T., 2019. Re-evaluation of hematocrit as a determinant of thrombotic risk in erythrocytosis. Haematologica 104 (4), 653!658. Gordeuk, V.R., Miasnikova, G.Y., et al., 2020. Thrombotic risk in congenital erythrocytosis due to up-regulated hypoxia sensing is not associated with elevated hematocrit. Haematologica 105 (3). Hopfeld-Fogel, A., Kasirer, Y., Mimouni, F.B., Hammerman, C., Bin-Nun, A., 2020. Neonatal polycythemia and hypoglycemia in newborns: are they related? Am. J. Perinatol. 10.1055/s-0040-1701193. Kucine, N., 2020. Myeloproliferative neoplasms in children, adolescents and young adults. Curr. Hematol. Malig. Rep. 15 (2), 141!148. Lorenzo, F.R., Yang, C., et al., 2013. Novel compound VHL heterozygosity (VHL T124A/L188V) associated with congenital polycythaemia. Br. J. Haematol. 162 (6), 851!853. Pasquier, F., Marty, C., Balligand, T., et al., 2018. New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis. Haematologica 103, 575. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 205 Patnaik, M.M., Tefferi, A., 2009. The complete evaluation of erythrocytosis: congenital and acquired. Leukemia 23, 834. Sergueeva, A.I., Miasnikova, G.Y., Polyakova, L.A., et al., 2015. Complications in children and adolescents with Chuvash polycythemia. Blood 125 (2), 414!415. Stembridge, M., Williams, A.M., et al., 2019. The overlooked significance of plasma volume for successful adaptation to high altitude in Sherpas and Andean natives. Proc. Natl. Acad. Sci. U.S.A. 116 (33), 16177!16179. Teofili, L., Giona, F., et al., 2007. Markers of myeloproliferative diseases in childhood polycythemia vera and essential thrombocythemia. J. Clin. Oncol. 25, 1048. Uslu, S., Ozdemir, H., Bulbul, A., Comert, S., Can, E., Nuhoglu, A., 2011. The evaluation of polycythemic newborns: efficacy of partial exchange transfusion. J. Matern. Fetal Neonatal Med. 24 (12), 1492!1497. Zhuang, Z., Yang, C., Lorenzo, F., et al., 2012. Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. N. Engl. J. Med. 367 (10), 922!930. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 11 Disorders of white blood cells Kelly Walkovich1 and James A. Connelly2 1 Pediatric Hematology/Oncology, Department of Pediatrics, C.S. Mott Children’s Hospital, University of Michigan, Ann Arbor, MI, United States 2Pediatric Hematology/Oncology, Monroe Carell Jr Children’s Hospital, Vanderbilt University Medical Center, Nashville, TN, United States The most inclusive term for white blood cells (WBCs), that is, leukocytes, refers to the colorless amoeboid blood cells of the immune system involved in counteracting foreign substances and disease. Among these cells are granulocytes, a term that most accurately denotes the presence of granules in their cytoplasm, that is, neutrophils, eosinophils, basophils, and mast cells. While often used interchangeably with granulocytes, the term phagocyte refers to cells capable of engulfing and digesting microbes, foreign material, and debris such as neutrophils, monocytes, and macrophages. In addition to granulocytes, mononuclear cells, including monocytes and lymphocytes, [i.e., B, T, and natural killer (NK) cells], are key members of the leukocyte family. Together these cells form the innate and adaptive immune systems to protect children and adults from a variety of external and internal insults, including infections, trauma, and cancer. Knowledge of their normal development and function is essential to understanding how their absence and/or dysfunction results in disease. Many patients with leukocyte disorders present in infancy or early childhood, although diagnosis may be delayed until adulthood with many of the acquired conditions more commonly seen in adolescence or adulthood. Leukocytosis Leukocytosis is defined as a total WBC count that is more than two standard deviations greater than the mean for age. The normal WBC range is 4400!11,000 cells/µL in adults but is age dependent in children, with the highest total WBCs seen within the first 2 weeks of life followed by a gradual decrease to adult normal ranges by early adolescence. Most often, leukocytosis is secondary to an excess of mature neutrophils; however, it may be due to a marked increase in lymphocytes, monocytes, eosinophils, and/or basophils. The clinical evaluation of leukocytosis is strongly influenced by which cell type is involved, the duration of the leukocytosis, and any associated findings. Leukemia and lymphomas are of particular concern when the leukocytosis is predominantly from immature cells. Hyperleukocytosis with a WBC . 100,000 cells/µL is almost exclusively associated with neoplastic causes and is an oncologic emergency due to the risk of leukostasis (see Chapter 31: Management of Oncologic Emergencies). A leukemoid reaction represents an exaggerated nonmalignant leukocytosis with a WBC count .50,000 cells/µL consisting of primarily mature neutrophils accompanied by increased numbers of bands, metamyelocytes, and myelocytes. Leukemoid reactions are more frequently seen in neonates and children and may be associated with severe bacterial infections, certain medications, or other physiologic stressors. Similarly, a leukoerythroblastic reaction caused by myelophthisis secondary to myelofibrosis, granulomatous or neoplastic invasion of the marrow space or osteopetrosis, can also present with leukocytosis accompanied by significant elevations of nucleated red blood cells (RBCs). Of note, a left shift wherein .5% of immature neutrophils, primarily bands, are in the blood indicates significant marrow stress with a depletion of the reserve pool of neutrophils and is often seen in serious bacterial infections, burns, hemorrhage, massive hemolysis, trauma, or major surgery. Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00002-7 207 © 2022 Elsevier Inc. All rights reserved. 208 11. Disorders of white blood cells Prior to extensive evaluation of leukocytosis, review of the blood smear should be undertaken to exclude spurious elevations in the WBC count. Processes that interfere with the automated enumeration of WBCs are the most likely causes of spurious elevations, including platelet clumping (most often due to insufficient sample anticoagulation and pseudothrombocytopenia from EDTA-dependent agglutinins), a high fraction of nucleated RBCs frequently encountered in newborns and in hemolytic anemias that may be miscounted as leukocytes, or the presence of cryoglobulins. Leukopenia Leukopenia refers to an abnormally low WBC that results from a decrease in one or more subclasses of leukocytes, typically either neutrophils and/or lymphocytes, in the blood. Leukopenia is frequently transient and secondary to infections, medications, and/or nutritional status. However, persistent leukopenia can herald an underlying malignancy, bone marrow failure syndrome, primary immune deficiency, autoimmune/immune dysregulation disorder, or metabolic syndrome. Neutrophil disorders WBCs arise from hematopoietic stem cells in the bone marrow. Each day roughly 1.6 billion leukocytes per kilogram of body weight are produced by the bone marrow and more than half of those cells are neutrophils. Ninety percent of neutrophils are retained in the bone marrow within reserve pools and 7!8% are located in peripheral tissues, leaving only 2!3% of the total WBC neutrophils to be detected in freely circulating blood. Hence, the total WBC count and absolute neutrophil count (ANC) can be greatly influenced by changes in myelopoiesis, size, and/or rate of release of cells from the marrow reserve pools, alterations in the balance of marginated versus freely circulating cells in the blood and the egress of WBCs into tissues. Normal neutrophil development and function Neutrophils have six distinct phases of proliferation and maturation that occur over 12!14 days. The first three phases, myeloblast, promyelocyte, and myelocytes, constitute the proliferative pool, while the latter three phases, metamyelocytes, bands, and polymorphonuclear neutrophils, are postmitotic and account for the majority of the neutrophils in the marrow space. Neutrophil maturation is characterized by the progressive development of granules within the cytoplasm, transitioning from a rich blue cytoplasm to a predominantly pink cytoplasm on hematoxylin and eosin (H&E) stain upon reaching maturity and increasing nuclear condensation with segmentation until the typical 3!5 lobed nucleus of a polymorphonuclear neutrophil is achieved. Cytoplasmic granules are central to neutrophil function as they contain key degradative enzymes and other components needed for effective microbial killing. • Primary granules, the azurophilic granules most notable in promyelocytes, contain elastase and myeloperoxidase. • Secondary granules first seen in myelocytes contain plentiful lactoferrin as well as other chemotactic, opsonic, and adhesion protein receptors, for example, CD11b/CD18, and the gp91phox/p22phox component of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. • Tertiary granules first evident in bands and secretory granules also contain adhesion protein receptors and components of the NADPH oxidase. Notably, secretory granules also contain alkaline phosphatase. Once fully matured and released from the marrow, neutrophils in the vasculature or peripheral tissues survive for only a few hours up to 5 days. Neutrophil cell death is via a combination of apoptosis and macrophage engulfment. Mature neutrophils have several distinct functions, including the ability to egress from the marrow space into the peripheral vasculature mediated by granulocyte colony-stimulating factor (G-CSF) and the interaction between CXCL12/SDF-1 and CXCR4, adhesion to and subsequent migration through the vasculature wall through the utilization of integrins (CD11a, CD11b, CD11c, and CD18) and selectins, chemotaxis, phagocytosis, and microbial killing largely reliant on functional granule fusion and NADPH oxidase activity. The most recently appreciated neutrophil function is NETosis, a form of neutrophil cell death characterized by the release of Lanzkowsky’s Manual of Pediatric Hematology and Oncology Neutrophil disorders 209 intracellular contents into the extracellular space that serves to physically ensnare microbes and enhance the overall localized immune response. Disruption in the normal neutrophil maturation leading to either neutrophilia or neutropenia, and/or neutrophil dysfunction results in clinically apparent disease as detailed next. Neutrophilia By definition, neutrophilia is an increase in the ANC that is more than two standard deviations above the mean for age. ANC 5 ðpercent of segmented neutrophils 1 bandsÞ 3 the total WBC count: Bands are included in the ANC calculation as they are fully functional phagocytes. In adults the normal WBC range is 4400!11,000 cells/µL, with the typical neutrophil predominance of 60!70% of the total WBC count attained during puberty. Hence, neutrophilia in adults is generally an ANC . 7700 cells/µL. The normal ANC ranges for children vary based on age with the highest ANCs seen in the first 24 hours after birth (range 5000!28,000 cells/µL). Neutrophilia may occur either through: • enhanced granulopoiesis, • accelerated mobilization of neutrophils from the bone marrow to the blood, • release of neutrophils from the marginated pool, or • impaired neutrophil egress into tissues. Neutropenia Similarly, neutropenia is a decrease in the absolute number of peripherally circulating segmented neutrophils and bands. Mild neutropenia is defined as an ANC between 1001 and 1500 cells/µL, moderate between 501 and 1000 cells/µL, and severe # 500 cells/µL. An ANC , 200 cells/µL is also referred to as agranulocytosis. The ANC must be considered in the context of both age and ethnicity. Neonatal ANC values are affected by the surge of neutrophils released from the marrow secondary to the stress of delivery and the subsequent decrease in the marrow proliferative pool postbirth. The lower limit of normal for an ANC during the first 24 hours after birth is 5000 cells/µL, then 1500 cells/µL during the first 2 weeks, followed by 1000 cells/µL between 2 weeks and 1 year. After 1 year the lower limit of normal for the ANC is 1500 cells/µL. In addition to age, many healthy individuals of African and Middle Eastern descent have a baseline lower ANC as compared to their peers with Western European or Asian heritage. The difference in the ANC is linked to a polymorphism in the Duffy antigen receptor chemokine (DARC) gene that encodes the Duffy antigen (FyA/B), a chemokine receptor expressed on the surface of RBCs used by Plasmodium vivax to infect RBCs. Patients with homozygous DARC null polymorphisms are often regarded as having benign ethnic neutropenia (BEN), although Duffy-null status is not required for the diagnosis of BEN. Patients with BEN are not at risk for infection or malignancy. Heterozygous patients have normal mean neutrophil counts. Neutrophil dysfunction Neutrophil dysfunction is defined as the partial or complete failure of the neutrophil to execute one or more of the key neutrophil functions. The International Union of Immunological Societies (IUIS) divides disorders of neutrophil and other phagocyte function into three broad categories: 1. defects of motility 2. defects of respiratory burst 3. other nonlymphoid defects Additionally, several other primary immunodeficiencies and immune dysregulation disorders have neutrophil dysfunction as a key element of their phenotype despite being classified under alternate IUIS categories. Support for neutrophil dysfunction can be gleaned from careful review of the blood smear, for example, identification of giant azurophilic granulocyte granules in Chediak!Higashi syndrome (CHS) or the absence of Lanzkowsky’s Manual of Pediatric Hematology and Oncology 210 11. Disorders of white blood cells granules and bilobed neutrophils in neutrophil-specific granule deficiency. More specific testing through flow cytometry for members of the beta-2 (β2) integrin family crucial to leukocyte adhesion to and migration through vasculature walls, namely, CD11b and CD18, is readily accessible. Similarly, assessment of the respiratory burst via flow cytometry with dihydrorhodamine (DHR), which has largely replaced the older, qualitative nitroblue tetrazolium (NBT) test, is also widely available. DHR relies on the oxidation of the test substrate DHR by hydrogen peroxide to produce rhodamine 123, a florescent compound detectable by flow cytometry, and the NBT relies on reduction by the NADPH oxidase complex to produce a colorimetric change that can be identified by microscopy. Other assays of neutrophil dysfunction, for example, chemotaxis and bactericidal activity, are performed in highly specialized laboratories, but interpretation of these tests is often hindered by the technical difficulty in performing the assay as well as methods of blood drawing, age of the specimen, influences of shipping/travel conditions, and need for normal controls. With the limitations of functional testing of neutrophils, genetic testing for pathogenic variants is being increasingly utilized. Approach to suspected neutrophil disorders Signs of neutrophil disorders include: 1. recurrent fevers, 2. oral ulcers, 3. chronic gingival or dental problems, 4. poor wound healing, 5. recurrent or unusual infections, 6. lack of pus, and 7. abnormal blood counts. The increasing recognition of neutropenia as a feature of primary immunodeficiency disorders and autoimmune/immune dysregulatory disorders, such as ADA2 deficiency, reinforces the need for a broad differential in the evaluation of patients with quantitative and qualitative neutrophil defects. Approach to neutrophilia By definition, 2.5% of the normal population will have baseline mild!moderate neutrophilia. Neutrophilia is often a physiologically normal response of the hematopoietic system to: • stress from acute or chronic infections, • underlying inflammatory disease states, • medication exposures, and • nonhematologic malignancies. A careful medical history to ascertain symptoms of infection (e.g., fever, cough, diarrhea, and rash), inflammation (e.g., periodic fevers, photosensitive rashes, joint pain, and chronic diarrhea), malignancy (e.g., fevers, weight loss, fatigue, and night sweats), drug exposures (including cigarette smoking), and physiologic stress (e.g., obesity, exercise, and pregnancy) can often determine the underlying etiology of the neutrophilia. Screening questions for sickle cell anemia, chronic hemolytic anemias, trisomy 21, and leukocyte adhesion deficiency are also appropriate, particularly in younger patients. Physical examination for localized infection (e.g., otitis media), joint swelling, rashes, or lymphoproliferation should be undertaken. Both the complete blood count (CBC) with differential and review of the blood smear can offer helpful insights to support infection/inflammation (e.g., toxic granulation, Döhle bodies), hemolysis, or raise concern for dysplastic processes. Additional labs to test for specific microbes, screen for autoimmunity, assess inflammation, and/or tumor lysis may be appropriate but should be guided by the patient-specific presentation. For patients with significant or persistent neutrophilia in the absence of overt infection, inflammation or medication exposure, or with coincident anemia and/or thrombocytopenia, strong consideration should be given to bone marrow aspirate and biopsy with flow cytometry, cytogenetics, and molecular testing to assess for myeloproliferative neoplasms (MPN), hematologic malignancy, or other infiltrative marrow processes. Additionally, flow cytometry for CD11b/CD18 to screen for leukocyte adhesion deficiency can be considered in patients with a concerning personal or family history. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Neutrophil disorders 211 Approach to neutropenia The majority of patients presenting with neutropenia will have transient neutropenia secondary to acute infections or medication exposures. Nonetheless, a complete history should be ascertained, focusing on the presence or absence of: • recurrent fevers, • oral ulcers or gingivitis, • frequent or unusual skin or deep-seated infections, • failure to thrive or malabsorption, • dietary/nutritional deficiencies, • prior or family history of malignancy, • liver or lung fibrosis, • early graying, • significant viral infections [e.g., warts, Epstein!Barr virus (EBV)], • lymphedema, and • evidence of autoimmune disease. On physical examination the oral cavity should be thoroughly assessed for oral ulcers, gingival inflammation, as well as dental abnormalities. Additional focus should include skeletal anomalies, rashes, warts, and lymphoproliferation. Patient height should also be obtained as many of the bone marrow failure syndromes and primary immunodeficiencies that manifest with neutropenia also have short stature. The blood smear should be reviewed for signs of marrow stress and a CBC with differential should be obtained to assess for other cell lineage involvement as multilineage involvement may prompt further evaluation for autoimmune disease (i.e., Evans syndrome), malignancy, or bone marrow failure. Depending on the patient history, additional testing with pancreatic enzymes (serum isoamylase, fecal elastase) and pancreatic ultrasound for Shwachman!Diamond syndrome (SDS), chromosomal breakage for Fanconi anemia, telomere length for dyskeratosis congenita, or lymphocyte subsets and quantitative immunoglobulins to screen for immunodeficiency may be appropriate. If a satisfactory etiology of the neutropenia is not identified, a bone marrow aspirate and biopsy is warranted. Given the overlap of neutropenia with primary immunodeficiency disorders, genetic sequencing that includes both inborn errors of myelopoiesis and immunity is increasingly being utilized to secure a molecular diagnosis to support biologically rationale therapy selection, provide an informed framework for supportive care and prognosis as well as influence decisions regarding definitive therapy, for example, hematopoietic stem cell transplant (HSCT). Approach to neutrophil dysfunction For patients with a normal ANC but with symptoms and signs consistent with neutrophil dysfunction, a thorough history should be ascertained with a focus on: • infection history with unusual organisms (e.g., catalase-positive organisms, nontuberculous mycobacteria, salmonella, severe viral infections); • oral health (e.g., ulcers, gingivitis, periodontitis); • rashes [e.g., pyoderma gangrenosum!like lesions that may raise concern for leukocyte adhesion deficiency type 1 (LAD-1), extensive warts concerning for GATA binding protein 2 (GATA2) haploinsufficiency or eczema consistent with Wiskott!Aldrich syndrome (WAS) or STAT3 LOF]; • delayed umbilical cord detachment; • history of omphalitis; and • lack of neutrophilic tissue infiltration and pus formation in the setting of infection. Similar to neutropenia, the physical examination should focus on the oral cavity for evidence of oral ulcers and chronic gingival inflammation and skeletal anomalies as well as skin findings, lymphedema, and lymphoproliferation. Review of the blood smear along with functional testing with flow cytometry for CD11b/CD18 or DHR testing can be pursued, although the majority of patients will rely on identification of pathogenic genetic variants for diagnosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 212 11. Disorders of white blood cells Primary neutrophilia disorders Hereditary neutrophilia An extremely rare benign hereditary neutrophilia caused by an activating heterozygous variant in the G-CSF receptor gene, CSF3R, has been described. Clinical presentation • Most patients with hereditary neutrophilia are asymptomatic but may have hepatosplenomegaly. • One patient is reported to have had a systemic inflammatory response syndrome and later to have developed myelodysplastic syndrome (MDS) with significant dysgranulopoiesis. • The median ANC reported is 16,900 cells/µL. Diagnosis • Genetic sequencing can confirm a suspected diagnosis of hereditary neutrophilia via identification of pathogenic variants in CSF3R. • It is plausible that additional genes not yet recognized may contribute to a similar phenotype as a cohort of healthy 34 patients with ANC ranging from 11,000 to 40,000 cells/µL (genetics unknown) is reported without an identified molecular etiology. Management and treatment • There is no standard approach to management of patients with hereditary neutrophilia. • Vigilance for hyperinflammatory syndromes and MDS/malignancy should be maintained. Primary neutropenia disorders ELANE!related neutropenia ELANE is the gene that encodes neutrophil elastase, a protease expressed early in neutrophil development. Disruption in neutrophil elastase structure results in initiation of the unfolded protein response, acceleration of apoptosis of developing myeloid cells, and ineffective myelopoiesis. Pathogenic variants in ELANE clinically result in the spectrum of severe congenital neutropenia (SCN) and cyclic neutropenia (CN). Roughly 45!60% of cases of SCN are due to underlying ELANE pathogenic variants, while 90% of CN patients have pathogenic variants in ELANE identified. Clinical presentation • Patients with the SCN phenotype present in early infancy with fevers, recurrent or deep-seated infections, and a persistently low ANC , 500 with a “promyelocyte arrest” on bone marrow evaluation. • Patients with the CN phenotype generally present in the first year of life with fevers, mouth ulcers, and infections classically recurring every 21 days. • Reciprocal monocytosis is a laboratory hallmark of CN. • Clostridial species-related and Gram-negative rod infections were commonly seen in CN patients prior to the advent of G-CSF. • SCN patients are at high risk for MDS/acute myeloid leukemia (AML) with a cumulative incidence of 22% at 15 years. • Only rare cases of leukemia have been reported in the context of CN. Diagnosis • Identification of a pathogenic variant in the ELANE confirms the diagnosis of ELANE-related neutropenia. • Both SCN and CN are transmitted in an autosomal dominant fashion. • Of note, several other genetic causes of the SCN phenotype associated with promyelocytic arrest (e.g., HAX1, CLPB, G6PC3, or GCSF3R) may also be identified by genetic sequencing. Management and treatment • The mainstay of treatment for ELANE-related neutropenia is G-CSF. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Neutrophil disorders 213 • Patients with SCN require higher doses (start B5 µg/kg/day; the majority of patients respond to 3!10 µg/kg/ day but the most refractory cases may require upward of 100 µg/kg/day) than those with CN (start B2.5 µg/ kg/day) to achieve a low normal ANC. • Treatment with G-CSF in CN patients does not abolish the neutrophil nadir but instead hastens the cycle and reduces the period of profound neutropenia from 7!10 to 1!3 days. • Vigilance for the development of MDS/AML is of particular importance in SCN with most patients undergoing annual bone marrow evaluations and quarterly blood counts. • Supportive care with regular dental care, oral rinses, aggressive antibiotic use for documented infections, and monitoring for osteopenia/osteoporosis is recommended. • HSCT remains the only definitive therapy but is often reserved for patients with minimal response to G-CSF and/or malignant transformation. • Oral elastase inhibitors and gene editing techniques are also under investigation as potential therapies for ELANE-mediated neutropenia. Shwachman!Diamond syndrome (SDS) SDS is an autosomal recessive disorder most commonly due to pathogenic variants in the SBDS gene that encodes a protein important for ribosome biogenesis and RNA processing. Clinical presentation • Neutropenia typically presents in the first year of life, although may be detected until adulthood and the degree of neutropenia may fluctuate and/or progress to bone marrow failure. • Anemia and thrombocytopenia may also be present. • Exocrine pancreatic insufficiency and skeletal abnormalities are common but may be subtle and are not required for diagnosis. • Pancreatic ultrasound will often show pancreatic lipomatosis. • Infections are frequently seen as patients with SDS secondary to neutropenia and/or neutrophil chemotactic defects, low numbers of B cells, and/or abnormal T-cell proliferation. • Leukemic transformation with MDS/AML is common in SDS with an overall incidence of 8.1% at 10 years. Diagnosis • Patients may be diagnosed with SDS either by identification of biallelic pathogenic variants in SBDS or via clinical manifestations based on consensus guidelines. • Roughly 90% of patients with SDS have an SDBS pathogenic variant identified. • Additional genes associated with ribosome assembly or protein translation, for example, DNAJC21, ELF1, and SRP54, also demonstrate an SDS-like phenotype. Management and treatment • Hematopoietic stem cell transplant is the only curative option for the hematologic abnormalities in patients with SDS but it is often reserved for patients refractory to G-CSF, severe and intractable cytopenias, or with malignant transformation. • Supportive care with G-CSF, antibiotics, and pancreatic enzyme replacement is common. Primary disorders of neutrophil dysfunction Warts, hypogammaglobulinemia, infections, and myelokathexis syndrome (WHIM) While not formally classified by the IUIS under disorders of phagocytes or phagocyte function, the syndrome of warts, hypogammaglobulinemia, infections, and myelokathexis, that is, WHIM syndrome, does include phagocyte deficits. WHIM is rare with an estimated incidence of 0.23 cases per million live births. In most patients the disorder is due to an autosomal dominant gain of function pathogenic variant in CXCR4 that results in dysfunction of the CXCR4 chemokine receptor, a G protein!coupled receptor expressed on progenitor cells of the hematopoietic, cardiovascular, nervous, and reproductive systems, that binds the ligand CXCL12/SDF-1. In WHIM syndrome, ligation of CXCL12 to mutated CXCR4 leads to prolonged intracellular signaling secondary to failed downregulation/internalization of CXCR4. The persistent signaling downstream of CXCL12!CXCR4 results in retention of neutrophils in the bone marrow and subsequent myelokathexis. CXCL12!CXCR4 signaling Lanzkowsky’s Manual of Pediatric Hematology and Oncology 214 11. Disorders of white blood cells is also critical for migration and maturation of lymphocytes. Patients with WHIM syndrome also have adaptive immune defects leading to poor immunoglobulin production and persistent and severe human papillomavirus (HPV) infections in some patients. Clinical presentation • Recurrent bacterial infections affecting the ear, skin, oral cavity, and sinopulmonary tract are common. • Bronchiectasis can develop from repeated infectious insults. • HPV skin infections are common (but not universal) and are frequently extensive and/or refractory to therapy, often leading to disfiguration and functional limitations. • HPV-driven dysplasia and squamous cell cancers are also seen. • Severe neutropenia, with an ANC , 500 cells/µL, monocytopenia, severe lymphopenia, and hypogammaglobulinemia are common. • Bone marrow evaluation classically shows hypercellularity with retention of numerous bands and polysegmented neutrophils. The myeloid:erythroid ratio is increased. • Neutrophils within the bone marrow also have a distinct morphologic appearance with apoptotic features of vacuolization and a hypersegmented pyknotic nucleus; very long filaments connecting the nuclear lobes are characteristic of WHIM. • Tetralogy of Fallot is associated with 10% of WHIM patients. Diagnosis • WHIM syndrome is most readily diagnosed in patients with the appropriate clinical phenotype who are identified to have a pathogenic heterozygous variant in CXCR4. • Patients without a documented pathogenic variant in CXCR4 but with the full WHIM syndrome clinical phenotype have been described. Management and treatment • Ensure early HPV vaccination to help minimize the risk of HPV-driven dysplasia/malignancies. • Intravenous immunoglobulin is a mainstay of therapy to reduce the risk of recurrent infections and thereby minimize the risk of bronchiectasis. • Many patients also receive prophylactic clotrimazole or azithromycin (or equivalent) to help protect against frequent skin and lung infections. • Pulmonary function tests and imaging are used at diagnosis to assess lung function and to monitor for complications. • G-CSF can be useful for patients who fail to mobilize neutrophils from the bone marrow during periods of acute infection (although many patients do so independent of exogenous G-CSF) and presurgical settings to reduce the risk of poor wound healing and postoperative infections. • CXCR4 receptor antagonists are being increasingly utilized to increase neutrophil and lymphocyte counts. Leukocyte adhesion defect, type 1 LAD-1 is a rare autosomal recessive immunodeficiency estimated to occur in 1:1 million live births that is characterized by integrins containing defective CD18. Leukocytes have defective immune cell adhesion to and migration through the vascular endothelium. Chemotaxis, phagocytosis, degranulation, and respiratory burst activity are also abnormal. Of note, deficient expression of CD18 also leads to impaired T-cell function. The degree of disease severity correlates with the degree of CD18 deficiency. Clinical presentation • Newborns may present with omphalitis and/or delayed umbilical cord detachment. • Significant gingivitis and periodontal disease are seen with most patients experiencing complete loss of adult teeth by late adolescence secondary to IL-17/IL-23 axis dysfunction. • Nonhealing ulcers or pyoderma gangrenosum!like lesions, poor wound healing with “cigarette paper” scars, periodontitis, HPV infection, and inflammatory bowel disease are also commonly encountered. • Basal neutrophilia with the absence of pus at sites of infection are hallmarks of the disease. • Profound leukocytosis .100,000 cells/µL can be seen with infections. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Neutrophil disorders 215 Diagnosis • Flow cytometry to assess for CD11b/dysfunctional CD18 expression on the surface of leukocytes can confirm a diagnosis of LAD-1. • Sequencing for the identification of biallelic pathogenic mutations in ITGB2, the gene encoding the β2 integrin CD18, that lead to impairment in β2 integrin expression or heterodimer formation is recommended. Management and treatment • Ensure early vaccination against HPV. • Encourage aggressive oral hygiene. • Ustekinumab, a monoclonal antibody of the p40 subunit common to IL-12 and IL-23, has been documented to improve periodontitis and wound healing in limited numbers of patients. Further studies are underway to evaluate overall efficacy in LAD-1. • For patients with mild disease, prophylactic and/or event-driven antimicrobials may be sufficient to manage infections. • For severely affected patients, HSCT is frequently indicated. Chediak!Higashi syndrome (CHS) CHS is a disorder of abnormal organelle protein trafficking that results from autosomal recessive pathogenic variants in CHS (previously LYST1). The disorder is rare with fewer than 500 cases described worldwide. Clinical presentation • Variable oculocutaneous albinism accompanied by recurrent infections, coagulopathy, progressive neurologic deterioration, and high risk of developing hemophagocytic lymphohistiocytosis (HLH) during the accelerated phase of disease are key disease characteristics. • Progressive neurologic deterioration is seen in the third decade of life even with HSCT and may include cerebellar ataxia, tremor, peripheral neuropathy, and low cognitive abilities that can be functional debilitating. • Neutropenia and impaired bactericidal activity are common as are abnormal platelet aggregation studies and bleeding times. • Giant azurophilic granules in neutrophils and eosinophils are easily identified upon review of the blood smear. Other granule-containing cell types are similarly affected. Diagnosis • A definitive diagnosis requires identification of pathogenic variants in the CHS gene. Management and treatment • Although imperfect, HSCT remains the primary treatment modality. • Supportive care with antimicrobials and G-CSF is often utilized. • For patients in the accelerated phase, standard HLH therapy approaches have been used with modest success. Chronic granulomatous disease Chronic granulomatous disease (CGD) is a genetically heterogeneous disorder caused by defects in the phagocyte NADPH oxidase that occurs with an estimated frequency of 1 in 200,000 live births. The NADPH oxidative complex is responsible for the generation of the oxidative burst in phagocytes. Dysfunction of the NADPH oxidase results in failure of neutrophils, monocytes, and macrophages to kill certain organisms, most notably catalase-positive bacteria, including Staphylococcus aureus, Burkholderia cepacia, Serratia marcescens, and Nocardia, and fungi such as Aspergillus. Clinical presentation • Recurrent, serious, or unusual infections frequently present in infancy or childhood, although the clinical presentation of the patient may be disproportionately underwhelming compared to the degree of infection. • Immune dysregulatory complications, including inflammatory bowel disease and systemic lupus erythematosus, are being increasingly recognized. • Most often the skin, lungs, liver, gastrointestinal (GI) tract, and/or lymph nodes are involved from both infectious and inflammatory pathology. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 216 11. Disorders of white blood cells • Some X-linked CGD patients may also present with the McLeod phenotype (low/absent expression of the Kell RBC antigen associated with anemia, elevated creatine phosphokinase, and neurologic symptoms) or Duchenne muscular dystrophy due to contiguous deletions on the X chromosome. • X-linked carriers frequently manifest with autoimmune/rheumatologic findings. Diagnosis • In patients suspected of having CGD, a DHR should be obtained to assess the respiratory burst. • Abnormal DHR results should prompt genetic testing to identify pathogenic variants in the genes encoding subunits of the NADPH oxidase, that is, CYBB, CYBA, NFC1, NFC2, NFC4, and the gene CYBC1 that encodes the endoplasmic reticulum cytochrome b-245 chaperone, EROS. Of note, there is significant phenotypic overlap with CGD in patients with pathogenic variants in RAC2. Management and treatment • Vaccination with the bacillus Calmette!Guerin (BCG) and live bacterial vaccines should be avoided. Live viral vaccines are recommended. • Lifetime prophylactic antibiotics, that is, trimethoprim!sulfamethoxazole, and antifungals (historically itraconazole although many authorities are utilizing newer generation azoles secondary to emergence of itraconazole-resistant organisms) are key to infection prevention. • Monitoring of inflammatory markers and early responsiveness to subtle examination changes/patient complaints with imaging can help to identify infections such as liver abscesses early. Attempts should be made to identify causative organisms when feasible. • Interferon-gamma therapy is also utilized frequently in the United States, but less so elsewhere. • HSCT is curative for patients with CGD and is being increasing utilized. • Gene therapy is also being explored for CGD treatment. Secondary neutrophil disorders Secondary neutrophilia Reactive acute neutrophilia is often secondary to: • Bacterial or viral infections, and can be seen in association with toxic granulation (prominent primary granules), Döhle bodies (prominent secondary granules), cytoplasmic vacuoles, and/or atypical lymphocytes on blood smear. Generally, there are accompanying symptoms, for example, fever, and laboratory support such as elevated inflammatory markers and/or microbiologic studies to support an active infection. Commonly encountered infections, namely, Pneumococcal spp., Staphylococcus spp., Clostridial spp., as well as mononucleosis, herpes simplex, and varicella can result in neutrophilia, but as the infection resolves the ANC normalizes. • Inflammatory conditions such as trauma/surgery or physiologic insults like heat stroke, myocardial infarction, or vigorous exercise. In these cases, as well as with infection, the excess of neutrophils is primarily due to accelerated release of neutrophils from the reserve pool in the marrow. • Exposure to medications: the most common culprits include corticosteroids, which accelerate the release of mature neutrophils and bands from the marrow reserve pool while also impairing neutrophil migration into tissues, and myeloid recombinant growth factors, for example, G-CSF and granulocyte!monocyte CSF (GMCSF), that stimulate neutrophil production and mobilize neutrophils from the marrow reserves. • Underlying autoimmune or inflammatory conditions. Kawasaki disease is often associated with a frank leukemoid reaction. Juvenile idiopathic arthritis, adult-onset Still’s disease, and acute febrile neutrophilic dermatosis (i.e., Sweet syndrome) are all characterized by a neutrophil-predominant leukocytosis. Other conditions such as chronic infections (e.g., Mycobacterium tuberculosis, hepatitis), inflammatory bowel disease, and cystic fibrosis with bronchiectasis can trigger neutrophilia during the onset or flares of disease. Other than persistent inflammatory conditions, chronic neutrophilia is associated with: • pregnancy, • cigarette smoking, • asplenia or hyposplenia, • sickle cell disease, • other forms of chronic hemolysis, Lanzkowsky’s Manual of Pediatric Hematology and Oncology Monocytes disorders 217 • familial cold urticaria, • periodic fever syndromes, • trisomy 21, • Myeloproliferative neoplasms (e.g., juvenile myelomonocytic leukemia, chronic myelogenous leukemia), and • malignancy. Secondary neutropenia Acquired or secondary neutropenia is also a common phenomenon. Transient neutropenia can be associated with infection, medications, nutritional deficits, or immune-mediated causes. • Viral infections are the most common cause of neutropenia with patients becoming neutropenic within the first 1!2 days of illness and throughout the period of viremia (3!8 days) as well as through the bone marrow recovery period (2!3 weeks). Other bacteria, protozoa, rickettsial, and fungal infections cause neutropenia through various mechanisms, including decreased granulocyte production, increased destruction, or sequestration. • Drug-induced neutropenia is common, and its frequency increases dramatically with age such that only 10% of cases are associated with children and young adults. Nearly any drug can trigger neutropenia, but common offenders include antimicrobial agents (e.g., beta-lactams, trimethoprim!sulfamethoxazole, chloramphenicol), antithyroid drugs, antiseizure agents (e.g., valproic acid), antipsychotics (e.g., clozapine), antipyretics, and antirheumatics. Withdrawal of the suspected offending agent will often result in correction of the neutropenia. However, G-CSF may be employed when drug withdrawal is not feasible and/or the neutropenia is prolonged. The mechanisms of drug-induced neutropenia are twofold, including direct marrow toxicity and/ or immune-mediated destruction of peripheral neutrophils via drug-dependent or drug-induced antibodies. Clinical testing to identify drug-specific antibodies is available for certain medications. • Nutritional deficiencies can contribute to neutropenia. Global deficits in calorie intake as seen in restrictive eating disorders, such as anorexia nervosa, or starvation can result in neutropenia and/or bone marrow hypocellularity. More commonly, specific defects in copper, vitamin B12, or folic acid deficiency from lack of adequate dietary intake, failure to adequately supplement parental nutrition, or lack of/poor absorptive capacity of bowel can lead to ineffective myelopoiesis. More indirectly, patients taking trimethoprim!sulfamethoxazole, which inhibits folic acid metabolism, or phenytoin, which impairs folate absorption in the small intestine, can trigger nutritionally related neutropenia. The neutropenia generally resolves with adequate calorie intake and/or nutrient supplementation. • Immune-mediated causes of neutropenia are often associated with circulating antineutrophil antibodies. Alloimmune neonatal neutropenia occurs after transplacental transfer of maternal alloantibodies directed against antigens on the infant’s neutrophils, analogous to Rh-hemolytic disease. Unlike Rh-hemolytic disease, however, alloimmune neonatal neutropenia can occur with the first pregnancy. The neutropenia is often severe and can result in fever, skin, and umbilical infections. As the maternal IgG wanes via natural decay, the neutropenia resolves; this occurs around 2 months of life. Treatment consists of antibiotics for infections and G-CSF for severe infections. Mothers with autoimmune disorders and circulating antineutrophil antibodies can similarly transmit IgG-mediated antineutrophil antibodies to the fetus as a more passive form of autoimmune neutropenia. In contrast, autoimmune neutropenia of infancy is a benign condition that occurs in infancy through early childhood. The severely low ANC is often detected incidentally through other routine screening around 1 year of age. Antineutrophil antibodies can often be detected; however, they are fraught with both falsepositive and false-negative results. Treatment for autoimmune neutropenia of infancy is generally not necessary as the marrow remains highly functional and can surge production of additional neutrophils during periods of stress. Antibiotics can be used for infections and G-CSF is used peri-operatively to optimize wound healing and/or to avert frequent emergency evaluations for febrile neutropenia. Most patients spontaneously resolve their neutropenia within a period of months to a few years. Secondary autoimmune neutropenia occurring in older children and adults is more frequently associated with other disorders, for example, systemic erythematous lupus or primary immunodeficiency and typically carriers a more significant infectious risk. Monocytes disorders Monocytes are large agranular cells that compose 1!9% of the leukocyte pool in peripheral circulation and serve multiple immune functions, including phagocytosis, antigen presentation, and cytokine production. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 218 11. Disorders of white blood cells Their role in tissues is site-specific and highly specialized due to their ability to mature into both dendritic cells and macrophages. Normal monocyte development and function Monocytes are derived from a common granulocyte!monocyte precursor and progress through the proliferative stages of monoblasts, promonocytes, and monocytes under the influence of cytokines, particularly IL-3, IL-6, and GM-CSF, within the marrow space or spleen. Once mature, monocytes irreversibly leave the bone marrow space to the blood and further migrate into tissues where they differentiate into tissue-specific macrophages or dendritic cells. Definition of monocytosis and monocytopenia While the overall percentage of monocytes in relation to other WBCs stays stable over the lifetime of healthy children as they mature into adults, the absolute monocyte count (AMC) varies by age. Once hematopoiesis in the fetus shifts to primarily bone marrow production around 24 weeks gestation, monocytes are reliably detectable and continue to gradually rise to a peak value of 1500 cells/µL until 2-week postbirth. Thereafter, the monocyte count decreases to below 1000 cells/µL, which is the World Health Organization threshold for absolute monocytosis, and has a gradual decline to achieve the adult plateau of 400 cells/µL. Approach to suspected monocyte disorders In evaluating a patient with a suspected monocyte disorder, it is important to calculate the AMC as the relative monocyte percentage is not as useful in determining normal versus abnormal monocyte counts. Additionally, review of the blood smear is critical for ensuring that the automated differential is valid as atypical lymphocytes, for example, hairy cells or Sezary cells, or hypogranular AML cells can be mistaken as monocytes. Once an abnormal AMC is verified, the remainder of the CBC with differential should be carefully reviewed for additional clues as abnormal monocyte values are rarely found in isolation. Approach to monocytosis History should include: • age of onset, • chronicity of the abnormal AMC, • prior relevant diagnoses (e.g., splenectomy, autoimmune or chronic inflammatory disorders, primary neutrophil disorders), and • potential infectious and drug exposures. Similarly, concurrent evidence of hemolytic anemia or thrombocytosis may provide context for the monocytosis secondary to overall enhanced marrow drive. With persistent monocytosis, particular care should be given to the assessment of other signs of potential neoplastic disease, including symptoms of fever, fatigue, weight loss, night sweats, or symptoms consistent with lymphoproliferation of the liver, spleen, or lymph nodes. Review of the blood smear provides the opportunity to assess for dysplastic changes and hypogranularity of the granulocytes, which would raise concern for a malignant process. Eosinophilia and basophilia may also raise suspicion for malignancy, while neutropenia or the presence of atypical lymphocytes may be consistent with various infections. Other laboratory values should be strongly considered with the suspicion of myeloproliferative disease or neoplasm, particularly a bone marrow aspirate and biopsy with cytogenetics, flow cytometry, special stains, and molecular genetics as appropriate. Approach to monocytopenia Concurrent chronic neutropenia and lymphopenia in association with monocytopenia may signal a rare primary immunodeficiency, that is, GATA2 haploinsufficiency, or be consistent with ELANE-related CN during the period of nadir for the monocyte oscillation. Similar to monocytosis a complete exposure and drug history should also be obtained. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Monocytes disorders 219 Primary monocyte disorders GATA2 haploinsufficiency While not solely a disorder of monocytes, monocytopenia is a prominent feature in GATA2 haploinsufficiency. Originally dubbed “MonoMac syndrome,” GATA2 haploinsufficiency is a primary immunodeficiency and leukemia predisposition syndrome due to autosomal dominant and sporadic heterozygous pathogenic variants in the transcription factor GATA2 that result in loss of function of the mutant allele and subsequent haploinsufficiency. Clinical manifestations • Median age of presentation is 20 years but is highly variable with some patients presenting in early childhood and asymptomatic older patients only identified as part of a family cohort. • Hematologic presentations include chronic neutropenia, bone marrow failure, MDS, AML, and chronic myelomonocytic leukemia. • Nonhematologic presentations include mycobacterial infections, severe or persistent viral infections (e.g., HPV, varicella, EBV), primary lymphedema, pulmonary alveolar proteinosis, and viral-driven solid tumors (e.g., HPV-related genital squamous cell carcinoma). • Hemograms demonstrate profound monocytopenia (78%), B-cell lymphopenia (86%), and NK-cell lymphopenia (82%) in the majority of patients. Tissue macrophages and plasma cells are present. More modest neutropenia (47%) and lymphopenia (51%) are also observed. • Patients with no major complications of disease often have normal blood counts that subsequently decline concurrent with the onset of disease complications. • Bone marrow examinations most frequently show hypocellularity for age, increased reticulin fibrosis, and atypical megakaryocytes. • Monosomy 7 and/or 8 are often noted on bone marrow cytogenetics in the setting of dysplastic or malignant transformation. Diagnosis • A diagnosis of GATA2 haploinsufficiency is achieved by identification of pathogenic variants in GATA2. Care must be taken to include sequencing of the conserved enhancer region of intron 5 in addition to coding sequences. Management and treatment options • HSCT is currently the only curative therapy for GATA2 haploinsufficiency. HSCT corrects the underlying immunodeficiency and abrogates the leukemia risk. • Other supportive care measures (mentioned next) can assist in minimizing disease-associated morbidity. • Ensure varicella vaccination as well as empiric early HPV vaccination. • Consider prophylactic azithromycin for mycobacterial prophylaxis with aggressive antimicrobial management of identified infections. • Screen for sensorineural hearing loss particularly in those patients receiving significant antimicrobial treatment with ototoxic regimens. • Monitor blood counts for the evolution of disease and malignant transformation. • Regular bone marrow evaluations should be undertaken to assess for dysplastic and cytogenetic changes. • Regular dermatologic and gynecologic monitoring for the development of dysplastic or premalignant changes should also be strongly encouraged. • For those with chronic lymphedema, maximize the use of compression stockings and other supportive measures. Secondary monocyte disorders The etiologies for secondary monocyte disorders are broad. The majority of cases of monocytosis are reactive and secondary to infections or inflammatory conditions, although monocytosis can herald underlying malignancy as detailed in Table 11.1 Secondary monocytopenia is less frequently encountered but is seen in hairy cell leukemia, after glucocorticoid administration and in infections associated with endotoxemia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 220 TABLE 11.1 11. Disorders of white blood cells Etiologies associated with secondary monocytosis. Infection Kala-azar Malaria Mycobacterium tuberculosis Rocky Mountain spotted fever Subacute bacterial endocarditis Syphilis Inflammatory bowel disease Myositis Rheumatoid arthritis Sarcoidosis Systemic lupus erythematosus Bone marrow recovery from transient neutropenia Cyclic neutropenia Hemolytic anemia Severe chronic neutropenia Sickle cell anemia Acute myeloid leukemia Cutaneous myeloid dendritic dysplasia Histiocytic medullary reticulosis Lymphoid and plasma cell malignancies, esp. Hodgkin lymphoma Myelodysplastic syndromes Myeloproliferative neoplasms, esp. CMML and JMML Solid tumors, for example, carcinoma Antipsychotics, for example, ziprasidone Corticosteroids Cytokine therapy, for example, G-CSF, GM-CSF Radiation therapy Chronic stress Lipoidoses, for example, Niemann!Pick disease Myocardial infarction Postsplenectomy Tetrachloroethane poisoning Autoimmune and inflammatory disorders Marrow stress Malignancy Iatrogenic Causes Other Abbreviations: G-CSF, Granulocyte colony-stimulating factor; GM-CSF, granulocyte!monocyte colony stimulating factor; CMML, chronic myelomonocytic leukemia; JMML, juvenile myelomonocytic leukemia. Eosinophil disorders Eosinophils are terminally differentiated granulocytes identified on blood smear by their bilobed nuclei and large, cytoplasmic eosinophilic-staining granules. Mature eosinophils are primarily present in the tissues where they can reside for several weeks and play important roles in innate immune function and tissue remodeling and repair. However, the role of eosinophils in health maintenance has been challenged by animal models and recent monoclonal antibodies against the eosinophil granulocyte!stimulating factor, IL-5, resulting in severe eosinopenia but relative safety and tolerance with long-term administration. The role in eosinophilic protection against helminths has even been challenged by some studies suggesting that eosinophils may contribute to the longevity of some parasites in tissue. In contrast to the beneficial roles of eosinophils, dysregulated eosinophil production and activation can promote disease and contribute to destructive tissue pathology. Normal eosinophil development and function Eosinophils differentiate in the human bone marrow from an eosinophil lineage!committed progenitor that develops from a common myeloid progenitor or an upstream multipotent progenitor under the influence of multiple transcription factors, including GATA1. Cytokines critical for maturation of eosinophilic granulocytes in the bone marrow include IL-3, IL-5, and GM-CSF. IL-5 is responsible for egress of mature eosinophils from the bone marrow into the peripheral circulation where they are subsequently activated and recruited by chemokines and cytokines, including IL-5, and the family of eotaxins, which are chemokines with greater specificity for eosinophils. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Eosinophil disorders 221 Eosinophils can be found in most tissues but have particular abundance in the bone marrow, GI tract outside of the esophagus, mammary gland, and uterus where the eosinophil may play a role in the estrous cycle. Eosinophils share many features with neutrophils, including chemotaxis ability, phagocytosis, degranulation, production of reactive oxygen species, and antibacterial and antifungal activity. The release of granules serves a major antimicrobial mechanism of eosinophils and there are four major proteins contained in eosinophil granules: eosinophil peroxidase, major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin. Additional proteins include a number of cytokines, growth factors, and enzymes. Upon confrontation with a pathogen, eosinophils can undergo degranulation, but unlike neutrophils that completely release granule contents, eosinophils can have a controlled release in a process termed “piecemeal degranulation.” By only releasing some of its granules, the eosinophil remains intact and is able to continue assault upon the pathogen. If the granules are released intact, they can persist in the extracellular matrix of the tissue, although the function and activity of these cell-free granules still remains under investigation. Eosinophils produce oxygen radicals through the NADPH oxidase multiprotein complex, but in contrast to hypochlorous acid produced from myeloperoxidase in neutrophils, eosinophils produce hypobromous acid from hydrogen peroxide via the enzyme eosinophil peroxidase. The oxidative burst of eosinophils is also more active with greater production of oxygen radicals in comparison to the neutrophil. For their antimicrobial activity, eosinophils provide immunity against multicellular parasites or helminths. However, the role of eosinophils in antihelminth immunity is now under question. The antibacterial properties are less controversial and include bactericidal properties of MBP and ECP as well as the release of eosinophil extracellular traps composed of mitochondrial DNA, MBP, and ECP. In contrast to neutrophil extracellular traps that are composed of nuclear DNA and neutrophil-specific granules and result in death of the cell, eosinophils can persist following release of the traps. Eosinophils may also have an antiviral role with animal models demonstrating eosinophil involvement in reducing the virulence of several respiratory pathogens, including RSV and parainfluenza. Like all leukocytes, eosinophils interact with each other and other lineages for coordinated immune response. Eosinophils interact with T cells through release of preformed interferons and cytokines, including IL-4 and IL-13. Eosinophils can also present antigen through major histocompatibility complex class II (MHCII) complexes to CD4 T cells, although the antigen-presenting potential of eosinophils is small compared to professional antigen-presenting cells. Eosinophils also play a role in priming B cells for IgM production and communicate with macrophages and mast cells in the tissue. In addition to their proinflammatory role, more recent investigations of eosinophils have demonstrated a homeostatic role in tissue repair and downregulation of inflammation. Much work is needed to understand the independent contributions of eosinophils to health and disease given modern approaches to chronically deplete eosinophil function in select asthmatic and eosinophilic patients. Definition of eosinophilia and eosinopenia Eosinophil level measures in the blood are age dependent with higher levels noted in toddlers. For children over 2 years of age and adults, the eosinophil count is typically 3!5% of the leukocyte differential for an absolute eosinophil count (AEC) between 300 and 500 cells/µL. Eosinopenia has been defined by eosinophil counts as low as 10 cells/µL while an AEC . 500 cells/µL is considered elevated. The term “blood eosinophilia” is defined as an AEC . 500 cells/µL and is categorized as mild (500!1500 cells/µL), moderate (1500!5000 cells/µL), and severe ( . 5000 cells/µL). The term “hypereosinophilia (HE)” is reserved for persistent elevations in eosinophil counts defined as an AEC . 1500 cells/µL on two separate occasions separated by $ 1 month or the presence of elevated tissue eosinophils. The criteria of elevated tissue eosinophils can be met by $ 20% of nucleated cells in the bone marrow of eosinophilic lineage or excessive eosinophilic cells in tissue as determined by a qualified pathologist. Hypereosinophilic syndrome (HES) is another classification applied to patients with HE who have evidence of organ damage and/or dysfunction attributed to tissue eosinophils. HES has classically been defined as an AEC . 1500 cells/µL for $ 6 months and evidence of eosinophilic associated end-organ disease. The requirement for an AEC . 1500 cells/µL is often dropped by some clinicians and investigators today as diagnostic methods are much improved, treatments are more nuanced and less toxic, and some patients will have endorgan damage with a lower AEC. HE is categorized as primary or myeloproliferative HE/HES (M-HE/HES) if a monoclonal disorder originating from a somatic genetic change within the bone marrow results in the eosinophil being the predominant expanding cell type or one of several proliferating cell lines. Secondary HE refers to reactive conditions in which eosinophil production is driven by disease states that result in production of eosinophilic cytokines (IL-3, IL-5, Lanzkowsky’s Manual of Pediatric Hematology and Oncology 222 11. Disorders of white blood cells GM-CSF) and subsequent polyclonal expansion of eosinophils. An international consensus group has categorized HES into six groups based on etiology with continued fine-tuning as the understanding and treatment of eosinophil disorders improves. 1. M-HE/HES Primary HE/HES driven by a presumed or proven somatic genetic change results in clonal eosinopoiesis. Oncogenic fusions in the tyrosine kinases PDGFRA, PDGFRB, or FGFR1 are the most commonly found oncogenic drivers resulting in constitutive activation and clonal production of eosinophils. PDGFRA!FIP1L1 is the most common fusion protein in M-HE/HES and has also been identified in AML and T-cell lymphoblastic lymphoma with associated eosinophilia. Activating mutations in PDGFRA has also resulted in HE. Over 30 fusion partners of PDGFRB and 14 fusion partners of FGFR1 have been identified, while PCM1!JAK2 is currently a provisional entity in the current WHO classification. 2. Lymphocytic-variant HE/HES (L-HE/HES) HE/HES is secondary to an abnormal immunophenotypic, frequently clonal T-cell population that produces eosinophilic growth factors causing reactive eosinophilia. The immunophenotype of the T-cell population is most commonly CD3 2 CD4 1 , often with elevated CD5 1 expression and loss of CD7 surface expression. A less common immunophenotype is identification of a double-negative T-cell (CD3 1 CD4 2 CD8 2 ) population. A clonal rearrangement of the T-cell receptor (TCR) is frequently identified, but not ubiquitously identified in patients with L-HE/HES. As the T-cell abnormalities are variable, definitive diagnostic criteria have not been established. But the identification of an abnormal T-cell immunophenotype and/or clonality by TCR rearrangement in the setting of HE/HES with absent genetic rearrangements seen in M-HE/HES are typically sufficient for a diagnosis of L-HE/HES. L-HE/HES is typically an indolent disorder, but progression to angioimmunoblastic T-cell lymphoma has been documented in a minority of patients. 3. Overlap HES HES is restricted to a single organ, such as eosinophilic GI, pulmonary, or dermatologic disease. The distinction between single organ and multisystem is often not clear leading to the designation of “overlap” with other systemic categories. An example is eosinophilic granulomatosis with polyangiitis (historically known as Churg!Strauss syndrome), which is considered an “overlap HES” as the eosinophilic involvement, consists of a single organ, the walls of small- and medium-sized vessels, but has multiple organ involvement given the extent of the circulatory system. 4. Associated HE/HES HES occurs in the setting of a distinct, secondary cause and treatment is directed at the associated cause and not directly at the eosinophilia itself. For instance, an infection secondary to helminths would be associated with HE, but the treatment and resolution of the eosinophilia will occur with antiparasitic treatment against the infection. 5. Familial HE/HES It includes some common forms such as eosinophilic esophagitis and rare multisystem disorders. Identification of the mechanism of genetic transmission has been difficult, but a large genome scan linked the chromosome location 5q31!33 to be associated with familial eosinophilia. 6. Idiopathic HES This is the most common category that patients with HES fall into as they do not fulfill the criteria for any of the abovementioned classifications. An additional classification system for malignant disorders with clonal eosinophilia by WHO was proposed in 2008 and again in 2016 based on molecular and histopathology in the myeloid malignancy classification. A major category in the WHO classification is “Myeloid/lymphoid neoplasms associated with eosinophilia and rearrangement of PDGRA, PDGFRB, or FGFR1, or with PCM1-JAK2,” the last fusion being a provisional entity in the 2016 classification. Chronic eosinophilic leukemia, not otherwise specified, is also included in the major category of MPN and is defined by increased blasts (but ,20% to exclude acute leukemia) in the bone marrow or blood, the absence of the Philadelphia chromosome or rearrangements involving PDGFRA, PDGFRB, FGFR1, and lack of other marrow neoplasms associated with eosinophilia. HE may also be present in several other malignancies, including BCR!ABL1-negative MPN, MDSs, chronic myeloid leukemias (CMLs), AMLs (especially the M2 and M4 Eo French!American!British historic classification with core binding factor translocations), and systemic mastocytosis. Lymphoid malignancies such as T-cell lymphomas, Hodgkin lymphoma, and acute lymphoid leukemias may also present with secondary eosinophilia secondary to production of IL-3, IL-5, and GM-CSF. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Eosinophil disorders 223 Approach to suspected eosinophil disorders Similar to other leukocyte disorders, a thorough history should focus on: • infectious exposure; • travel; • diet; • medications; • end-organ manifestations of illness such as fatigue, cough, rash, and GI symptoms; and • underlying hematologic (pallor, petechiae, bruising) or immunologic symptoms (asthma, allergy, frequent or rare infections). A thorough physical examination with particular attention to fever, tachypnea, tachycardia, angioedema, skin findings, dyspnea, cardiac murmur, lymphadenopathy or hepatosplenomegaly, and general well-being will help direct the differential and testing as well as determine the urgency of evaluation and treatment. Inclusion of specialists in allergy, immunology, infectious disease, pulmonology, gastroenterology, cardiology, and dermatology may be necessary to assist with the evaluation, perform diagnostic procedures, and determine treatment depending on the ultimate diagnosis. The next step is to rule out secondary causes, which will account for most cases and can typically be elucidated by a good history and physical examination. Additional evaluation will be dependent on either a low or high eosinophil count. Approach to eosinophilia An informative study conducted by the NIH segregated patients with HE and HES into pediatric (n 5 32 patients) and adult cohorts (n 5 254 patients) for comparison. The median age of presentation in pediatric patients was 10.5 years with a median AEC of 1376 cells/m and peak AEC of 9376 cells/µL. For patients with HES, dermatologic, allergic, hematologic, and constitutional symptoms were common with similar occurrence between cohorts, but children more frequently had GI symptoms (62% vs 34%) and less pulmonary disease other than asthma (34% vs 59%) and less cardiac involvement (3% vs 16%). Idiopathic HES was the most common diagnosis for both age cohorts (46% in children, 47% in adults) and M-HE/HES was also similar in frequency between groups (8% in children, 11% of adults). A secondary treatable cause was found in 14% of children and 10% of adults. The most common cause of secondary HE was helminth infection, with 60% of cases in children (n 5 3) and 40% in adults (n 5 5). The pathogenic parasite differed between the populations with all cases (three) in pediatrics secondary to Toxocara species and adults with strongyloidiasis, schistosomiasis, onchocerciasis, or intestinal helminth. Children were more likely to have a primary immunodeficiency, while adults had a malignancy-associated neoplasm in 3% (n 5 7) and none in children. Transient eosinophilia is commonly encountered in pediatric practice and generally insignificant, but persistent elevation and/or the presence of eosinophil-related manifestations requires evaluation. Thus all children who meet the criteria for HE or HES should be evaluated for an underlying cause. Patients with organ toxicity, particularly cardiac, pulmonary, and neurologic eosinophilic disease, require urgent evaluation and treatment. Differentiation between primary (clonal) and secondary (reactive) HE/HES may not be apparent on initial evaluation of the pediatric patient. Clues to M-HE/HES can include the degree of HE, with AEC . 100,000 cells/ µL, evidence of cytopenias or dysplastic cells on the blood smear, and the presence of hepatosplenomegaly as features that would be more consistent with a primary, clonal HE. Evaluation in both primary and secondary HE/ HES should include: • CBC with differential. • Chemistries, including kidney and liver function. • Bone marrow aspirate and biopsy if there is a concern for a primary bone marrow process driving the HE. Studies, including morphology (dysplastic, increased blasts), immunophenotyping, cytogenetics, and molecular studies to evaluate for common oncogenic fusions involving PDGFRA, PDGFRB, and FGFR1 as well as JAK2 and KIT should be conducted by PCR, fluorescence in situ hybridization (FISH), or other institutionally available methods. • Screening for a primary immunodeficiency should include T-, B-, and NK-cell subsets and quantitative IgG, IgA, IgM, and IgE. Specialized flow cytometry for immunophenotypic aberrant T cells (CD3 2 CD4 1 , CD3 1 CD4 2 CD8 2 ) and TCR rearrangement studies should be sent for L-HE/HES. • Tryptase and Vitamin B12 are nonspecific, but often elevated in M-HE/HES. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 224 11. Disorders of white blood cells • Testing for specific secondary causes, including antineutrophil cytoplasmic antibodies, antigen and serology testing for infection, and HIV screening should be conducted based on history and physical. Evaluation for end-organ manifestations is crucial, particularly cardiac impact, and can be a lifesaving screen while working up the etiology of the HE/HES. It should include: • Serum troponin, electrocardiogram, and echocardiogram to evaluate for myocarditis, although cardiac MRI is more sensitive to evaluate for eosinophilic involvement. • Pulmonary function tests. • CT imaging of the chest abdomen, and pelvis. • Biopsy of affected tissue, such as skin or lymph nodes, may provide additional diagnostic information if less invasive tests are unrevealing. • Genetic sequencing for primary immunodeficiency genes is appropriate if additional signs of an immune disorder are apparent or suspected. Management of HE/HES is dependent on the underlying driver. The chronicity and presence of end-organ disease will be important in making decisions for inpatient versus outpatient evaluation and observation and timing of treatment initiation as some of the tests for HE/HES may take several days to result. Approach to eosinopenia Eosinopenia is almost exclusively seen as a secondary phenomenon. Primary or constitutional eosinopenia is incredibly rare with only a handful of cases reported in the literature, including some with concomitant basopenia. The few patients with primary eosinopenia have very infrequent clinical consequences from absent eosinophils, mirroring what has been seen in animal models and iatrogenic depletion of eosinophils with anti-IL5 therapy. When approaching a patient with eosinopenia, an extensive evaluation for secondary causes, including medications and other medical conditions, should be undertaken before proceeding in identifying a constitutional cause. However, if unsuccessful in finding an external driver of eosinopenia, a bone marrow aspirate and biopsy should be performed with high consideration of broad genetic testing to identify variants specific to eosinophil maturation, proliferation, and activation. Primary eosinophil disorders (M-HE/HES) Clinical manifestations • Epidemiology demonstrates a male predominance. • Findings typically seen in other bone marrow disorders, including anemia, thrombocytopenia, and hepatosplenomegaly. • Invasion of eosinophils into tissue can result in damage and fibrosis leading to findings such as endomyocardial dysfunction and restrictive lung disease. • HE typically resistant to glucocorticosteroids. Diagnosis • Identification of chromosomal fusions in the tyrosine kinases PDGFRA, PDGFRB, or FGFR1 by FISH or PCR. • Diagnosis with negative oncogenic fusions can be confirmed by meeting at least four of the following criteria: dysplastic eosinophils on blood smear, serum vitamin B12 level .1000 pg/mL, serum tryptase .12 ng/mL, anemia and/or thrombocytopenia, bone marrow cellularity .80%, myelofibrosis, or spindle-shaped mast cells in the bone marrow. Management and treatment options • M-HE/HES with imatinib-sensitive mutations is treated first-line with tyrosine kinase inhibitors. • Imatinib is the most well-studied tyrosine kinase inhibitor and is typically the first agent used in M-HE/HES. • High-dose glucocorticosteroids should be initiated a few days before tyrosine kinase inhibition to prevent imatinib-induced myocardial necrosis, which is a severe, although rare complication of imatinib therapy in patients with HES believed to be secondary to eosinophilic release of cytokines in the heart with therapy. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Eosinophil disorders 225 Secondary HE/HES As previously discussed, there are multiple categories of secondary (reactive) HE/HES to consider in the absence of a clonal eosinophil disorder. The history may indicate an environmental trigger related to dietary or medication intake leading to elevated eosinophil counts. Causes of secondary eosinophilia are listed in Table 11.2, with the most common etiology infections, including invasive helminths (strongyloidiasis, schistosomiasis, hookworm, filariasis, ascariasis, toxocariasis, trichinosis). Toxocara should be particularly considered in younger children with poor sanitation and strongyloidiasis in the southeastern United States as the parasite persists in hot, humid climates. Other infectious agents can include fungus (allergic bronchopulmonary aspergillosis, coccidioidomycosis, histoplasmosis) and HIV. Medications commonly associated with hypersensitivity and elevated eosinophil counts include antibiotics (penicillins, cephalosporins, vancomycin), antiepileptic medications, and NSAIDs. Allergic conditions may have eosinophilia, or mild HE, and HE may be a prominent feature of several primary immune deficiencies, including the hyper-IgE syndromes (STAT3 loss-of-function and DOCK8 immunodeficiency disorder), combined immunodeficiencies, SCN, WAS, autoimmune lymphoproliferative syndrome (ALPS), immunodysregulation!polyendocrinopathy!enteropathy!X-linked (IPEX), and autoinflammatory disorders. Autoimmune conditions, although less common in children, can also have elevated eosinophil counts. Patients with “associated HE/HES” by definition are treated for the underlying cause without directed therapy targeting eosinophils. Treatments for secondary causes of HE/HES are diverse and complex depending on the mechanism and patient but include: TABLE 11.2 Etiologies associated with secondary eosinophilia. Infection Medications Immune disorders Autoimmune and inflammatory disorders Allergy Malignancy Other Bacterial (Mycobacterium tuberculosis) Ectoparasites (scabies, myiasis) Fungal (allergic bronchopulmonary aspergillosis, coccidioidomycosis, histoplasmosis) Invasive helminths (strongyloidiasis, schistosomiasis, hookworm, filariasis, ascariasis, trichinosis, toxocariasis) Viral (HIV, HTLV) Antibiotics (penicillins, cephalosporins, vancomycin) Antidepressants Antiepileptics Antihypertensives (ACE inhibitors, beta blockers) NSAIDs ALPS Combined immunodeficiencies, esp. Omenn syndrome DOCK8 immunodeficiency disorder IPEX Severe congenital neutropenia STAT3 loss-of-function (Job syndrome) Wiskott!Aldrich syndrome EGPA Inflammatory arthritis Inflammatory bowel disease Sarcoidosis SLE Allergic rhinitis Asthma Atopic dermatitis ALL Adenocarcinoma CML Hodgkin lymphoma Adrenal insufficiency GVHD IL-2 therapy Radiation Sickle cell disease Abbreviations: ALL, Acute lymphoblastic leukemia; ALPS, autoimmune lymphoproliferative syndrome; CML, chronic myeloid leukemia; EGPA, eosinophilic granulomatosis with polyangiitis; GVHD, graft-versus-host disease; HTLV, human T-lymphotropic virus; IPEX, immunodysregulation!polyendocrinopathy!enteropathy!X-linked; SLE, systemic lupus erythematosus. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 226 11. Disorders of white blood cells • glucocorticosteroids (mainstay of therapy), • hydroxyurea, • tyrosine kinase inhibition, • interferon-alpha for downregulation of Th2 cytokines, • alemtuzumab, and • monoclonal antibody therapy against IL-5. This last option is gaining particular popularity with more clinical experience given that IL-5 is very specific for eosinophil development and activation with relatively few short- and long-term side effects. HSCT has also been used with success in patients with high-risk, aggressive disease. Basophil disorders Basophils are found in low frequency on a blood smear but are readily identified by purple-black granules that often obscure the cell nucleus. They are roughly the same size as neutrophils (10!14 µm) and the nucleus is band-shaped or segmented into two lobes. Basophils serve a role in allergy and anaphylaxis, but more recent studies have demonstrated a myriad of important roles in the immune system. Basophils also share many features with the mast cell, but unlike mast cells that reside in the tissue, basophils are circulating leukocytes that can be recruited to tissues for allergic or immunologic action. Although basophil abnormalities are the rarest of the leukocyte disorders, increased basophil counts in the blood may accompany and even be the first sign of a malignant disorder. Several benign disorders can also give rise to abnormal basophil counts. Normal basophil development and function Basophils develop along the lineage of common myeloid progenitors, granulocyte!monocyte progenitors, and possibly granulocyte progenitors in the bone marrow. The next stage of development can occur in the bone marrow as a prebasophil mast cell progenitor or in the spleen as a basophil mast cell progenitor. The transcription factors CCAAT enhancer-binding protein alpha (C/EBPalpha) and GATA2 help direct basophil maturation and relative levels of C/EBPalpha and GATA2 in these intermediate precursors help determine the fate of the cell toward mast cell or basophil development. Once fully mature, the basophil will migrate into the bloodstream with a relatively short life span with in vivo survival of 2!3 days under homeostatic conditions. Additional actions of the basophil in the peripheral circulation are dependent on the interaction with several cytokines, chemokines, and other mediators. Basophils fill multiple roles to promote health and disease, including allergy, anaphylaxis, protection against infections, and potential role in malignancy. Central to basophil activity is the cytokine IL-3, which is important for many basophil functions for signaling, growth, release of inflammatory mediators, and combined action when combined with other signaling molecules. A major effector role of basophils is the rapid release of histamine and leukotriene CR (LTCR) upon interaction with IgE!antigen complexes. The IgE!antigen complex cross-links high-affinity IgE receptors (Fc epsilon RI) on the surface of the basophil resulting in intracellular signaling and granule release of preformed mediators, including histamine and proteoglycans, a process termed “anaphylactic degranulation.” Degranulation is largely restricted through crosslinking of Fc epsilon RI, but the anaphylatoxin C5a and IL-3 have also been implicated in this process. Granule release is accompanied by rapid synthesis and release of LTCR, secretion of preformed IL-4, and production and secretion of IL-13 and additional IL-4. Basophils, along with mast cells, elicit immediate hypersensitivity reaction, through anaphylactic degranulation and rapid generation of LTCR and platelet-activating factor, resulting in a range of symptoms from mild allergy to life-threatening anaphylaxis. Late-phase hypersensitivity reactions such as allergic rhinitis and asthma can occur hours after encounter with an allergic trigger and result from basophils promoting an environment for allergy by secreting cytokines such as IL-4. Basophils also play a role in delayed hypersensitivity reactions, a process that occurs 2!3 days after challenge with an allergen, likely from continued production of cytokines, namely, IL-4 and IL-13. Basophils provide a protective function against infections, including helminths, ticks and tick infestation in guinea pigs, and possible bacterial respiratory infections through IgD-dependent interactions with B cells resulting in class switching. Basophils have been indicated to play a role in autoimmune disorders, including autoimmune urticaria and lupus nephritis. Elevated basophils are also associated with a number of malignancies, including AML, CML, and MDSs. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Basophil disorders 227 Definition of basophilia and basopenia The prevalence of basophils on a blood smear is about 0.5% of the total leukocyte population in healthy individuals. Gender and age can have an impact on the basophil count but given the low number of basophils in the blood, this variation is not clinically impactful. Interpretation of basophil counts must also be conducted with some degree of caution given the sensitivity, and specificity of some hematology analyzers in identifying basophils may be lower compared to other leukocyte subsets given the small number of basophils in the blood. Flow cytometry should be used for measuring the basophil count, particularly for basopenia, or basophil counts ,10 cells/µL, which may be difficult to find by automated analyzer or manual inspection of the blood smear. Absolute basophilia is defined as .100 cells/µL and relative basophilia as percentage of basophils .1!2% of total leukocytes. The term “hyperbasophilia” has also been a proposed nomenclature to refer to persistent absolute basophil count (ABC) . 1000 cells/µL over $ 8 weeks. Approach to suspected basophil disorders When evaluating basophil disorders, it is important to note the degree of quantitative abnormality and the chronicity of the findings. Transient abnormalities of basophil number are more likely to be a reactive process, while persistent basophil counts above 1000 cells/µL should lead to suspicion of a neoplastic process. The history should focus on: • medications, • diet, • atopy, • recent contacts, • travel, • infectious symptoms, • past medical history, • symptoms seen in leukemia such as: • fatigue, • bone pain, and • bleeding. Physical examination should include attention to atopic conditions such as urticaria, allergic rhinitis, and bronchial wheezing, infectious signs, and evidence of cytopenias such as pallor, bruising, or petechiae. Approach to basophilia When confronted with a patient with basophilia, it is essential to rule out the possibility of malignancy. Basophilia is a common finding in CML and persistent and significantly elevated (i.e., hyperbasophilia) will raise the concern for this disorder. Other malignant conditions include AML and MDS. Reactive, benign processes that can also cause elevated basophil counts include atopic conditions, including urticaria, allergic rhinitis, and acute allergy, iron deficiency anemia, and diabetes. Increased basophils may be a component of some infections, including parasitic infections, but this association has been contradicted in some reports. Other infections implicated include Mycobacterium tuberculosis, chicken pox, and smallpox. Evaluation of the patient with basophilia includes exclusion of atopic and infectious conditions that can lead to elevation of the basophil count. Bone marrow biopsy and aspiration with morphology, flow cytometry, cytogenetic, and molecular evaluation for myelodysplastic and myeloproliferative disorders, including BCR!ABL1, JAK2, CALR, and MPL, should be conducted in concerning patients. Patients that are more likely to have a reactive basophilia not associated with malignancy may be watched with repeated CBCs with persistent, rising, or other findings concerning a bone marrow clonal process prompting malignancy evaluation. Most cases of abnormal basophil number are reactive and benign and require no targeted therapy against the basophil. Treating the underlying condition should result in improvement in the count abnormality. Patients with malignant conditions should undergo appropriate therapy, including tyrosine kinase inhibition, chemotherapy, and/or HSCT. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 228 11. Disorders of white blood cells Approach to basopenia Patients with low basophil counts are hard to catalog given the poor reproducibility of identifying basopenia of routine hematology analyzers. Conditions that have been associated with basopenia include: • chronic urticarial, • glucocorticosteroid administration or Cushing disease, and • chronic inflammation. Congenital disorders of basopenia are incredibly rare and may be associated with the absence of eosinophils. Lymphocyte disorders The class of leukocytes known as lymphocytes consists of a diverse population of WBCs involved in both the innate and adaptive immune systems. The term “lymphocyte” stems from the fact that this cell type is the predominant leukocyte found in “lymph,” although these cells are perverse throughout the human body where they are intricate to development, activation, regulation, and communication within the immune system. While an elevated lymphocyte count (i.e., lymphocytosis) often triggers an evaluation to rule out monoclonal or malignant causes, reactive lymphocytosis is much more common. Lymphocytopenia may be a component of disorders of hematopoiesis but is also a frequent finding in inborn errors of immunity. Normal lymphocyte development and function Lymphocytes include T cells and B cells, which populate the arms of the cellular and humoral adaptive immune systems, respectively, and NK cells that reside in the innate immune system. Under physiologic conditions, T cells are the most populous cell type on peripheral flow cytometry evaluation, followed by B- and NK cells. Each lymphocyte has unique development with strict mechanisms of regulation to avoid the development of autoreactive lymphocytes resulting in autoimmune disorders. Because most development is compartmentalized outside the blood, our understanding of lymphocyte development is based on murine lymphocyte biology. However, the rapid expansion of genetic discovery resulting in lymphocyte dysfunction has aided our understanding of human lymphocyte biology. T cells T lymphocytes originate within the bone marrow before emigration to the thymus as T-cell precursors with intact germline TCR loci and lacking the coreceptors CD4 and CD8. Within the thymus, T-cell precursors undergo rearrangement of TCR loci and begin to express both CD4 and CD8 on the surface of the developing lymphocyte to earn the nomenclature of a “double-positive thymocyte.” The newly rearranged TCR will undergo a process known as “positive selection” within the cortex of the thymus by interacting with MHCs on epithelial cells. In order for the double-positive thymocyte to survive and undergo additional maturation, it must successfully recognize the self-MHC. If recognition is sufficient, the double-positive thymocyte will downregulate either CD4 or CD8 depending on the class of MHC interaction. If the double-positive thymocyte successfully recognizes a self-MHC class I, it will downregulate CD4; if the interaction is through self-MHC class II, it will downregulate CD8. The thymocyte is now termed a “single-positive thymocyte” and migrates to the medulla where it must undergo a second round of screening known as “negative selection.” In this scenario, single-positive thymocytes interact with thymic dendritic cell self-MHCs. If the interaction is deemed too strong, the T cell may become activated by self-MHCs in the extrathymic environment and drive the development of autoimmune disease. To avoid this possibility the thymus serves a central regulatory purpose by eliminating these thymocytes before they leave the thymus to serve as functional T cells. Thymic production of new T cells in humans is not static but declines with age. The thymus is largest at birth, but with age, there is reduction in volume and function. The greatest decline begins after puberty and thymic function effectively stops after the age of 40 years. Upon leaving the thymus, each naı̈ve T cell has a different TCR with unique antigen specificity and will express CD4 for MHC class II or CD8 for MHC class I interaction. Activation of a naı̈ve T cell requires ligation of the TCR with its specific antigen in the correct MHC along with costimulatory ligands and cytokines. Successful activation results in massive proliferation and differentiation to an effector cell population that then disseminate to local and distant locations to clear the pathogenic intruder. Following clearance of the infection, most of the Lanzkowsky’s Manual of Pediatric Hematology and Oncology Lymphocyte disorders 229 effector cells are eliminated, but a small fraction persists as memory T cells that have the capacity for rapid activation and proliferation if the same pathogen is encountered again. The ratio of naı̈ve-to-memory T cells is high at birth, but through rapid exposure to foreign antigens during childhood, the number of memory T cells accumulates up until adulthood, reducing the naı̈ve-to-memory ratio. The two main categories of T cells are CD4 or helper T cells and CD8 or cytotoxic T cells. • CD4 T cells are critical to orchestrating the immune response amongst leukocyte populations, hence earning the title of the helper T cell. Important functions of CD4 T cells include interaction with B cells to drive maturation and production of mature immunoglobulins, cross-talk with macrophages for activation and infection clearance, recruitment of inflammatory cells, including neutrophils, eosinophils, and basophils to the site of infection, and production of cytokines and chemokines. CD4 T cells are further divided into subtypes, including Th1, Th2, Th3, Th9, Th17, T follicular (Tfh), and regulatory T cells. Regulatory T cells deserve special mention given their important role in immune tolerance. The absence of regulatory CD4 T cells is the hallmark of IPEX syndrome that presents in young male infants with severe autoimmune disease. • CD8 T cells secrete cytokines and exert cellular death upon target cells, earning the title of the cytotoxic T cell. During an infection the repertoire of unique, naı̈ve CD8 T cells that can respond to a specific antigen ranges from 10 to 1000 cells in the mouse. However, activation of a single T cell with its cognate antigen can result in massive proliferation resulting in as much as a 500,000-fold clonal expansion. Cytotoxicity from CD8 cells can occur through exocytosis of granules containing perforin and granzymes or through the death receptor pathway in which fas ligand on CD8 cells ligates with the fas receptor on the target cell to initiate cellular apoptosis. Defects in cytotoxic granule production, trafficking, and exocytosis can result in profound cytotoxic defects and resultant hyperinflammation seen in familial HLH. Abnormal fas!fas ligand signaling can result in uncontrolled lymphocyte proliferation, persistence, and autoreactivity resulting in the clinical disorder ALPS. B cells B cells and antibodies constitute the humoral immune system, a term originating from the term humors, or body fluids, that contain the protective immunoglobulins. Unlike T cells that require production in the thymus, functional B cells, although immature, are produced in the bone marrow. Within the bone marrow, B-cell precursors undergo successive rearrangements of the immunoglobulin heavy and light chain loci to produce a diverse set of BCRs/ immunoglobulins with antigen specificity. Successful rearrangement results in the immature B cell expressing an IgM molecule as the BCR. Similar to T cells, B cells undergo a positive selection process in which the immature B cell requires some level of tonic signals from the BCR for maturation. Negative selection is necessary to avoid the production of autoreactive B cells and occurs when the BCR reacts with significant avidity to self-antigen resulting in one of several fates, including clonal deletion through apoptosis for high-affinity binding, anergy for low-affinity binding, or receptor editing of the BCR away from autoreactivity. Immature B cells that survive central selection migrate to the spleen where they await further maturation. Upon recognition of their specific antigen, B cells present the antigen on the MHC class II molecule to CD4 cells for additional signals in the form of CD40 ligand and cytokines from the helper T cell to undergo proliferation and differentiation. The proliferating B cells form germinal centers where they continue to interact with CD4 helper T cells, specifically follicular T cells, to produce B cells with more robust immunoglobulins through the processes of immunoglobulin class-switch recombination and somatic hypermutation to change the isotype and strengthen the avidity of the immunoglobulin, respectively. The success of these processes can result in either long-lived plasma cells that produce IgG, IgA, or IgE or memory B cells. T cell!independent B-cell activation can also occur through pathogen-associated molecular pattern interaction with Toll-like receptors on B cells to produce short-lived, IgM-producing antibodies that assist with the early immune response against pathogens. Although best known for the production of antibodies, B cells have several additional roles in the immune system. As previously mentioned, B cells can act as antigen-presenting cells to CD4 T cells. The absence of this interaction through defects in CD40 or CD40 ligand result in inadequate CD4 activation and a severe combined immunodeficiency and excessive production of IgM (hyper-IgM syndrome). B cells are important producers of both inflammatory and regulatory cytokines for coordinated and controlled immune responses. This latter function includes the role of B10 cells that can modulate T-cell inflammatory responses through production of IL-10. NK cells NK cells develop in the bone marrow, but secondary lymphoid tissue can also be a site of NK-cell production. They are identified by expression of the Fc receptor, CD16, in a subset of NK cells, and/or CD56, which is expressed by most Lanzkowsky’s Manual of Pediatric Hematology and Oncology 230 11. Disorders of white blood cells NK cells. Unlike T- and B cells, NK cells are part of the innate immune system and in general lack antigen specificity. However, they are tightly regulated through an education process involving the expression of activation and inhibitor receptors known as killer immunoglobulin-like receptors (KIR). This education process that is mediated by interaction with self MHC class I proteins promotes tolerance of NK cells toward cells expressing self-MHC proteins. NK cells exert their effector function similar to CD8 T cells through cellular degranulation and fas receptor ligation. NK and CD8 cells use the same pathways for granule exocytosis and defects in this process result in familial HLH. NK cells also produce a variety of inflammatory cytokines to enhance the immune response. NK cells play important roles in multiple infectious processes but have particular importance in herpesviruses and surveillance of damaged or malignant cells. Definition of lymphocytosis and lymphopenia Given the important role of antigen recognition for T- and B-cell proliferation, the relative and absolute numbers of lymphocytes are quite dynamic during the first few years of life with the massive introduction of neopeptides for immune activation. • Total Lymphocytes: the absolute number of lymphocytes increases 1.3-fold immediately after birth and are very high during the first 2 years of life, followed by a gradual decrease by threefold to adult levels. • T cells: the relative frequency of T cells remains between 60 and 75% throughout life, but the absolute number of T cells reflects the rise of total lymphocytes afterbirth and decreases from 2 years of age into adulthood. CD4 and CD8 percentages also show relative stability with age with CD4 38!53% and CD8 16!24% of total lymphocytes. However, the ratio of CD4:CD8 cells decreases from 2.0 to 3.0 in the first 2 years of life followed by lower ratios of 1.0!2.0 in later childhood and adults. • B cells: the relative number of B lymphocytes increases twofold during the first 5 months of life to a median value of 12!24% of total lymphocytes, followed by stability until 5 years of life, followed by a decrease to a median value of 13%. • NK cells: unlike the other lymphocyte subsets, NK cells show a relative decline from a median of 20 to 7% after birth, although absolute values are stable, followed by a steady increase to a median value of 13% as an adult. Given the age-dependent changes in lymphocyte count, the definition of low or high lymphocyte count varies with the age of the patient. In general, lymphocytopenia is defined when the lymphocyte count is ,1500 cells/µL in adults and ,4500 cells/µL before 8 months of age. Lymphocytosis is defined as a lymphocyte count .8000 cells/µL in young children and .4000 cells/µL in adolescents and adults. Approach to lymphocytosis Lymphocytosis can be segregated into primary and secondary causes. However, unlike lymphocytopenia, there are very few cases of primary or inherited lymphocytosis, so leukemic transformation must be considered. The history should focus on: • infectious symptoms; • recent stress or trauma; • B symptoms (weight loss, night sweats, fever); • medications; and • surgery, including splenectomy. A physical examination focused on signs of malignancy such as lymphadenopathy and hepatosplenomegaly is important. Laboratory testing should include: • CBC, differential and blood smear. The presence of blasts or concurrent cytopenias raises the concern for a malignancy. The appearance of lymphocytes such as atypical large lymphocytes or cleaved nuclei may be associated with EBV/CMV/early HIV or Bordetella pertussis, respectively. • Flow cytometry will help determine the subtype of origin and help assess for the presence of clonality. • Appropriate infectious studies should be conducted if the lymphocytosis is concurrent with other signs and symptoms of infection. • Bone marrow or lymph node biopsy with morphology, flow cytometry, cytogenetics, and molecular studies should be conducted when concerns for malignancy are entertained. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Lymphocyte disorders 231 Primary lymphocytosis B-cell expansion with NF-(kappa)B and T-cell anergy syndrome The absence of secondary causes and consistent elevation of lymphocytes should prompt evaluation for a congenital cause. The classic disorder associated with B-cell lymphocytosis secondary to a germline gain-of-function mutation in CARD11 results in a B-cell lymphoproliferative disease and immunodeficiency termed BENTA for Bcell expansion with NF-(kappa)B and T-cell anergy. Although CARD11 is necessary for both B- and T-cell signaling, the GOF defect results in lymphocytosis in B cells but hyporesponsiveness in T cells to TCR stimulation. Clinical manifestations • In addition to B-cell lymphocytosis, patients have splenomegaly, lymphadenopathy, and recurrent sinopulmonary and viral infections. • Autoimmunity and B-cell malignancies have also been reported in adults. • Immunoglobulin levels may be low or normal, but polysaccharide and persistent response to proteinconjugate vaccines are poor. Diagnosis • Identification of a pathogenic heterozygous germline variant in CARD11 Management and treatment • Treatment options for BENTA are limited but have included close monitoring for infection, autoimmunity, and B-cell malignancy • Infectious supportive care may include immunoglobulin supplementation and antibiotic administration Secondary lymphocytosis A variety of benign and malignant causes of lymphocytosis can be seen in pediatric patients (see Table 11.3). • Of the infectious causes, EBV and CMV infections are the most common. Normal response to EBV results in lymphocytosis consisting of CD8 cell proliferation in response to infected B cells and is present in up to twothirds of cases. In both CMV and EBV the blood smear may show atypical lymphocytes with large size, open chromatin, and increased cytoplasm. Primary HIV infection can lead to an acute febrile, mono-like illness associated with lymphocytosis, while chronic HIV patients have CD4 lymphocytopenia. Other viruses include adenovirus, HHV6, HHV8, rubella, varicella, human T-lymphotropic virus type 1, hepatitis viruses, influenza, mumps, coxsackie A and B6, and echovirus. • Infection: atypical lymphocytes with elevated lymphocyte count can be seen in Toxoplasma gondii. B. pertussis toxin blocks migration of lymphocytes in the lymph tissue resulting in elevated blood lymphocyte counts. The TABLE 11.3 Etiologies associated with secondary lymphocytosis. Bacterial (Bordetella pertussis, Mycobacterium tuberculosis, rickettsia, Bartonella henselae, brucellosis, syphilis) Parasite (Toxoplasma gondii, malaria) Viral (EBV, CMV, acute HIV, coxsackie A and B6, echovirus, adenovirus, HHV6, HHV8, rubella, VZV, HTLV-1, hepatitis viruses, influenza, mumps) Medications Allopurinol Antiepileptics Sulfa drugs Sulfonamides Vancomycin Malignancy Acute lymphoblastic leukemia and lymphoma Chronic lymphocytic leukemia Large granular lymphocyte leukemia Non-Hodgkin lymphoma Other Persistent polyclonal B-cell lymphocytosis Splenectomy Stress (exercise, surgery, trauma) Infection Abbreviations: CMV, cytomegalovirus; EBV, Epstein!Barr virus; HHV, human herpes virus; HIV, human immunodeficiency virus; HTLV, human Tlymphotropic virus; VZV, varicella zoster virus. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 232 11. Disorders of white blood cells lymphocytes are also small for size with a deeply cleaved nucleus. Other infections include Bartonella henselae, brucellosis, syphilis, malaria, Mycobacterium tuberculosis, rickettsia infection, and shigellosis. • Drug reaction: lymphocytosis may accompany severe drug reactions, including drug reaction with eosinophilia and systemic symptoms (DRESS), which is mediated by T cells. Drugs associated with DRESS syndrome include allopurinol, carbamazepine, vancomycin, and sulfa drugs. Significant stress from exercise, surgery, and trauma can also raise the lymphocyte count secondary to catecholamine and steroid hormone release. • Malignant disorders are identified by monomorphic lymphocytosis and in pediatric patients may be secondary to acute lymphocytic leukemia and non-Hodgkin lymphoma. Splenectomy is also often associated with lymphocytosis. Malignant disorders resulting in lymphocytosis require prompt cancer therapy as outlined in other chapters in this book. Infections should similarly be addressed quickly with appropriate antimicrobials and avoidance of medications suspected to be associated with severe systemic symptoms should be advised. Approach to lymphocytopenia A decreased lymphocyte count is a common occurrence on CBCs with 1.5!3% of samples having lymphocytopenia. Given the majority of lymphocytes are T cells, identification of lymphocytopenia on a blood differential typically requires a reduction in T-cell numbers with or without other subset declines. Also, isolated lymphocyte subset deficiency may be masked by an increase in other lymphocyte subsets on routine differential. Primary or inherited disorders are secondary to an error in lymphocyte production and homeostasis and can be accompanied by recurrent, severe, or unusual infections, autoimmune or autoinflammatory conditions, or malignancy. Secondary or reactive lymphocytopenia is common but can also result in severe complications, such as the CD4 lymphocytopenia seen in chronic HIV infection or may be a transient disorder without repercussions. Secondary causes of lymphocytopenia may cause reduced production, increased destruction, or shifting of lymphocytes to the lymphoid tissues giving a reduced lymphocyte count on blood count differential. Patients may first come to medical attention by identification of low T cell receptor excision circles (TRECs) on the newborn screen. The screen consists of identifying TRECs by qRT-PCR on a dried blood spot, which are circular DNA remnants of the excised alpha loci that are produced during normal TCR loci rearrangement during T-cell maturation in the thymus. The TREC is not duplicated during normal T-cell replication and, therefore, will be a direct measure of naı̈ve T-cell production through the thymus. Some infants with severe combined immune deficiency (SCID) may have T lymphocytes detected in the blood made by oligoclonal proliferation of a small number of T cells or persistence of maternal T cells transferred through the placenta. As the T-cell load consists of poorly functioning, replicated T cells that lack reciprocal replication of TRECs, the TREC will still be low in this clinical scenario and detected by the newborn screen. Infants identified to have a low TREC should be evaluated immediately for diagnosis and institution of infectious preventive measures while awaiting confirmation of a T-cell deficiency. Evaluation in these newborns should include: • maternal history of infections, • medications, • autoimmune complications, and • additional medical and family history with a specific focus on early or unexplained deaths in the family. Blood work for the infant should include a CBC and flow cytometry for lymphocyte subsets with markers for naı̈ve and mature T cells (CD45RA 1 and CD45RO 1 isoforms are utilized to identify naive and memory T cells, respectively). Additional testing includes diversity studies such as TCR-Vbeta spectratyping and T-cell proliferation in response to mitogen stimulation. Detection of maternal T cells in the blood of the infant can be conducted through DNA profiling techniques such as variable number tandem repeat analysis to differentiate the DNA origin as maternal or infant. Protective measures should include: • avoidance of infectious contacts, • testing for maternal CMV exposure, and • discontinuation of breastfeeding to avoid CMV transmission if the mother is identified to be CMV IgG positive. Sanger or next-generation sequencing panels screening for SCID genetic defects is important for treatment options such as HSCT versus gene therapy and identification of extraimmunologic findings such as radiation sensitivity. For patients identified by symptoms or lymphocytopenia on blood count differential, a thorough history should be conducted with a specific focus on infections, medications, past medical and family history, vaccine history, and thorough physical examination evaluating for signs of immune deficiency (failure to thrive, general poor health), Lanzkowsky’s Manual of Pediatric Hematology and Oncology Lymphocyte disorders 233 autoimmunity (skin rashes, lymphoproliferation), and infection. Flow cytometry to identify affected lymphocyte subsets should be conducted in all patients with concerning lymphocytopenia without readily identifiable secondary cause. For significant T-cell lymphopenia, further evaluation of T cells as listed earlier for low TREC patients can be pursued. B-cell function can be difficult to assess during the first few months of life secondary to placental transfer of IgG, physiologic poor production of immunoglobulin, and incomplete vaccine status. However, between 6 and 12 months of age, measurement of quantitative immunoglobulins is more accurate and vaccine responses can be sent as functional analysis of B-cell function. For patients with normal NK-cell numbers but concern for NK-cell dysfunction, NK-cell killing and degranulation assays can be sent, although the former test is plagued by poor sensitivity. Primary lymphocytopenia disorders Severe combined immunodeficiency The most severe and critical lymphocytopenia disorder to identify in newborns is severe combined immunodeficiencies (SCIDs) that are defects in T-cell production with or without deficiency in B-cell and/or NK-cell production. There have been over 20 genetic defects associated with SCID that are either X-linked (IL2RG/ common!gamma chain deficiency) or autosomal recessive. The subtypes of SCID can be divided based on the phenotypic pattern of deficiency on lymphocyte flow cytometry (i.e., the presence or absence of B- and NK cells) or by the pathogenic mechanism that includes (1) V(D)J recombinations and alterations in the TCR, (2) cytokinesignaling abnormalities, (3) toxic metabolite accumulation, (4) defective survival of hematopoietic precursors, (5) TCR abnormalities, and (6) severe thymic abnormalities. Clinical manifestations • Before the era of newborn screening, infants would present with atypical or severe manifestations of infection, disease following live virus vaccine, chronic diarrhea, recurrent fevers, and/or failure to thrive. • Patients identified by newborn screen are typically normal in appearance and asymptomatic. • Newborn screen will not identify infants with severe T-cell deficiencies if the defect allows sufficient thymic production of TRECs, which can occur in conditions such as Zap70 deficiency, MHC class II deficiency, NF(kappa)B essential modulator deficiency, late-onset adenosine deaminase (ADA) deficiency, and hypomorphic mutations in some classic SCID genes. • Patients with Omenn syndrome, a disorder in which the genetic defect allows the production of a limited number of oligoclonal T cells that are autoreactive, can have skin rash, hepatosplenomegaly, adenopathy, and elevation of eosinophil and IgE levels. Diagnosis • Typical SCID is defined as a total CD3 1 count ,300 cells/µL and functional testing with a mitogen stimulation assay with phytohemagglutinin (PHA) , 10% of the lower limit of normal. • Definitive diagnosis of typical SCID requires an absolute CD3 1 count ,300 or absence of naı̈ve T cells (CD3 1 CD45RA 1 ), along with identification of a pathogenic variant in a known SCID gene, very low ADA type 1 enzyme activity, or engraftment of maternal T cells in the infant. • Leaky SCID refers to combined immunodeficiencies that allow the production of a limited, but dysfunctional T cell population with limited diversity. • Leaky SCID is defined by a T-cell count above 300 cells/µL and a PHA mitogen stimulation between 10 and 30% of the lower limit of normal. T cells at birth may be much higher than typical SCID but are typically memory T cells (CD3 1 CD45RO 1 ). Management and treatment • For patients with SCID, isolation and infectious preventive measure, including antimicrobial prophylaxis, immunoglobulin replacement, and cessation of breastfeeding for CMV-positive mothers is necessary. • Cure with HSCT is necessary, unless the patient has a genetic disorder amenable to gene therapy such as IL2RG or ADA deficiency SCID, which can be an alternate to allogeneic transplant. X-linked agammaglobulinemia In contrast to T-cell deficiencies, isolated disorders of B cells are not immediately life-threatening and can be often be treated successfully with supportive care measures such as immunoglobulin infusions and antibacterial prophylaxis. The Lanzkowsky’s Manual of Pediatric Hematology and Oncology 234 11. Disorders of white blood cells classic pure B-cell deficiency is Bruton’s agammaglobulinemia, also known as X-linked agammaglobulinemia (XLA). XLA is secondary to a deficiency in the X-linked gene, BTK, which is necessary for B-cell production and the absence of the gene product is responsible for 85% of congenital agammaglobulinemia. Efforts are being made to include screening for B-cell deficiencies with kappa-recombining excision circles, a circular remnant produced following V(D)J recombination of the IGK locus, on the newborn screen to mirror the success in early identification of T-cell defects. Clinical manifestations • Male child with frequent respiratory infections with viral and bacterial pathogens • Small adenoids and tonsils are common features on physical examination • Lymph nodes may also be small but can also be normal size from hypertrophy of T-cell areas Diagnosis • Absent B cells on lymphocyte flow cytometry • Low quantitative immunoglobulins across all isotypes and lack of serologic response to vaccines • Identification of a pathogenic variant in the BTK gene Management and treatment • Disorders of B-cell dysfunction require lifelong immunoglobulin replacement and isolated B-cell deficiencies, such as XLA, are not typical candidates for HSCT secondary to the risk imposed by such a treatment measure NK-cell lymphopenia Isolated NK-cell deficiency is incredibly rare and usually low NK cells are associated with combined immune deficiency disorders. Disorders in NK cytotoxic function can result in severe infections, particularly with herpesviruses, and/or severe lymphoproliferative disease such as seen in HLH. Severe defects in NK function require HSCT for cure. Secondary lymphocytopenia disorders Secondary lymphocytopenia is common and can be found associated with a variety of infections, medications, autoimmune diseases, systemic disorders, and malignancies (see Table 11.4). The degree of lymphocytopenia can also be a biomarker of severity, including pediatric patients with RSV and adult patients with SARS-CoV-2. TABLE 11.4 Etiologies associated with secondary lymphocytopenia. Infection Medications Autoimmune and inflammatory disorders Malignancy Other Bacterial (ehrlichiosis, Salmonella typhi, leptospirosis, Mycobacterium tuberculosis, bacterial sepsis) Viral (HIV, influenza, including H1N1 and H5N1, RSV, SARS-CoV-1 and SARS-CoV-2, measles, West Nile, HHV6, parvovirus B19, dengue virus) Chemotherapy Glucocorticosteroids Monoclonal antibodies targeting lymphocytes Radiation Crohn’s disease Inflammatory arthritis MIS-C Primary vasculitides Sarcoidosis Sjogren’s syndrome SLE Type I diabetes mellitus Lymphoma (Hodgkin lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma) Solid tumors (breast, colon) Soft tissue sarcomas Burn victims End-stage renal disease Lymphatic malformations Protein!energy malnutrition Zinc deficiency Abbreviations: HHV6, human herpesvirus 6; HIV, human immunodeficiency virus; MIS-C, Multisystem inflammatory syndrome in children; RSV, respiratory syncytial vrius; SLE, systemic lupus erythematous. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Further reading and references 235 Secondary lymphocytopenia is typically benign, with the exception being HIV infection, and typically resolves with treatment of the primary cause. Dedication To our mentor and friend, Laurence A. Boxer, MD: May this chapter kindle the same sense of curiosity and determination to uncover the intricacies of the phagocyte in others that you so generously sparked in us. Further reading and references Almarza Novoa, E., Kasbekar, S., Thrasher, A.J., et al., 2018. Leukocyte adhesion deficiency-I: a comprehensive review of all published cases. J. Allergy Clin. Immunol. Pract. 6 (4), 1418!1420.e10 (in English). Available from: https://doi.org/10.1016/j.jaip.2017.12.008. Badolato, R., Donadieu, J., Group, W.R., 2017. How I treat warts, hypogammaglobulinemia, infections, and myelokathexis syndrome. Blood 130 (23), 2491!2498 (in English). Available from: https://doi.org/10.1182/blood-2017-02-708552. Berezné, A., Bono, W., Guillevin, L., Mouthon, L., 2006. Diagnosis of lymphocytopenia. Presse Med. 35 (5 Pt 2), 895!902 (in French). Available from: https://doi.org/10.1016/s0755-4982(06)74709-1. Bousfiha, A., Jeddane, L., Picard, C., et al., 2020. Human inborn errors of immunity: 2019 update of the IUIS phenotypical classification. J. Clin. Immunol. 40 (1), 66!81 (in English). Available from: https://doi.org/10.1007/s10875-020-00758-x. Cerny, J., Rosmarin, A.G., 2012. Why does my patient have leukocytosis? Hematol. Oncol. Clin. N. Am. 26 (2), 303!319, viii (in English). Available from: https://doi.org/10.1016/j.hoc.2012.01.001. Connelly, J.A., Choi, S.W., Levine, J.E., 2012. Hematopoietic stem cell transplantation for severe congenital neutropenia. Curr. Opin. Hematol. 19 (1), 44!51 (in English). Available from: https://doi.org/10.1097/MOH.0b013e32834da96e. Feriel, J., Depasse, F., Geneviève, F., 2020. How I investigate basophilia in daily practice. Int. J. Lab. Hematol. 42 (3), 237!245 (in English). Available from: https://doi.org/10.1111/ijlh.13146. Gennery, A.R., 2020. Progress in treating chronic granulomatous disease. Br. J. Haematol. 192, 251!264 (in English). Available from: https:// doi.org/10.1111/bjh.16939. Kaplan, J., De Domenico, I., Ward, D.M., 2008. Chediak-Higashi syndrome. Curr. Opin. Hematol. 15 (1), 22!29 (in English). Available from: https://doi.org/10.1097/MOH.0b013e3282f2bcce. Klion, A., 2018. Hypereosinophilic syndrome: approach to treatment in the era of precision medicine. Hematol. Am. Soc. Hematol Educ. Program. 2018 (1), 326!331 (in English). Available from: https://doi.org/10.1182/asheducation-2018.1.326. Kumrah, R., Vignesh, P., Patra, P., et al., 2020. Genetics of severe combined immunodeficiency. Genes. Dis. 7 (1), 52!61 (in English). Available from: https://doi.org/10.1016/j.gendis.2019.07.004. Lawrence, S.M., Corriden, R., Nizet, V., 2017. Age-appropriate functions and dysfunctions of the neonatal neutrophil. Front. Pediatr. 5, 23 (in English). Available from: https://doi.org/10.3389/fped.2017.00023. Lynch, D.T., Hall, J., Foucar, K., 2018. How I investigate monocytosis. Int. J. Lab. Hematol. 40 (2), 107!114 (in English). Available from: https://doi.org/10.1111/ijlh.12776. Makaryan, V., Zeidler, C., Bolyard, A.A., et al., 2015. The diversity of mutations and clinical outcomes for ELANE-associated neutropenia. Curr. Opin. Hematol. 22 (1), 3!11(in English). Available from: https://doi.org/10.1097/MOH.0000000000000105. Myers, K.C., Furutani, E., Weller, E., et al., 2020. Clinical features and outcomes of patients with Shwachman-Diamond syndrome and myelodysplastic syndrome or acute myeloid leukaemia: a multicentre, retrospective, cohort study. Lancet Haematol. 7 (3), e238!e246(in English). Available from: https://doi.org/10.1016/S2352-3026(19)30206-6. Schwartz, J.T., Fulkerson, P.C., 2018. An approach to the evaluation of persistent hypereosinophilia in pediatric patients. Front. Immunol. 9, 1944 (in English). Available from: https://doi.org/10.3389/fimmu.2018.01944. Segel, G.B., Halterman, J.S., 2008. Neutropenia in pediatric practice. Pediatr. Rev. 29 (1), 12!23; quiz 24 (in English). Available from: https:// doi.org/10.1542/pir.29-1-12. Shillitoe, B., Gennery, A., 2017. X-linked agammaglobulinaemia: outcomes in the modern era. Clin. Immunol. 183, 54!62 (in English). Available from: https://doi.org/10.1016/j.clim.2017.07.008. Shomali, W., Gotlib, J., 2019. World Health Organization-defined eosinophilic disorders: 2019 update on diagnosis, risk stratification, and management. Am. J. Hematol. 94 (10), 1149!1167 (in English). Available from: https://doi.org/10.1002/ajh.25617. Snow, A.L., Xiao, W., Stinson, J.R., et al., 2012. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations. J. Exp. Med. 209 (12), 2247!2261 (in English). Available from: https://doi.org/10.1084/jem.20120831. Spinner, M.A., Sanchez, L.A., Hsu, A.P., et al., 2014. GATA2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity. Blood 123 (6), 809!821(in English). Available from: https://doi.org/10.1182/blood-2013-07-515528. Valent, P., Sotlar, K., Blatt, K., et al., 2017. Proposed diagnostic criteria and classification of basophilic leukemias and related disorders. Leukemia 31 (4), 788!797 (in English). Available from: https://doi.org/10.1038/leu.2017.15. Walkovich, K., Boxer, L.A., 2013. How to approach neutropenia in childhood. Pediatr. Rev. 34 (4), 173!184 (in English). Available from: https://doi.org/10.1542/pir.34-4-173. Williams, K.W., Ware, J., Abiodun, A., Holland-Thomas, N.C., Khoury, P., Klion, A.D., 2016. Hypereosinophilia in children and adults: a retrospective comparison. J. Allergy Clin. Immunol. Pract. 4 (5), 941!947.e1 (in English). Available from: https://doi.org/10.1016/j. jaip.2016.03.020. Yu, H.H., Yang, Y.H., Chiang, B.L., 2020. Chronic granulomatous disease: a comprehensive review. Clin. Rev. Allergy Immunol. (in English). Available from: https://doi.org/10.1007/s12016-020-08800-x. Lanzkowsky’s Manual of Pediatric Hematology and Oncology This page intentionally left blank C H A P T E R 12 Disorders of platelets Catherine McGuinn and James B. Bussel Division of Hematology-Oncology, Department of Pediatrics, Weill Cornell Medical College, New York, NY, United States Platelets are an important component in primary hemostasis. Defects in platelet number or function may lead to bleeding and bruising. Bleeding due to platelet disorders usually involves skin and mucous membranes, presenting as petechiae, purpura, ecchymosis, epistaxis, hematuria, menorrhagia, as well as gastrointestinal or intracranial hemorrhage (ICH). Platelet characteristics include: 1. Size: a. 1!4 µm (younger platelets are larger) b. Mean platelet volume (MPV): 8.9 6 1.5 fL 2. Distribution: a. 1/3 in the spleen b. 2/3 in circulation 3. Average life span: a. 9!10 days Table 12.1 lists the causes of thrombocytopenia based on platelet size. Table 12.2 lists the causes of thrombocytopenia according to pathophysiology. Table 12.3 lists the common and uncommon causes of thrombocytopenia by age. Thrombocytopenia in the newborn Neonatal thrombocytopenia is relatively common, occurring in 1!3% of healthy term infants and in 20!30% in the neonatal intensive care unit population. Thrombocytopenia in sick neonates is often secondary to an underlying pathology such as sepsis, disseminated intravascular coagulation (DIC), and respiratory distress syndrome or secondary to maternal factors such as pregnancy-induced hypertension, gestational diabetes, and intrauterine growth retardation (IUGR). Table 12.4 lists the causes of neonatal thrombocytopenia. Neonatal alloimmune thrombocytopenia Neonatal alloimmune thrombocytopenia (NAIT) is the most common cause of severe thrombocytopenia in the newborn, with an overall incidence of approximately 1 in 500!1000 births if the definition is expanded to identify mild cases with platelet count ,100,000/µL as a threshold. NAIT is perhaps as frequent as 1:3000!5000 births when defined as a platelet count ,50,000/µL and perhaps 1 in 5000!10,000 if bleeding manifestations leading to clinical identification in the newborn nursery are required. NAIT typically resolves in 2!4 weeks. Firstborn infants represent approximately 50% of those severely affected, and subsequent affected pregnancies may have increasingly severe presentations and require antenatal management. Lanzkowsky’s Manual of Pediatric Hematology and Oncology. DOI: https://doi.org/10.1016/B978-0-12-821671-2.00016-7 237 © 2022 Elsevier Inc. All rights reserved. 238 TABLE 12.1 12. Disorders of platelets Platelet diseases based on platelet size. Macrothrombocytes (elevated MPV) ITP Conditions with increased platelet turnover (e.g., DIC) Bernard!Soulier syndrome MYH9-related diseases Gray platelet syndrome Velocardiofacial syndrome Various mucopolysaccharidoses GATA1 XLT with thalassemia TUBB1-related thrombocytopenia Normal size (MPV normal) Conditions in which marrow is hypocellular or infiltrated with malignant disease CAMT TAR syndrome Microthrombocytes (MPV decreased) WAS/XLT FYB-related thrombocytopenia ARCP1B-related thrombocytopenia Some storage pool diseases Cytomegalovirus infection Inherited thrombocytopenias are not well characterized and may present with normal/large platelet size. Abbreviations: CAMT, congenital amegakaryocytic thrombocytopenia; DIC, disseminated intravascular coagulation; ITP, idiopathic thrombocytopenic purpura; MPV, mean platelet volume (as determined by automated electronic counters); MYH9, nonmuscle myosin heavy chain 9 gene; normal, 8.9 6 1.5 µm3; TAR, thrombocytopenia-absent radius; WAS, Wiskott!Aldrich syndrome; XLT, X-linked thrombocytopenia. Pathophysiology NAIT can be thought of as a platelet analog of Rh incompatibility [i.e., hemolytic disease of the fetus and newborn (HDFN)]. It differs from Rh incompatibility because many cases are firstborn infants, suggesting the antigenic exposure occurs early in pregnancy unlike in Rh, which occurs primarily at the time of delivery. NAIT occurs when fetal platelets that express platelet-specific antigens inherited from the father are the target of maternal alloantibodies. Mothers who lack the paternally inherited platelet-specific surface antigen, and who possess the immunologic predisposition to make antibodies to it, can become sensitized when they are expressed on fetal platelets, or possibly also from the AV /BIII receptor on the syncytiotrophoblast that may explain the presentation in first pregnancies. The most common antigen involved is HPA-1a, which accounts for approximately 75!80% of cases. A further 10!20% of cases are due to maternal sensitization to HPA-5b. More than 30 other antigens are known to be involved in NAIT. HPA-4 is important in Asian populations that do not have the HPA-1a/b polymorphism. Mothers who possess the Human Leukocyte Antigen (HLA)-DR-type DRB30101 have a 25-fold increase risk of alloimmunization to HPA-1a. These IgG antibodies cross the placenta and attach to the surface of fetal platelets, causing platelet destruction and likely inhibition of platelet production. Clinical features 1. Typically infants are otherwise healthy full-term babies, who manifest symptomatic thrombocytopenia with generalized petechiae, ecchymosis, cephalohematoma, umbilical bleeding, oozing from skin puncture sites, and/or gastrointestinal or renal tract bleeding. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thrombocytopenia in the newborn TABLE 12.2 239 Pathophysiological classification of thrombocytopenic states. 1. Increased platelet destruction (normal/increase megakaryocytes in the marrow) a. Immune thrombocytopenia i. ITP ii. Drug induced iii. Secondary to systemic autoimmune disorder • Autoimmune hemolytic anemia (Evans syndrome) • ALPS • SLE • Posttransfusion purpura • DiGeorge • Hyperthyroidism iv. Neonatal • Neonatal autoimmune thrombocytopenia • FNAIT • HDFN v. Infection related • Viral—HIV, CMV, EBV, varicella, rubella, rubeola, mumps, measles, hepatitis, parvovirus • Bacterial: tuberculosis, typhoid, pertussis b. Consumption i. TMA • HUS • Complement-mediated—aHUS • TTP • TA-TMA ii. DIC iii. Kasabach!Merritt syndrome iv. HLH (see Chapter 15, Histiocytosis syndromes) v. Cyanotic congenital heart disease vi. Malignant hypertension vii. Drugs (e.g., ristocetin, protamine, bleomycin) c. Mechanical i. ECMO ii. VAD 2. Disorders of platelet distribution a. Hypersplenism i. Secondary to portal hypertension, Gaucher disease, cyanotic congenital heart disease, neoplastic, infections b. Hypothermia 3. Decreased platelet production (decreased or absent megakaryocytes) a. Drug i. (e.g., chlorothiazides, estrogenic hormones, ethanol, tolbutamide) b. Infections i. Viral • HIV, EBV, CMV, hepatitis, parvovirus, varicella ii. Bacterial • Sepsis, Rickettsia c. Infiltrative marrow disease i. Benign • Osteopetrosis • Storage disease (e.g., Gaucher) ii. Malignant • Leukemia/lymphoma • Metastatic disease (solid tumors) • MPN • LCH d. Congenital i. CAMT ii. TAR syndrome iii. RUSAT iv. Paris!Trousseau syndrome v. WAS/XLT vi. BSS vii. MYH-related disorders viii. Inherited bone marrow failures (e.g., Fanconi, dyskeratosis, SDS) (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 240 TABLE 12.2 12. Disorders of platelets (Continued) e. Nutritional i. B12 ii. Folate iii. Iron deficiency f. Constitutional i. Trisomy 18, 13, 21 g. Metabolic disorders i. Infants born to hypothyroid mothers ii. Methylmalonic academia iii. Ketotic glycinemia iv. Holocarboxylase synthetase deficiency v. Isovaleric academia vi. Idiopathic hyperglycinemia h. Acquired i. Aplastic anemia ii. PNH iii. Drug induced (e.g., dose-related: antineoplastic agents; benzene, organic and in organic arsenicals, Mesantoin, Tridione, antithyroids, antidiabetics, antihistamines, phenylbutazone, insecticides, gold compounds; idiosyncrasy: chloramphenicol) iv. Radiation induced i. Pseudothrombocytopenia i. Platelet activation during blood collection ii. Undercounting of macrothrombocytes iii. In vitro agglutination of platelets due to EDTA iv. Monoclonal antibodies that bind to platelet glycoprotein receptors such as abciximab, eptifibatide, tirobifan Abbreviations: aHUS, Atypical hemolytic!uremic syndrome; ALPS, autoimmune lymphoproliferative syndrome; BSS, Bernard!Soulier syndrome; CAMT, congenital amegakaryocytic thrombocytopenia; CMV, cytomegalovirus; DIC, disseminated intravascular coagulation; EBV, Epstein!Barr virus; ECMO, extracorporeal membrane oxygenation; FNAIT, fetal and neonatal alloimmune thrombocytopenia; HDFN, hemolytic disease of the fetus and newborn; HLH, hemophagocytic lymphohistiocytosis; HUS, hemolytic!uremic syndrome; ITP, idiopathic thrombocytopenic purpura; LCH, langerhans cell histiocytosis; MPN, myeloproliferative neoplasm; MYH, nonmuscle myosin heavy chain; PNH, paroxysmal nocturnal hemoglobinuria; RUSAT, radioulnar synostosis with amegakaryocytic thrombocytopenia; SDS, Shwachman!Diamond syndrome; SLE, systemic lupus erythematosus; TAR, thrombocytopenia-absent radius; TATMA, transplant-associated thrombotic microangiopathy; TMA, thrombotic microangiopathy; TTP, thrombotic thrombocytopenia purpura; VAD, ventricular assist device; WAS, Wiskott!Aldrich syndrome; XLT, X-linked thrombocytopenia. A bone marrow biopsy, in addition to marrow aspiration, should always be carried out to avoid sampling errors and to establish the presence of a decreased number of megakaryocytes in the marrow. 2. Affected neonates have rates of ICH up to 10!20% and, when present, the ICH tends to be severe and intraparenchymal. When ICH occurs, it most frequently occurs in utero, not at delivery, and may be detected on ultrasonography during apparently uncomplicated pregnancies. Death in utero may occur. 3. The platelet count is very low at birth, usually ,50,000/µL. Cases of HPA-5b incompatibility are less markedly thrombocytopenic. Diagnosis NAIT should be considered in all newborns with thrombocytopenia. Precisely, 90% of cases of HPA-1a incompatibility NAIT have a platelet count of ,50,000/µL, making it a reasonable screening threshold for identifying NAIT. Two other reasons to suspect NAIT, even if the neonatal count is .50,000/µL, include: 1. no clinically apparent etiology of thrombocytopenia and 2. family history of transient neonatal thrombocytopenia. Response to random platelet transfusion or lack thereof is not diagnostically useful. It is important to investigate and establish the diagnosis because of the impact on subsequent pregnancies and hence their management. Laboratory evaluation should include HPA typing of the mother, father, and (only if needed) the child to establish incompatibilities. Additionally, maternal serum should be investigated to identify platelet-specific antibodies with inclusion HPA-1, 3, and 5 antibodies, as well as HPA-4 antibodies in those of Asian descent. HPA-9b and 15 are the next most common antigen incompatibilities. To confirm the diagnosis of NAIT, testing should ideally show both parental platelet antigen incompatibility and antibodies to the discordant antigen. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thrombocytopenia in the newborn TABLE 12.3 Neonate 241 Classification of thrombocytopenic purpura by age and frequency. Common Uncommon Sepsis Cardiac (prosthetic heart valves, repair of intracardiac defects, left ventricular outflow obstruction) Asphyxia Alloimmune thrombocytopenia Maternal hypertension Necrotizing enterocolitis Infections (rubella, CMV, HIV, hepatitis B, syphilis) Maternal ITP Amegakaryocytic thrombocytopenias CAMT without anomalies Congenital amegakaryocytic TAR syndrome ATRUS Wiskott!Aldrich and X-linked thrombocytopenia Bernard!Soulier syndrome MYH9 disorders Quebec syndrome Gray platelet syndrome Inborn errors of metabolism Congenital leukemia (trisomies 13, 18, 21) Child ITP Type IIB von Willebrand disease Drug induced Autoimmune diseases such as SLE, JIA Immunodeficiency Infections Inherited bone marrow failures (e.g., Fanconi anemia) Leukemia and other malignancies with bone marrow involvement Autoimmune hemolytic anemia (Evans syndrome) TTP/HUS Hyperthyroidism Megaloblastic anemias (folate and vitamin B12 deficiencies) Severe iron-deficiency anemia Cyclic thrombocytopenias Lymphoproliferative disorders Aplastic anemia Abbreviations: ATRUS, Amegakaryocytic thrombocytopenia with radioulnar synostosis; CAMT, congenital amegakaryocytic thrombocytopenia; CMV, cytomegalovirus; HUS, hemolytic!uremic syndrome; ITP, idiopathic thrombocytopenic purpura; JIA, juvenile idiopathic arthritis; MYH9, nonmuscle myosin heavy chain 9 gene; SLE, systemic lupus erythematosus; TAR, thrombocytopenia with bilateral absence of radii; TTP, thrombotic thrombocytopenic purpura. The useful clinical criteria for the diagnosis, in addition to severe congenital thrombocytopenia (,50,000/µL), include: 1. normal nonpregnant maternal platelet count and negative history of maternal immune thrombocytopenic purpura (ITP), 2. exclusion of alternate diagnoses, 3. recovery of normal platelet count within 2!3 weeks, 4. history of NAIT in a prior pregnancy, and 5. the absence of bone marrow aplasia upon bone marrow examination (if performed). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 242 TABLE 12.4 12. Disorders of platelets Causes of neonatal thrombocytopenia. Normal or increased megakaryocytes in the marrow (consumptive thrombocytopenia) 1. Immune disorders a. Autoimmune (passive transfer of platelet antibody) Neonatal Immune Thrombocytopenia (NITP) i. Maternal ITP ii. Maternal drug-induced thrombocytopenia iii. Maternal SLE b. Alloimmune (NAIT) i. Isolated platelet antigen incompatibility 2. Infection a. Bacterial: Gram-negative and Gram-positive septicemia, listeriosis b. Viral: cytomegalovirus, rubella, herpes simplex, coxsackievirus, HIV c. Protozoal: toxoplasmosis d. Spirochetal: syphilis 3. Drugs a. Immune: drug-hapten disease (e.g., quinine, quinidine, sedormid) b. Nonimmune: thiazide, tolbutamide (given to mother) c. Prolonged antibiotics or ganciclovir d. Chemotherapy 4. Disseminated intravascular coagulation a. Antenatal causes i. Preeclampsia and eclampsia ii. Abruptio placentae iii. Amniotic fluid embolism b. Intranatal causes i. Breech delivery ii. Fetal distress c. Postnatal causes i. Infections ii. Hypoxia and acidosis iii. Respiratory distress syndrome iv. Renal vein thrombosis v. Indwelling catheters vi. Vascular anomalies with KMP 5. Inherited thrombocytopenia a. Sex-linked i. GATA1 ii. Wiskott!Aldrich syndrome b. Autosomal i. Bernard!Soulier syndrome ii. MYH9-RD (was called May!Hegglin and other rarer types) Decreased or absent megakaryocytes in the marrow (amegakaryocytic thrombocytopenia) 1. Isolated megakaryocytic hypoplasia a. Congenital amegakaryocytic TAR syndromea b. Congenital RUSATa c. Congenital megakaryocytic hypoplasia without anomalies (CAMT)a d. Congenital hypoplastic thrombocytopenia with microcephaly e. Rubella syndrome f. Congenital hypoplastic thrombocytopenia associated with trisomy syndromes g. Thrombocytopenia agenesis of corpus callosum h. Fanconi anemia i. Hoyeraal!Hreidarsson syndrome 2. Generalized bone marrow disorders a. Bone marrow aplasia 3. Fanconi anemia 4. Pancytopenia without congenital anomalies 5. Osteopetrosis 6. Bone marrow infiltration a. Congenital leukemia b. Langerhans cell histiocytosis c. Congenital neuroblastoma (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thrombocytopenia in the newborn TABLE 12.4 243 (Continued) 7. Metabolic causes a. Associated with acidosis and ketosis b. Hyperglycinemia c. Methylmalonic acidemia d. Isovaleric acidemia e. Propionic acidemia 8. Maternal hyperthyroidism Abbreviations: CAMT, Congenital amegakaryocytic thrombocytopenia; ITP, immune thrombocytopenic purpura; KMP, Kasabach!Merritt phenomenon; MYH9, nonmuscle myosin heavy chain 9 gene; MYH9-RD, MYH9-Related Disorders NAIT, neonatal alloimmune thrombocytopenia; RUSAT, radioulnar synostosis with amegakaryocytic thrombocytopenia; SLE, systemic lupus erythematosus; TAR, thrombocytopenia associated with bilateral absent radii. a Characterized by severe thrombocytopenia at presentation. Treatment 1. Platelet transfusion 10!20 mL/kg body weight. Maternal or matched platelets are infrequently necessary for effective therapy since there is often good efficacy with transfusion of unmatched platelets. 2. Matched donor platelets or concentrated maternal platelets may be used, if available, especially if unrelated donor platelet transfusions are ineffective. 3. Intravenous immunoglobulin (IVIG) 1 g/kg per day for 1!3 days can be considered in combination with random platelet transfusions, depending on the response, to maintain the platelet count above 30,000/µL, and 50!100,000/µL in the presence of life-threatening hemorrhage. 4. Corticosteroids have been used in conjunction with platelet transfusions and IVIG, despite limited evidence, using a regimen of methylprednisolone (1 mg IV) every 8 hours on days IVIG is administered. 5. Urgent head ultrasound is mandatory for the thrombocytopenic neonate. If there are any abnormal neurological findings and the ultrasound is unrevealing or equivocal, an MRI should be done using the feed and swaddle technique to avoid anesthesia. If ICH is present in NAIT on ultrasound, the target platelet count should be .100,000/µL and an MRI may also be performed to better define the hemorrhage. Imaging should be repeated to document stabilization/improvement and then monthly for 3 months to identify early hydrocephalus, along with head circumference measurements. 6. With or without treatment and with or without hemorrhage, following the infant until the platelet count is within the normal range will avoid missing inherited causes of thrombocytopenia. Management of subsequent pregnancies Identification of a family at risk for NAIT is critical to help stratify the antenatal management of future pregnancies with the goal of ameliorating fetal thrombocytopenia and thus preventing fetal and neonatal ICH. The previous stratification included the sampling of fetal blood to determine antigen expression and platelet count, but given the invasiveness of this procedure and the risks of serious complications, current approaches are based on noninvasive information. Management of subsequent pregnancies should be undertaken by experienced specialists in maternal!fetal medicine. In cases where there is identification of paternal incompatibility, but not active alloimmunization, the recommendation is for heightened antibody screening. In these potential cases of NAIT, serial crossmatching of paternal platelets and maternal serum for anti-HPA antibodies can be performed with initiation of therapy if antiHPA antibodies are detected (rarely). With certain incompatibilities (i.e., HPA-3a and -b and HPA-9b), antibodies are more difficult to detect. Clinical judgment in conjunction with the expertise of the laboratory needs to be combined in these cases. If a previous affected sibling was serologically diagnosed with severe NAIT, the likelihood of the next fetus being affected depends on the father’s platelet typing. If the father is homozygous for the antigen responsible (as is the case in 75% of men with HPA-1a), essentially all later fetuses will be affected. If the father is heterozygous, or if paternal typing is unavailable or uncertain, fetal HPA typing should be performed, preferentially using noninasive prenatal testing by cell-free fetal DNA (cffDNA) (or amniocentesis if cffDNA is not available) to determine whether the fetus is at risk and to help guide recommendations for initiation of stratified antenatal therapy. For mothers with previous NAIT pregnancy without ICH, recommended treatment starts later (20 weeks) and is less intensive than for those in which a previous pregnancy had an ICH (12 weeks). In some cases, even without a history of ICH, IVIG 1 g/kg per week by itself may not be sufficient treatment and the addition of increased doses of IVIG or corticosteroids can be useful depending on input from maternal!fetal medicine. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 244 12. Disorders of platelets Neonatal autoimmune thrombocytopenia Neonatal autoimmune thrombocytopenia is due to a passive transfer of autoantibodies from mothers with ITP to their fetus. It may also be seen in association with other conditions such as maternal systemic lupus erythematosus (SLE) and lymphoproliferative states. Neonates born to mothers who have autoimmune thrombocytopenia are typically well after an uncomplicated delivery. Maternal history and platelet counts can help to distinguish autofrom alloimmune thrombocytopenia, but such a distinction can be confusing in the presence of gestational thrombocytopenia (GTP). A history of maternal thrombocytopenia during the pregnancy is not diagnostic of maternal ITP depending upon its severity. GTP occurs in 5!10% of pregnancies, which is almost always mild (70!100,000/µL), is not associated with neonatal thrombocytopenia, and the maternal platelet count normalizes after delivery. Neonatal thrombocytopenia in infants born to mothers with autoimmune thrombocytopenia is usually less severe than that seen in NAIT. Only 10!15% of these newborns have a platelet count ,50,000/µL. There is a lower risk of bleeding and only rare reports of ICH. The platelet count may be “adequate” at delivery (e.g., 90,000/µL), but then fall to a clinically significant nadir over the next 1!3 days. Table 12.5 lists the pathogenesis and clinical differences between NAIT and autoimmune thrombocytopenia. Pathophysiology Neonatal ITP occurs as a result of passive transfer of maternal antibodies across the placenta, as is seen in NAIT. However, the target of the antibodies is an antigen present on maternal platelets that is also on fetal platelets (in contrast to alloimmune thrombocytopenia, where the antigen is not present on maternal platelets). The most frequently targeted antigens are the GPIIB/IIIA or GPIb/IX complexes. Differences in glycosylation of fetal and maternal platelets may explain mothers with normal or near-normal counts having thrombocytopenic neonates. Mothers who have previously undergone splenectomy for ITP may still pass antibodies transplacentally even if their platelet counts are normal. Diagnosis Pregnant women with the following conditions may give birth to a neonate with autoimmune thrombocytopenia: 1. History of previously affected infant. 