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  2. 2. Introduction <ul><li>Definition and sites </li></ul><ul><li>Haematopoiesis or haemopoiesis is the formation of blood cells. haemopoietic organs are bone marrow, thymus, lymph nodes and lymph follicles, spleen and liver. </li></ul>
  3. 3. Prenatal and neonatal haemopoiesis <ul><li>haemopoietic cells in mammalian and avian embryos first appear in yolk sac wall. </li></ul><ul><li>Later the liver and spleen are seeded. </li></ul><ul><li>Towards term and post-natally bone marrow become major site of haemopoiesis. </li></ul>
  4. 4. <ul><li>Progressive haemopoiesis in these organs results from in situ differentiation of circulating stem cells. </li></ul><ul><li>Stem cells in the bursa of Fabricius in birds and thymus in mammals and birds are supplied through circulation. </li></ul><ul><li>Post-natally haemopoiesis is restricted to bone marrow. </li></ul><ul><li>The liver and spleen are inactive but retain potential to revert to haemopoiesis in diseases of bone marrow </li></ul>
  5. 5. Functions of haemopoietic organs and related tissues <ul><li>Bone marrow </li></ul><ul><li>Produces erythrocytes, granulocytes, monocytes, platelets and B-lymphocytes. </li></ul><ul><li>Supplies stem cells for lymphocyte production in thymus and spleen. </li></ul><ul><li>Stores iron </li></ul>
  6. 6. <ul><li>Thymus </li></ul><ul><li>Central lymphoid organ where bone marrow derived precursor cells differentiate into immunologically competent T-lymphocytes </li></ul>
  7. 7. <ul><li>Lymph nodes </li></ul><ul><li>Produce lymphocytes and plasma cells </li></ul><ul><li>Produce antibodies. </li></ul><ul><li>Spleen </li></ul><ul><li>Produces lymphocytes and plasma cells </li></ul><ul><li>Synthesizes antibodies. </li></ul><ul><li>Reservoir of erythrocytes and thrombocytes </li></ul><ul><li>Destroys senescent and abnormal erythrocytes </li></ul><ul><li>Degrades haemoglobin </li></ul><ul><li>Stores iron </li></ul>
  8. 8. <ul><li>Pitting function; removes Howel-Jolly bodies, Heinz bodies, nuclei and parasites from erythrocytes. </li></ul><ul><li>Potential of haemopoiesis </li></ul>
  9. 9. <ul><li>Liver </li></ul><ul><li>Stores vitamin B12, foliate and iron </li></ul><ul><li>Produces coagulation factors, albumin, and some globulins. </li></ul><ul><li>Converts free bilirubin to bilirubin glucuronide for excretion into bile </li></ul><ul><li>Participates in entero-hepatic circulation of urobilinogen </li></ul><ul><li>Produces α-globulin, a precursor of erythropoietin </li></ul>
  10. 10. <ul><li>Produces erythropoietin </li></ul><ul><li>Embryonic potential of haemopoiesis </li></ul><ul><li>Stomach and intestines </li></ul><ul><li>Produce HCl for release of iron from complex organic molecules. </li></ul><ul><li>Produce intrinsic factor to facilitate absorption of vitamin B12. </li></ul><ul><li>Absorb vitamin B12 and folic acid through intestinal epithelial cells. </li></ul><ul><li>Control the rate of iron absorption in relation to body needs </li></ul>
  11. 11. <ul><li>Kidneys </li></ul><ul><li>Produce erythropoietin and thrombopoietin </li></ul><ul><li>Degrade excessive haemoglobin to bilirubin for urinary excretion </li></ul><ul><li>Mononuclear phagocyte system (reticulo-endothelial system) </li></ul><ul><li>Major phagocytic system of the body for cellular defense against microbial infection </li></ul><ul><li>Destroys various blood cells </li></ul>
  12. 12. <ul><li>Degrade haemoglobin into iron, globin and free bilirubin </li></ul><ul><li>Store iron </li></ul><ul><li>Secrete biologically active macromolecules; colony stimulating factor, complement </li></ul>
