Hematopathology
Ruth Padmore MD, FRCPC, PhD
Staff Hematopathologist
The Ottawa Hospital
1
Lecture/Laboratory Outline
• Hematopathology Lecture (9 - 10:15am)
– Basic Histology, text and atlas, LC Junqueira and J
Carnerio, 11th edition, McGraw-Hill, 2005, ISBN 0-07144091-7
– Chapters 12 (Blood cells), 13 (Hematopoiesis) and 14
(Lymphoid organs)
– Stem cell transplantation
10:15 – 10:30 BREAK
• Histology Laboratory (10:30am - noon), Room 2236
– Review glass slides of:
– Normal peripheral blood and bone marrow
– Selected diseases
2
Lecture Outline
• Hematopathology Lecture
• Basic Histology, text and atlas, LC Junqueira and J
Carnerio, 11th edition, McGraw-Hill, 2005, ISBN 0-07144091-7
• Chapter 12: Blood Cells
• Chapter 13: Hematopoiesis
• Chapter 14: Lymphoid Organs
3
Peripheral Blood
• About 55% of blood is liquid
• The liquid portion of blood
(before clotting) = plasma
• The liquid portion of blood
(after clotting) = serum
• The cellular part of blood (45%
of volume) is made up of a
variety of different cell types
• The hematocrit (Hct) measures
the volume occupied by the cells
in the blood
Figure 12-1
4
Cellular Components of Blood
• Erythrocytes
– Also called red blood cells
(RBCs)
• Leukocytes
– Also called white blood
cells (WBCs)
– 5 majors types:
•
•
•
•
•
Neutrophils (60%)
Lymphocytes (30%)
Monocytes (7%)
Eosinophils (2%)
Basophils (1%)
• Platelets
– Also called thrombocytes
5
Figure 12-3. Scanning Electron Micrograph
of normal human erythrocytes. Note their biconcave
shape for maximum oxygen exchange. x 3300
Wright-Giemsa stained
Peripheral blood film
In mammals, RBCs in peripheral blood
lack a nucleus; whereas those of birds
and reptiles have a nucleus.
6
Erythrocytes (Red Blood Cells)
• RBCs contain mostly
hemoglobin, to carry
oxygen to the tissues
• One hemoglobin
molecule can carry 4
oxygen molecules
7
Figure12-4. Scanning electron micrograph of a sickle cell, from a
person homozygous for the sickle cell mutation (Glu to Val in position
6 of beta chain of the hemoglobin molecule)
8
Figure 12-5. The five types of
Human Leukocytes
• The 5 types of WBCs
• Can be identified by
automated hematology
analyzer (5 part
differential count)
•
•
•
•
•
Neutrophils (60%)
Lymphocytes (30%)
Monocytes (7%)
Eosinophils (2%)
Basophils (1%)
9
Figure 12-6. Neutrophils,
Giemsa-stained
• Neutrophils are also called
polymorphonuclear
leukocytes, due to the
multiple number of
nuclear lobes
• Barr body (drumstick like
nuclear appendage) =
inactivated X chromosome
in females (see diagram
fig 12-5)
10
Abnormal Neutrophils
• Hypersegmented
neutrophils: a sign of
Vitamin B12 or folate
deficiency (interferes
with DNA synthesis)
• Hyposegmented
neutrophils (Pelger-Huet
like morphology) a sign
of myelodysplasia)
11
Neutrophil Granules
• Substances present in both primary and secondary
granules:
– Collagenase, lysozyme
• Primary (azurophilic) granules
– Larger (0.5um) than secondary granules
– Contain myeloperoxidase, acid phosphatase and other
enzymes
• Secondary (specific) granules
– Lactoferrin (binds iron), alkaline phosphatase
12
Destruction of Bacteria by Neutrophils
• Neutrophil engulfs bacteria
• Bacteria in vacuoles (phagosomes) in neutrophils
• Specific granules fuse with and discharge contents
into phagosome
• pH of phagosome lowered to 5.0, for maximal
activity of lysosomal enzymes
• Azurophilic granules discharge contents into
phagosomes, killing and digesting bacteria
• Absolute neutrophil count (ANC) is normally
between 2 to 5 x 109/L