2. Mother who was previously splenectomized for ITP (mother may have platelet antibodies without being thrombocytopenic). TABLE 12.5 Pathogenesis and clinical differences between alloimmune thrombocytopenia (neonatal alloimmune thrombocytopenia) and autoimmune thrombocytopenia. Alloimmune thrombocytopenia Autoimmune thrombocytopenia Platelet antigens Antigens found on fetal platelets not present on maternal platelets (usually HPA-1A or HPA-5b) Antigens common to both maternal and fetal platelets (usually GPIIB/IIIA and GPIb/IX complexes) Platelet count Often ,20,000/µL Birth counts often .50,000/µL Time of presentation Birth Platelet count can be near normal at birth, and then fall Maternal history Low platelet counts (unless mother is splenectomized) Normal platelet count, no history of ITP, SLE, or hypothyroidism, may have GTP (unrelated) History of ITP, SLE, hypothyroidism Intracranial hemorrhage 10!20% ,1!2% Treatment Random donor platelets IVIG IVIG 6 Methylprednisolone 1/! Methylprednisolone Random platelets (if hemorrhage) Matched platelets Resolution of thrombocytopenia Usually in 2!4 weeks Usually 3!12 weeks Abbreviations: GTP, Gestational thrombocytopenia; ITP, immune thrombocytopenic purpura; IVIG, intravenous immunoglobulin; SLE, systemic lupus erythematosus. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thrombocytopenia in the newborn 245 3. Mother with thrombocytopenia (,100,000/µL) in current pregnancy especially if the platelet count is ,50,000/µL. 4. Mother with SLE, hypothyroidism, preeclampsia—HELLP syndrome (hemolytic anemia, elevated liver enzymes, and low platelets). 5. Maternal drug ingestion (e.g., thiazide). Treatment Follow the platelet count (as the initial count may be near normal) until 3!7 days of life to capture nadir and monitor until stable or a rising count to .150,000/µL without intervention. All infants with severe thrombocytopenia should have a head ultrasound to exclude ICH. Treatment is required when the infant’s platelet count falls below 30,000/µL or if significant bleeding is present. The regimen is similar to that of NAIT, utilizing IVIG and IV methylprednisolone but unrelated donor platelet transfusion is used only if indicated by severe bleeding symptoms or a platelet count that remains ,30,000/ µL despite maximal care. The duration of neonatal thrombocytopenia is usually about 1!3 weeks. If persistent in an infant of a breastfeeding mother, or worsening past day 5, a trial of discontinuation of breastfeeding should be considered. Unlike NAIT, there is no need for maternal treatment during pregnancy except under extraordinary circumstances. General diagnostic approach to a newborn with thrombocytopenia If the maternal platelet count is low, especially ,50,000/µL, this suggests maternal ITP or another etiology for the maternal thrombocytopenia such as inherited thrombocytopenia (IT). If the maternal platelet count is normal but the mother has had a splenectomy for ITP, then neonatal ITP can still be seen from circulating antiplatelet antibodies. Gestational thrombocytopenia, which complicates up to 10% of all normal deliveries, may present with a maternal platelet count as low as 50!70,000/µL, but hardly ever results in neonatal thrombocytopenia. If the platelet count is ,50,000/µL on the first day of life in an otherwise normal neonate, NAIT should be considered and appropriate laboratory testing performed. Imaging is required based on the platelet count and clinical findings. Platelet transfusion and possibly IVIG should be given. The next most common cause of early severe neonatal thrombocytopenia is a TORCH infection [toxoplasmosis, rubella, cytomegalovirus (CMV) or herpes virus], which presents as a variably sick newborn with associated fever, microcephaly, IUGR, conjunctivitis, hearing loss, hepatosplenomegaly, and/or blueberry muffin rash. If the baby is sick and the platelet count is .50,000/µL, it is likely that the underlying illness (e.g., respiratory distress syndrome, sepsis, DIC) is the cause of the low platelet count. The underlying condition should be treated with the expectation that with a resolution of the underlying illness, resolution of the thrombocytopenia will occur, although monitoring until resolution is indicated. If there is persistent severe thrombocytopenia, congenital amegakaryocytic thrombocytopenia (CAMT) and other ITs should be considered (see Chapter 6, Bone Marrow Failure). Thrombocytopenia associated with hemolytic disease of the fetus and neonate HDFN is rarely seen. Thrombocytopenia is secondary to many etiologies, for example, dilution, hepatosplenomegaly. Thrombocytopenia secondary to chronic fetal hypoxia, maternal diabetes, pregnancy-induced hypertension, or intrauterine growth retardation Neonatal thrombocytopenia may be caused by chronic intrauterine hypoxia resulting in placental insufficiency in association with pregnancy-induced hypertension, preeclampsia, HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome, maternal diabetes mellitus, and IUGR. These may be due in part to increased platelet destruction, but typically there is impaired megakaryopoiesis and an elevated thrombopoietin level. Neonates can increase the number of megakaryocytes but typically do not increase their size, which may limit their platelet-producing capability in an emergency setting. Thrombocytopenia from hypoxia is usually not severe and is self-limited. The nadir tends to occur around days 3!4 with recovery by days 7!10. Often no treatment is required. In the setting of preeclampsia, even with maternal thrombocytopenia, neonatal leukopenia is more common than neonatal thrombocytopenia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 246 12. Disorders of platelets Thrombocytopenia secondary to congenital infections Perinatal viral, bacterial, and fungal infection may present with early- or late-onset thrombocytopenia. Often the presentation may be in an ill neonate with accompanying low birth weight, microcephaly, hepatosplenomegaly, chorioretinitis, and impaired hearing. Infections to consider include toxoplasmosis, rubella, CMV, or herpes simplex (TORCH), group B Streptococcus, Listeria monocytogenes, Escherichia coli, or HIV. Of the TORCH infections, CMV infection most commonly causes severe thrombocytopenia, with 50!77% infants affected. TORCH infections may cause a “blueberry muffin” rash, which appears different from bruising because it represents subcutaneous islands of extramedullary hematopoiesis. Thrombocytopenia in early-onset neonatal sepsis can occur because of the following: 1. Platelet consumption associated with DIC. 2. Bacteria, viruses, or immune complexes that adhere to platelets and are cleared by the mononuclear phagocyte system. 3. Sequestration secondary to hepatosplenomegaly. 4. Impaired thrombopoiesis—often there is insufficient compensation for platelet destruction or increased platelet clearance. In these infants, thrombocytopenia resolves with effective treatment of the underlying infection. Late-onset thrombocytopenia secondary to late-onset infections, necrotizing enterocolitis, or thrombosis Thrombocytopenia occurring at .72 hours after birth is more likely to be related to late-onset sepsis, necrotizing enterocolitis (NEC), thrombosis, or liver disease. NEC presents with feeding intolerance, abdominal distension, bloody stools, and pneumatosis intestinalis. Severe thrombocytopenia, when present, has been associated with poor outcomes. The causes of late-onset thrombocytopenia are varied and often multifactorial, including: 1. consumption related to infection (as in NEC), 2. deficient platelet production, 3. DIC, 4. consumption secondary to thrombosis (e.g., renal vein or hepatic vein thrombosis), and 5. liver disease due to the reasons listed previously. Thrombocytopenia due to aneuploidy Thrombocytopenia is common in Down syndrome, occurring in up to 85% of cases. Cases may indicative of transient myeloproliferative disorder (TMD). TMD occurs in 5!10% of trisomy 21 cases and is associated with mutations in the second exon of GATA1 transcription factor. TMD typically presents in the first month of life with thrombocytopenia, leukocytosis, hepatosplenomegaly, and/or hepatic fibrosis from megakaryocytic infiltration appearing as if it is the onset of leukemia. The great majority of TMD cases resolve with observation during the neonatal period. However, 20!30% will recur, typically within 1!2 years, to develop Acute megakaryoblastic leukaemia (AMKL) (see Chapter 19: Acute Myeloid Leukemia). Thrombocytopenia may also be seen in trisomies 13 and 18, triploidy, and Turner syndrome. In these cases, other congenital anomalies, for example, midline abnormalities suggest the underlying diagnosis and other medical problems in these patients far outweigh thrombocytopenia in their importance. Rare bone marrow disease or inborn errors of metabolism The following marrow diseases may be associated with thrombocytopenia in the newborn: 1. Osteopetrosis—generalized hyperostosis of bone and obliteration of bone marrow cavity resulting in extramedullary hematopoiesis and pancytopenia. 2. Metastatic neuroblastoma. 3. Gaucher disease, often including hypersplenism and other coagulopathies. 4. Niemann!Pick disease. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Thrombocytopenia in the newborn 247 5. Hemophagocytic lymphohistiocytosis (HLH) (see Chapter 15, Histiocytic Disorders). 6. Congenital leukemia—leukemia in the first 4 weeks of life is rare (1!5 per million live births) and carries a very poor prognosis (unless it is actually TMD). Acute myelogenous leukemia (AML) with KMT2A rearrangements is most common. It may be associated with skin infiltrates, hepatosplenomegaly, pallor, nephromegaly, and megakaryoblasts in the blood smear. These skin lesions, which may resemble blueberry muffin lesions, may also be seen in other etiologies of bone marrow infiltration and replacement, TORCH infections, or severe hemolysis. Metabolic causes Hyperglycinemia with ketosis and the closely related metabolic disorder of methylmalonic acidemia may cause periodic thrombocytopenia, as well as neutropenia, during infancy. Infants with these metabolic disorders present with lethargy, vomiting, and ketosis during the neonatal period. A similar disorder, isovaleric acidemia, is associated with a “sweaty feet odor” but may present with a generalized marrow hypoplasia causing thrombocytopenia and neutropenia. Thrombocytopenia has also been reported in rare infants born with neonatal Graves’ disease, resulting from maternal transfer of thyroid-stimulating antibodies with associated symptoms of tachycardia, cardiac dysfunction, liver disease, and hepatosplenomegaly. Vascular anomalies Vascular anomalies are heterogeneous disorders involving the skin, subcutaneous tissues, and visceral organs (see Chapter 14, Vascular Anomalies). Current classification from the International Society for the Study of Vascular Anomalies (www.issva.org/classficiation) divides lesions broadly into proliferative lesions (tumors) or lesions, the growth of which parallels the patient (malformations). Included within this classification of complicated vascular tumors are kaposiform hemangioendothelioma (KHE) and Tufted angioma. KHE is a rare locally aggressive, rapidly proliferating unifocal cutaneous or soft tissue lesion usually presents as a .5-cm red-purple indurated plaque with a pebbled texture and ill-defined margins typically occurring on the extremities, trunk or retroperitoneum most commonly occurring in infants. The morditity and mortality seen with these specific vascular tumors correlated with size ( . 5!8 cm) and degree of infiltration (muscle, bone, mediastinal or retroperitoneal sites) of the lesion associated with increasing risk for Kasabach!Merritt phenomenon (KMP). KMP is a form of localized intravascular microangiopathic hemolytic anemia presenting with severe thrombocytopenia, hypofibrinogenemia, and elevated fibrin split products or D-dimers, which is seen in B70% of KHE cases. Intralesional platelet trapping and fibrinogen consumption have been demonstrated through immunohistochemistry and radiolabeling lead to increased risk for hemorrhage and high-output cardiac failure. MRI of the lesion is used to determine the extent of dermal, lymphatic, and muscular involvement as well identifying the arterial supply and venous drainage that may help to direct therapy. CT, X-ray, and ultrasound are of limited utility in determining the extent of the lesion and directing appropriate therapy. Since these vascular anomalies often grow after birth, thrombocytopenia may not present until after the first month of life. Infantile hemangiomas, the most common tumor of infancy, are not associated with the complication of KMP. Additional reports of thrombocytopenia have been seen with other types of vascular anaolmies, including: 1. Vascular tumor: benign: rapidly involuting congenital hemangioma. 2. Vascular malformation: lymphatic: kaposiform lymphangiomatosis. 3. Unclassified vascular anomalies: multifocal lymphangiomatosis with thrombocytopenia/cutaneovisceral angiomatosis with thrombocytopenia. Treatment Supportive care with transfusions of platelets and other coagulation factors (e.g., fibrinogen concentrates, fresh frozen plasma, cryoprecipitate, and antifibrinolytic drugs) have only transient effects. These modalities may help with acute management of bleeding and stabilization in the cases of intralesional or systemic bleeding. Prophylactic platelet transfusions are NOT recommended as they may lead to increased trapping and pain with tumor enlargement. Antiplatelet agents (aspirin, ticlopidine, and clopidogrel) have been used Lanzkowsky’s Manual of Pediatric Hematology and Oncology 248 12. Disorders of platelets with mixed results. If there is ongoing DIC, which may be local, for example, intralesional, anticoagulation may be crucial. Surgical resection, the gold standard for cure, is often difficult given the extent of tumor infiltration and coagulopathy at presentation. Embolization may be an adjunctive therapy to minimize bleeding but is often a temporizing measure that may improve the thrombocytopenia rather than being a curative modality. Radiation therapy is not recommended as frontline therapy secondary to long-term complications. Frontline pharmacologic therapy for this tumor has been evolving. Historically, steroids have been used in combination with vincristine, but increasing reports of response to mammalian target of rapamycin inhibitors have been evaluated in prospective clinical trials. Currently, there is a randomized control trial of steroid in combination with either vincristine or sirolimus (rapamycin) for KHE with KMP enrolling. Interferon was previously utilized but it had important toxicities that prevented its routine use, including deafness, neutropenia, hypothyroidism, locked-in syndrome, and others. Sirolimus targets antiangiogenesis pathways and results in apoptosis as the proposed mechanism of action. Used at a dose of 0.8 mg/kg per m2 twice per day with therapeutic monitoring of levels was shown in case series and a prospective phase II study to be well tolerated. Advantages include the ability to titrate to a therapeutic level, minimizing typical side effects such as mucositis, myelosuppression, and hyperlipidemia. Steroids: corticosteroids are the most commonly used first-line therapy at high oral doses (2 mg/kg per day of oral prednisone) or equivalent intravenous doses. There is limited evidence supporting their effectiveness as a single agent therapy. Chemotherapy: vincristine, a vinca alkaloid and inhibitor of endothelial proliferation, has shown successful response in tumor regression. Doses of vincristine 0.05 mg/kg weekly have been used. Barriers to its use include the need for IV access and risk of neurologic side effects. Other reports have looked at cyclophosphamide and actinomycin D in refractory disease, with less clear data for efficacy, safety, and dosing. Propranolol, a nonselective beta-blocker, has been used successfully in the management of infantile hemangioma. The proposed mechanism is multifactorial with vasoconstriction, antiangiogenesis, and apoptosis of capillary endothelial cells contributing to the clinical outcome. Isolated case reports have demonstrated successful patient outcomes in KHE. All these treatments may ameliorate thrombocytopenia in proportion to their reduction in tumor size and perhaps their reduction of intratumoral consumption of platelets. Inherited thrombocytopenias ITs are heterogenous group of disorders distinguished by a decreased platelet number with wide variability in pattern of inheritance, platelet morphology, and association with syndromic/congenital defects resulting from other facets of gene expression, including predisposition to malignancy. The understanding and classification of IT has expanded greatly with the incorporation of genomic sequencing (next-generation sequence targeted panels, whole-exome sequence, and whole-genome sequencing—see Chapter 1, Molecular and Genomic Methodologies for Clinicians). There are currently well over 30 ITs with relatively frequent identification of new ones. Applications of new platforms have allowed improved classification and characterization of spectrum and phenotype, in addition to highlighting the mechanism of megakaryopoiesis and platelet production contribution to clinical presentation. Some of these defects have severe bleeding and striking thrombocytopenia recognized in the neonatal period, whereas others remain undiagnosed well into adulthood. There is overlap in that some syndromes are also characterized by abnormal platelet function (e.g., Wiskott!Aldrich, Bernard!Soulier) but clinically this is infrequent. Previous classifications of IT have focused on platelet morphology, familial inheritance, and associated syndromic findings or using a framework of genetics and biology to understand the defect at a molecular level. Below is a clinically relevant classification of isolated thrombocytopenia, syndromic/congenital defects, and increased predisposition to disease development (renal, malignancy, myelofibrosis). It is important to realize that diagnosis of a specific IT allows better anticipation of other problems that can arise with a given syndrome beyond those directly related to the platelet. Table 12.6 lists the characteristics of congenital thrombocytopenic syndromes, including their underlying mutation, mode of inheritance, platelet size, and any findings on blood smear, common platelet count and degree of bleeding, and any nonhematologic manifestations of the syndrome. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 249 Inherited thrombocytopenias TABLE 12.6 Characteristics of congenital thrombocytopenic syndromes. Disease Inheritance Genetic Platelet size Additional Functional defect Gp1bIX Thrombocytopenia (platelet defect) AR GP1BA m AD GP9 m Gray platelet syndrome AR NBEAL2 m Pale “ghost platelets.” Functional defect—α-granules Platelet-type VWD AD GPIBA m Thrombocytopenia worsens with stress ACTN1-related thrombocytopenia AD ACTN1 m TUBB1-related thrombocytopenia AS TUBB1 m Spherocytic platelets CYCS-related thrombocytopenia AD CYCS GF1b-related thrombocytopenia AS GF1B m RBC anisocytosisPlatelet function defect (α-granule) ITGA2B/ITGB3 AD ITGA2BITGB3 m Functional defect PRKACG-related thrombocytopenia AR PRKACG m Functional defect SLFN14-related thrombocytopenia AD SLFN14 m Functional defect FYB-related thrombocytopenia AR FYB m FLI1-related thrombocytopenia AR FLI1 m Functional defectGiant α-granule THPO mutation AD THPO TRPM7-related thrombocytopenia AS TRPM7 m Aberrant distribution of granules Tropomyosin 4!related thrombocytopenia AD TPM4 m Bernard!Soulier syndrome Thrombocytopenia with congenital defect (syndromic) Wiskott!Aldrich syndrome XL WAS k X-linked thrombocytopenia XL WAS k Paris!Trousseau syndrome AD 11q23 Del FLNA-related thrombocytopenia XL FLNA GATA1 related XL GATA1 X-linked thrombocytopenia with thalassemia; X-linked thrombocytopenia with dyserythropoietic anemia Thrombocytopenia-absent radius AR RBM8A Bilateral absent radius. Cow’s milk intolerance. Upper/lower limbs abnormalities Stormorken syndrome AD STIM1 Myopathy, asplenia, ichthyosis, facial dysmorphism, cognitive impairment York platelet syndrome AD STIM1 Myopathy, platelet structure abnormalities Eczema, autoimmunity, immunodeficiency Functional defect. Large granules, growth and cognitive impairment, CNS, GI, kidney and urinary malformations m Periventricular nodular heterotopia (Continued) Lanzkowsky’s Manual of Pediatric Hematology and Oncology 250 TABLE 12.6 12. Disorders of platelets (Continued) Disease Inheritance Genetic Platelet size Additional Thrombocytopenia with increased risk (acquired disease) MYH9-related disease AD MYH9 m Sensorineural deafness, nephropathy, cataracts DIAPH1-related thrombocytopenia AD DIAPH1 m Infantile sensorineural deafness CAMT AR MPL Absent megakaryocytes. Evolution to severe aplasia RUSAT AD/AR HOXA11 MECOM RUNX1 Bilateral radioulnar synostosis. Risk of progression to aplasia Functional defect. Risk of AML or T-cell ALL Familial platelet disorder with AD propensity to AML ANKRD26-related thrombocytopenia AD ANKRD26 Reduced α-granules. AML m Hgb and WBC ETV6-related thrombocytopenia AD ETV6 ALL, AML. MDS risk SRC-related thrombocytopenia AD SRC Abundant vacuoles. Juvenile myelofibrosis, splenomegaly, osteoporosis, and facial dysmorphism Abbreviations: AML, Acute myelogenous leukemia; CAMT, congenital amegakaryocytic thrombocytopenia; CNS, central nevous system; GI, gastroenterology; RUSAT, radioulnar synostosis with amegakaryocytic thrombocytopenia; WAS, Wiskott!Aldrich syndrome; VWD, Von Willebrand disease. Adapted and modified from Noris, P., Pecci, A., 2017. Hereditary thrombocytopenias: a growing list of disorders. Hematology Am. Soc. Hematol. Educ. Program. 2017 (1), 385!399; Lambert, M.P., 2019. Inherited platelet disorders: a modern approach to evaluation and treatment. Hematol. Oncol. Clin. North. Am. 33 (3), 471!487. Bernard!Soulier syndrome Bernard!Soulier syndrome (BSS) is a relatively rare autosomal recessive disorder with a bleeding phenotype; monoallelic cases have variable clinical phenotypes. BSS has an estimated incidence of 1 per million and is typically characterized as biallelic (bBSS) when two mutations in GP1BA, GP1BB, or GP9 are found. There are monoallelic (mBSS) cases that are dominant (Bolzano mutation) and often have a less severe presentation. The gene encodes for the GPIb!IX complex that binds von Willebrand factor (vWF). Clinically, it is characterized by the following: 1. Moderate-to-large macrothrombocytopenia: BSS is associated with automated counting underestimating the platelet count due to undercounting of very large platelets. The typical true platelet range is 60!100,00/µL. 2. Prolonged Platelet Functional Analyzer (PFA) closure times (historically prolonged bleeding time). 3. Platelet aggregation studies with reduced response specifically to ristocetin agonist but normal responses to other agonists. 4. Flow cytometry with decreased CD42a (GPIX) and CD42b (Gp1b-alpha). 5. Characteristic platelet morphology on blood smear. Platelets, even in the monoallelic cases, are very large, equaling or exceeding the size of a red cell (macrothrombocytes). These platelets contain two to four times the normal protein and dense granule content. The dense granules can gather and give the appearance of a pseudonucleus. Morphologically, the megakaryocytes have an abnormality in the demarcation membrane system with impaired proplatelet formation, explaining the large platelets and the thrombocytopenia. There can be the complete absence of GPIb glycoprotein complex or a point mutation in the GPIb-alpha subunit known as the Bolzano variant, all of which lead to inability of vWF binding. Platelets fail to agglutinate in response to ristocetin, despite normal aggregation and secretion in response to adenosine diphosphate (ADP), epinephrine, thrombin, and collagen. Because vWF acts as a bridge in adhesion of platelets to exposed subendothelium, the absence of the vWF receptors inhibits normal platelet adhesion and causes a significant degree of bleeding, irrespective of the degree of thrombocytopenia. Mucocutaneous bleeding can begin in early infancy, including: 1. Unexplained bruising and purpura. 2. Gingival or mucosal bleeding (especially severe epistaxis). 3. Excessive hemorrhage following trauma or invasive procedure. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited thrombocytopenias 251 Treatment 1. No routine prophylaxis is recommended. 2. Antifibrinolytic therapy is recommended for mild!moderate dental bleeding, epistaxis, and menorrhagia (as well as hormonal treatment for the latter). 3. Platelet transfusions, while the most reliable therapy, are reserved for managing severe bleeding episodes, prophylaxis for major surgery, and failure of adjuvant, local, and antifibrinolytic measures. There is a risk of isoimmunization because these patients lack the GPIb/IX complex and readily make antibodies to it upon transfusion. 4. Recombinant activated factor VII (rFVIIa), while not licensed for BSS, has been used in emergency situations of platelet refractoriness. DDAVP may be useful, but its effect is weak. Gray platelet syndrome It is a storage pool disorder resulting from defective formation of α-granules with macrothrombocytopenia and typical morphologic identification of gray platelets (no purple-stained α-granules) on peripheral smear. The absence of α-granules results in functional manifestations of lack of platelet adhesion, hemostasis, and wound healing. A genetic defect in the NBEAL2 gene has been identified in some patients with an autosomal recessive inheritance pattern. Patients have bleeding symptoms from the impaired platelet function and risk of progression to splenomegaly. In particular, the leakage in the marrow of TGF-beta (instead of being sequestered in α-granules) leads to bone marrow myelofibrosis. Myosin heavy chain 9 disorders Myosin heavy chain 9 (MYH9)-Related Disease (RD) encodes the nonmuscle myosin heavy chain expressed only in neutrophils and platelets (myosin IIa) and plays a central role in the cytoskeletal formation of proplatelets and their release. This autosomal dominant (monoallelic) disorder is the most prevalent IT. Clinically, these patients have mild-to-severe macrothrombocytopenia (very infrequently ,20!30,000) and, depending upon the mutation site, may have a risk of developing nephrotic syndrome with risk of progression to end-stage renal disease (30%), sensorineural deafness most, and cataracts (20%). Previously, these disorders comprised May!Hegglin anomaly, Sebastian, Fechtner, and Epstein syndromes (and Alport which does not have platelet manifestations). Now with greater understanding of the underlying genetic mutation, they are collectively classified as MYH9-related diseases. The site and type of mutation in the gene may determine the findings with a genotype!phenotype correlation. Diagnosis is typically based on: 1. macrothrombocytopenia with mild bleeding symptoms, 2. morphology review of smear with large bluish cytoplasmic inclusions in granulocytes and monocytes known as Döhle bodies (only in what we would have called May!Hegglin), and 3. immunofluorescence on blood smears of MYH9 protein aggregates. Clinical presentation can include: 1. Mucocutaneous bleeding symptoms (variable, rarely severe). 2. Elevated liver enzymes (up to 30%, usually mild). 3. Hearing loss (primarily high tone; onset in first!sixth decade in those with certain mutations in the MYH9 gene). 4. Glomerular nephropathy, proteinuria, and microscopic hematuria, which often result in renal failure. These cases will be seen first and followed by nephrologists. Treatment Symptom surveillance is recommended for renal, eye, and ear symptoms, although exactly what to monitor, how to monitor it, and how frequently to monitor it remains to be established. It would ideally be dependent upon the mutation and clinical presentation. There is not a set schema of which we are aware as to how to carry out follow-up. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 252 12. Disorders of platelets At least two trials with thrombopoietic (TPO) agonists (eltrombopag) demonstrated platelet response in 80% of cases using short courses of treatments in the preoperative setting. Another use is in the third trimester of pregnancy to increase the platelet count for delivery perhaps starting 1!2 weeks prior to planned induction. A recent study demonstrated the correlation of in vitro platelet production stimulated by TPO agents and in vivo effects of TPO agents. Reasons for nonresponse have not been well elucidated. Additionally, there may be abnormal natural killer (NK) activity because of the dysfunctional actin!myosin interaction in the cytoskeleton. Wiskott!Aldrich syndrome This syndrome has X-linked inheritance with classic but variable features of thrombocytopenia, eczema, recurrent bacterial and viral infections secondary to abnormalities in antibody and T-cell function, and a propensity to develop autoimmune disorders (ITP and Autoimmune Hemolytic Anemia [AIHA]) and malignancy (lymphoma and leukemia). Pathophysiology The molecular defect in this syndrome is the absent and/or abnormal Wiskott!Aldrich syndrome protein (WASp) resulting from a mutation of the WAS gene located on band Xp11!12. WAS is a scaffold protein known to be involved in signal transduction. It regulates actin filament assembly that explains the abnormalities in platelet and lymphocyte cytoskeleton and signaling. Thrombocytopenia is due to the combination of decreased platelet production, suggested by the therapeutic response to TPO agonists, and increased platelet destruction, suggested by the therapeutic response to splenectomy. There is debate about “platelet dysfunction” in this syndrome. Many past studies were hampered by attempting to assess platelet function in the setting of severe thrombocytopenia. Several studies concluded that WAS included platelet dysfunction. A more recent study using flow cytometry to assess single platelet function identified normal function of the individual platelets. The increased rate of major bleeding at very low platelet counts can be explained to be a consequence of the very low platelet mass at a given platelet count rather than being caused by nonfunctioning platelets. Diagnosis 1. Mild to often severe thrombocytopenia. 2. Microthrombocytopenia—small platelets noted on blood smear. 3. Genetic testing along with WASp expression testing via flow cytometry. Clinical manifestations 1. Male infants present with thrombocytopenia and bruising symptoms in the first few months of life (only two females with WAS have been reported and two infants with neutropenia). There is also a very rare syndrome with a similar presentation in which there is absence of WASp-binding protein. 2. Bleeding frequently presents with bright red blood and/or melena in the GI tract during the neonatal period, later followed by purpura. GI bleeding may be more frequent and out of proportion to thrombocytopenia and may be aggravated by milk allergy. 3. The clinical course is punctuated by recurrent pyogenic infections, including otitis media, pneumonia, and skin infections. There is also lowered resistance to nonbacterial infections, including herpes simplex and Pneumocystis jirovecii (formerly carinii) pneumonia. 4. Without proper management the disease may often be fatal by the early teens due to infection, lymphoproliferative malignancy, and/or bleeding. 5. Differentiation of WAS from milder X-linked thrombocytopenia (XLT) is done both clinically and via laboratory testing (XLT details discussed later). Hematologic findings 1. Thrombocytopenia (platelet count 1000!100,000/m3); microthrombocytes; very low MPV. This may not be obvious in the newborn and the MPV is unreliable when the platelet count is very low. In addition, it is possible that the platelets are of more normal size in the immediate newborn period because of immaturity of splenic function. 2. Platelets have reduced survival to half of normal. 3. Ineffective megakaryocytopoiesis reflected by a platelet turnover 25% that of normal megakaryocyte mass. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited thrombocytopenias 253 4. Anemia (due to blood loss). 5. Leukocytosis (due to infection). 6. Normal or increased megakaryocytes. 7. Absent isohemagglutinins, reduced IgM, and normal or elevated IgG and IgA. 8. Inability to make antibody to polysaccharide antigens, for example, pneumococcus. 9. Defective cell-mediated immunity in some cases. 10. X-linked inheritance. Treatment 1. Allogeneic stem cell transplantation is the treatment of choice when there is a fully matched donor available. 2. If no matched donor is available, alternative donors of gene therapy clinical trials should be considered. Gene therapy with a lentiviral vector in ongoing clinical trials, after the original retroviral gene therapy trials were halted with development of leukemia. 3. The patient should be managed with supportive therapy consisting of platelet transfusions for hemorrhagic episodes not managed with local measures or antifibrinolytic therapy. 4. Autoimmune thrombocytopenia with platelet consumption can infrequently develop but be managed with steroids, IVIG, TPO agents, and rituximab therapy. 5. Splenectomy: reserved only for severe cases because of risk of overwhelming postsplenectomy infection. Usual preand postsplenectomy precautions should be performed, including appropriate vaccinations with conjugated vaccines, as well as IVIG or subcutaneous Ig prophylaxis in those without antipneumococcal antibodies or low IgG levels. 6. Topical management of eczema (emollient, steroids, and topical tacrolimus). 