  13. 13. Postnatal haemopoiesis <ul><li>Bone marrow </li></ul><ul><li>In early postnatal life bone marrow of all bones performs haemopoiesis. As demand for erythrocytes decreases at maturity haemopoiesis recedes from shaft of long bones. </li></ul><ul><li>Red haemopoietically active marrow is replaced by resting yellow marrow. </li></ul><ul><li>Active haemopoiesis continues throughout life in epiphysis of long bones and all flat bones; sternum, ribs, pelvis, vertebrae and skull </li></ul>
  14. 14. <ul><li>Bone marrow consists of various haemopoietic cells and their precursors, reticular cells and reticular fibers, endothelial lined sinusoids and adipocytes. Yellow marrow is limited to three types of cells; reticular cells, connected with endosteum and blood capillaries, endothelial cells of capillary walls and sinusoids and adipocytes </li></ul>
  15. 15. <ul><li>Adipocytes occupy space as haemopoiesis recedes and give up space as the demand for expansion of red marrow occurs in response to continuous blood loss or haemolytic anemia. Transition from yellow to red marrow takes place in response to the hormone erythropoietin </li></ul>
  16. 16. <ul><li>Figure 1: Sequence and contribution of haemopoietic organs to prenatal blood cell formation in cats </li></ul>
  17. 17. <ul><li>Bone with central vein, sinuses, haemopoietic space </li></ul>Cortical bone Trabecula bone Adventitial process Vascular sinus Haemopoietic space Central vein
  18. 18. <ul><li>Transit cells from bone marrow to blood </li></ul><ul><li>Thin walls of sinuses lined by fenestrated endothelium </li></ul><ul><li>Cells slide out of adventitial tissue into sinus in response </li></ul><ul><li>Endothelial cells have many organelles, show endocytotic activity </li></ul><ul><li>Maturing cells egress across sinus wall trans-cellularly, not intercellular </li></ul><ul><li>Cell migration selective for maturity </li></ul>
  19. 19. <ul><li>Terminal stage of erythrocyte maturation occurs next to venous sinus wall </li></ul><ul><li>Metarubricytes and reticulocytes press against sinus wall, nucleus faces sinus and is extruded and phagocytosed into endothelium, then to sinus </li></ul><ul><li>Reticulocytes held in bone marrow because surfaces are stick due to transferrin and sialic acid </li></ul>
  20. 20. <ul><li>On maturation stick substances diminish </li></ul><ul><li>Erythropoietin enhances erythropoiesis and accelerates delivery of reticulocytes into circulation </li></ul><ul><li>Bone marrow diseases; granulomatous inflammation, tumor metastases, leukemia, result in release of nucleated erythrocytes </li></ul>
  21. 21. <ul><li>Leukocytes produced from interior of bone marrow move into sinus by own </li></ul><ul><li>Leukocytes release into circulation affected by surface charge, which decreases with cell maturation, & hormonal factors (neutrophil releasing factor) </li></ul>
  22. 22. <ul><li>Excessive need for granulocytes leads to release of mature and immature neutrophils into circulation (shift to left) </li></ul><ul><li>Breakage of sinus wall leads to massive immature and mature granulocytes released into circulation </li></ul>
  23. 23. <ul><li>Megakaryocytes are near sinus wall or where wall is lacking </li></ul><ul><li>They shed platelets into sinus lumen. Platelets begin as extensions (proplatelets) through sinus wall </li></ul><ul><li>Megakaryocytes also enter circulation, shed their platelets </li></ul>
