The physiologic process of formation of blood cells is known as haematopoiesis. It proceeds through different stages starting from early embryonic life:
- Mesoblastic stage (yolk sac)
- Hepatic stage (liver)
- Myeloid stage (bone marrow)
Bone marrow becomes the chief site of blood cell production at about 6 months of intrauterine life and exclusive site of blood cell formation by birth. In adults, there are two types of marrow: red or active marrow and yellow (fatty) or inactive marrow. In adults, the main sites of haematopoiesis are the vertebrae, ribs, sternum, skull bones, pelvis, and the proximal ends of the femur and humerus. At these sites, half the marrow is actively producing blood cells (red marrow) and the other half is inactive (yellow fatty marrow). Haematopoiesis occurring in the bone marrow is called as medullary haematopoiesis; that occurring outside the marrow is called as extramedullary haematopoiesis. In severe chronic cases of anaemia (e.g.,thalassaemia, myelofibrosis) extramedullary haematopoiesis can resume in adults as a compensatory mechanism.
HAEMATOPOIETIC STEM CELL
- All bood cells are derived from a common population of haematopoietic stem cells in the bone marrow.
- It is the cell from which two distinct cell lines originate: myeloid and lymphoid. The term used for all non-lymphoid cells is myeloid (includes erythroid, granulocytic, monocytic, and megakaryocytic cells). Myeloid stem cells produce different committed stem cells (colony forming units or CFU) that give rise to precursor cells which in turn proliferate and differentiate into mature red cells, monocytes, granulocytes, and platelets (Fig. 1.1).
- It is believed that there is 1 stem cell for every 10,000 blood cells in bone marrow. Stem cells also circulate in peripheral blood.
- Stem cells are morphologically indistinguishable from small lymphocytes. They express CD 34 antigen.
- These cells have the ability of self-renewal, proliferation, and differentiation.
Haematopoiesis is regulated by cytokines and other growth factors. They are glycoproteins formed in the bone marrow, liver, or kidneys. They interact with specific receptors on the surface of haematopoietic cells and regulate their proliferation, differentiation, and maturation.
Site of action
1. c-kit ligand (stem cell factor)
Pleuripotent stem cells
Myeloid stem cell
3. Granulocyte macrophage
colony stimulating factor
4. Granulocyte colony
5. Macrophage colony
Stages in the formation of red cells (erythropoiesis) are shown in Figure 1.2. In the bone marrow this occurs in islands (Fig. 1.3).
Fig. 1.3: Erythroid island in bone marrow stained with iron stain. Erythropoiesis occurs in erythroid islands which consist of a macrophage (stained blue) surrounded by erythroblasts. Macrophage (called a ‘nurse cell’) provides iron to erythroid progenitor cells through cytoplasmic processes and also phagocytose extruded nuclear material. Erythroid islands are located in intertrabecular (‘central’) spaces in the bone marrow. The cytoplasmic processes of the ‘nurse cell’ also stain for iron
Haemoglobin present in large amounts in red cells carries oxygen from lungs to the tissues and carbon dioxide (generated by cell activity) from tissues to the lungs. Haemoglobin is composed of haem (iron + protoporphyrin) and globin (two pairs of polypeptide chains). Each globin chain is linked to a haem moiety. Different variants of haemoglobin exist at different developmental stages.
- Hb Gower I: ζ2 ε2
- Hb Gower II: α2 ε2
- Hb Portland: ζ2 γ2
The above three haemoglobins are embryonic haemoglobins.
- Hb F: α2 γ2: Predominates in foetal life and for the first few months of life (<1% in adults)
- HbA2: α2 δ2: Comprises small amount in adults (<3%).
- HbA: α2 β2: Predominates in adult life (97%)
The α-like polypeptide chains (ζ and α) and β-like polypeptide chains (ε, γ, β and δ) are encoded by α and β globin gene clusters on chromosomes 16 and 11 respectively (Fig. 1.4).
