BONE MARROW EXAMINATION Presentor- Dr. Manju kumari Moderator- Dr. A.S Nigam BONE MARROW Bone marrow examination is an indispensable adjunct to the study of diseases of the blood and may be the only way in which a correct diagnosis can be made. HISTORY 1905 Pianese Trephine bx in an infant with Leishmaniasis. 1909 Pianese Tibia & femur marrow aspiration with attached syringe. 1923 Seyfaith Surgical trephine to obtain marrow from ribs & sternum.But there was excessive bleeding. 1927 Airinkin Eliminated trephine complication by using short lumbar needle. 1945 Vanderberg First obtained marrow from iliac creast. 1952 Bierman Used posterior Iliac creast for biopsy 1. Bone marrow aspiration 2. Clot section 3. Bone marrow biopsy 4. Biopsy imprint smears INDICATIONS OF BONE MARROW ASPIRATION • Red cell disorders • Leucocytic disorders • Megakaryocyte and platelet disorders • Myeloproliferative disorders and myelodysblastic • • • • • syndrome with BMB Paraproteinemias Infection Storage disorders Iron assessment Metastasis Unexplained heptosplenomegaly Marrow harvesting for transplant. INDICATIONS OF BONE MARROW BIOPSY 1.To accurately assess marrow cellularity. 2. To diagnose Aplastic anaemia Hypoplastic myelodysplastic syndrome. Hypoplastic leukemia. 3. Lymphoproliferative disorders Hairy cell leukemia CLL 4. Myeloproliferative disorders 5. Unexplained leukoerythroblastic picture. 6. In suspected cases of multiple myeloma and serum paraproteins. 7. Diagnosis and staging of Non hodgkin’s lymphoma Hodgkin’s lymphoma Malignancy Metastatic carcinoma Small round cell tumors of childhood 8. Stromal changes Fibrosis Necrosis Gelatinous marrow transformation 9. Pyrexia of unknown origin 10. Focal lesions –Metastasis, Granuloma 11.Amyloidosis 12.Metabolic bone diseases 13.To assess the mineralisation front and appositional growth after tetracycline labeling CONTRADICATIONS Sternal aspirate-osteoporosis and children Biopsy in coagulopathies (For aspiration factor replacement therapy prior to procedure and observation should be done for next 24-48 hrs.) COMPLICATIONS Hemorrhage (Risk factors- coagulopathies, myeloproliferative disorders, aspirin and warfarin therapy, thrombocytopenia, DIC, liver disease and disease) Pain Infection Perforation of major vessels Risk of general anaesthesia and sedation. BONE MARROW ASPIRATE V/S BIOPSY Aspiration biopsy and trephine biopsy are complementary to each other. Better cytological detail. More range for cytochemical stains, flowcytometry and IHC. Ideal for cytogenetics and molecular genetics. Topographical details, cellularity and infiltration. Less range. Can be used for both. Dry tap in fibrosis Can be performed alone in iron deficiency anemia, anemia of chronic disease , megaloblastic anemia and acute leukaemia. Less painful Essential for diagnosis in dry tap. Helpful for aplastic hypoplastic anaemia, lymphoma, metastatic carcinoma, myeloproliferative neoplasms and diseases of the bones. More painful IMPORTANCE OF TOUCH/ IMPRINT SMEAR Gives cytological details when aspirate is not obtained. Shows more neoplastic cells than aspirate. Can show marrow infiltration , not seen in aspirate IMPORTANCE OF CLOT SECTION Assessment of bone marrow cellularity. For detecting granuloma and tumour infiltrates complementary to biopsy. No decalcification associated nucleic acid or protein damage. SITE FOR ASPIRATION 1. Posterior superior iliac spine- most preferred. 2. Anterior superior iliac spine. (The iliac spines have the advantage that if no material is aspirated, a trephine biopsy can be performed immediately.) 3. Sternum (abt 1cm above the sternomanubrial angle,to one side of midline). (avoided in babies) 4. Medial aspect of tibia just below tibial tubercle (small babies). 5. Spinous process of vertebrae 1. Posterior superior iliac spine- most preferred. 2. Anterior superior iliac spine. The posterior iliac spine is said to provide samples that are longer and larger, and the aspiration is less uncomfortable for the patient. Needles should be stout and made of hard stainless steel, about 7-8 cm in length, with a well-fitting stilette, and they must be provided with an adjustable guard. 