  24. 24. Hemopoiesis in avian bone marrow <ul><li>Erythopoiesis and thrombopoiesis occur intravascularly in sinuses while granulopoiesis takes place extravascularly </li></ul><ul><li>Developing granulocytes contain eosin colored granules common in heterophils </li></ul><ul><li>Sinus walls lined by cells lacking basement membrane </li></ul><ul><li>Cells continuous, but open at places where granulocytes pass through </li></ul>
  25. 25. <ul><li>Immature erythroid cells adhere to sinus wall </li></ul><ul><li>Mature cells with hemoglobin occur more in center of sinus </li></ul><ul><li>No megakaryocytes in avian marrow, instead have nucleated cell lines producing nucleated thrombocytes </li></ul><ul><li>Hemopoietic population of bursa of Fabricius, bone marrow, thymus derived from blood-borne extrinsic stem cells of yolk sac origin </li></ul>
  26. 26. Hemopoietic stem cells <ul><li>Stimulants of hemopoiesis </li></ul><ul><li>Low erythrocyte numbers (hemorrhage or hemolysis) </li></ul><ul><li>Increased utilization or destruction of neutrophils (inflammation) </li></ul><ul><li>Increased destruction of platelets (immune mediated thrombocytopenia) </li></ul>
  27. 27. <ul><li>Stimulatory feedback mechanism exists for each cell </li></ul><ul><li>Compartment of primitive stem cells which responds to stimuli exist in bone marrow </li></ul>
  28. 28. <ul><li>There is structural hierarchy of multipotential, oligopotential and unipotential stem cells of formed elements of blood </li></ul><ul><li>Most primitive pluripotential hemopoietic stem cells are capable of differentiating into oligopotential progenitor cells (cells producing progenitors of two or more cell lines) </li></ul>
  29. 29. <ul><li>Oligopotential progenitor cells in turn differentiate into unipotential cells committed to produce single lineage under stimuli </li></ul><ul><li>Stem cells are lymphocytic like </li></ul><ul><li>Unipotential progenitor cells develop into morphologically recognizable precursor cells such as rubriblasts, myeloblast megakaryoblast, which give rise to mature cells under specific stimuli </li></ul>
  30. 30. <ul><li>There exist subsets of the pluripotential stem cells and committed progenitor cells of various cell lineages </li></ul><ul><li>Myeloid and lymphoid cells originate separately but from a common progenitor cell </li></ul><ul><li>Erythrocytes, all leukocytes, macrophages, mast cells and megakaryocytes originate from pluripotential stem cell, pluripotential hemopoietic stem cell, capable of differentiation into myeloid stem cell and lymphoid stem cell. </li></ul>
  31. 31. <ul><li>Myeloid and lymphoid stem cells in turn give rise to committed cell units called colony forming units (CFU) leading to specific series that produce mature cells </li></ul>
  32. 32. Pluripotential Stem Cell Myeloid stem cell Lymphoid stem cell
  33. 33. Myeloid stem cell CFU-MEG CFU-E CFU-GM CFU-EOS CFU-BAS Megakaryocyte Rubriblast Monoblast Myeloblast Myeloblast Myeloblast Platelets Erythroblasts Monocyte/Macrophage Neutrophils Eosinophils Basophils
  34. 34. Lymphoid stem cell T- Cell precursors B- Cell precursors Lymphoblast Lymphoblast T-lymphocyte B-lymphocyte/plasma cell
  35. 35. Pluripotential stem cell <ul><li>Primitive mesenchymal cell from yolk sac endoderm </li></ul><ul><li>Seeds liver spleen, bone marrow </li></ul><ul><li>In adults pluriotential hemopoietic stem cells found in bone marrow </li></ul><ul><li>Mobilized by dextran sulfate injection </li></ul>