Fig. 1.4: α and β globin gene clusters. Open boxes represent pseudogenes while filled boxes represent active genes. Normal genotype is shown below each gene cluster
Each globin gene (Fig. 1.5) consists of 3 exons and 2 introns. There is one β globin locus on each chromosome 11, and therefore β genes are two in number. β chain has 146 amino acids. β gene mutations are responsible for sickle cell disorders and β thalassaemias.
The regions of DNA strand which code for amino acids in the protein product are called as exons, while non-coding regions which interrupt the coding sequences are known as introns or intervening sequences. Sequences at the junction of exons and introns are called as splice junction sequences and are necessary for precise removal of introns during the formation of mRNA. The promoter region is required for correct initiation of transcription.
Globin Chain Synthesis
Globin chain synthesis (Fig. 1.6) involves three steps-
- Transcription: Synthesis of a single strand of RNA from DNA.
- Processing of mRNA: Consists of addition of a poly-A tail, a cap structure, and removal of introns.
- Translation: Synthesis of a polypeptide chain on ribosomes according to mRNA template.
Red Cell Metabolism
The main source of energy for red cells is glucose. Glucose is metabolised by glycolysis (Embden-Meyerhof pathway) and hexose monophosphate shunt (pentose phosphate shunt). In the middle of the glycolytic pathway, a Rapoport-Luebering shunt exists in red cells for the synthesis of 2, 3-diphosphoglycerate (2, 3-DPG). Glycolytic pathway generates ATP. Energy is required for maintenance of red cell shape and osmotic pressure. 2, 3-DPG generated by Rapoport-Luebering shunt is an important determinant for oxygen affinity of haemoglobin. In hexose monophosphate shunt, two NADPH (phosphorylated nicotinamide adenine dinucleotide) molecules are produced for every molecule of glucose-6-phosphate entering the shunt. NADPH reduces oxidized glutathione (GSSG). Reduced glutathione (GSH) along with glutathione peroxidase detoxifies hydrogen peroxide and protects haemoglobin from oxidant damage. This process of neutralization of oxidant stress in red cells is dependent on glucose 6 phosphate dehydrogenase (G6PD) enzyme (G6PD is necessary for generation of NADPH in the hexose monophosphate shunt) (Fig 1.7).
Red Cell Membrane
The normal red cell is biconcave in shape and consists of haemoglobin that is surrounded by a cytoskeleton and a lipid bilayer. The proteins of the red cell membrane constitute the cytoskeleton. The cytoskeleton maintains red cell shape and permits flexibility enabling the red cells to pass through the capillaries of small diameter; the biconcave shape also provides large surface area for gaseous exchange. The important cytoskeletal proteins are spectrin (two dissimilar chains intertwined together), ankyrin, protein 4.1, and actin (Fig. 1.8).
White Blood Cells
White blood cells include neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Their development and salient features are shown in Figures 1.9 to 1.14.
In B cells, initially there is rearrangement of heavy chain genes which is followed by rearrangement of light chain genes. The earliest antigens expressed during B cell development are TdT and HLA-DR. There is a sequential appearance of antigens on developing B cells: CD19, CD10, and CD20. Plasma cells express a specific antigen CD38.
B Lymphocyte Development
Fig. 1.11: Normal stages of B cell development showing sequential expression of various antigens and heavy and light chain gene rearrangement. As shown at the bottom, lymphoid neoplasms represent cells arrested at various stages of normal development
T Lymphocyte Development
Fig. 1.13: Stages of T cell development. Correlation of stages with T cell neoplasms is shown at the bottom
About 2/3rd of T lymphocytes in peripheral blood are CD4+ helper cells and about 1/3rd are CD8+ cytotoxic cells (Fig. 1.14).
Initially immature cortical thymocytes express CD7, TdT, and cytoplasmic CD3 (cCD3). At first, both CD4 and CD8 antigens are acquired; with further maturation cells retain either CD4 or CD8 antigen.