7. Aggressive treatment of infections and prevention with prophylactic antibiotics. X-linked thrombocytopenia A variant of WAS, known as inherited XLT, has thrombocytopenia, which is less severe than WAS and does not have the associated features of eczema or immunodeficiency. Like WAS, there is an abnormality in the WASp. However, the mutations in XLT occur more likely as point mutations in exons 1 and especially 2 of WASp, which give rise to this milder phenotype, because only the megakaryocyte binding domain is affected. Patients with XLT may be mistaken for chronic “refractory” ITP. As described, it is important to realize that the distinction of ITP from XLT is not always straightforward but rather may be subtle. Patients with “XLT” have a higher incidence of postsplenectomy sepsis than patients with ITP. The distinction of the XLT form from WAS is not always clinically clear; XLT and WAS are on a spectrum and not two distinct entities. The severity of a given patient and their clinical manifestations may depend in part where their mutation is and on how much WASp is in circulation. In addition, there are forms of XLT in which the thrombocytopenia improves (substantially) with puberty. Anemia and thrombocytopenia with GATA1 mutation Several families have been identified with variable microcytic anemia and thrombocytopenia with GATA1 mutations recognized as the underlying cause. GATA1 is a transcription factor important in erythrocyte and megakaryocyte development. Bone marrow shows large megakaryocytes with nuclei pushed to the side and with disorganized granular content. XLT with thalassemia and dyserythropoietic anemia is caused by GATA1 mutations; the latter may resemble dyskeratosis congenita (DC). Paris!Trousseau syndrome/Jacobsen syndrome This syndrome consists of mild thrombocytopenia with a subpopulation of platelets with giant α-granules. There is an expansion of immature megakaryocyte progenitors in the bone marrow with normal erythroid and granulocytic maturation. A subset of these patients will have Jacobsen syndrome, which includes the same platelet defects as Paris!Trousseau with additional abnormalities such as trigonocephaly, facial dysmorphism, cardiac defects, syndactyly, and prominent psychomotor retardation. The genetic defect is deletion in 11q23 associated with deletion of FLI1 transcription factor, which has a role in regulation of platelet granule development. Platelet function is variable. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 254 12. Disorders of platelets Thrombocytopenia with absent radii syndrome 1. Thrombocytopenia with absent radii (TAR) syndrome is a rare disorder with autosomal recessive inheritance. 2. Most cases are diagnosed in utero by routine ultrasound; the rest are almost always identified on the first day of life. 3. There is bilateral absence of the radii manifesting as a shortening of the forearms and flexion at the elbows such that the hands end up near to each other. 4. Both thumbs are present, which helps to distinguish TAR from Fanconi anemia. Other defects of phalanges, ulna, humeri, scapuli, vertebrae, and lower limbs (knees and hips) are frequently present when sought out radiologically, as well as cardiac anomalies. 5. White blood cell count elevation can be seen and, when extreme, is called “leukoerythroblastic.” Anemia is also part of the clinical picture. 6. Cow’s milk allergy is highly prevalent in patients with TAR. 7. Significant bleeding episodes, such as gastrointestinal and even intracerebral hemorrhage, occur in the first 3!12 months of life with severe thrombocytopenia ,20!30,000/µL at a relatively high rate, for example, 30%. 8. Death can occur in the first year of life due to hemorrhage, especially intracranial or gastrointestinal but can be mitigated by platelet transfusions. Typically, the thrombocytopenia improves with time and the platelet count can be .50,000/µL or often normal beyond the first year of life. 9. There is a group of patients who continue to have low counts that may fall back to very low levels in adulthood. 10. Autosomal recessive, compound inheritance of a rare null allele and one of two low-frequency single nucleotide polymorphisms in the regulatory regions of RBM8A, encoding the Y14 subunit of the exon junction complex (EJC) causes TAR. The EJC performs essential RNA processing tasks. TAR is the first human disorder caused by deficiency in one of the four EJC subunits. 11. There have been several case reports of acute myeloid leukemia in early childhood in these patients, but there is no associated chromosomal fragility as seen in Fanconi anemia. There is concern about the use of TPO agents since these patients have an increased risk of malignancy later in life. Discrimination of “leucoerythroblastosis” from leukemia can be difficult especially because leucoerythroblastosis appears to be a myeloproliferative state. Treatment 1. Management is based on the use of prophylactic single-donor platelet transfusions when counts are very low until they improve. Transfusion of red cells for anemia may be necessary. 2. Allogeneic stem cell transplantation may be required for symptomatic patients especially if they “relapse,” for example, have their counts fall as they get older. 3. Orthopedic surgery to correct the upper extremity defects for patients with TAR remains complicated and usually involves multiple procedures. Continued platelet transfusions may be needed for these patients. 4. Avoidance of cow’s milk. 5. Corticosteroids and splenectomy are of no long-lasting benefit. Congenital amegakaryocytic thrombocytopenia 1. CAMT is an autosomal recessive inherited bone marrow failure syndrome. 2. Patients often present with isolated thrombocytopenia in the neonatal period and almost all present thrombocytopenia within the first month, because of petechiae and other bleeding symptoms. 3. The thrombocytopenia is severe and, unlike a disease it mimics, NAIT, does not resolve. 4. The diagnosis of CAMT is not usually made until the infant is several weeks or months old when the bone marrow is examined or genetic diagnosis is considered for failure to improve. 5. Bone marrow evaluation initially reveals absent or greatly reduced numbers of megakaryocytes with normal granulopoietic and erythroid elements. Due to the role of MPL in stem cell maintenance, many patients progress to severe aplastic anemia from stem cell depletion by 5 years of age. 6. Thrombopoietin levels are very high as a result of the markedly decreased megakaryocytes and their progenitors. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Inherited thrombocytopenias 255 7. Physical anomalies may occur in a minority of patients: orthopedic, renal, and/or cardiac but none are diagnostic. 8. CAMT is caused by mutations in the MPL gene (thrombopoietin receptor). Both frameshift and nonsense mutations have been described, which result in loss of MPL function. The type of mutation, such as frameshift or nonsense (Group I) versus missense mutation (Group II), may determine the degree of clinical severity, and whether megakaryocytes are present in small numbers or altogether absent. 9. Recombinant thrombopoietin would likely play no role in therapy for these patients, as its receptor is unable to signal normally although there is a case report of a Group II patient who responded to a TPO agent. 10. Current treatment for CAMT is supportive, using platelet transfusion. 11. The only curative and really only effective treatment is allogeneic stem cell transplantation. 12. Gene therapy is being developed. THPO-related thrombocytopenia 1. Biallelic mutations in the THPO gene that encodes TPO cause a trilineage aplastic anemia clinically overlapping with CAMT. Unlike CAMT, these patients have low TPO levels despite the absence of megakaryocytes in the bone marrow. 2. In contrast to CAMT, these patients do not respond to allogenic stem cell transplant, but rather benefit from treatment with TPO agonist, romiplostim (or eltrombopag and avatrombopag). 3. Monoallelic mutations of THPO result in mild IT. Radioulnar synostosis with amegakaryocytic thrombocytopenia Patients present at birth with thrombocytopenia (similar to CAMT), although the thrombocytopenia is not as severe and the presentation may be later in infancy. They have the following characteristic physical examination findings that can identify this specific syndrome: 1. Proximal radioulnar synostosis (fusion of the radius and ulnar at the elbow) that is not usually evident until at least the second half of the first year of life if not later. It may be unilateral or bilateral. 2. Clinodactyly (minor). 3. Shallow acetabulae (minor). Thus far two separate mutations have been identified: three cases (two families) of HOXA11 mutation and multiple cases of MECOM mutation. The disease is associated with aplastic anemia and possible leukemia and managed similar to CAMT with allogenic stem cell transplantation if needed. MECOM mutations have been more severe with presentation to marrow failure. However, at least one such patient improved with puberty. Platelet function has not been well studied but may be abnormal, for example, bruising and petechiae at platelet counts of 30!50,000/µL or even a little higher. Familial platelet syndrome with predisposition to Acute Myelogenous Leukemia 1. Familial platelet syndrome with predisposition to AML involves a mild-to-moderate thrombocytopenia (a variable platelet count that may be as high as 100,000/µL) with dysfunctional platelets (aspirin-like defect) and decreased dense granules. 2. The cause is a genetic defect in Runt-related transcription factor 1 (RUNX1), a transcription factor previously known as AML1 or CBFA2, related to regulating hematopoiesis and associated with a high risk of myeloid malignancy inherited in an autosomal dominant pattern. The congenital thrombocytopenia is due to a single mutation in all cells, and the leukemia develops when a cell develops a second hit, resulting in a mutation in the RUNX1 gene of the previously unaffected chromosome. 3. There is a 30!40% chance of malignancy, including myelodysplastic syndrome (MDS)/AML, T-cell ALL, hairy cell leukemia, and chronic myelomonocytic leukemia. In addition, approximately 1/3 of patients may develop solid tumors. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 256 12. Disorders of platelets Inherited thrombocytopenia syndromes with predisposition to malignancies ANKRD26-related thrombocytopenia (familial thrombocytopenia type-2) 1. Autosomal dominant mutation with mild!moderate thrombocytopenia with normal-to-mild bleeding phenotype. Bone marrow morphology with small dysmorphic megakaryocytes (micromegakaryocytes) with hypolobulated nuclei. Germline predisposition to myeloid and lymphoid malignancies. 2. This is a relatively common form of IT and does not have additional manifestations consistently—percentage of progression to malignancy is not clear but definitely lower than RUNX1. ETV6-related thrombocytopenia It is autosomal dominant mutation with an increased risk of hematologic malignancies (B-cell ALL/lymphoma) and solid neoplasms. The rate of malignancy is not clear but certainly not the majority of patients. Patients have mild-to-moderate thrombocytopenia and bleeding symptoms out of proportion to the platelet count. Women who are menstruating may manifest iron deficiency. Malignancy can present in childhood with this germline mutation and mimic chronic ITP. Platelet response to steroids has been seen. Von Willebrand Disease Type 2B (VWDIIB) This is an unusual IT with certain characteristic features. 1. It is clinically indistinguishable (except possibly by response to treatment) from platelet-type von Willebrand disease but VWDIIB appears to be approximately 10 times as common. 2. Both diseases involve excessive binding of vWF to the platelet GPIb!IX receptor on platelets. In VWDIIB the vWF is abnormal (too avid) and in platelet-type VW, there is a gain of function mutation in GPIb (too avid). 3. Characteristic features of VWDIIB are that the platelet count decreases markedly with stress and, in particular, as gestation progresses. The latter is thought to be caused by the higher levels of vWF over time. With stress the platelet count may be near normal and suddenly decrease to very low levels mimicking the onset of de novo ITP. If this happens, distinguishing VWDIIB from ITP can be very difficult clinically at the onset: VWDIIB will rarely be suspected initially. Furthermore, only certain types of VWDIIB will have platelet clumps visible on blood smear. If they are present, however, it will first suggest pseudothrombocytopenia. While the conventional approach is to draw blood in citrate and perform a Complete Blood Count (CBC), another alternative is to place a drop of blood from a fingerstick directly on a glass slide and look at it after H and E staining under the microscope. Pseudo-thrombocytopenia should appear normal, whereas VWDIIB would still demonstrate clumps. 4. The primary reason to distinguish VWDIIB from platelet-type VW is treatment. For VWDIIB the goal is to give large quantities of normal vWF. This will ideally outcompete the abnormal vWF, especially the vWF multimers, and reverse the disease. For platelet-type VW the goal is to give high doses of normal platelets. Again, the goal is to have the normal platelets (with normal GPIb) outnumber the affected ones. Both diseases, especially platelet-type VW, are so rare that to the best of our knowledge, novel therapies have not been specifically investigated to treat them. Treatments of ITP would not be expected to impact VWDIIB; however, bleeding and even bruising with VWDIIB (and platelet-type VWD) is usually minor to absent. Miscellaneous diseases in which thrombocytopenia may be prominent but not the sole manifestation Gaucher disease may present with isolated thrombocytopenia, typically not severe, often in the setting of splenomegaly. The ethnic association with Jews of Ashkenazi origin should raise suspicion and lead to genetic testing. MDS is rare in children. If ITP is suspected, inadequate response to treatment should lead to a diagnostic bone marrow if other cell lines and the mean corpuscular volume (MCV) are normal (see Chapter 17, Myelodysplastic Syndromes and Myeloproliferative Disorders). Fanconi anemia can present initially with isolated thrombocytopenia. While overt abnormal radial ray findings are not universal in Fanconi anemia, careful consideration of this diagnosis may reveal hypothenar hypoplasia, renal abnormalities on ultrasound, or increased MCV (which can be missed because the upper limit of normal in a toddler is approximately 90 fL, not 100 as it is in teenagers and adults). Lanzkowsky’s Manual of Pediatric Hematology and Oncology Immune thrombocytopenic purpura 257 Consideration of DC is similar to Fanconi anemia although cytopenias may present at later ages. Testing of telomere length is now widely available if the diagnosis is suspected. SLE has a large overlap with ITP. The diagnosis of SLE requires 4 diagnostic criteria out of 11, of which ITP is 1. Obtaining at least an ANA (other considerations would be a partial thromboplastin time (PTT), Lupus Anticoagulant (LA) test, anti-double standed DNA (DS-DNA), and thyroid testing) is especially useful in teenage women with ITP. While as many as 5% of women can have a 1:40 ANA, it is found in only 2% for 1:80 and essentially no women without autoimmunity have titers higher than this. Reports from France suggest that ITP that is so-called lupoid, for example, with two or three but not four criteria for SLE may be optimally managed with hydroxychloroquine. Liver disease has multiple ways in which thrombocytopenia can occur, one of which is ITP. Immune thrombocytopenic purpura Immune thrombocytopenia is a disorder caused by antiplatelet antibodies and autoreactive T cells leading to accelerated destruction of platelets and inhibition of the production of platelets. ITP is the most common cause of thrombocytopenia in children of any age (except for possibly chemotherapy induced) with a peak occurrence between 2 and 5 years of age. In most children the disease is self-limited, with resolution in 80% of patients within 12 months from diagnosis. North American studies report an incidence of 7.2!9.5/100,000 children between 1 and 14 years of age. There is a seasonal pattern to ITP with a peak in late winter and early springtime, presumably mimicking the seasonality of viral illnesses. There does not appear to be a sex predilection in younger children although, as in adults of child-bearing age, it is more common in adolescent women. Pathophysiology ITP is a heterogeneous disease with a complex pathogenesis, the different types of which are not readily distinguishable at diagnosis. An acute infection may be the initial trigger but may only potentiate an already established autoimmune disturbance. Mechanisms: 1. Antibody-mediated platelet destruction: a. Most identified autoantibodies are directed against GPIIb!GPIIIa, GPIb!GPIX, and GPIa!IIa; follicular B cells in the spleen are heavily involved in synthesis. b. Anti-CPIIB/IIIA antibody-coated platelets bind activated Fc receptors on reticuloendothelial cells (mostly splenic) via recognition of the IgG Fc region of the antiplatelet antibody. This results in their internalization and destruction. Recent work has shown that antibodies directed at GPIb/IX may result in desialylation with platelet clearance by carbohydrate receptors in the liver. c. Antiplatelet antibodies rarely have a significant effect on platelet function although platelets are often shown to be activated. 2. Impaired megakaryopoiesis and reduced platelet production: a. Antibody, cellular cytotoxicity, and/or immune cell!derived cytokines have been implicated in impairment of megakaryocytes. b. Platelet kinetic studies have shown that autologous platelets labeled with indium have a 2- to 3-day halflife (longer than expected for platelet count) suggestive of reduced platelet production. c. Absolute platelet reticulocyte counts are reduced, which is also suggestive of reduced platelet production. d. Thrombopoietin levels are minimally elevated as compared to normal. 3. T-cell activity against platelets is seen but its effects and the frequency of this phenomenon is uncertain especially in children a. Glycoprotein-specific antibodies are absent in 40% of cases of ITP. b. There is an upregulation of genes involved in cell-mediated toxicity (e.g., granzyme B, perforin) in CD31CD81 T cells in ITP patients. c. CD41 T-helper cells stimulate antiplatelet antibody!secreting B cells. d. Th1-associated cytokines predominate in ITP. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 258 12. Disorders of platelets Infections in immune thrombocytopenic purpura Acute infections have been implicated in the initiation of ITP, yet also may cause an acute decrease (or paradoxically an acute increase) in the platelet count in patients with ongoing ITP. 1. Virus-specific antibodies that cross-react with platelets have been demonstrated in several children with varicella but how often this is the cause of de novo ITP remains uncertain. 2. HIV a. A clear relationship between platelet count and viral load has been demonstrated; suppressing the virus increases the platelet count in .90% of cases. b. In the United States, HIV-TP is increasingly infrequent. The pathogenesis of HIV-related ITP may be different from non-HIV-related ITP. First, severe T-cell depletion and immune dysregulation not only occurs in HIV, but also antiplatelet antibodies may cause intravascular platelet lysis more frequently than FcR-mediated clearance. c. Megakaryocytes express receptors for HIV, including CD4, CXCR4, and CCR5, suggesting that direct viral infection may play a role and reduce platelet production. 3. Helicobacter pylori—studies indicate that low platelet counts are often increased with H. pylori treatment in countries with high background incidence of H. pylori, for example, Italy and Japan (but not the United States). This seems to be less common in children 4. Hepatitis C and its treatment with interferon are each associated with thrombocytopenia, which may prevent successful eradication of the virus. Newer hepatitis C treatments that do not cause thrombocytopenia have made low platelet counts associated with hepatitis C more rare complications. Interferon use has been almost fully discontinued. TPO agents may raise the platelet counts in these cases (e.g., eltrombopag 50!100 mg orally daily). The effect of eradication of hepatitis C in patients with cirrhosis on thrombocytopenia is not clear. 5. ITP refractory to therapy may be caused or exacerbated by CMV infection (reactivation or de novo). 6. SARS-CoV-2 causes thrombocytopenia in adults. The induction of de novo ITP is distinctly uncommon in adults and rare in children. However, many cases of ITP in which the patient has become infected with SARS-CoV-2 have manifested acute dramatic decreases in their platelet count. How to manage the ITP in these SARS-CoV-2 infected patients is not yet clear. Whether to treat them differently from classical ITP patients has not been established. Clinical manifestations Typically, many patients are otherwise well and present with petechiae, purpura, and nonpalpable ecchymoses often 1!3 weeks after a viral infection or after rubella, rubeola, chickenpox, Epstein!Barr virus (EBV), or live virus vaccination. Occasionally, patients may present with mucosal bleeding (hematuria, hematochezia, menometrorrhagia, or epistaxis). In a recent study of 200 children, approximately 20% had grade 3 bleeding at presentation. Most often, bleeding symptoms are mild, but rarely patients may develop severe bleeding, including ICH, protracted epistaxis, hematuria, hemoptysis, menometrorrhagia, and gastrointestinal bleeding. ICH occurs in approximately 0.5% of children not always at diagnosis. (Table 12.7) lists the clinical manifestations of ICH in ITP. With the exception of hemorrhagic manifestations, the physical examination is normal. Pallor is usually absent unless there has been significant bleeding. The spleen tip is palpable in fewer than 10% of patients. The finding of splenomegaly suggests the probability of leukemia, SLE, infectious mononucleosis, or liver disease with hypersplenism. Cervical lymphadenopathy is not present unless the precipitating factor is a viral illness. There are no congenital anomalies suggestive of an inherited bone marrow failure syndrome nor findings consistent with a congenital immunodeficiency. In children, secondary ITP may be less frequent than in adults. Table 12.8 lists the features of newly diagnosed, persistent, and chronic ITP. Diagnosis The diagnosis of ITP remains one of exclusion. Demonstrating antiplatelet antibodies has not been shown to be of diagnostic or prognostic importance since antiplatelet antibodies are only present in approximately 60!80% of cases and specificity may be poor as well. There are three diagnostic criteria: 1. Isolated thrombocytopenia (platelet count ,100,000/µL) with otherwise completely normal CBC and blood smear (including morphology of platelets, red cells, and white cells) and otherwise normal Lanzkowsky’s Manual of Pediatric Hematology and Oncology 259 Immune thrombocytopenic purpura TABLE 12.7 Characteristics of intracranial hemorrhage in immune thrombocytopenic purpura. Incidence 0.2!0.8% Age 13 months!16 years Platelet count ,20,000 in 90% of cases ,10,000 in 75% of cases Interval between diagnosis of ITP and ICH At presentation (25% of cases) ,1 week (45% of cases) 1 week!6 months (25% of cases) .6 months (30%) Identifiable risk factors for ICH include • Head injuries (33%) (vs 1% in ITP without ICH) • Hematuria (22%) (vs 0% in ITP without ICH) • Hemorrhage more than petechiae and bruises (63%) (vs 44% in ITP without ICH) • Arteriovenous malformation • Aspirin treatment Site of ICH • Intracerebral (77% of cases)—87% supratentorial; 13% posterior fossa • Subdural hematoma (23% of cases) Prior treatment • 70% had prior treatment Survival • 75% survive, but 1/3 have neurologic sequelae Abbreviations: ICH, intracranial hemorrhage; ITP, immune thrombocytopenic purpura. Adapted from Butros, L.J., Bussel, J.B., 2003. Intracranial hemorrhage in immune thrombocytopenic purpura: a retrospective analysis. J Pediatr Hematol Oncol 25 (8), 660!4. https://doi.org/10.1097/00043426-200308000-00017. 12902925. (Psaila et al., 2009)(Medeiros and Buchanan, 1996). TABLE 12.8 Features of newly diagnosed and chronic immune thrombocytopenic purpura. Feature Newly diagnosed/persistent Chronic Age Children 2!6 years old Adults Sex distribution Equal Female:male 5 2:1 Preceding infection B80% Unusual Seasonal predilection Springtime None Associated autoimmunity Uncommon More common Onset Acute Insidious Platelet count ,20,000/µL ,20,000!80,000/µL Eosinophilia-lymphocytosis Not uncommon Rare IgA/IgG levels Normal Infrequently low Duration 2!8 weeks/3!12 months 1 to many years Prognosis Spontaneous remission in 70!80% of cases Ongoing thrombocytopenia with occasional remission physical examination [the absence of hepatosplenomegaly, lymphadenopathy, and congenital anomalies such as radial ray anomaly (oligodactyly, aplasia or hypoplasia of the thumb or radius, radioulnar synostosis)]. a. The blood smear in ITP often has large platelets but a large number of macrothrombocytes suggest other disorders, for example, Bernard!Soulier, MYH9 disorders (Table 12.1). b. Artifactual low platelet counts (pseudothrombocytopenia) must be excluded. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 260 12. Disorders of platelets c. Secondary ITP such as Evans syndrome and SLE and also thrombotic thrombocytopenic purpura (TTP) (anemia, high reticulocyte count, schistocytes) must be considered and excluded. d. Other possible entities need to be excluded by specific testing directed and based on history and physical examination. 2. Platelet response to ITP therapy (especially IVIG or anti-D, possibly steroids) is the only finding that positively supports the diagnosis of ITP (including both primary and secondary ITP). Exactly what degree of response is required is not clear. Coagulation screening tests (prothrombin time (PT), PTT, fibrinogen) if performed, are all normal (unless there is an incidental finding or antiphospholipid syndrome). Other causes of thrombocytopenia must be excluded: 1. IT by: a. Family history, duration, response to therapy. b. The presence of congenital defects (skeletal, cardiac, renal, neurologic). c. Usually large platelet size on smear (MPV is unreliable in severe thrombocytopenia). 2. Pregnancy. 3. Chronic infection with HIV, hepatitis C, H. pylori, and CMV. 4. Immunodeficiency [e.g., common variable immunodeficiency (CVID) (hypogammaglobulinemia), Wiskott!Aldrich], other syndromes. 5. Lymphoproliferative disease (benign or malignant) (Chapter 16, Lymphoproliferative Disorders) 6. Type IIB von Willebrand disease (see Chapter 13, Disorders of Coagulation). 7. Medications, especially quinine, valproate, heparin, estrogen in any form, diphenylhydantoin and dietary supplements. Many drugs have been proven or suspected to induce drug-dependent antibody-mediated immune thrombocytopenia (http://www.ouhsc.edu/platelets/DITP.html is a current reference source). Additional laboratory analyses may be required for nonresponsive, persistent or chronic cases or for those cases which have specific clinical indications: 1. Autoimmune screening—ANA and antidouble-stranded DNA antibodies, RA, C3, C4. 2. Thyroid screening (TSH, free T4, thyroid antibodies). 3. Immune globulin measurements (IgG, IgA, and IgM) and possibly specific antibody levels, for example, to pneumococcal serotypes. 4. Liver function tests. 5. EBV, CMV, parvovirus, hepatitis C, and HIV testing by polymerase chain reaction. 6. H. pylori testing (in countries with a high background incidence). 7. Bone marrow aspiration and biopsy. Patients with isolated thrombocytopenia, who fit the diagnostic criteria mentioned earlier, generally do not require bone marrow sampling to rule out leukemia; however, abnormalities on examination or the blood count (WBC or Hgb) or smear may suggest an alternative diagnosis and the need for a bone marrow examination. Failure to respond (not even a transient increase in platelets) to ITP therapy (e.g., anti-D and/or IVIG) also strongly suggests the need for a diagnostic bone marrow aspirate and biopsy. 8. Antiphospholipid antibodies (present in up to 15% of cases), mixed. PTT, and lupus anticoagulant in cases of prolonged PTT, persistent headache, and/or thrombosis. Treatment The goal of therapy in ITP is to increase the platelet count enough to prevent serious hemorrhage and possibly also to alleviate fatigue or difficulty with activities of daily living. Treatment decisions should be based on the potential for bleeding, including the physical activity level, patient’s history of bleeding, current platelet count, signs and symptoms, and several other factors as follows: 1. Patients who have a platelet count .20,000/µL with no signs of bleeding generally do not need treatment, although certain risk factors (Table 12.7) and surgery may necessitate treatment. At diagnosis, children without serious (grade 3) bleeding on the modified Adix!Buchanan scale often do not need treatment at any platelet count and can be observed carefully with a high expectation of spontaneous improvement within 3!6 months. However, the “watch and wait” approach has never been validated for the long term (i.e., .3!6 months). 2. Patients with a platelet count ,30,000/µL and moderate (grade 3) bleeding at diagnosis should be treated in the interest of preventing more serious bleeding. This is thought to be approximately 20% of children at Lanzkowsky’s Manual of Pediatric Hematology and Oncology Immune thrombocytopenic purpura 261 diagnosis of their ITP. Treatment should consist of IVIG (0.8!1.0 g/kg per infusion) and/or high-dose shortterm steroids (dexamethasone 24 mg/m2/dose 3 4 daily doses OR methylprednisolone 30 mg/kg/dose 3 3!4 doses OR prednisone 4 mg/kg per day for 3!7 days); IV anti-D (75 µg/kg/dose) could be used if available in Rh1, DAT negative, nonsplenectomized patients also with high-dose steroid premedication. 3. The goal of this therapy is to immediately increase the platelet count to a safe level, that is, one that is sufficient to stop bleeding. Patients who have substantial bleeding to require urgent treatment may not respond well to single treatment and may require combination treatments. Other agents, beyond steroids, IVIG, and IV anti-D could be used, including TPO agents, platelet transfusion, vincristine, rituximab, and/or even other agents. It is important to realize that while “watch and wait” is appropriate for most patients, children who are actively bleeding may often need not just standard treatment but aggressive treatment, which may require multiple agents in combination. Supportive care 1. Quality of life can be a major concern in ITP (in particular fatigue and depression) and this may be a reason to treat patient even in the absence of moderate-to-severe bleeding. 2. Competitive contact sports should be avoided when the platelet count is known to be ,30,000/µL. Discussion with the child and the parents may be appropriate to consider treatment to allow participation in certain circumstances. 3. Heavy menstrual bleeding may need intervention for symptom relief with antifibrinolytics, Depo-Provera (medroxyprogesterone acetate), daily oral Provera, or intrauterine device with progestin such as Mirena. 4. Aspirin, nonsteroidal antiinflammatory agents, fish oil, and any other drugs that interfere with platelet function should be avoided. Pharmacologic therapy for ITP is as follows. Corticosteroids Prednisone 2!4 mg/kg per day in divided doses orally for 5!7 days, recognizing a more prolonged course or slow taper may be necessary for some children with newly diagnosed disease. The initial response rate is 50!90% (children tend to be at the higher end of the response range but ones needing treatment may be at the lower end of the range). As with any initial therapy of newly diagnosed ITP, many children go into sustained remission after a single course. Dexamethasone, which is used with more evidence in the adult ITP population, has a less durable response in pediatric but nonetheless may be effective. Prolonged use of steroids in ITP is undesirable. Large doses and prolonged usage might possibly perpetuate the thrombocytopenia and depress platelet production. Primarily longer term higher dose steroids also lead to side effects, including gastritis, ulcers, weight gain, Cushingoid facies, fluid retention, acne, hyperglycemia, hypertension, mood swings, pseudotumor cerebri, cataracts, growth retardation, and avascular necrosis especially of the femoral heads. Mechanism of action of steroids: 1. inhibits the phagocytosis of antibody-coated platelets, 2. inhibits platelet antibody production, and 3. suppresses activation of T cells driving the autoimmune response, Intravenous Immuneglobulin (IVIG) Intravenous immuneglobulin (IVIG) can be administered in a dose of 0.4!1 g/kg per day for 1!5 days at any time from diagnosis to chronic disease when an urgent substantial platelet increase is required. IVIG is preferred over steroids in children ,2 years of age because they tend to have lower response rate to steroids and more challenging behavioral risk factors for bleeding. Metaanalysis of a number of randomized controlled trials has shown a more rapid response to IVIG in children compared to corticosteroids. In addition, a large, retrospective study has suggested that there may be a lower rate of chronic disease in patients initially treated with IVIG compared to those treated with prednisone; however, a more recent study only partially confirmed this: there were significantly more children in remission up to and including 3 months from IVIG treatment at diagnosis and a trend to less children with active ITP at 6 months but by 12 months from diagnosis (development of chronic ITP). Although there were only 10 and 12 children in the two groups who had chronic ITP, the authors suggested that a polymorphism in FcgRIIa was a strong Lanzkowsky’s Manual of Pediatric Hematology and Oncology 262 12. Disorders of platelets indication of chronic ITP. This awaits confirmation, as do studies of RNA expression and other testing, as predictors of chronic ITP. IVIG as an alternative therapy to corticosteroid therapy is much more expensive and has significant side effects (see next). Mechanism of action of intravenous immunoglobulin Early studies demonstrated that IVIG inhibits clearance of Ig-coated platelets. Recent studies of mouse models of ITP suggested that this may happen via IVIG upregulation of FcγRIIb, which may inhibit FcγR on phagocytes but this has not yet been confirmed in people. Saturation of FcRn by IVIG with more rapid catabolism of antiplatelet antibodies is a second likely effect of IVIG given the clinical efficacy of FcRn inhibitors in ITP. Adverse effects of intravenous immunoglobulin 1. Postinfusion headache in .50% of patients. It is transient but occasionally severe (in severe cases, administer IV steroids, for example, dexamethasone 0.15- to 0.3-mg/kg IV up to 40 mg). Severe headache in ITP may suggest the presence of ICH and, if clinically indicated, may require a CT scan, although almost all post-IVIG headaches occur with good platelet counts. Prevention and amelioration of this adverse effect with acetaminophen and prednisone is thus important. 2. Fever and chills in 1!3% of patients. Prophylactic acetaminophen (10!15 mg/kg, 4 hourly, as required) and diphenhydramine (1 mg/kg, 6!8 hourly, as required) may be useful to reduce the incidence and severity. Difficult to resolve infusion reactions is a sign of hypogammaglobulinemia or IgA deficiency. 