  36. 36. <ul><li>Migration of granulocytic progenitor cells into blood induced by exercise, ACTH, dexamethasone, epinephrine, endotoxin, antigens, hypoxia, radiation, neoplasm; erythroleukemia, essential thrombocythemia, polycythemia vera </li></ul><ul><li>Or non-neoplastic disorders; cyclic hematopoiesis (gray collie dogs), red cell aplasia, aplastic anemia </li></ul>
  37. 37. Committed Hemopoietic cells <ul><li>Committed unipotential progenitor cells an intermediate stage between primitive multipotential stem cell and differentiated blast cell of specific series </li></ul>
  38. 38. <ul><li>These stages explains the selective depression of specific cell series by some chemical agents. Vinblastine suppresses erythropoiesis and granulopoiesis but not megakaryopoiesis </li></ul><ul><li>Both multipotential and unipotential stem cell compartments are self perpetuating </li></ul>
  39. 39. <ul><li>Stem cells are resting, require serious insult to hemopoietic tissue to increase their mitotic activity to provide more progenitor cells </li></ul><ul><li>Neutrophils, monocytes and macrophages have a common progenitor, the colony forming unit – granulocyte-macrophage (CFU-GM). </li></ul>
  40. 40. <ul><li>Erythrocytes come from two progenitors </li></ul><ul><li>1. Burst forming unit – erythroid (BFU-E) </li></ul><ul><li>2. Colony forming unit-erythroid (CFU-E). BFU-E is progenitor of CFU-E </li></ul><ul><li>BFU-E produces large erythroid colonies in large amounts of erythropoietin or humoral factor called burst promoting activity (BPA). CFU-E forms small colonies in 48 hours in a small amount of erythropoietin </li></ul>
  41. 41. <ul><li>Progenitor for megakaryocytes is colony forming unit-megakaryocyte (CFU-M or CFU-Meg) </li></ul><ul><li>Progenitors for eosinophils, basophils and mast cells are colony forming unit-eosinophil (CFU-EOS) and colony forming unit-basophil (CFU-BAS). </li></ul>
  42. 42. Factors regulating hemopoiesis <ul><li>Both endogenous and exogenous </li></ul><ul><li>Microenvironment and humoral substances that stimulate or suppress proliferation of single or multiple cell lineages </li></ul><ul><li>Interleukin 3 (IL-3) is multispecific growth factor that stimulates early progenitor cells </li></ul>
  43. 43. <ul><li>Erythropoietin acts on differentiated cell lineage of erythropoietic series </li></ul><ul><li>Other specific poietins; thrombopoietin, eosinophilopoietin, granulocyte poietin monocytopoietin, lymphocyte mitogenic factor, T cell growth factor (IL-1) </li></ul>
  44. 44. <ul><li>Stem cell differentiation is stimulated by hemopoietic inductive microenvironment </li></ul><ul><li>Erythropoiesis is stimulated by </li></ul><ul><li>Burst promoting activity (IL-3) </li></ul><ul><li>Erythropoietin </li></ul><ul><li>Certain lymphocyte interleukins </li></ul><ul><li>Macrophages </li></ul><ul><li>Prostaglandin E (PGE 1 ) and PGE 2 , PGI 2 </li></ul>
  45. 45. <ul><li>Erythroblast enhancing factor </li></ul><ul><li>Erythropoietic stimulating Cofactor </li></ul><ul><li>Androgen </li></ul><ul><li>Corticosteroids </li></ul><ul><li>Growth hormone </li></ul><ul><li>Thyroxine </li></ul><ul><li>Cobalt </li></ul><ul><li>GM-colony stimulating factor </li></ul>
  46. 46. <ul><li>Erythropoiesis is inhibited by </li></ul><ul><li>Estrogen </li></ul><ul><li>A-factor in urine </li></ul><ul><li>A-factor in plasma </li></ul><ul><li>Lithium </li></ul><ul><li>Feline leukemia virus </li></ul><ul><li>Suppressor T-lymphocytes (CD8+) </li></ul>
