1

Hematology
Table of Contents

Chapter 1: Embryology of Hematology...................................................................................................................................................4

Fetal Erythropoiesis...............................................................................................................................................................................4
Blood Groups..........................................................................................................................................................................................5

Chapter 2: Hematology Anatomy & Histology.......................................................................................................................................6

Hematopoiesis........................................................................................................................................................................................6
Blood Cell Morphology & Functions.....................................................................................................................................................6
Neutrophils.........................................................................................................................................................................................6
Erythrocytes (RBC)............................................................................................................................................................................7
Platelets..............................................................................................................................................................................................8
Monocytes..........................................................................................................................................................................................8
Macrophages......................................................................................................................................................................................8
Dendritic cells....................................................................................................................................................................................8
Eosinophils.........................................................................................................................................................................................9
Basophils............................................................................................................................................................................................9
Mast Cells..........................................................................................................................................................................................9
Plasma Cells.....................................................................................................................................................................................10
Lymphocytes....................................................................................................................................................................................10

Chapter 3: Physiology of Hematology...................................................................................................................................................11

Hemoglobin Electrophoresis................................................................................................................................................................11
Coombs Test (Anti-globin Test)............................................................................................................................................................11
Function:..........................................................................................................................................................................................11
Direct Coombs test:..........................................................................................................................................................................11
Indirect Coombs test:.......................................................................................................................................................................11
Platelet Plug Formation (1o Hemostasis).............................................................................................................................................12
Synthesis:.........................................................................................................................................................................................12
Drugs of thrombogenesis:................................................................................................................................................................12
Coagulation Cascade and Kinin Pathway (2o Hemostasis).................................................................................................................13
Activation requires:..........................................................................................................................................................................13
Clinical:............................................................................................................................................................................................13
Drugs:...............................................................................................................................................................................................13
Vitamin K-dependent Components:.................................................................................................................................................14
Blood Film Interpretation.....................................................................................................................................................................15
RBC Shape.......................................................................................................................................................................................15
RBC Inclusions................................................................................................................................................................................16
Anemias (RBC Size and Color).......................................................................................................................................................16
Reticulocyte Production Index (Corrected Reticulocyte Count)..........................................................................................................16
2
3
4

Chapter 1: Embryology of Hematology

Fetal Erythropoiesis
 Site (occurs in):
o Yolk sac (3–8 weeks)
o Liver (6 weeks–birth)
o Spleen (10–28 weeks)
o Bone marrow (18 weeks to adult)
 Hb Type:
o Embryonic globins: ζ and ε.
o Fetal hemoglobin (HbF) = α2γ2.
o Adult hemoglobin (HbA1) = α2β2.
 Clinical application:
o HbF has higher affinity for O2 due to less avid binding of 2,3-BPG, allowing HbF to extract O2
from maternal hemoglobin (HbA1 and HbA2) across the placenta.
o HbA2 (α2δ2) is a form of adult hemoglobin present in small amounts.
5

Blood Groups

 Anti-Kell Antibody (usually IgG):

o Complex blood group system with high immunogenicity.
o Associated with hemolytic transfusion reactions and hemolytic disease of newborn.
o If produced, Anti-Kell Ab of Kell (+) pregnant pt. causes hemolytic disease of newborn following
previous sensitization to Kell antigen and transferred through placenta.
o It targets fetal RBC precursors ( RBC production) and mature RBCs ( hemolysis) resulting in
severe fetal anemia.
 Hemolytic disease of fetus and newborn / Erythroblastosis fetalis

Chapter 2: Hematology Anatomy & Histology

6

Hematopoiesis
Blood Cell Morphology & Functions

Neutrophils
 Morphology:
o Acute inflammatory response cells (phagocytic) with multilobed nucleus (2-5 lobes).
o Specific granules contain leukocyte alkaline phosphatase (LAP), collagenase, lysozyme, lactoferrin
o Azurophilic granules (lysosomes) contain proteinases, acid phosphatase, myeloperoxidase, and
β-glucuronidase.
 Clinical applications:
o Inflammatory states (eg, bacterial infection) cause neutrophilia and changes in neutrophil
morphology such as cytoplasmic vacuoles, left shift, and
 toxic granulation (dark blue, coarse granules)
 Döhle bodies (light blue, peripheral inclusions, arrow in B)
o Left shift is  neutrophil precursors (eg, band cells, metamyelocytes) in peripheral blood which
reflects states of myeloid proliferation (eg, inflammation, CML).
o Leukoerythroblastic reaction is left shift accompanied by immature RBCs which suggests bone
marrow infiltration (eg, myelofibrosis, metastasis).
o Hyper-segmented neutrophils (nucleus has 6+ lobes) are seen in vitamin B12/folate deficiency.
 Chemotactic factors:
o Chemotaxis is the process of neutrophil migration towards site of cell injury and inflammation
o Neutrophil chemotactic agents: C5a, IL-8, LTB4, 5-HETE (leukotriene precursor), kallikrein,
platelet-activating factor, N-formylmethionine (bacterial proteins).
7