Comparison of B and T lymphocytes is presented below:
% in peripheral blood
Location in lymph nodes
T cell receptor
The HLA System
The HLA or human leucocyte antigens are encoded by a cluster of tightly linked genes on the short arm of chromosome 6 called as major histocompatibility complex (MHC). These proteins help the immune system in recognizing foreign substances. There are two main types of HLA antigens: Class I (present on almost all nucleated cells) and Class II (present on cells of immune system i.e. lymphocytes and macrophages). They are important in organ transplantation and in transfusion medicine. Structure of HLA antigens is presented in Figure 1.15.
Significance of HLA antigens: (1) Important in organ transplantation as histocompatibility antigens; (2) In transfusion medicine, HLA antigens are responsible for alloimmunization against platelet antigens, febrile transfusion reactions, and graft vs. host disease; (3) There is a relationship between some HLA antigens and susceptibility to certain diseases; (4) HLA antigen typing can be used for paternity testing.
Tests for detection of HLA antigens: (1) Lymphocytotoxicity test, (2) Mixed lymphocyte culture (MLC) or mixed lymphocyte reaction (MLR), (3) Primed lymphocyte typing (PLT), and (4) DNA analysis.
Thrombocytes (platelets) are produced in the bone marrow from megakaryocytes. A humoral factor, thrombopoietin, controls the maturation of megakaryocytes.
Platelets are small (2-3 µ) disc-shaped, anucleate cell fragments of megakaryocytes. Upon maturation, mega-karyocytes extend pseudopods through the walls of marrow sinusoids and individual platelets break off into the peripheral circulation (Fig. 1.16). The lifespan of platelets is 7-10 days. Platelets play a major role in the formation of a haemostatic plug in haemostasis.
Haemostasis is the physiologic process by which loss of blood from the vascular system is arrested by interaction of blood vessel wall, platelets, and plasma proteins. Primary haemostasis is the initial stage during which blood vessels and platelets interact to limit the blood loss from the damaged vessel. During secondary haemostasis, a stable fibrin clot is formed from coagulation factors by sequential enzymatic reactions. After the arrest of bleeding, fibrin clot is dissolved to resume the normal blood flow; the process of dissolution of blood clot is called as fibrinolysis.
Role of Vascular Wall
Vascular wall can favour or inhibit haemostasis (Fig. 1.17).
Tissue factor activates extrinsic pathway of coagulation. von Willebrand factor mediates adhesion of platelets to subendothelium. Platelet activating factor induces aggregation of platelets. Thrombomodulin helps in activation of protein C that in turn inactivates factors V and VIII. Protein S is a cofactor for protein C. Prostacycline inhibits platelet aggregation. Tissue plasminogen activator activates fibrinolytic system. Heparin-like substances potentiate the action of antithrombin III.
Role of Platelets
Structure of a platelet is shown in Figure 1.18.
Alpha granules contain: platelet derived growth factor, platelet factor 4, beta-thromboglobulin, fibrinogen, von Willebrand factor, factor V, and thrombospondin.
Dense granules contain: ADP, ATP, serotonin, and calcium.
Important platelet membrane glycoproteins:
- GpIb: Mediates adhesion of platelets to subendothelial collagen via von Willebrand factor
- GpIIb/IIIa: Binds fibrinogen and mediates aggregation of platelets.
The main functions of platelets in haemostasis are adhesion, aggregation, and release reaction.
1. Adhesion: This refers to binding of platelets to subendothelial collagen following vascular injury. This requires von Willebrand factor in plasma that mediates adhesion via gpIb receptor on platelet surface (Fig. 1.19).
Fig. 1.19: Platelet adhesion to subendothelial collagen. GpIb receptor on platelets and von Willebrand factor are necessary for attachment of platelets to subendothelial collagen
2. Release reaction: In this process, contents of platelets are released to the exterior, e.g. ADP and thromboxane A2 (Fig. 1.20).