1. Jamshidi needle 2. Islam needle 3. Salah needle 4. Klima needle If larger specimens are needed, trephine needles that have bores of 4-5 mm may be used. Other needles occasionally used for trephine biopsy specimens are a 2-mm bore “microtrephine” needle and a Vim–Silverman needle. However, compared with other needles, these yield smaller specimens of marrow that are prone to fracturing. JAMSHIDI NEEDLE a: STYLET b : BORE c : PROBE The tip gradually widens for next 2cm and then uniform bore the needle.The marrow biopsy does not get compressed because of narrow cutting edges and hence cell morhology is well made out. ISLAM A modified version of the Islam needle has multiple holes in the distal NEEDLE portion of the shaft in addition to the opening at the tip to overcome sampling error when the marrow is not uniformly involved in a pathological lesion. A.Salah needle B. Klima needle These are most common reusable needles. PROCEDURE Consent- an written consent should be taken from patient. • An appropriate clinical history should accompany the bone marrow, as they relate to possible findings within the bone marrow examination. It is useful to know relevant laboratory data such as Iron studies, Folate or Vitamin B-12 studies, transfusion therapy, hematinics or history of chemotherapy. The physician’s clinical impression should be included on the form. Lignocaine sensitivity test should be done. Either aspirate or biopsy may be performed first. But the two should be performed through the same incision, approx. 0.5-1 cm away from the other. This is done to avoid clotting of aspirate and hemorrhagic or damaged biopsy. It is recommended that the aspirate and biopsy be obtained using respective needles separately, and not through a trephine needle. • Operator should wear Surgical gloves • Skin at the site should be cleaned with 70% alcohol or 0.5% chlorhexidine. • Infiltrate the skin, s/c tissue and periosteum over the site with 2-5ml of lignocaine. • Children are either sedated or given general anaesthesia. With boring movement pass the needle perpendicularly at center of PSIS. When bone has been penetrated, remove the stilette, attach 30 ml syringe and suck up marrow contents (0.3ml) for making smears immediately. If large sample is needed for cytogenetics and immunophenotyping attach another 5 or 10 ml syringe and aspirate. (should be kept in preservative free heparin than in EDTA) • Fix the slides in absolute methanol (20 min) as soon as they are thoroughly dry for subsequent staining by a Romanowsky method or Perls' stain for iron. The preparation can be considered satisfactory only when marrow particles and free marrow cells can be seen in stained films. These films are also suitable for cytochemical staining. Some material (clots) can be preserved in fixative rather than anticoagulant for preparation of histological sections. (clot section). Clot is processed same as bone marrow biopsy but without decalcification • Peripheral blood sample is also taken along with it. • Dry tap- Failure to aspirate marrow (suggests bone marrow fibrosis or infiltration.) • If there has been a “dry tap,” insert the stilette into the needle and push any material in the lumen of the needle onto a slide; in lymphomas and carcinomas, especially, sufficient material can be obtained to make a diagnosis. • CT–guided marrow sampling-obese, in whom it is difficult to locate the iliac spine. Look for marrow fragments while making the smears EXAMINATION OF ASPIRATED BONE MARROW Low power (10x) Determine cellularity Identify megakaryocytes and note morphology and maturation sequence (higher power may be needed for smaller immature megakaryocytes and micromegakaryocytes). Look for clumps of abnormal cells (higher power needed to examine content and morphology of clumps) Identify macrophages (higher power for evidence of haemophagocytosis, malaria pigment, and bacterial or fungal infections that may be present in the cytoplasm) High power (40x,100x oil immersion) Identify all stages of maturation of myeloid and erythroid cells Maturation abnormalities are noted Determine the myeloid:erythroid ratio Perform a differential count using the categories erythroid, myeloid, lymphoid, plasma cell, and “others,” simultaneously noting any morphological abnormalities. Look for areas of bone marrow necrosis Assess the iron content Area for myelogram near fragment Cellurarity Normocellular bone marrow on aspirate High cellularity of 95-100% Hypercellular for adult and normocellular for child Cellularity of 40-50% Hypocellular bone marrow (10-20%) Normal ranges for differential counts on aspirated bone marrow (500 cells should be counted) 95% Range Mean Mean Myeloblasts 0–3 1.4 0.4 Promyelocytes 3–12 7.8 13.7[*] Myelocytes (neutrophil) 2–13 7.6 – Metamyelocytes 2–6 4.1 – Neutrophils 22–46 32.1M; 37.4W 35.5 Myelocytes (eosinophil) 0–3 1.3 1.6 Eosinophils 0.3–4 2.2 1.7 Basophils 0–0.5 0.1 0.2 Lymphocytes 5–20 13.1 16.1 Monocytes 0–3 1.3 2.5 Plasma cells 0–3.5 0.6 1.9 Erythroblasts[†] 5–35 28.1M; 22.5W 23.5 Megakaryocytes 0–2 0.5 Macrophages 0–2 0.4 2.0 Myeloid series of cells Erythroid cells Erythroid island on aspirate. Macrophage is surrounded by developing normoblast, hemosiderin form of iron is stored in it. Plasma cell has an eccentric nucleus,cartwheel nuclear chromatin,basophilic cytoplasm with perinuclear hoff. Present in pericapillary location. Plasma cells present in pericappilary region OSTEOBLAST have basophillic