Erythrocytes (RBC)
 Morphology:
o Biconcave, anucleate and lack organelles
o large surface area-to-volume ratio for rapid gas exchange.
o Life span of ~120 days in healthy adults; 60–90 days in neonates.
 Functions:
o Carry O2 to tissues and CO2 to lungs.
o Source of energy is glucose (90% used in glycolysis, 10% used in HMP shunt).
o Membranes contain Cl−/HCO3− antiporter, which allow RBCs to export HCO3− and transport CO2
from the periphery to the lungs for elimination.
 Clinical applications:
o Reticulocyte = immature RBC; reflects erythroid proliferation.
o Bluish color (polychromasia) on Wright-Giemsa stain of reticulocytes represents residual
ribosomal RNA.
o Erythrocytosis is a sign of polycythemia (Hct).
o Anisocytosis is varying size while poikilocytosis is varying shape.
8

Platelets
 Morphology:
o Thrombocytes are anucleate with small cytoplasmic fragments; derived from megakaryocytes.
o 1/3 of platelet pool is stored in spleen; life span of 8–10 days (pl8lets).
o Contains
 dense granules (Ca2+, ADP, Serotonin, Histamine; CASH)
 α granules contains vWF, fibrinogen, fibronectin, platelet factor 4
 Functions:
o Involved in 1° hemostasis. When activated by endothelial injury, aggregate with other platelets
and interact with fibrinogen to form platelet plug.
o vWF receptor: GpIb.
o Fibrinogen receptor: GpIIb/IIIa.
 Clinical applications:
o Thrombocytopenia or  platelet function results in petechiae.
o Thrombopoietin stimulates megakaryocyte proliferation.
9

Monocytes
 Mono = one (nucleus); cyte = cell.
 Large, kidney-shaped nucleus with extensive “frosted glass” cytoplasm.
 Found in blood, differentiate into macrophages in tissues.
Macrophages
 Morphology: (Macro = large; phage = eater)
o An antigen-presenting cell which phagocytose bacteria, cellular debris, & senescent RBCs.
o Have long life in tissues.
o Differentiate from circulating blood monocytes.
o Name of macrophage in specific tissue = Kupffer cells in liver, histiocytes in connective tissue,
osteoclasts in bone, microglial cells in brain.
 Functions:
o Activated by γ-interferon and use MHC II to present the antigens on its surface.
o Also engage in antibody-dependent cellular cytotoxicity.
 Clinical applications:
o Important cellular component of granulomas (TB, sarcoidosis) where they fuse to form giant cell.
o Septic shock starts if lipid A from bacterial lipopolysaccharide binds to CD14 on macrophages.
Dendritic cells
 Highly phagocytic antigen-presenting cells (APCs)
 Function as link between innate and adaptive immune systems (eg, via T-cell stimulation).
 Express MHC class II and Fc receptors on surface.
 Can present exogenous antigens on MHC class I (cross-presentation).

Eosinophils
 Morphology: Bilobate nucleus packed with large eosinophilic granules of uniform size.
 Functions:
o Highly phagocytic for antigen-antibody complexes.
o Defends against helminthic infections (major basic protein).
o Eosinophilic granules produces histaminase, major basic protein (MBP, a helminthotoxin),
eosinophil peroxidase, eosinophil cationic protein, and eosinophil- derived neurotoxin.
 Causes of Eosinophilia: (Pacman eats eosinophils; Eosin = pink dye; philic = loving)
o Parasites
o Asthma
o Chronic adrenal insufficiency
o Myeloproliferative disorders
o Allergic processes
o Neoplasia (eg, Hodgkin lymphoma)
10