3. DAT-positive hemolytic anemia in patients with blood group A because of the presence of blood group antibodies (especially anti-A) has occurred much less often because of recent reduction of acceptable anti-A titers in IVIG. 4. Anaphylaxis in IgA-deficient patients because of preexisting IgA antibodies that react with small amounts of IgA present in commercially available gammaglobulin; this is vanishingly rare. 5. Aseptic meningitis is rare and presents as a severe headache within 24 hours after IVIG. 6. Acute renal failure (very uncommon in children and much less overall since elimination of higher osmolality IVIG preparations). 7. Pulmonary insufficiency (although very rare). 8. Thrombosis was demonstrated in certain cases in the past to be caused by increased levels of FXIa in IVIG; however, FXIa is currently screened for and eliminated from IVIG and the incidence of thrombosis has been greatly reduced. 9. No viral transmission is known to currently occur and there are at least two antiviral steps in the processing of every preparation of IVIG; prions are filtered out in processing IVIG. This, of course, does not guarantee that no infections will be transmitted by IVIG but none are known to be transmitted currently. Anti-D therapy IV anti-D is used in a dose of 50!75 µg/kg for initial therapy or recurrent disease. Approximately 70% of patients have a good initial response to 75 µg/kg of anti-D therapy within 1 day (comparable to high-dose IVIG). The effect is more pronounced after 48!72 hours. Anti-D is plasma-derived, hyperimmune globulin with high titers against the Rhesus D antigen. It can be used only in Rh 1 (and in DAT negative and nonsplenectomized) patients for treatment of ITP. Hemolysis is expected when anti-D is used, and hemoglobin levels usually decrease by 0.5!2 g/dL. Mechanism of action Anti-D works by binding to Rhesus D antigen expressed on red blood cells, which leads to their recognition by Fc receptors on cells of the reticuloendothelial system. The antibody-coated red cells slow clearance of antiplatelet antibody!coated platelets. Adverse effects (largely preventable by premedication with high-dose steroids, acetaminophen and diphenhydramine, or other antihistamines) include: 1. Fever and chills. 2. Intravascular hemolysis—this is universal and severe in just over 1 of 1000 cases. The use of high-dose steroid premedication largely prevents this. 3. Headache, vomiting. 4. Anaphylaxis (rare). Lanzkowsky’s Manual of Pediatric Hematology and Oncology Immune thrombocytopenic purpura 263 Splenectomy 1. Splenectomy is rarely indicated in children with ITP because of the increasing number of effective medical therapies that have been developed combined with the favorable natural history of ITP in children. 2. Splenectomy is indicated for severe ITP with acute life-threatening bleeding that is nonresponsive to medical treatment or in patients with chronic ITP with bleeding and/or limitation of a patient’s activities, for example, contact sports (because of potential danger of ICH), and nonresponsive to medical treatment. It is very rarely performed within 1 year of diagnosis and in children ,5 years of age; it is rarely performed in even chronic ITP patients. Splenectomy can restore platelet counts in at least two-thirds of patients. Other modalities should be tried before splenectomy in pediatric patients because of: a. perioperative complications (thrombosis or infection or bleeding), b. long-term risk of infection with encapsulated organisms postsplenectomy (splenectomy should be proceeded by appropriate immunizations that may need repeating at 5!10 year intervals), c. unpredictable response to splenectomy (given the unavailability of autologous indium-labeled platelet scans), d. 1.5-fold increased rate of stroke after splenectomy, and e. unknown late side effects of splenectomy. Rituximab Rituximab is a chimeric human!mouse monoclonal antibody directed against the transmembrane CD20 antigen present on B cells, licensed for the treatment of B-cell non-Hodgkin lymphoma and also rheumatoid arthritis. Currently, there are a number of other anti-CD20 molecules available both humanized rituximab, other anti-CD20s, and generic anti-CD20s. Any that have been tested, for example, veltuzumab appear to have similar efficacy. Rituximab has shown a substantial initial response rate (40!50%) in children with chronic ITP after a four-dose course, but long-term response rates in children after 2 years are 25!30%. A recent randomized controlled study in adults suggested very limited benefit, although there was a substantial reduction of steroid use in the first year after treatment and a longer time until “relapse.” One study in pediatric patient demonstrated that adolescent women with ITP are most likely by far to achieve a “cure” with rituximab, a finding that was also seen in two adult studies. Dosage The standard dosage of Rituximab is 375-mg/m2 IV weekly for 4 weeks. Very limited studies of lower doses in adults have suggested comparable efficacy but shorter duration of response and thus the optimal dose has not yet been determined. Other doses have been explored in ITP, including the standard dose adult dose for rheumatoid arthritis (1000 mg twice separated by a 2-week interval) and maintenance therapy, as utilized in adults with non-Hodgkin lymphoma. There are limited data using this approach in ITP, only 1 preliminary study with 12 adults and 4 children. Mechanism of action Rituximab works by attaching to CD20 antigen on B cells causing: 1. cell lysis via induction of apoptosis, antibody-dependent cellular cytotoxicity, and complement deposition on antibody-coated B cells and 2. antibody-mediated opsonization resulting in phagocytosis of circulating B cells. Adverse effects 1. Fever and chills—common with first infusion only due to antibody-mediated clearance of circulating B cells; one infusion is enough to clear essentially all circulating B cells. 2. Serum sickness—5!10% in children with persistent and chronic ITP; this is more common in children than in adults. 3. Hypogammaglobulinemia—this is seen only if rituximab is combined with high-dose dexamethasone or if the levels are low to begin with. We believe that treatment can be initiated without knowing immunoglobulin levels but that the levels should be sent to allow prevention of hypogammaglobulinemia by administration of Ig if needed. 4. Headache, nausea, and vomiting. 5. Hypotension (rare). 6. Tachycardia (rare) 7. Mucocutaneous reactions, including hives during the first infusion, and rarely Stevens!Johnson syndrome, lichenoid dermatitis, vesiculobullous dermatitis, toxic epidermal necrolysis (all very rare). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 264 12. Disorders of platelets 8. Profound and prolonged B-cell depletion (hepatitis B reactivation has been seen so hepatitis B carriers should not receive rituximab). 9. Progressive multifocal leukoencephalopathy that is activation for the JC virus in a carrier: one case has been reported in an adult with ITP that was .3 years after rituximab treatment. 10. Late-onset neutropenia. 11. Inability to respond to primary antigens (do novo ones whether via a vaccine or an infection) while B cells are absent. 12. Fetal B-cell toxicity if given to a mother while she is pregnant. Thrombopoietic receptor agonists TPO receptor agonists (e.g., eltrombopag and romiplostim), platelet growth factors, have recently been approved for pediatric use in ITP. There is significant experience in the adult ITP population for greater than a decade in the United States, and expanding data about efficacy in children with eltrombopag and romiplostim. The initial randomized controlled phase I/II trial and phase III study of pediatric ITP patients with romiplostim given subcutaneously weekly at doses of 1!10 µg/kg showed responses in 72!88% children with chronic ITP. Higher doses, for example, 7!10 µg/kg tended to be needed. In a recent study the clear majority of patients treated with romiplostim had platelet counts .50,000/µL and 75!90% of the time, 23% (15 patients) were successfully able to discontinue romiplostim and nonetheless maintain very good platelet counts without bleeding. For eltrombopag, an orally administered medication, there were two randomized placebo-controlled trials (PETIT and PETIT2). In the PETIT study a single count .50,000/µL in the first 6 weeks was considered a response and was seen in 64% of treated patients compared to 32% of placebo patients. In the second trial PETIT2 with 92 children with chronic ITP, durable platelet count $50,000/µL was seen more frequently with eltrombopag (40% vs 3% with placebo). During the open-label extension study, 81% of patients in each of PETIT and PETIT2 achieved a platelet count $50,000/µL. Eltrombopag package insert recommends a starting dose of 50 mg/day with dose modifications of 25mg increment for age $6 years, while the starting dose for children #5 years is 25 mg. In pediatric trials the most common adverse events were headache, upper respiratory infection, and gastrointestinal complaints (mild). Thrombosis, bone marrow fibrosis, and risk of myelodysplasia are concerns extrapolated from the adult population but rarely seen in children. Eltrombopag has additional monitoring for liver toxicity and cataracts. In pediatric patients the issues of thromboembolic events are very uncommon and often associated with additional risk factors. There is considerable information on long-term follow-up with romiplostim while there is very little such information with eltrombopag. Furthermore with eltrombopag, there are confusing features with iron status: longer term eltrombopag usage leads to iron deficiency and eltrombopag works better in the setting of iron deficiency. Drugs that may be effective but are infrequently to rarely used in pediatric immune thrombocytopenic purpura 1. Danazol—if given before puberty, will shorten final height; in adolescent females will abrogate menses. Leads to acne and hirsutism, aggressive behaviors, and has liver toxicity. 2. Dapsone—used more commonly in underdeveloped countries because of cost considerations. Recommended to test for G6PD deficiency prior to use to avoid aggravated hemolysis and anemia. Mechanism of effect remains unclear. Good treatment for patient of average severity. 3. Azathioprine (or 6MP)—used in ITP for .40 years, can be combined with danazol. Liver test monitoring required. Best administered at bedtime. 4. Cyclophosphamide—seemingly more effective IV than PO, often last resort because of multiple toxicities. Hydration required to prevent bladder fibrosis. 5. Vinca alkaloids (vincristine, vinblastine)—efficacy usually short term. Neurotoxicity more pronounced with vincristine; cytopenias with vinblastine. Ideally give as prolonged infusion. 6. Cyclosporine A—relatively effective but may not be curative; levels need to be monitored and targeted to trough level of 100!200 ng/mL (below transplant levels to minimize toxicity especially renal and neurologic). 7. Mycophenolate mofetil—in class with azathioprine and cyclosporine; effective in Evans syndrome. 8. Sirolimus (rapamycin) is of uncertain use in isolated thrombocytopenia but appears more useful in combined cytopenias such as Evans syndrome where it currently is the treatment of choice. Plasmapheresis Plasmapheresis may rarely be useful to accelerate the effect of other therapies by the removal of previously synthesized platelet antibodies when treatment has been given to prevent the synthesis of additional antiplatelet antibody. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Other causes of thrombocytopenia 265 Platelet transfusions Platelet transfusions may be transiently effective and required for rare emergency situations, including ICH, internal bleeding, and emergency surgery. Some short-term hemostatic activity is usually obtained. If necessary, a single-donor unit platelet bolus given over 1 hour followed immediately by a continuous infusion initially of 1 unit over 4 hours. Emergency therapy Patients with profound mucosal bleeding or internal bleeding or ICH require immediate therapy. Combination therapy is optimal: 1. IV methylprednisolone 30 mg/kg per day for 1!3 days. 2. IVIG 1 g/kg per day for 2!3 days with or without. 3. Anti-D 75 µg/kg (one dose). 4. Vincristine 0.03 mg/kg 3 1. 5. Romiplostim 10 µg/kg 3 1 subcutaneous. 6. Rituximab 375 mg/m2. 7. Platelet transfusion (bolus followed by continuous infusion). 8. Recombinant human factor VIIa (rFVIIa). 9. Emergency splenectomy in urgent, life-threatening bleeding is not usually recommended but can be pursued. Chronic immune thrombocytopenic purpura Chronic ITP is currently defined as thrombocytopenia that persists beyond 12 months from diagnosis. Patients with ITP of 3!12 months’ duration are defined as persistent disease. It is more common in older children, those with autoimmune disease, and those with altered immunity or other causes of an underlying disease. Other causes of thrombocytopenia HIV-associated thrombocytopenia Thrombocytopenia is common in patients with HIV not on antiretroviral therapy and can be an early finding in the disease process. Currently, it is reported primarily in underdeveloped countries without wide availability of highly active antiretroviral therapy (HAART). HIV-associated thrombocytopenia should be considered in a child with known HIV or a patient who presents with thrombocytopenia in conjunction with a compatible family or transfusion history and on physical examination has axillary or inguinal lymphadenopathy. The thrombocytopenia in this group of patients is multifactorial but includes immune-mediated thrombocytopenia, sequestration, and ineffective thrombopoiesis. The antibodies are most commonly to GPIIb/IIIa, as in ITP without HIV, but have been shown to often be directed at a specific peptide of GPIIIa (amino acids 49!66) leading to platelet intravascular lysis. The virus may also invade megakaryocytes and precursors, leading to reduced thrombopoiesis. Aggressive treatment of the underlying HIV infection with HAART has almost eliminated HIV-related thrombocytopenia as a clinical problem. Treatments used in non-HIV ITP are often effective. Steroids, however, should be avoided, as they may contribute to the development of secondary infections although high-dose 4-day dexamethasone may be effective. Additionally, specific attention needs to be paid to consideration of non-HIV-related thrombocytopoenia such as HCV coinfection or drugrelated thrombocytopenia specifically with trimethoprim!sulfamethoxazole if used for P. jirovecii prophylaxis. Drug-induced thrombocytopenia Many drugs have been proven or suspected to induce drug-dependent antibody-mediated immune thrombocytopenia. http://www.ouhsc.edu/platelets/DITP.html is a current reference source containing a list of drugs that have been implicated in causing thrombocytopenia. Almost any drug can, but very few frequently do, cause thrombocytopenia. The most common ones, in addition to heparin that is discussed separately later, include valproic acid, Dilantin, and vancomycin. Selective serotonin reuptake inhibitor drugs used as antidepressants may affect platelet function, thereby leading to bleeding symptoms without thrombocytopenia. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 266 12. Disorders of platelets Heparin-induced thrombocytopenia Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening cause of thrombocytopenia, more common in adults than children. HIT is an immune complication of exposure to unfractionated heparin or, less frequently, low-molecular-weight heparin. HIT is caused by antibodies directed against complexes of heparin and platelet factor 4. The antibodies attached to the complex bind to and activate FcγRIIa on the platelet surface and thus cause platelet activation. This in turn creates an increased risk for both venous and arterial thromboses. HIT is a challenging clinical diagnosis because of the combination of the frequency of heparin use and thrombocytopenia in hospitalized patients and the low incidence in pediatrics. HIT occurs at a rate of 1!2% in pediatric patients who are in a pediatric ICU or who have recently undergone cardiac surgery. Features of HIT include (four Ts): 1. Thrombocytopenia: platelet count drops below 150,000/µL or 30!50% drop from baseline. Moderate thrombocytopenia with mean platelet counts 50!70,000/µL. 2. Timing: onset of thrombocytopenia usually 5!10 days following first heparin exposure; may be 3!5 days, for example, if sensitization occurred previously, that is, at cardiac catheterization. 3. Thrombosis: venous or arterial thrombosis. Less commonly, skin necrosis at subcutaneous heparin injection sites. 4. Other: the absence of alternative explanation for thrombocytopenia. To confirm diagnosis, in the setting of high clinical probability, heparin-dependent antibodies are screened for by immunologic testing for antibodies against heparin-PF4 by ELISA. This is sensitive but not always specific so, if required, confirmation can be obtained by functional antibody testing using control platelets with the serotonin release assay demonstrating platelet activation. When a child is receiving heparin, no matter how small the dose (e.g., to keep a catheter patent or in total parenteral nutrition), a watchful eye should be kept on the daily platelet count. When HIT is suspected to be a likely cause of thrombocytopenia, heparin must be discontinued immediately and alternative, immediately active (not warfarin) anticoagulation initiated (see Chapter 13, Disorders of Coagulation). A separate entity called nonimmune heparin-associated thrombocytopenia can also occur. Its exact pathophysiology is not known but may be mediated by platelet clumping. It is mild and occurs on the first day of heparin administration. Similarly, recently there is recently described a form of HIT in which the endogenous glycosaminoglycans substitute for heparin is implicated in causing the syndrome. Recent studies have also demonstrated the role of inflammation mediated by neutrophils and/or monocytes and the possible role of downstream endothelial damage and activation of the HIT complex. These two descriptions have been only in adults thus far but provide a rationale for consideration of the role of antiinflammatory treatment in addition to anticoagulation for optimal immediate cessation of symptoms. Thrombotic microangiopathies Thrombotic microangiopathies (TMAs) are an infrequent, related group of syndromes that are more common in adults. They have overlapping clinical features of microangiopathic hemolytic anemia (see Chapter 8, Extracorpuscular Hemolytic Anemia), thrombocytopenia, and organ injury. Pathologic features of TMA include vascular endothelial damage with microthrombi of the arterial and capillary vessels, leading to a final common pathway of end-organ damage and tissue ischemia. Thrombotic thrombocytopenic purpura TTP is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic symptoms, renal impairment, fever, and elevated LDH. Both the acquired and congenital forms are a consequence of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats) deficiency-mediated TMA. ADAMTS13 is a protein responsible for cleaving unusually large multimers of vWF into biologically less active multimers. The absence of this protein, either due to antibody binding (acquired) or decreased production (congenital), results in an increase in ultralarge multimers that cause “spontaneous” platelet adhesion and aggregation. A recent study has suggested that there is an “active” (open) and “inactive” (closed) conformation of ADAMTS13 that is related to enzyme activity; this may explain any discrepancy between ADAMTS13 level and its activity. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Other causes of thrombocytopenia 267 Clinical features The spectrum of systemic involvement is heterogeneous in severity and varies from patient to patient and from time to time and may be acute, chronic, or relapsing. Clinical features include fever, bleeding, pallor, jaundice, malaise, nausea, vomiting, abdominal pain, chest pain, seizures, fluctuating neurologic signs and symptoms, progressive renal failure (may occur in 25% of chronic patients). Laboratory features 1. Microangiopathic hemolytic anemia (blood smear reveals polychromasia, basophilic stippling, schistocytes, microspherocytes, and nucleated red blood cells). 2. DAT negative. 3. Thrombocytopenia (often more severe than the degree of hemorrhage). 4. DIC (prolonged PT/PTT, elevated D-dimer, and hypofibrinogenemia). 5. Haptoglobin level reduced. 6. Hemoglobinuria and hemosiderinuria usually present. 7. Unconjugated bilirubin increased. 8. Lactate dehydrogenase levels very increased. 9. Elevated BUN/creatinine. Acquired thrombotic thrombocytopenic purpura In the majority of cases of acquired TTP, there is an antibody-mediated inhibition of ADAMTS13 activity. Incidence is 4!11 cases per million with additional risk factors that include female sex, African!American descent, history of autoimmune disease, pregnancy, or infection. One-third of patients successfully treated relapse, and the mortality is approximately 10!20%. The disease is far more prevalent in adults compared to children who are typically adolescents. Diagnostic evaluation CBC, reticulocyte count, blood smear, BUN/Cr, bilirubin, LDH, and direct antiglobulin test (DAT; and direct Coombs). A diagnosis is suspected in the setting of microangiopathic anemia (based on smear review) and thrombocytopenia and confirmatory testing with an ADAMTS13 assay. The diagnosis is made clinically and treatment should not await the results of an ADAMTS13 assay that involves identification of severe ADAMTS13 activity ,10% and documentation of the presence of inhibitory anti-ADAMTS13 IgG autoantibodies. There are additional patients who present with a clinical picture of acquired TTP, who do not demonstrate ADAMTS13 deficiency who may nonetheless benefit from TTP-directed therapy. A clinical scoring system, the PLASMIC score, uses platelet count, hemolytic anemia, macrocytosis, coagulopathy, renal failure, history of organ transplantation or malignancy to identify patient with higher likelihood of a low ADAMTS-13 who should be managed as presumptive TTP patients. Treatment Management of acquired TTP is in flux. A number of years ago it was exchange plasmapheresis combined with high-dose steroids. Plasmapheresis provides a 50!80% remission rate by the combination of removing antibodies to ADAMTS13 and the donor plasma replacing the missing enzyme. Given the mortality and possibility of stroke, there is urgency to begin plasma exchange and steroids prior to confirmation of diagnosis. Platelet transfusion is not generally recommended in TTP as it has the potential to worsen the consumptive coagulopathy but can be used in emergent situations. Current management includes rituximab. Rituximab has been traditionally added upfront and in refractory cases to target ADAMTS13 antibody production. There is benefit both in administration to refractory patients and in early administration to reduce the number of plasmaphereses required, morbidity, and potentially rates of relapse. Rituximab is administered at 375 mg/m2 weekly for 2!8 weeks, although the schedule will vary with plasma exchange schedule and clinical response. Ideally, it is not given within 48 hours of plasmapheresis but this may be very difficult especially within the first infusion. The great majority of patients respond to serial plasma exchange with high-dose steroids with the addition of rituximab, but refractory or relapsing patents may require additional therapies. In patients who relapse, it could be further rounds of steroids, rituximab, and plasma exchange as needed. But in refractory patients, adjuvant therapies include caplacizumab and other immunosuppressive agents. Caplacizumab is a humanized monoclonal antibody fragment that inhibits the platelet interaction with vWF and was approved for management of TTP in Lanzkowsky’s Manual of Pediatric Hematology and Oncology 268 12. Disorders of platelets 2019. There are limited case reports in pediatric patients, but adult data from double-blind, controlled trials demonstrated a shorter time to platelet count normalization and decreased composite outcome (TTP-related death, recurrence of TTP, refractory TTP, normalization of organ damage markers), although the main side effect of mucocutaneous bleeding should be noted. Immunosuppressive agents like cyclosporine, cyclophosphamide, vincristine, mycophenolate mofetil, and azathioprine have been used, but no systematic evaluation of these agents has been performed nor is it clear when to add these agents. Congenital thrombotic thrombocytopenic purpura (Upshaw!Shulman syndrome) Congenital TTP occurs secondary to a genetic mutation in the gene for ADAMTS13 on chromosome 9q34. This is a rare disorder and occurs much less frequently than the acquired form. Patients typically present with neonatal jaundice and thrombocytopenia. Some patients will not have episodes of overt TTP until late childhood or adulthood and then only following an environmental trigger (i.e., pregnancy or infection). Patients with congenital TTP have low levels of ADAMTS13 activity, but no antibodies. Because there are no circulating antibodies to ADAMTS13 and a small amount of the enzyme is needed for function; plasma infusion is the mainstay of therapy. Many patients require plasma therapy only with symptomatic episodes, while others are maintained on a prophylactic schedule administered at 2- to 3-week intervals. An additional therapeutic option for patients with severe reactions to plasma is administration of plasma-derived FVIII concentrate that contains ADAMTS13 or recombinant ADAMTS-13 concentrate that is in phase III clinical trials for congenital TTP. Studies of FEIBA had some preliminary efficacy but were not completed. Hemolytic!uremic syndrome Hemolytic!uremic syndrome (HUS) is the clinical triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. HUS is often associated ( .90%) with the production of Shiga-like toxin from E. coli 0157:H7 or Shigella dysenteriae 1. These Shiga toxin!producing bacteria are common in cattle and outbreaks often occur secondary to contaminated beef, water, or vegetables. The bacteria first bind to the gut endothelium and penetrate the lining so that Shiga toxin can enter the bloodstream. The Shiga toxin then binds to CD77 (global ceramide 3) on endothelial cells and renal mesangial cells resulting in apoptosis and a proinflammatory state, enhancing the secretion of vWF and creating a thrombogenic microenvironment. Clinical presentation initially includes abdominal pain and classically bloody diarrhea; the latter is from the penetration of the gut wall by the Shiga toxin plasmid!containing organism, for example, E. coli. This is followed by several days of apparent improvement, including cessation of bloody diarrhea and then development of symptomatic anemia, thrombocytopenia, and renal failure. HUS treatment consists of supportive care (e.g., dialysis that can be peritoneal and antihypertensive medication). Red cell transfusions are commonly needed, but platelet transfusions should be used only when necessary, for example, for placement of a catheter for peritoneal dialysis. HUS is a common cause of renal failure in children and approximately 50% of children who develop the disorder will require dialysis, but fortunately, it is rare that a patient does not recover and develops end-stage renal disease (,5%). Despite the severe thrombocytopenia, bleeding is very rare. Whether IVIG has any use, that is, with anti-Shiga-like toxin remains to be seen; likely too late once renal failure ensues. Complement-mediated hemolytic-uremic syndrome Complement-mediated HUS (also previously referred to as atypical HUS or aHUS) is TMA mediated by dysregulation of the alternative complement pathway. The alternative complement pathway is a constitutively active part of the innate immune system with unregulated activation from a mutation in a critical component. This results in deposition of C3b in tissue leading to increased formation of the C5b-9 membrane attack complex. Ineffective, or inhibition of, regulatory proteins of this alternative pathway through genetic mutations or antibody inhibition have been identified in 60!70% of aHUS patients. Genetic mutations have been identified in complement factor H, membrane cofactor protein, complement factor I, C3, complement factor B, thrombomodulin, and complement factor H-related proteins. The clinical presentation of aHUS is similar to that other TMA syndromes described with microangiopathic anemia, thrombocytopenia, and predominance of acute kidney injury and hypertension. Suspicion of aHUS, other than by laboratory findings indicated below, is based on either recurrence or the known occurrence of HUS in family Lanzkowsky’s Manual of Pediatric Hematology and Oncology Other causes of thrombocytopenia 269 members or culture not revealing a plasmaid containing E. coli in the stool. Central Nervous System (CNS) manifestations may occur in 10% of patients with seizures, diplopia, irritability, cortical blindness, hemiparesis, or hemiplegia. Similar to HUS, diarrheal symptoms may proceed clinical diagnosis in 24% of cases. Additional triggers may include respiratory illness or pregnancy. Given the limited ability to test the complement system inhibition at the time of presentation, this is often a diagnosis of exclusion with identification of normal ADAMTS13 . 5!10%, negative stool studies for Shiga-producing organisms in the setting of elevated serum creatinine and TMA triad (microangiopathic anemia, thrombocytopenia, and elevated LDH). Complement genetic testing can be performed for the known mutations, but measurement of C3, C4, and complements H, I, and B at normal levels does not exclude this diagnosis. Recognition of aHUS is important as there is targeted anticomplement therapy available. Eculizumab is a recombinant humanized monoclonal immunoglobulin targeting C5 to prevent the cleavage of C5!C5a, thereby preventing the downstream formation of the membrane attack complex. This therapy was developed for management of paroxysmal nocturnal hemoglobinuria but has been used in some patients with TTP and aHUS. A side effect of this therapy is increased risk for infection with encapsulated organisms, particularly Neisseria meningitidis. Given the difficulty of diagnosis and the expense of eculizumab, the role as adjuvant to plasma exchange therapy or a first-line therapy in children is still debated. A microangiopathic picture following hematopoietic stem cell transplant is very likely a variant aHUS, typically referred to as transplant associated microangiopathy (TMA). Disseminated intravascular coagulation Thrombocytopenia is seen in several syndromes associated with DIC, including purpura fulminans, overwhelming sepsis, and Kasabach!Merritt syndrome. In an unusual case of thrombocytopenia, PT, PTT, fibrinogen, and fibrin split products or D-dimers should be determined to exclude DIC as the cause of the thrombocytopenia. Autoimmune disorders Thrombocytopenia is often associated with a variety of autoimmune disorders. 1. SLE: thrombocytopenia occurs in 15!25% of patients with SLE. The initial treatment is similar to that of ITP, including steroids, IVIG, and immunosuppressive agents. TPO agents may be effective but should be avoided as they may lead to a higher incidence of thrombosis in SLE. Hydroxychloroquin has been reported to not only manage the SLE but also the thrombocytopenia in SLE ITP. In the absence of data, immunosuppressive treatment, for example, rituximab, is thought to be useful also. TPO agents have been reported in adults to be both safe and effective but there should be increased concern for thrombosis in these patients. 2. Autoimmune lymphoproliferative syndrome (ALPS): this syndrome is characterized by lymphadenopathy, hepatosplenomegaly, hypergammaglobulinemia, and autoimmune cytopenias (i.e., Evans syndrome) and ITP. It is discussed in detail in Chapter 16: Lymphoproliferative Disorders. Ideal treatment is sirolimus (rapamycin) with mycophenolate mofetil (MMF) as a backup; rituximab may lead to hypogammaglobulinemia. 3. Antiphospholipid antibody syndrome: antiphospholipid antibodies enhance platelet activation. These patients have recurrent arterial or venous thrombi. The treatment is anticoagulation; steroids and/or immunosuppressive agents may be used in severe presentations. Thrombocytopenia is often consumptive and responds to anticoagulation. Thrombocytopenia in steady-state patients is often milder and above any treatment threshold. 4. Evans syndrome: Evans syndrome is a combination of at least two of autoimmune hemolytic anemia, thrombocytopenia, and/or neutropenia, classically ITP and AIHA. Patients typically have poor responses to steroids, IVIG, or splenectomy. A combination of these therapies is often required and rituximab and MMF seem to be particularly effective as described earlier under ALPS; Evans syndrome is often associated with ALPS and CVID. 5. Other secondary ITP in autoimmune processes: Hodgkin lymphoma, non-Hodgkin lymphoma, juvenile rheumatoid arthritis, dermatomyositis, Graves’ disease, Hashimoto thyroiditis, myasthenia gravis, inflammatory bowel disease, sarcoidosis, and protein-losing enteropathy may be associated with autoimmune thrombocytopenia. Treatment of the underlying autoimmune disease usually may, but may not, improve the secondary ITP. Hypothyroidism is by far the most common association with ITP and thyroid hormone may need to be replaced with uncertain effects on the platelet count. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 270 12. Disorders of platelets Cyanotic congenital heart disease Thrombocytopenia frequently occurs in children with severe cyanotic congenital heart disease, usually when the hematocrit levels are .65% and when the arterial oxygen saturation is ,65%. This may be due to margination of platelets in the small blood vessels, which may occur in the presence of a high hematocrit level, although it may often be at least partly artifactual. The balance between bleeding and thromboembolism in these children may need to be considered with clinical decision making. Additionally, with right-to-left shunting, there is a proposed mechanism that megakaryocytes may be delivered into arterial circulation bypassing the lungs favoring larger, but fewer platelets and contributing to the thromboembolism that may be seen in congenital cyanotic cardiac patients. Cyanotic congenital heart disease may also be associated with impairment of platelet aggregation by ADP, norepinephrine, and collagen. This impairment is correlated with the severity of hypoxia. Hypersplenism A variety of conditions characterized by splenomegaly are associated with thrombocytopenia, presumably resulting from the sequestration or also destruction of platelets by the enlarged spleen. This is usually associated with neutropenia and anemia but. Thrombocytopenia may be more marked. Megakaryocytes are plentiful in the marrow. Hypersplenism occurs in patients who have splenomegaly, irrespective of cause. Fortunately, the platelet counts are often low but not severely low. There are data for longer term use of eltrombopag in cirrhotic patients (on interferon). Short term, there are data on the use of avatrombopag and lusutrombopag in adults to increase platelet counts for procedures. Treatment with platelet transfusion must be timed exactly to the invasive intervention required. Bleeding associated with other coagulation issues due to impairment of protein synthesis in liver disease is severe. Thrombocytosis Thrombocytosis is defined as a platelet count greater than two standard deviations above the mean or .450,000/µL. The severity of thrombocytosis can be classified into the following groups: 1. Mild (450!700,000/µL) 2. Moderate (700!900,000/µL) 3. Severe (900,000!1 million/µL) 4. Extreme ( . 