• If neutrophil count less than 0.5 x 109/L, at high
risk for bacterial sepsis (febrile neutropenia)
• Increased neutrophils in peripheral blood as
13
response to infection = neutrophilia
Eosinophils: Figures 12-8 and 12-9
• Bilobed nucleus
• Prominent eosinophilic (reddish)
granules
• Same size or just slightly larger
than neutrophils (12-15um in
diameter)
• Eosinophilia:
– Allergic reactions
• Asthma
• Drug reactions
– Parasitic reactions
14
Figure 12-12 Basophils
• Rare in peripheral blood
(less than 1%)
• Similar in size to
neutrophil (12-15um)
• Nucleus divided into
irregular lobes which are
obscured by overlying
specific granules, which
contain heparin and
histidine
• Noted in hypersensitivity
reactions
15
Figure 12-15: Lymphocytes
• Range from small
lymphocytes, 6-8um
diameter, to large
lymphocytes up to 18um
diameter
• Round nucleus, with
coarsely clumped
chromatin
• scant blue cytoplasm, with
rare azurophilic granules
• Function in immune
reactions, defending
against microorganisms,
foreign macromolecules,
and cancer cells
16
Flow Cytometric Immunophenotypic
Analysis of Leukocytes
Cells gated based on Forward
Scatter, FS (cell size) and Side
Scatter, SS (cell granularity)
Fluorescent-tagged antibodies
identify lineage of cells (T-cell,
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B-cell, myeloid)
Flow Cytometric Immunophenotypic
Analysis of Leukocytes
• T-cells:
• CD2, CD3, CD4, CD5, CD7, CD8
• Kill virus infected cells
• Recruit B-cells in immune response
• B-cells
• CD19, CD20, CD22, surface kappa, lambda
• Differentiate into plasma cells and make antibodies
• NK-cells
• CD2, CD56
• Killing of tumour and virus-infected cells
18
Figure 12-17: Monocytes
• Large cells, 12-20um,
with kidney-shaped
nucleus and abundant
bluish-grey cytoplasm
• Enter tissue and
differentiate into
macrophages
• Phagocytic function
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Platelets
• Small, non-nucleated
fragments of cytoplasm
formed from
megakaryocytes in bone
marrow
• 2-4um diameter, discshaped
• 200-400 x 109/L in
peripheral blood
• Life span 10 days in
peripheral blood
20
Figure 12-19: Electron micrograph
on human platelet
• Prevent bleeding
• Repair gaps in blood
vessel walls
• Promote blood clotting
• Open canalicular system
for rapid release of active
molecules in clotting
• Microtubules, actin,
myosin function in
changes of shape (discoid
to ameboid)
21
Figure 12-19: Electron micrograph
on human platelet
• Alpha granules
– Fibrinogen, PDFG
• Dense granules
– ADP, ATP, Ca++, serotonin
• Clotting:
–
–
–
–
Platelet aggregation
Blood coagulation
Clot retraction
Clot removal
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Hematopoiesis
• Hematopoiesis: making blood cells
• Location of hematopoiesis:
– Embryo: yolk sac, then liver/spleen
– After birth: bone marrow
• Types of hematopoiesis:
– Erythropoiesis: red cells
– Granulopoiesis: granulocytes
– Megakaryopoiesis: megakaryocytes
23
Hematopoietic cells during differentiation
• Stem cells:
Figure 13-1
– Rare cells, proliferate at low level, self-renew
– Pluripotential stem cell
• Lymphoid multipotential cell, Myeloid multipotential cell
• Progenitor Cells
– Source of differentiated cells, influenced by growth
factors, reduced multipotentiality
– Colony forming cells ( or units, CFC or CFU),
– eg. CFU-E: erythrocyte-colony forming cell or unit
• Precursor Cells (blasts)
– High mitotic activity, lineage committed
– Eg Lymphoblast, erythroblast etc.