  47. 47. <ul><li>Granulopoiesis is stimulated by </li></ul><ul><li>GM-colony stimulating factor (GM-CSF) CSF 2 , 23kd glycoprotein </li></ul><ul><li>G-colonony stimulating factor (G-CSF) 24 kd glycoprotein </li></ul><ul><li>Colon stimulating factor–1 or M-CSF (Macrophage-CSF) 40–60kd glycoprotein </li></ul><ul><li>Antichalone </li></ul><ul><li>Lymphocyte interleukins ? </li></ul><ul><li>Eosinophilopoietin </li></ul><ul><li>Basophilopoietin </li></ul><ul><li>Lithium </li></ul><ul><li>PGI 2 </li></ul><ul><li>Conticosteroids </li></ul>
  48. 48. <ul><li>Granulopoiesis is inhibited by </li></ul><ul><li>Colony – inhibiting factor </li></ul><ul><li>Chalone </li></ul><ul><li>Lactoferrin – transferrin </li></ul><ul><li>Acidic isoferritins </li></ul><ul><li>Lymphocyte interleukins </li></ul><ul><li>PGE 1 PGE 2 </li></ul><ul><li>Macrophage products (IL-12?) </li></ul>
  49. 49. <ul><li>Monocytopoiesis is stimulated by </li></ul><ul><li>M-Colony stimulating factor </li></ul><ul><li>Monocytopoietin </li></ul><ul><li>  </li></ul><ul><li>Monocytopoiesis is inhibited by </li></ul><ul><li>PGE 1 and PGE 2 </li></ul><ul><li>Conticosteroids </li></ul><ul><li>A serum inhibitor </li></ul>
  50. 50. <ul><li>Megakaryocytopoiesis and thrombocytopoiesis is stimulated by </li></ul><ul><li>Meg-Colony stimulating factor </li></ul><ul><li>Thrombocytopoetin </li></ul><ul><li>Iron, lithium, a factor in bovine bile </li></ul><ul><li>  </li></ul><ul><li>Megakaryocytopoiesis and thrombocytopoiesis is inhibited by </li></ul><ul><li>A splenic factor </li></ul><ul><li>Iron in different amounts  </li></ul>
  51. 51. <ul><li>Lymphopoiesis is stimulated by </li></ul><ul><li>Specific microenvironment </li></ul><ul><li>Thymic hormones </li></ul><ul><li>Antigens </li></ul><ul><li>IL-I and IL-3 </li></ul><ul><li>Lymphocyte interleukins such as IL-2, B-cell growth factor, B-cell differentiation factor. </li></ul><ul><li>Bone marrow is engaged primarily in granulopoiesis, while the spleen takes part in lymphopoiesis and erythropoiesis. </li></ul>
  52. 52. Regulation of Committed Progenitor Cells <ul><li>committed progenitor cells of the erythrocyte, granulocyte-macrophage, eosinophil, and megakaryocyte are regulated by specific stimulations, which are glycoproteins containing neuraminic acid. Erythroid progenitor cells are stimulated by erythropoietin. Differentiation of pluripotential stem cell into BFU-E is enhanced by BPA and T-cell product’s IL-I, IL-3 </li></ul>
  53. 53. <ul><li>Soluble substances of macrophages, probably IL-12, stimulate erythrocyte production by promoting growth in early (BFU-E) and late (CFU-E) committed erythroid progenitors and through cell to cell contacts </li></ul>
  54. 54. <ul><li>Production of B– and T-lymphocytes is influenced by specific microenvironment, regulated by humoral factors specific for T- or B- types; including thymic hormones, interleukin 1, IL-2, IL-12. </li></ul><ul><li>IL-1, a 12-16 kDa non protein cell secretion increases proliferation of both T- and B-lymphocytes. IL-2, a 15-26 kDa molecule is essential for proliferation of activated T-lymphocytes </li></ul>
  55. 55. <ul><li>IL-3, a 28 kDa cell secretion induces maturation of T-cells and differentiation of many myeloid cells </li></ul><ul><li>B-cell growth factor stimulates proliferation of activated B-lymphocytes </li></ul><ul><li>B-cell differentiation factors act at terminal stage of B-cell differentiation to increase synthesis and release of immunoglobulins </li></ul><ul><li>Corticosteroids stimulate lymphopoiesis, but are also lympholytic </li></ul>