cytoplasm,extruding nucleus and regular chromatin with 1-4 nucleoli. Can be distinguished from plasma cells by their larger size and the position of the Golgi zone, which is not immediately adjacent to the nucleus. An osteoclast; note the highly granular cytoplasm and the multiple nuclei (2100)which are uniform in size and have indistinct, medium - sized, single nucleoli. MGG × 100. Mast cells on bone marrow. With PAS stain they display magenta coloured granules Mature megakaryocyte having loblated nucleus and pink granular cytoplasm..platelets are formed by budding of the cytoplasm which are shed in the circulation. Aspirate of normal BM: a macrophage containing granular and refractile debris and several normoblast nuclei. MGG × 100. Aspirate of non - infiltrated BM from a patient with Hodgkin lymphoma: a mature megakaryocyte exhibiting emperipolesis. MGG× 100. Grading of bone marrow storage iron 0 1+ 2+ 3+ 4+ 5+ 6+ No stainable iron Small iron particles just visible in reticulum cells using an oil objective Small, sparse iron particles in reticulum cells, visible at lower power Numerous small particles in reticulum cells Larger particles with a tendency to aggregate into clumps Dense, large clumps Very large clumps and extracellular iron Grade 1 Grade 2 GRADE 3 Grading for iron on bone marrow aspirate 1+ Small iron particles just visible in reticulum cells using an oil objective 2+ Small, sparse iron particles in reticulum cells, visible at lower power 3+ Numerous small particles in reticulum cells GRADE 4 GRADE 5 GRADE 6 Iron grading 4+ Larger particles with a tendency to aggregate into clumps 5+ Dense, large clumps 6+ Very large clumps and extracellular iron Perl’s stain sideroblast Macrophage with iron Perl’s stain Ringed sideroblast Example of report form for bone marrow films BIOPSY (Procedure) • The trephine specimen is obtained by inserting the biopsy needle into the bone and using a toand-fro rotation to obtain a core of tissue. • If an aspirate has been performed first the needle should be inserted through the same incision but the needle should be advanced at a slightly different angle. • The bony core is gently dabbed or rolled across the slide to form imprint smears, which is then fixed and stained as for bone marrow aspiration smears. This allows immediate examination of cells that fall out of the specimen onto the slide and may provide a diagnosis several days before the trephine biopsy specimen has been processedaspirate. and also useful in dry • Bilateral trephine biopsies may be performed to increase the yield of detecting focal lesions. Length at least 1.5 cm At least 10 partialy preserved trabecular spaces seen Sections of 3-4 micron in thickness cut at a distance of 50 micron each. (WHO Classification of tumours of haematopoietic and lymphoid tissue 2008) FIXATION: Biopsy is fixed in 10% neutral buffered formalin for 6 hrs.(ICSH preferred) Other fixatives: Zinc formaldehyde Acetic acid formalin Isotonic buffered formalin B5: not used due to presence of mercuric chloride (poisonous) Bouin’s fixative: not used in some countries, due to presence of picric acid. It is important to ensure that the formol - saline is not left for long periods at ambient or high temperature before being used because formic acid and formalin pigment may be produced. The reactivity of antibodies used for immunohistochemical staining may also be affected by the choice of fixative (use of Bouin’s solution is particularly limiting) and Zenker’s fixative can destroy chloroacetate esterase activity. B5 fixative- if fixation lasts for more than 6 hours hardening of the tissue can make it difficult to cut sections. Decalcifier agent volume to tissue volume should be 20:1 1. 10-15% EDTA( 1-3 days) preserves morphology, enzymes and immunological epitopes. ICSH recommended 2. 10% Formic acid( 2-3 days) causes more tissue distortion. 3. 5-10% nitric acid(3-6 hrs) used for urgent processing. (nitric acid can cause megakaryocytes to give positive reactions with antibodies to CD34 and affect IHC.) 4. In our lab-EDTA. DECALCIFICATION :PITFALLS It chelates storage iron. Affects the morphology and cytological details. Affects ability to perform immunohistochemistry and to retrieve material for molecular analysis. Factors affecting decalcification Conc. of decalcifying agent. Temperature Agitation Age of patient Type of bone Size of spicules Decalcification endpoint Specimen radiography (FAXITRON,most reliable) Chemical method Calcium oxalate test (5ml of used decalcifying agent+Ca(OH)+ 5 mlsat. ammonium oxalate) Physical method Bubble test : Causes shrinkage and loss of some cellular detail but better for IHC. : Glycol methacrylate or methyl methacrylate used Finer sections with better cellular details. No decalcification required so used for metabolic bone diseases. SECTIONING At least 6 sections should be cut at 3 levels into cross sectional diameter of core25% 50% 75% Additional sections needed for IHC or histochemical stains 1. H&E 2. GIEMSA-helpful for plasma cells,mast 3. 