o Eosinophilic granulomatosis with polyangiitis

Basophils
 Morphology: Densely basophilic granules contain heparin (anticoagulant) and histamine (vasodilator).
 Functions: Synthesizes & releases leukotrienes on demand thus mediates allergic reactions (inflam.)
 Basophilia: sign of myeloproliferative disorders esp. CML (Basophilic—stains readily with basic stains)
Mast Cells
 Morphology:
o Originate from same precursor as basophils but are not the same cell type.
o Contain basophilic granules (same as basophils).
 Functions:
o Mediates local tissue allergic reactions thus can bind the Fc portion of IgE to membrane.
o Activated by tissue trauma, C3a and C5a, surface IgE cross-linking by antigen (IgE receptor
aggregation) ➝ degranulation ➝ release of histamine, heparin, tryptase, and eosinophil
chemotactic factors.
 Clinical applications:
o Involved in type I hypersensitivity reactions.
o Cromolyn sodium = prevents mast cell degranulation (used for asthma prophylaxis).
o Vancomycin, opioids, radiocontrast dye = elicit IgE-independent mast cell degranulation.
o Mastocytosis (rare)
 proliferation of mast cells in skin and/or extracutaneous organs.
 Associated with c-KIT mutations and  serum tryptase.
  histamine ➝ flushing, pruritus, hypotension, abdo. pain, diarrhea, peptic ulcer disease

Plasma Cells
 Morphology:
o Found in bone marrow and normally do not circulate in peripheral blood.
o Eccentric nucleus with “Clock-face” chromatin distribution,  RER & well-developed Golgi.
 Functions: Produce large amounts of antibody specific to a particular antigen.
 Clinical applications: Multiple myeloma is a plasma cell dyscrasia.
Lymphocytes
 Round, densely staining nucleus with small amount of pale cytoplasm.
 NK cells mediate innate immunity while B & T cells mediate adaptive immunity
Natural killer cells (NK cells)
 Morphology: Larger than B & T cells w cytoplasmic lytic granules containing perforin & granzymes.
 Functions:
o NK cells are part of innate immune response esp. against intracellular pathogens.
o When released, they act on target cells to induce apoptosis (killer) in cells that do not express
class I MHC cell surface molecules eg, virally infected cells where these molecules are
downregulated.
11

 Clinical applications:
o stress, malignant transformation, or microbial infections induces cell surface proteins which are
identified to distinguish between healthy and infected cells.
B cells
 Lifecycle:
o Originate from stem cells in bone marrow & matures in marrow (B = bone marrow)
o Migrate to peripheral lymphoid tissue (LN follicle, white pulp of spleen, unencapsulated
lymphoid tissue)
 Functions:
o Mediates humoral immune response.
o When antigen is encountered, B cells differentiate into plasma cells (which produce antibodies)
and memory cells. Can function as an APC.
T cells
 Lifecycle:
o Originate from stem cells in bone marrow but mature in the thymus (T = thymus).
o Differentiate into
 cytotoxic T cells (express CD8, recognize MHC I),
 helper T cells (express CD4, recognize MHC II),
 regulatory T cells.
o CD28 (costimulatory signal) necessary for T-cell activation.
 Functions:
o Mediate cellular immune response.
o CD4+ helper T cells are the 1o target of HIV (Rule of 8 ➝ MHC II × CD4 = 8 or MHC I × CD8 = 8)

Chapter 3: Physiology of Hematology

Hemoglobin Electrophoresis

Interpretation & Use:

 Hb migrates from -ve charged cathode to + charged anode during gel electrophoresis.
 HbA migrates the farthest followed by HbF, HbS, HbC.
 Missense mutation in HbS and HbC replace glutamic acid ⊝ w valine (neutral) & lysine ⊕, respectively.
o Thus, it makes HbC and HbS  +vely charged than HbA.
12

Coombs Test (Anti-globin Test)

Function: Detects the presence of antibodies against circulating RBCs.