ADP released from dense granules promotes platelet aggregation. Platelet factor released from alpha granules neutralizes anticoagulant action of heparin while platelet derived growth factor stimulates proliferation of vascular smooth muscle cells and skin fibroblasts.
Platelets synthesize and secrete thromboxane A2 when activated by agonists such as ADP and epinephrine (Fig. 1.21).
Fig. 1.21: Synthesis of thromboxane A2. Modes of action of certain antiplatelet drugs are also shown
3. Aggregation: This refers to binding of platelets to each other. The clump of platelets formed is called as primary platelet plug (Fig. 1.22).
Fig. 1.22: Platelet aggregation. This requires binding of fibrinogen molecules to GpIIb/IIIa receptors on platelets
Binding of fibrinogen molecules to GPIIb/IIIa receptors on adjacent platelets causes platelet aggregation. Fibrin and aggregated mass of platelets at the site of injury constitute the haemostatic plug.
Role of Plasma Proteins
Plasma proteins in haemostasis can be divided into following groups:
- Coagulation system: Factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, prekallikrein, high molecular weight kininogen (HMWK) (Table 1.1)
- Fibrinolytic system: Plasminogen, plasmin, tissue plasminogen activator (t-PA), α2-antiplasmin, and plasminogen activator inhibitors called PAI-1, PAI-2
- Inhibitor system: Protein C, protein S, antithrombin-III.
Coagulation Proteins can be divided into following categories:
- Serine proteases:
- vitamin K-dependent factors II, VII, IX, and X,
- contact factors-XI, XII, high molecular weight kininogen, prekallikrein,
- Cofactors-V, VIII, tissue factor, and (4) Transglutaminase: F XIII.
Mechanism of Blood Coagulation
Scheme of blood coagulation is divided into intrinsic, extrinsic, and common pathways. The mechanism consists of a cascade of reactions leading eventually to conversion of fibrinogen to fibrin.
- Intrinsic pathway: It is uncertain whether intrinsic pathway plays any role in vivo. Intrinsic pathway is of importance mainly in in vitro blood clotting. Intrinsic pathway is initiated when plasma comes in contact with a negatively charged surface.
- Extrinsic pathway: Extrinsic pathway is initiated by tissue injury with the release of tissue thromboplastin which causes activation of Factor VII; the enzyme that is formed activates Factor X.
- Common pathway: Both intrinsic and extrinsic pathways proceed to common pathway which begins with activation of Factor X, involves interaction of F X, F V, prothrombin, fibrinogen, phospholipid, calcium, and F XIII, and leads to the formation of fibrin clot (Fig. 1.23).
Fig. 1.23: Scheme of blood coagulation. Solid arrows indicate transformation. Broken lines indicate action. Abbreviations: HMWK: High molecular weight kininogen; TF: Tissue factor; PL: Phospholipid; Ca++: Calcium
Fibrinolysis is the process of dissolution of blood clots which is necessary to maintain free flow of blood in the vascular system. The major enzyme of the fibrinolytic system is plasmin, which is generated from plasminogen. This reaction occurs on the surface of fibrin. Plasmin can degrade both fibrin as well as fibrinogen to form fibrinogen/fibrin degradation products (Fig. 1.24).
The important physiologic inhibitors of coagulation present in normal plasma include antithrombin III (AT III), protein C, protein S, and tissue factor pathway inhibitor. Actions of AT III, protein C, and protein S are shown in Figure 1.25.
Fig. 1.25: (A) Antithrombin III binds with thrombin and other serine proteases (FXa, XIa, XIIa, and IXa) to form a stable complex. (B) Protein C is activated by thrombin and thrombomodulin. Activated protein C causes proteolytic destruction of factors V and VIII. Protein S functions as a cofactor in this reaction