Hematopathology

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Hematopathology

• Ruth Padmore MD, FRCPC, PhD
• Staff Hematopathologist
• The Ottawa Hospital

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Lecture/Laboratory Outline

• Hematopathology Lecture (9 - 1015am)
• Basic Histology, text and atlas, LC Junqueira
  and J Carnerio, 11th edition, McGraw-Hill, 2005,
  ISBN 0-07-144091-7
• Chapters 12 (Blood cells), 13 (Hematopoiesis) and
  14 (Lymphoid organs)
• Stem cell transplantation
• 1015 1030 BREAK
• Histology Laboratory (1030am - 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 0-07-144091-7
• 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
  response to infection neutrophilia

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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 ImmunophenotypicAnalysis 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, B-cell, myeloid)
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Flow Cytometric ImmunophenotypicAnalysis 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, disc-shaped
• 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
Figure 13-1

• Stem cells
• 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
Table 13-2

• G-CSF (Neupogen, Filgastrin)
• 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
Polychromatic Orthochromic
erythroblast
erythroblast
erythroblast
(two examples)
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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 13-16Megakaryopoiesis 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 in bone marrow biopsy
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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 responseB-cells (Humoral
response)

• B-cells are activated by foreign proteins
  (antigens) and differentiate into plasma cells
• Plasma cells make immunoglobulin

Figure 14-24A Plasma cells in medulla of lymph
node
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Table 14-1Classes 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 responseT-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
  T-helper 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 responseT-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 T-cytotoxic
  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
• Peyers 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 T-cell differentiation, into
  CD4 or CD8 T-cells
• 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 Hematoxylin
Eosin stain (HE)
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Figure 14-27. Spleen
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Figure 14-28Schematic of Spleen
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Figure 14-35. Spleen macrophages with
phagocytosis of red cells (erythrophagocytosis)
Figure 14-33. Scanning electron micrograph of
sinusoids, red pulp cords and macrophages
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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 TransplantationGraft-
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 GVHDPassenger 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 GVHDTransfusion related
Graft-versus-host 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

• 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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Smudge cell
Slide 12 Chronic Lymphocytic Leukemia
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Slide 13Chronic Myelogenous Leukemia

• High WBC
• Left-shift in peripheral blood, with increased
  myelocytes, metamyelocytes, and a few blasts
• Basophilia
• t(922) (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