Direct Coombs:
 anti-Ig Ab (Coombs reagent) added to pt. RBCs ➝ RBCs agglutinate if RBCs are coated with Ig.
 Used for AIHA diagnosis.
Indirect Coombs:
 Normal RBC + Coomb reagent added to pt. serum ➝ RBC agglutinate if serum has anti-RBC surface Ig
 Used for pretransfusion testing.
Platelet Plug Formation (1o Hemostasis)

Synthesis:
 vWF
o derived from Weibel-Palade bodies of endothelial cells and alpha granules of platelets.
o carries/protects factor VIII (volksWagen Factories make gr8 cars)
 ADP is released from platelet dense granules which promotes exposure of GPIIb/IIIa R’ on platelets.
 TXA2 is synthesized by platelet cyclooxygenase (COX) and released ➝ promotes platelet aggregation.
13

Drugs of thrombogenesis:
 Aspirin irreversibly inhibits COX thereby inhibiting TXA2 synthesis.
 Clopidogrel, prasugrel, ticagrelor, and ticlopidine block P2Y12 receptor thereby inhibiting ADP-induced
expression of GpIIb/IIIa.
 Abciximab, eptifibatide, and tirofiban inhibit GpIIb/IIIa directly.
 Desmopressin promotes the release of vWF and factor VIII from endothelial cells.
 Ristocetin:
o activates vWF to bind GpIb.
o Failure of aggregation with ristocetin assay occurs in von Willebrand disease & Bernard-Soulier.

Coagulation Cascade and Kinin Pathway (2o Hemostasis)

 Cascade stabilizes weak platelet plug by generating thrombin which converts fibrinogen to fibrin.
 Cross linking of fibrin yields a stable platelet-fibrin thrombus.
Activation requires:
 Phospholipid surface of platelets.
 Calcium (derived from platelet dense granules).
 Exposure to an activating substance.
o Tissue thromboplastin activates factor VII (extrinsic pathway).
o Subendothelial collagen activates factor XII (intrinsic pathway).
Clinical:
 PT monitors extrinsic and common pathway, reflecting activity of factors I, II, V, VII, and X.
 PTT monitors intrinsic and common pathway, reflecting activity of all factors except VII and XIII.
 Hemophilia A: def. of factor 8 (XR); Hemophilia B: def. of 9 (XR); Hemophilia C: def. of 11 (AR)
 C1-esterase inhibitor deficiency ➝ hereditary angioedema
 Regular anti-coagulant protein: proteins C and S
Drugs:
 ACEi inhibits ACE thus activates bradykinin ➝ dry cough & inflammation ( pain, vasodilation)
 Thrombolytics: USTAR (urokinase, streptokinase, Tenecteplase, alteplase, reteplase)
14

 Anti-fibrinolytics: aminocaproic acid, tranexamic acid

Anti-coagulants of Xa Anti-coagulants of IIa (thrombin)
 LMWH (dalteparin, enoxaparin)  Heparin
 Heparin, Direct Xa inhibitors  LMWH, Direct thrombin inhibitors
 Fondaparinux o Argatroban, dabigatran, bivalirudin
15

Vitamin K-dependent Components:

Source: liver produces factors of coagulation cascade in an inactive state.
Pro-coagulation:
 The mature carboxylated clotting factors (2, 7, 9, 10) convert the fibrinogen to fibrin.
 Factor VII (seven)—shortest half-life.
 Factor II (two)—longest (too long) half-life.
 Clinical:
o Neonates lack enteric baKteria, which produce vitamin K.
o Vitamin K deficiency causes  synthesis of factors II, VII, IX, X, protein C, protein S.
o Early administration of vitamin K overcomes neonatal vitamin K deficiency/coagulopathy.
 Warfarin:
o inhibits vitamin K epoxide reductase.
o Vitamin K reverses inhibitory effect of warfarin on clotting factor synthesis (delayed).
o FFP or PCC adm. reverses warfarin action immediately thus given with vit K if severe bleeding.
Anti-coagulation:
 The mature protein C (anti-coagulant) activates when combined with thrombin-thrombomodulin
complex in endothelial cells. This cleaves and inactivates factor 5a, 8a.
 Anti-thrombin mainly inhibits thrombin (IIa) and factor Xa but also inhibits factors 7, 9, 11, 12.
 Factor V Leiden mutation produces a factor V resistant to inhibition by activated protein C.
 Drugs:
o Heparin enhances the activity of antithrombin.
o tPA is used clinically as a thrombolytic.
16

Blood Film Interpretation

RBC Shape

 a
17

RBC Inclusions

RBC Size and Color (Anemia)

Reticulocyte Production Index (Corrected Reticulocyte Count)

 Used to correct falsely elevated reticulocyte count in anemia.
 Measures appropriate bone marrow response to anemic conditions (effective erythropoiesis).
 High RPI (> 3) indicates compensatory RBC production.
18

 low RPI (< 2) indicates inadequate response to correct anemia.

 (
RPI = % reticulocytes x )
actual Hct
normal Hct
/ maturation time