• Mature cells
– No mitotic activity, abundant in peripheral blood
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25
Hematopoiesis: Recombinant Growth Factors in Clinical Use
• G-CSF: (Neupogen, Filgastrin)
Table 13-2
– Stimulated formation and function of neutrophils and neutrophil
precursors
– Used in cases of febrile neutropenia following chemotherapy for
cancer
– Avoid use in acute myeloid leukemia, stimulates leukemia blasts to
grow
• Erythropoietin: (epoetin alpha, EPO, darbepoetin alpha)
– Produced by kidney in response to hypoxia
– Stimulates erythropoiesis, prevents apoptosis of erythroid
precursors
– Used in renal dialysis patients to prevent anemia and cancer
patients to improve quality of life
– side effects: hypertension, thrombosis, pure red cell aplasia due to
anti-EPO antibodies, changed formulation to reduce antigenicity
26
Figure 13-3. Section of
Hematopoietically Active Bone Marrow
• Connective tissue
stroma
• Hematopoietic cords
• Sinusoids
27
Stages in Red cell (erythroid)
Maturation
Proerythroblast
Basophilic
erythroblast
Polychromatic
erythroblast
(two examples)
Orthochromic
erythroblast
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Figure 13-7: Erythropoiesis
Aspirate
Biopsy
29
Stages in Granulocyte
Maturation
Blast cell
Promyelocyte Myelocyte Metamyelocyte Band cell Segmented
Neutrophil
30
Figure 13-9. Granulopoiesis
Aspirate
Biopsy
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Figure 13-16:
Megakaryopoiesis and Platelet Formation
• Mature
megakaryocytes form
by cell division
without nuclear
division
• Megakaryocytes are
polyploid, 8 to 16
ploidy and very large
35-150um
Megakaryocyte in aspirate
Several megakaryocytes in32
bone marrow biopsy
The Immune System
• Function of the immune system: to eliminate
foreign molecules/cells (tumor cells, virally
infected cells, bacteria, foreign bodies)
• Two basic types of immune response:
• (1) innate response:
–
–
–
–
Neutrophils
Macrophages
Mast cells
Natural killer cells
• (2) adaptive response: (lymphocytes)
– B-cells
– T-cells
33
Chapter 14: Lymphoid Organs
• Cells of the immune
system are organized into
lymphoid organs
• (1) distributed through
body in blood, lymph, and
epithelial and connective
tissues
• (2) lymphoid nodules
• (3) lymphoid organs
–
–
–
–
Lymph nodes
Spleen
Thymus
Bone marrow
34
The adaptive immune response:
B-cells (Humoral response)
• B-cells are activated
by foreign proteins
(antigens) and
differentiate into
plasma cells
• Plasma cells make
immunoglobulin
Figure 14-24A: Plasma35cells
in medulla of lymph node
Table 14-1:
Classes of Immunoglobulins (Ig)
• IgG: monomer, lots of it in
plasma, neutralizes antigens
• IgM: pentamer, initial immune
response, high levels seen in
acute infection
• IgA: dimer, present in
secretions (saliva, breast milk,
tears), protects mucosal
surfaces
• IgE: monomer, allergic and
anti-parasite responses
• IgD: monomer, small amount
in plasma, triggers initial B-cell
activation
36
Antibodies
• Microorganism is covered by antibodies that
recognize it (opsonization)
– Antibody-coated microorganism is ingested by
macrophages, neutrophils, eosinophils, which have
receptors for the FC part of the immunoglobulin
molecule
• IgM activates the complement system
– C3 binds to microorganism, and is ingested by
phagocytic cells with C3 receptors
– Complement cascade produces membrane attack
complex, makes holes in cell membrane, resulting
in cell lysis
37
The adaptive immune response:
T-cells (Cellular response)
• T-helper Cells
– CD4+, receptor for class II MHC molecules
– Class II MHC molecules expressed by antigen
presenting cells (B-cells, macrophages, dendritic
cells), which present exogenous antigen to the Thelper cell
– T-helper cell is stimulated to:
• Make B-cells differentiate into plasma cells
• Activate cytotoxic CD8+ T-cells
• Induce an inflammatory reaction
38
The adaptive immune response:
T-cells (Cellular response)
• T-cytotoxic Cells
– CD8+, receptor for class I MHC molecules
– Class I MHC molecules expressed by almost all
cells in the body
– Class I MHC molecules complex with abnormal
endogenous proteins (eg virally infected cell or
tumour cell)
– This complex is presented to the CD8+ Tcytotoxic cell, resulting in:
• Release of perforins with destruction of abnormal cell
• Induce apoptosis of abnormal cell
39
Mucosa-Associated Lymphoid Tissue
(MALT)
• Lungs
• Peyer’s patches (small
bowel)
• Tonsils
• Sites of IgA secretion
to protect mucosal
surfaces from
infection
Figure 14-12: Palatine Tonsil
40
Figure14-14.
