Hematopathology

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Transcript Hematopathology

Hematopathology
Ruth Padmore MD, FRCPC, PhD
Staff Hematopathologist
The Ottawa Hospital
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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
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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
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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
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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
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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.
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Erythrocytes (Red Blood Cells)
• RBCs contain mostly
hemoglobin, to carry
oxygen to the tissues
• One hemoglobin
molecule can carry 4
oxygen molecules
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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)
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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%)
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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)
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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)
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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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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
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Figure 13-3. Section of
Hematopoietically Active Bone Marrow
• Connective tissue
stroma
• Hematopoietic cords
• Sinusoids
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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
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Stages in Granulocyte
Maturation
Blast cell
Promyelocyte Myelocyte Metamyelocyte Band cell Segmented
Neutrophil
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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
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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
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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
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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
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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
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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
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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
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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
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Figure 14-20.
Schematic of Lymph Node
Figure 14-21.
Section of Lymph Node
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Hematoxylin & Eosin stain (H&E)
Figure 14-27. Spleen
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Figure 14-28
Schematic of Spleen
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Figure 14-33.
Scanning electron micrograph of
sinusoids, red pulp cords and macrophages
Figure 14-35. Spleen macrophages
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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
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Stem Cell Transplantation
•
•
•
•
Complications of Stem Cell Transplantation
Failure of engraftment
Relapse of malignancy
Graft-versus-host disease
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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)
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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
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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
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Laboratory
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•
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•
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
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Slide 1: Normal Peripheral Blood
Best area of slide
to look at
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Slide 2: normal marrow aspirate
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Best area of slide to look at
Slide 2: normal marrow aspirate
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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)
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Slide 12: Chronic Lymphocytic Leukemia
Smudge
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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
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Slide 14: Acute Lymphoblastic Leukemia
Lymphoblast
Lymphoblast
Lymphoblast
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Slide 18: Follicular Lymphoma
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