1 million/µL) Thrombocytosis is relatively common in young children but is usually a transient, benign finding occurring secondary to an underlying infection or inflammatory process. Platelets are an acute-phase reactant and thrombocytosis can be part of the inflammatory response with upregulation of thrombopoietin (TPO receptors) and interleukin-6. Reactive thrombocytosis may also occur secondary to iron deficiency, major trauma, surgery, and postsplenectomy. Table 12.9 lists the conditions associated with thrombocytosis in infants and children. Primary thrombocytosis Primary thrombocytosis is not well characterized in the pediatric population, due to the limited number of identified genetic mutations and small case series of long-term clinical information reported. Essential thrombocythemia Essential thrombocythemia (ET) is a well-characterized myeloproliferative neoplasm (MPN) in adults associated with a mutation in JAK2V617F or relevant JAK!STAT and TPO proliferation pathway genes (cMPL PRV-1 and CALR). It is characterized by a sustained thrombocytosis, hyperplasia of megakaryocytes in the bone marrow in the presence of a pathogenic mutation, and the absence of evidence for reactive thrombocytosis. ET diagnosis in children is rare, occurring in approximately 1/1,000,000 children with identified JAK/STAT/CALR mutations or in the absence of any of these (so-called triple negative). Lanzkowsky’s Manual of Pediatric Hematology and Oncology 271 Thrombocytosis TABLE 12.9 Conditions associated with thrombocytosis in infants and children. Hereditary Asplenia Myeloproliferative disorder in Down syndrome Nutritional Iron deficiency (chronic blood loss) Vitamin E deficiency Megaloblastic anemia Autoimmune diseases or chronic inflammation Kawasaki disease Inflammatory bowel disease Rheumatoid arthritis Henoch!Schönlein purpura Polyarteritis nodosa Myelodysplastic states 5q-syndrome Sideroblastic anemia Traumatic Surgery Fractures Hemorrhage Burns Metabolic Hyperadrenalism Immune Graft-versus-host reaction Nephrotic syndrome Miscellaneous Splenectomy Caffey disease Pulmonary embolism Thrombophlebitis Cerebrovascular accident Sarcoidosis Acute blood loss Hemolytic anemia Exercise Ankylosing spondylitis Spurious Idiopathic Infectious Viral (e.g., CMV) Bacterial Mycobacterial Fungal Drug-induced Vinca alkaloids Citrovorum factor Corticosteroid therapy Epinephrine Neoplastic Chronic myeloid leukemia Polycythemia vera Essential thrombocythemia Histiocytosis Lymphoma (Hodgkin and non-Hodgkin) Carcinoma of colon, lung Hepatoblastoma Wilms’ tumor Neuroblastoma Leukemia Abbreviation: CMV, cytomegalovirus. In adult populations, there is a well-described risk stratification system predicated on the risk of the development of vascular events (arterial and venous thrombosis) and the progression to MDS/AML that stratifies treatment and interventions in ET and MPNs. While the molecular pathogenesis is less well characterized in the pediatric population, the clinical presentation of primary thrombocytosis in children also has limited characterization. In children, there appears to be a more benign course compared to adults, with a decreased risk of thrombosis but it may be that cases reported in adults have already had long runup as children. Diagnosis of ET requires the following criteria (World Health Organization 2008 Criteria): 1. persistent thrombocytosis ( . 450,000/µL); 2. bone marrow biopsy with megakaryocyte proliferation without increased neutrophil granulopoiesis or erythropoiesis; 3. the absence of criteria for PV*, PMF, CML, MDS, or myeloid neoplasm; 4. the presence of JAK2V617F, MPL1515, CALR, or another clonal marker; and 5. no known cause for reactive thrombocytosis. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 272 12. Disorders of platelets Genetic testing may confirm the diagnosis, but the absence of mutations does not rule out ET in a pediatric patients. There are lower reported rates of JAK2V617F mutation, PRV-1 expression, and alternatively an increase in MPL and possibly CALR mutations identified. Hereditary thrombocytosis Familial or hereditary thrombocytosis is reported in families with causative mutations in the TPO (THPO) or TPO receptor (cMPL) gene leading to constitutive activation or upstream regulation leading to increased TPO expression. Mutations in the TPO gene lead to increased translation and subsequent thrombocytosis, whereas the mpl mutation (S505N) leads to constitutive activation of the protein. The course of the disease had seemed to be largely benign with rare thrombosis; however, recently, three case reports have each identified upper limb abnormalities in these cases. Treatment of pediatric primary thrombocytosis Adults are stratified for treatment using risk factors or age, thrombosis history, and cardiovascular risk factor into low-, intermediate-, and high-risk groups. In pediatrics, given the lower risk of thrombosis, there are no clear consensus guidelines for management but the general approach is adding observation for asymptomatic patients, adding antiplatelet agents for lower risk patients with additional thrombophilia risk factors and escalating to cytoreductive therapy in high-risk patients with extreme thrombocytosis or symptoms of thrombosis/bleeding. In these patients the risk of bleeding can be associated with acquired von Willebrand Disease (VWD) and testing for ristocetin cofactor activity can be important prior to starting therapy. Antiplatelet agents 1. Low-dose acetylsalicylic acid (ASA) (risk for Reye syndrome should be considered in pediatric patients.). 2. Additional antiplatelet agents: clopidogrel, prasugrel, dipyridamole, or ibuprofen can be considered. Cytoreductive therapy (platelet-lowering drugs) 1. Hydroxyurea can be used as a daily oral medication to lower platelet count to between 30,000 and 600,000/L. Continued maintenance is necessary to sustain the effect. The use of hydoxyurea (HU) hydri in children with sickle cell disease suggests the risk of induction of malignancy is very low when the marrow cells are intrinsically normal. 2. Pegylated-interferon, has limited data in children, but is generally well tolerated for consideration for upfront therapy based on risk/benefit discussion with patients and families. 3. Anagrelide hydrochloride is effective in adults as a daily oral medication to lower platelet count between 300,000 and 600,000/µL with maintenance therapy necessary to sustain response, but it was inferior to interferon. Furthermore, there is limited experience in children. 4. Possibly the best therapy (limited data in children): ruxolitinib (targeted JAK1/2 inhibitor) is standard treatment in adults with ET. It improves symptoms but does not create a curative effect. Findings in children appear to demonstrate similar efficacy and safety. Qualitative platelet disorders Qualitative platelet disorders result from a variety of congenital or acquired conditions but usually demonstrate a bleeding tendency of variable degrees, including petechiae, purpura, and mucosal bleeding as well as bleeding following surgery or trauma. Various tests may demonstrate abnormalities depending upon the particular condition and the platelet count that may be normal or low. Table 12.10 provides a classification by laboratory finding of congenital platelet function disorders, Table 12.11 lists the laboratory findings in inherited platelet function disorders, and Table 12.12 lists the genetic transmission and recommended treatment for commonly known hereditary disorders of platelet function. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Qualitative platelet disorders TABLE 12.10 273 Classification of congenital platelet function disorders. Defects in platelet!agonist interaction (receptor defects) 1. Selective epinephrine defect 2. Selective collagen defect 3. Selective thromboxane A2 defect 4. Selective ADP defect Defects in platelet!vessel wall interaction (disorders of adhesion) 1. von Willebrand’s disease (deficiency or defect in plasma von Willebrand factor) 2. Platelet-type von Willebrand disease 3. Bernard!Soulier syndrome (deficiency or defect in GP1b) Defects in platelet!platelet interaction (disorders of aggregation) 1. Congenital afibrinogenemia 2. Glanzmann’s thrombasthenia (deficiency or defect in GP11b/111a) Disorders of platelet secretion 1. Storage pool deficiency a. δ-SPD i. Hermansky!Pudlak syndrome ii. Chédiak!Higashi syndrome b. α-Storage pool deficiency (gray platelet syndrome) 2. Abnormalities in arachidonic acid pathway a. Impaired liberation of arachidonic acid b. Cyclooxygenase deficiency c. Thromboxane synthetase deficiency d. Altered nucleotide metabolism e. Glycogen storage disease f. Fructose 1,6, bisphosphatase deficiency 3. Primary secretion defect with normal granule stores and normal thromboxane synthesis a. Defects in calcium mobilization b. Defects in phosphatidylinositol metabolism c. Defects in myosin phosphorylation 4. Disorders of platelet!coagulant protein interaction 5. Defect in factor Va!Xa interaction on platelets Vascular or connective tissue defect 1. Ehlers!Danlos syndrome 2. Pseudoxanthoma elasticum 3. Marfan’s syndrome 4. Osteogenesis imperfecta 5. Hereditary hemorrhagic telangiectasia (Osler!Weber!Rendu disease) Abbreviations: ADP, adenosine diphosphate; δ-SPD, δ-storage pool deficiency. The treatments of these conditions may be unrelated to the specifics of the condition in many cases. There are certain specific treatments that may not work in certain conditions, for example, DDAVP in Glanzmann’s thrombasthenia (GT). There is an increasing likelihood that specific treatments may be developed, for example, gene therapy. However, the overall situation is still relatively primitive in regard to the absence of specific therapy for specific bleeding disorders. Overall, in some of these disorders, hemostasis will be improved by administration of DDAVP, as is used in management of mild hemophilia and von Willebrand disease. Antifibrinolytics are often very useful to prevent or treat minor and moderate bleeding. If a given person in a given situation is not responsive to DDAVP and/or antifibrinolytic, then platelet transfusion is often necessary for hemostasis if there is major bleeding or a risk of major bleeding. Leukodepleted single-donor platelets, HLA matched if available, are preferred in these patients to reduce the risk of platelet allo-sensitization. Isoantibodies Lanzkowsky’s Manual of Pediatric Hematology and Oncology 274 12. Disorders of platelets TABLE 12.11 Laboratory findings in inherited platelet function disorders. Glanzmann’s thrombasthenia Storage pool deficiencya Collagen receptor defect Release defectb Bernard!Soulier syndrome von Willebrand disease Platelet count Normal Normal Normal Normal Decreased Normal Platelet size Normal Microplatelets Normal Normal Giant platelets Normalc Bleeding time Prolonged Variable Variable Variable Prolonged Prolonged Platelet aggregation ADP Absent No second wave Normal No second wave Normal Normal Arachidonic acid Absent Variable Absent Decreased Normal Normal Collagen Absent Decreased Normal Absent Normal Normal Ristocetin Normal (1.5 mg/mL) Normal Normal Normal Absent Decreasedd Ristocetin Absent (0.5 mg/mL) variable Absent Absent Normal Decreasedd Storage nucleotide pool Normal Decreased Normal Normal Normal Normal Others HPA-1a absent; GPIIb and III deficient Dense bodies reduced; ATP/ADP ratio increased GPIa and IIa deficient Cyclooxygenase; thromboxane synthetase or TXA2 receptor deficient GPIb deficiency Decreased FVIII, vWF antigen, and ristocetin cofactor a In Hermansky!Pudlak, Chédiak!Higashi, and Wiskott!Aldrich syndromes. Classically occurs with acetylsalicylic acid, omega-3 ingestion, and other drugs affecting arachidonic acid and prostaglandin pathway. Platelet in type IIB will often be clumped together. d Types I and II, decreased; type IIB increased; type III absent. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; vWF, von Willebrand factor. b c TABLE 12.12 Genetic transmission and recommended treatment for commonly known hereditary disorders of platelet function. Disorder Transmission/ frequency Defect Primary treatment for platelets Alternative treatment Disorders of receptors Glanzmann’s thrombasthenia Autosomal recessive, rare GPIIb/IIIa complex Platelet transfusion rFVIIa in instances of platelet alloimmunization Bernard!Soulier syndrome Autosomal recessive, rare GPIb/V/IX complex Platelet transfusion rFVIIa in instances of platelet alloimmunization Defects in granule content, storage pool deficiency Gray platelet syndrome Rare Absent α-granules (platelets look gray) DDAVP (treatment rarely required) Platelet transfusion for nonresponders Chédiak!Higashi syndrome Autosomal recessive, rare Abnormal granules DDAVP Platelet transfusion for nonresponders Wiskott!Aldrich syndrome X-linked recessive WAS protein. Primarily a quantitative defect, storage pool deficiency may also be present Platelet transfusion Splenectomy TPO-R agonists HSCT Hermansky!Pudlak Autosomal recessive Absent dense granules DDAVP Platelet transfusion Storage pool release defects Impaired secondary wave of aggregation DDAVP Platelet transfusion Variable Abbreviations: HSCT, hematopoietic stem cell transplantation; TPO-R, thrombopoietin receptor; WAS, Wiskott!Aldrich syndrome. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Qualitative platelet disorders 275 against the platelet proteins absent from the patient are common in these disorders especially GT and Bernard!Soulier disease. If this occurs, it can be disastrous since it would render patients refractory to platelet transfusion. Administration of rFVIIa 90 µg/kg can effect hemostasis in some patients not responsive to platelet transfusion but response can be erratic. For nonresponders, even higher doses may be hemostatic. In addition to antifibrinolytic agents, topical treatments and hormonal therapy are useful adjuncts in patients with these disorders. Hormonal treatment is most useful for controlling menometrorrhagia. Defects in platelet receptor!agonist interactions Selective impairments in platelet responsiveness to epinephrine The interaction of platelets with epinephrine (adrenaline), in vitro in aggregation tests, is mediated by α2 -adrenargic receptors and results in several responses, including the exposure of fibrinogen receptors, an increase in intracellular ionized calcium, the inhibition of adenylate cyclase activity, and platelet aggregation. Some patients have impaired aggregation and secretion only to epinephrine, associated with a decrease in the number of platelet α2 -adrenargic receptors, with a history of easy bruising with minimally prolonged bleeding times. On the other hand, platelets in 10% of apparently normal people may fail to aggregate in response to epinephrine. Therefore the clinical significance of this finding is unknown. Selective impairment in platelet responsiveness to collagen Collagen is a substrate for platelet adhesion, binding site for vWF, and agonist for platelet secretion and aggregation. There are two major collagen receptors, glycoprotein VI (GPVI) and α2β1, that mediate these hemostatic interactions. Defects in GPVI structure and signaling have been identified in case reports in congenital deficiency and autoantibody development leading to mild!severe mucocutaneous bleeding symptoms. α2β1 Integrins allow for a firm platelet adhesion to collagen and in the absence of this interaction impaired aggregation and adherence to collagen. Very few patients have been shown to have bleeding due to abnormalities in α2β1. Recently, ibrutinib has been shown to impair platelet aggregation to collagen potentially explaining the infrequent but serious bleeding problems in certain Chronic Lymphocytic Leukemia (CLL) patients. Modified Btk inhibitors have been constructed to not have this defect. Defects in thromboxane A2 Defects in thromboxane A2 (TXA2) formation and TXA2 receptor function have been identified as causes of mild but lifelong bleeding symptoms, similar to the effects of aspirin or nonsteroidal antiinflammatory drug (NSAID) treatment. The use of several weeks of omega 3!enriched fatty acids may create an aspirin like defect (see next). Selective impairment in platelet response to adenosine diphosphate Patients have been identified with defects in P2Y12 (the ADP receptor responsible for macroscopic platelet aggregation), with associated bleeding symptoms. This should be suspected when ADP at high .10 µM concentrations fails to produce full aggregation. This receptor is targeted by clopidogrel and prasugrel. Defects in platelet!vessel wall interaction Bernard!Soulier syndrome BSS is a relatively rare disorder inherited as either an autosomal dominant or recessive. It is characterized by: 1. moderate thrombocytopenia (automated counting often underestimates the true platelet count because of undercounting of very large platelets), 2. prolonged bleeding time, and 3. characteristic very large platelet morphology. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 276 12. Disorders of platelets Platelets are very large, equaling or exceeding the size of a red cell. These platelets contain two to four times the normal protein content, and three times the usual number of dense granules. These dense granules can gather and give the appearance of a pseudonucleus. The findings are more severe in cases of rare homozygotes (or compound heterozygotes) than in the more common heterozygote inheritance. Morphologically, the megakaryocytes have an abnormality in the demarcation membrane system, likely explaining the large platelets and the thrombocytopenia. There can be complete absence of GPIb glycoprotein complex or a point mutation in the GPIb-alpha subunit known as the Bolzano variant; all of which leads to the inability of vWF binding. Platelets fail to agglutinate in response to high-dose ristocetin, despite normal aggregation and secretion in response to ADP, epinephrine, thrombin, and collagen. Because vWF acts as a bridge in adhesion of platelets to exposed subendothelium especially in high shear states, the absence of the vWF receptors prevents normal platelet adhesion and causes a significant degree of bleeding (even in cases of mild thrombocytopenia) with mucocutaneous bleeding that can begin in early infancy, especially severe epistaxis. Confirmation of the diagnosis is based on GP1b!IX!V deficiency by flow cytometry or immunoblotting or genetic diagnosis especially for the Bolzano variant. Type IIB von Willebrand disease and platelet-type (pseudo-von Willebrand) disease Type IIB von Willebrand and platelet-type von Willebrand both cause mucocutaneous bleeding out of proportion to any thrombocytopenia that may be present. Stress-induced worsening of thrombocytopenia is common, such as pregnancy when there are elevated levels of large vWF multimers. Type IIB von Willebrand disease is approximately 10 times more common than platelet-type von Willebrand disease. The management of these conditions is described in detail in Chapter 13: Disorders of Coagulation. Defects in platelet!platelet interaction Glanzmann’s thrombasthenia GT is an autosomal recessive bleeding disorder characterized by the following: 1. Normal platelet count, platelet size, and morphology. 2. Prolonged bleeding time. 3. Mild-to-severe bleeding symptoms, including petechiae looking just like a de novo ITP except for the normal platelet count. 4. Reduced clot retraction. 5. Defective platelet aggregation with all agonists except ristocetin. GT represents a family of disorders of the platelet GPIIb/IIIa receptor complex. As this complex functions in platelet aggregation in low shear states via fibrinogen, vWF, and fibronectin, disruption of this pathway leads to impaired platelet aggregation. GT platelets attach normally to damaged subendothelium but fail to spread normally to form platelet aggregates. Platelets from patients with GT fail to aggregate in response to most physiologic agonists like ADP, thrombin, and epinephrine; they do agglutinate in response to ristocetin, however. The treatment of GT is platelet transfusions; DDAVP is ineffective. Patients may become refractory to transfusions, and activated recombinant human factor VIIa (rFVIIa) may be used upfront to prevent this complication or in refractory cases. In rare refractory cases with antiplatelet antibodies and severe bleeding, hematopoietic stem cell transplantation may be needed for management. Females may require suppression of menses to avoid recurrent heavy bleeding. Disorders of platelet secretion The category of storage pool disease includes patients with deficiencies of dense granules [δ-storage pool deficiency (δ-SPD)], α-granules (α-SPD), or both types of granules (αδ-SPD). δ-Storage pool deficiency The total adenosine triphosphate and ADP platelet granule content in patients with δ-SPD are decreased as are other dense granule contents, including calcium, pyrophosphate, and serotonin. Patients tend to have mild-tomoderate bleeding symptoms. Platelet aggregation studies show an absent second wave of aggregation with Lanzkowsky’s Manual of Pediatric Hematology and Oncology Qualitative platelet disorders 277 ADP and epinephrine. The aggregation response to collagen is impaired or absent. Response to arachidonic acid is variable. Disorders of δ-granules are classically seen in not only Hermansky!Pudlak syndrome (HPS) and Chédiak!Higashi syndrome, but also may be seen in WAS and TAR syndrome. Hermansky!Pudlak syndrome HPS is an autosomal recessive disorder associated with defective lysosome-related organelles. The syndrome includes oculocutaneous albinism with photophobia and rotatory nystagmus, loss of visual acuity, and ceroidlike material accumulation in reticuloendothelial cells. Platelets have platelet dense-body granule storage pool deficiency and the bleeding tendency is usually mild (related to storage pool defect and not thrombocytopenia, which is not a feature of the syndrome). Patients generally present in childhood with mucosal bleeding symptoms, or prolonged bleeding after surgical procedures or dental extraction. HPS is a group of autosomal recessive disorders (HPS 1!10). The most common and severe form is HPS 1, which is a defect on chromosome 10q23. While rare worldwide, it is more common in northwest Puerto Rico, where 1 in 1800 individuals are affected. In this disorder, platelets contain very low levels of serotonin, adenine nucleotides, and calcium. The most serious complication and most common cause of death is pulmonary fibrosis, which may be due to ceroid-like lipofuscin deposits in the lungs. Granulomatous colitis is also seen in this variant; conversely pulmonary fibrosis does not occur in HPS 3 even though granulomatous colitis does. HPS has been reported to be associated with HLH. Treatment is DDAVP for bleeding and/or platelet transfusion if necessary with antifibrinolytics. Chédiak!Higashi syndrome This rare autosomal recessive syndrome includes partial oculocutaneous albinism, recurrent infections, mild coagulation defects, and progressive neurologic dysfunction. Mutations in lysosomal trafficking regulator on chromosome 1 are the cause of this disease. Leukocytes, lymphocytes, monocytes, and platelets have large peroxidase-positive intracytoplasmic granules. Severe immunologic deficiency with abnormal chemotaxis and NK function leads to HLH and death. Patients also develop lymphoproliferative infiltration of bone marrow and reticuloendothelial system in .85% of affected individuals. DDAVP is used for bleeding and platelet transfusions are indicated for nonresponders. Stem cell transplantation may cure patients of immune and hemostatic defects but does not improve the neurologic dysfunction. α-Granule storage pool deficiency (gray platelet syndrome) The term “gray platelet” describes the morphological appearance of platelets in Romanowsky-stained blood smears prepared from patients with a deficiency of α-granules. Electron microscopy reveals the virtual absence of α-granules and platelets appear gray because they are devoid of “purplish” cytoplasmic granulation on blood smear. Platelets from these patients contain absent or markedly reduced α-granule proteins, including PF4, vWF, fibronectin, and factor V. Affected patients have mild thrombocytopenia with a platelet count of around 100,000/ µL, prolonged bleeding times, and a lifelong bleeding diathesis. These patients tend to develop myelofibrosis due to an inability of megakaryocytes to store newly synthesized, platelet-derived granule proteins such as TGF-beta and PDGF-alpha, which “leak out” in the marrow. The most consistent laboratory abnormality has been impairment in thrombin-mediated aggregation and secretion. Aggregation responses to collagen and ADP are variable. Autosomal recessive, autosomal dominant, and often X-linked (GATA1 mutation) inheritance has been observed with the gray platelet syndrome gene mapping to chromosome 3p21. Additional observations have noted these patients often have mild β-thalassemia in the X-linked form. DDAVP is used if bleeding occurs and platelet transfusions are indicated for nonresponders. Quebec platelet disorder (platelet factor V Quebec) Quebec syndrome is an autosomal dominant inherited disorder with mild thrombocytopenia. However, it is associated with a significant degree of bleeding, including mucosal and joint bleeding often with a delayed presentation 12!24 hours from time of injury. In this disorder, there is an overproduction of the proteolytic enzyme urokinase-type plasminogen activator (uPA). This enzyme overexpression occurs in megakaryocytes and leads to degradation of several proteins, including factor V. There is a platelet aggregation deficiency, in the setting of epinephrine exposure in particular, for unclear reasons. The disorder is diagnosed by examination of platelet uPA levels and α-granule fibrinogen degradation products. Genetic analysis has shown random duplication of Lanzkowsky’s Manual of Pediatric Hematology and Oncology 278 12. Disorders of platelets the uPA gene. The treatment of choice is antifibrinolytic agents. Patients do not respond adequately to platelet transfusions. Arthrogryposis!renal dysfunction!cholestasis Arthrogryposis!renal dysfunction!cholestasis syndrome is a multisystem disorder that includes platelet dysfunction and low α-granule content associated with impaired vacuolar protein sorting 33homologue B (VPS33B) gene and IPAR encodes for a VPS33B interactor protein that regulates apico-basolateral polarity, which can result in a bleeding diathesis. Miscellaneous Platelet function abnormalities have been reported in WAS, TAR syndrome, hexokinase deficiency, and glucose-6-phosphate deficiency. Impaired liberation of arachidonic acid pathways A major response of platelets during activation is the release of arachidonic acid from membrane-bound phospholipids and its subsequent oxygenation to TXA2 . TXA 2 forms an important positive feedback that enhances platelet activation and vasoconstrictions. Ingestion of omega-3 fatty acids in sufficient quantity over a several-week period can reduce arachidonic release by replacing the natural omega-4 fatty acids in the platelet membrane with omega-3. Cyclooxygenase and thromboxane synthetase deficiency Defects in TXA2 due to deficiencies of cyclooxygenase and thromboxane synthetase have been reported. Platelet intracellular signaling defects Kindlin-3 (leukocyte adhesion defect III) Kindlin-3 is a combination of mild leukocyte adhesion deficiency and platelet dysfunction. The intracellular signaling molecules, kindlin-2 and -3, are important in the activation of integrin αIIBβ3. All patients identified with this disease have been shown to have mutations in the cytoskeleton linking protein kindlin-3 (FERMTS3) gene. The presentation is mucocutaneous bleeding symptoms and predisposition to infections without pus formation and with delayed wound healing, delayed umbilical stump separation, and variable osteopetrosis. Cure can only be achieved by stem cell transplantation. Dysregulated calcium signaling Calcium is an essential signaling molecule involved in platelet activation; several patients have been reported with defective calcium mobilization. Deficiency of platelet procoagulant activity Scott syndrome Scott syndrome is a rare autosomal recessive disorder involving mucosal and postsurgical bleeding. The disorder arises from impaired phospholipid reorganization with defects in translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. In turn, this results in impaired binding of coagulation proteins and subsequent decrease in thrombin generation and fibrin formation. In these patients, bleeding time, platelet aggregation, and secretion studies are normal. Mutations in TMEM16F, a protein associated with membrane scramblase activity, have been described in at least three patients. Isolated defect in membrane vesiculation Patients in four families have been reported with lifelong bleeding disorders who have impaired microvesicle secretion. Lanzkowsky’s Manual of Pediatric Hematology and Oncology Qualitative platelet disorders 279 Acquired qualitative platelet disorders Table 12.13 lists the acquired disorders causing defective platelet function. Medications ASA (or aspirin) impairs platelet aggregation usually attributed to inhibition of cyclooxygenase-1 and subsequent reduction in TXA2 production, although other mechanisms have been implicated. A dose of 81 mg of aspirin daily can permanently affect platelets in circulation, such that the length of drug effect is related to platelet life span, not drug half-life. Other NSAIDs impair TXA2 production as well but the effects are limited to drug half-life. Platelet dysfunction caused by aspirin is important in several clinical settings: 1. Excessive posttonsillectomy bleeding after ASA ingestion. 2. Development of purpura in children after ingesting aspirin. 3. Prolonged bleeding time in children with suspected hemostatic defect. 4. Misdiagnosing children with mild von Willebrand disease because of ASA/NSAID ingestion prior to testing. Platelet aggregation may be impaired in the newborn following maternal drug therapy. For this reason a history of drug ingestion is an essential part of the investigation of hemorrhagic states in the newborn period. Renal failure A generalized hemorrhagic state is known to occur in advanced renal failure. Thrombocytopenia is present in a minority of patients, and reduced platelet adhesiveness to glass and defective platelet factor 3 occurs with platelet dysfunction being the primary hemostatic problem. Treatment with estrogen and DDAVP can be useful but the former may take a week to have its effect and reversal of renal failure with dialysis may be required to control bleeding. TABLE 12.13 Acquired disorders causing defective platelet function. Vascular or connective tissue defects 1. Scurvy 2. Amyloidosis Adhesion defects 1. Acquired von Willebrand disease, for example, high-flow cardiac lesion/Wilms’ tumor 2. Renal failure 3. Drugs: dipyridamole Platelet aggregation defects 1. Fibrin or fibrinogen split products: DIC, liver disease 2. Macromolecules: paraproteins, dextran 3. Drugs: penicillin, semisynthetic penicillins, cephalosporins Release reaction defects 1. Storage pool deficiency a. ~ -Granules: cardiopulmonary bypass b. Dense granules: ITP, SLE c. Drugs: reserpine, tricyclic antidepressants, phenothiazines 2. Defective release a. Platelet dyspoiesis: myelodysplastic syndromes, acute leukemias, myeloproliferative syndromes b. Drugs: aspirin, other nonsteroidal antiinflammatory agents, furosemide, nitrofuradentin c. Ethanol 3. Altered nucleotide metabolism a. Drugs: phosphodiesterase inhibitors or stimulators of adenylyl cyclase Other defects 1. Drugs: heparin, sympathetic blockers, clofibrate, antihistamines 2. Infection: viral 3. Hypothyroidism Abbreviations: DIC, disseminated intravascular coagulation; ITP, idiopathic thrombocytopenic purpura; SLE, systemic lupus erythematosus. Lanzkowsky’s Manual of Pediatric Hematology and Oncology 280 12. Disorders of platelets Liver disease In addition to a deficiency of coagulation factors of the prothrombin complex and others in liver disease, an abnormality of platelet function has been described. Platelet aggregation by ADP and thrombin is significantly impaired in patients with cirrhosis and prolonged bleeding time. This is due to known inhibition of platelet function by fibrinogen degradation products, resulting from excessive fibrinolysis seen in advanced liver disease. Management of defects in platelet function The management of defects of platelet function may include: 1. Removing any exogenous cause of platelet dysfunction (e.g., drugs). 2. Treating the underlying disorder. 3. Using platelet transfusions for hemorrhagic episodes or surgery. 4. DDAVP or cryoprecipitate—this may shorten bleeding time in some patients with: a. renal failure, b. inherited or acquired defects in release reaction, and c. inherited or acquired von Willebrand disease. 5. Antifibrinolytic therapy—EACA (ε-aminocaproic acid) in a dose of 50!100 mg/kg orally or IV every 6 hours (tranexamic acid, if formulation available)—this may have some benefit in mucosal hemorrhage; there is no direct platelet effect. 6. Activated recombinant factor VIIa (rFVIIa). Inherited vascular and connective tissue disorders Disorders of the vascular system and connective tissues do not generally lead to clotting or platelet abnormalities; bleeding is instead caused by the fragility of the connective tissues of the skin, subcutaneous tissues, and vessel wall. Ehlers!Danlos syndrome Ehlers!Danlos syndrome (EDS) is a heterogeneous group of disorders of connective tissue characterized by skin hyperextensibility, delayed wound healing, joint hypermobility, bleeding tendency, and connective tissue fragility. Patients may present with easy bruising, bleeding from the gums after dental extraction, and prolonged menstruation. This condition may be associated with reduced aggregation with ADP, epinephrine, and collagen. Epistaxis, petechiae, hematuria, hemoptysis, and hemarthrosis are usually not seen. There are many subtypes of EDS. Vascular!type EDS is an autosomal dominant disorder with a defect in type III collagen. Molecular testing identifies a mutation in the COL3A1 gene with a generalized vascular fragility that may manifest as arterial rupture and sudden death. Type IV EDS carries the worst prognosis due to the vascular complications. Pseudoxanthoma elasticum Pseudoxanthoma elasticum is a rare disorder resulting in mineralization of elastic tissues in the skin, eyes, and blood vessels, and less frequently in other areas such as the digestive tract. It is inherited in an autosomal dominant fashion with mutations in ABCC6. Clinical presentation includes yellowish bumps called papules on the necks, underarms