Thymus with lobules composed of dark
cortical and light medullary zones
• Thymus is site of Tcell differentiation,
into CD4+ or CD8+ Tcells
• Thymic-blood barrier,
with non-fenestrated
endothelial cells
• No afferent lymphatics
41
Figure 14-20.
Schematic of Lymph Node
Figure 14-21.
Section of Lymph Node
42
Hematoxylin & Eosin stain (H&E)
Figure 14-27. Spleen
43
Figure 14-28
Schematic of Spleen
44
Figure 14-33.
Scanning electron micrograph of
sinusoids, red pulp cords and macrophages
Figure 14-35. Spleen macrophages
45
with phagocytosis of red cells (erythrophagocytosis)
Stem Cell Transplantation
• CD34+ hematopoietic stem cells used in
transplantation
• Three sources of stem cells:
– peripheral blood, bone marrow or cord blood
• Autologous Transplant:
– Stem cells are collected from the patient, and re-infused
after intensive chemotherapy and radiation therapy
• Allogeneic Transplant:
– Stem cells are collected from a donor, and are infused
into the patient after intensive chemotherapy and
radiation therapy
46
Stem Cell Transplantation
•
•
•
•
Complications of Stem Cell Transplantation
Failure of engraftment
Relapse of malignancy
Graft-versus-host disease
47
Complications of Stem Cell
Transplantation:
Graft-versus-host Disease (GVHD)
• Stem cell transplants are HLA-matched
• 6/6 match, identical for HLA-A, B and DR
• GVHD develops after successful engraftment, as
engrafted donor immune system recognizes
recipient tissues as foreign
• Can be acute or chronic
• Organs affect: skin, liver, gut, eye, oral (dry
mouth)
48
Other forms of GVHD:
Passenger Lymphocyte Syndrome
• Seen in solid organ transplant (e.g. kidney)
• Donor has antibodies to recipient red cell antigens
• Hemolysis develops after transplant, due to donor
lymphocytes present in the blood vessels of the
donated organ
• May result in transient hemolysis and anemia after
transplant
49
Other forms of GVHD:
Transfusion related Graft-versushost Disease
• Lymphocytes can engraft after blood transfusion
• If donor and recipient are haplo-identical for HLA
(3/6 match)
• Donor lymphocytes engraft because of haplotype
match, but then recognize recipient as foreign,
because of haplotype mismatch
• Recipient bone marrow cells are destroyed,
resulting in pancytopenia
• Is always fatal due to bone marrow failure
50
Laboratory
•
•
•
•
•
•
•
Slide 1: Normal Peripheral Blood
Slide 2: Normal Bone Marrow
Slide 8: Sickle Cell Anemia
Slide 12: Chronic Lymphocytic Leukemia
Slide 13: Chronic Myelogenous Leukemia
Slide 14: Acute Lymphoblastic Leukemia
Slide 18: Follicular Lymphoma
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Slide 1: Normal Peripheral Blood
52
Slide 1: Normal Peripheral Blood
Best area of slide
to look at
53
Slide 2: normal marrow aspirate
54
Best area of slide to look at
Slide 2: normal marrow aspirate
55
Leukemias: Cancer of the White Blood Cells
• Chronic Leukemias
– Chronic lymphocytic leukemia (CLL)
– Chronic myelogenous leukemia (CML)
• Acute Leukemias
– Acute lymphoblastic leukemia (ALL)
– Acute myeloid leukemia (AML)
56
Slide 12: Chronic Lymphocytic Leukemia
Smudge
57
cell
Slide 13
Chronic Myelogenous Leukemia
• High WBC
• Left-shift in peripheral blood,
with increased myelocytes,
metamyelocytes, and a few
blasts
• Basophilia
• t(9;22) (ABL/BCR) (ABL
tyrosine kinase)
• Therapy with Gleevec
(imatinib): signal transduction
inhibitor, binds to ATP binding
domain of ABL kinase and
inhibits downstream activation
58
Slide 14: Acute Lymphoblastic Leukemia
Lymphoblast
Lymphoblast
Lymphoblast
59
Slide 18: Follicular Lymphoma
60