4. 5. 6. cells,lymphoid cells,eosinophils,myeloblasts and proerythroblasts. RETICULIN(silver impregnation) TRICHROME PERL STAIN OTHERS: toluidine blue, congo red,ziehl neelson,PAS EVALUATION OF BONE MARROW BIOPSY 4x or 10x Adequacy Cellularity Pattern Presence of focal lesions Megakaryocyte number Abnormal cell clusters and location Bone structure Osteoclastic and osteoblastic activity. 40x Assess haemopoietic cells(erythroid, myeloid, megakaryocytes, lymphoid cells, plasma cells and macrophages) and cytological details. Oil immersion For finer cytological details (eg.intracellular granules, organisms.) Normal topography Myeloid cells Paratrabecular Mature cells towards centre Erythroid cells Centre in colonies Megakaryocytes Centre around sinusoids Monocyte precursors: Not recognizable. Some can be seen randomly distributed. Lymphoid precursors: Seen in periarteriolar region. Stroma Fat cells, fibroblasts, reticulin fibres Osteocytes: Seen in bony lacunae. . Osteoblasts: Seen lining the trabeculae. Osteoclasts: Seen in howship’s lacunae Topography of cells Bone marrow is highly organised structure with haemopoietic elements maturing in different micro-anatomical sites. Cellularity: Normal bone marrow cellularity In adults Hypocellular bone marrow Aplastic anaemia Hypercellular bone marrow In AML WITH MATURATION The bone marrow imprint smear on low power showing optimum cellularity Erythroid cells in the centre Myeloblast and promyelocytes paratrabecular Megakayocyte in bone marrow parasinusoidal Mast cells in immunoperoxidase stain Usually seen in giemsa . Present irregularly in medullary cavity Quantification of bone marrow reticulin and collagen 0 No reticulin fibres demonstrable 1 Occasional fine individual fibres and foci of fine fibre network 2 Fine fibre network throughout most of the section; no coarse fibres 3 Diffuse fibre network with scattered thick coarse fibres but no mature collagen 4 Diffuse often coarse fibre network with areas of collagenization grade 1 grade 0 grade 2, Occasional fine individual fibres and foci of fine fibre network Fine fibre network throughout most of the section; no Diffuse fibre network with scattered coarse fibres but no mature collagen grade 3, thick Diffuse often coarse fibre network withgrade areas 4, Marrow fibrosis Reticulin (silver impregnation method) SUPPLEMENTARY INVESTIGATIONS - Immunohistochemistry- for demonstration of antigens in biopsy Ex.- CD 34, CD 45, Lysozyme, MPO, CD 68, CEA etc. - Cytogenetic analysis- for chromosomal rearrangements - Molecular genetics- by PCR, RTPCR FISH Bone marrow biopsy report (ICSH guidelines 2008): Name of institution Unique specimen identifier Details of patient- name,age,gender, contact details Name of responsible physician Name of requesting doctor Date of procedure Clinical history, examination,therapy Indication of bone marrow examination Procedure performed Site of procedure Length of biopsy core Adequacy and appearance of the core Percentage and pattern of cellularity Bone architechture Location, number,morphology and pattern of differentiation of 1. Erythroid 2. Myeloid 3. Megakaryocytic 4. Lymphoid 5. Plasma cells 6. Macrophages Abnormal cells Reticulin stain IHC Other Ix Signature and date of report Turn around time Varies from lab to lab 24 hrs in case of microwave decalcification and as long as 1 week if EDTA based decalcifying agent is used. 5 days for less urgent cases Additional 1-2 days if IHC is required In our lab aspiration is reported within 24hrs and biopsy within 4-5 days. Storage No guideline BM slides should be stored for 20yrs or indefinitely if possible. Quality assurance North America and Europe have well trenched external quality assessment for histopathology which includes bone marrow biopsy. Situation in India is less advanced due to vast unorganised structure of private labs and highly variable training, technological facilities and reporting styles. TAKE HOME MESSAGE Biopsy of the bone marrow is an indispensable adjunct to the study of diseases of the blood and may be the only way in which a correct diagnosis can be made. A minimum definition of an adequate bone marrow biopsy specimen stipulates a length of atleast 1.5 cm, 5-6 marrow spaces and no aspiration, crush or processing artifacts. • Not to assess histology in isolation, ideally reported along with bone marrow aspirate and peripheral smear and preferrably reported by same pathologist. • There should be an integrated approach in reporting which includes Clinical history Laboratory investigations, Imaging information IHC wherever applicable.