  56. 56. Lymphoid Tissues <ul><li>Lymphatic tissues constitute the lymphoid organs and accumulations of lymphoid cells in loose connective tissues of the body </li></ul><ul><li>Thymus and bursar of Fabricius (birds) are primary lymphoid organs </li></ul><ul><li>Parenchyma consists of diffuse lymphoid tissues containing T-(thymus) and B- (bursa, bone marrow) lymphocytes </li></ul><ul><li>Secondary lymphoid organs (spleen, lymph nodes, hemal nodes hemal lymph nodes, tonsils, Peyer’s patches and gut associated lymphoid tissues contain nodular and diffuse lymphoid tissues. </li></ul>
  57. 57. <ul><li>Diffuse tissue consists of T-lymphocytes and nodular tissue B-lymphocytes </li></ul><ul><li>Stroma of secondary lymphoid tissues is rich in reticular fibers and phagocytic reticular cells (macrophages). </li></ul><ul><li>Primary lymphoid organs are well developed at birth </li></ul><ul><li>Secondary lymphoid organs develop fully after birth parallel with immuno-competence </li></ul><ul><li>Antigenic exposure stimulates lymphopoiesis </li></ul>
  58. 58. Erythropoiesis <ul><li>Erythron is the functional unit constituting the mass of circulating erythrocytes and the erythropoietic tissue in the bone marrow </li></ul><ul><li>Change of the erythron above (polycythemia) or below (anemia) may be relative or absolute and is caused by physiological or pathological factors </li></ul><ul><li>Daily erythrocyte production balance those lost by destruction and age </li></ul>
  59. 59. <ul><li>Erythrocyte production </li></ul><ul><li>  A lymphocyte-like primitive undifferentiated stem cell exists in bone marrow, pluripotential stem cell ( CFU-S ), gives rise to unipotential cells that commit to erythrocytic, granulocytic, monocytic or megakaryocytic series </li></ul>
  60. 60. <ul><li>Erythropoiesis begins when pluripotential stem cells differentiate into two erythroid progenitor cells, burst forming unit-erythroid ( BFU-E ) and its progeny, colony forming unit –erythroid ( CFU-E ), which under erythropoietin (Ep) gives rise to erythroid precursors, rubriblasts </li></ul><ul><li>Rubriblasts undergo 4-5 mitotic divisions to produce mature erythrocytes </li></ul>
  61. 61. <ul><li>Rubriblast is earliest recognizable immature cell of erythrocytic series </li></ul><ul><li>Divides and its daughter cells divide and mature to erythrocytes (sequential mitotic divisions and maturation of daughter cells in series) </li></ul><ul><li>In mitotic compartment the rubriblast undergoes four divisions to become polychromatic rubricyte, which gives rise to metarubricyte </li></ul>
  62. 62. <ul><li>These divisions occur at each stage of rubriblast, prorubricyte, basophilic rubricyte and polychromatic rubricyte </li></ul><ul><li>Metarubricytes do not divide </li></ul><ul><li>Sequential maturation involves loss of nucleoli and mitotic capacity, progressive decrease in size and cytoplasmic basophilia and increase in nuclear chromatin condensation, chromasia leading to normochromasia with hemoglobin synthesis </li></ul>
  63. 63. CFU-STEM BFU-E CFU-E Rubriblast series Erythrocyte Erythropoietin low conc. Erythropoietin low conc. Erythropoietin high conc. Macrophage factor Cell-cell interaction, microenvironment, Burst forming activity, macrophage products, lymphocyte interleukin 1,2,3
  64. 64. <ul><li>Fig: An erythrobastic island having corona of erythroid cells, encircling a reticulum cell (nurse cell) where hemosiderin occurs in its cytoplasm. Note nucleoli of early erythroid series and nucleus of reticulum cell (NRC). </li></ul>NRC Erythroid cells
  65. 65. <ul><li>BFU-E proliferation is enhanced by T-cell growth factors (IL-I) </li></ul><ul><li>Proliferation of late erythroid precursor cells in vitro and in vivo is stimulated by erythropoietin, erythroblast enhancing factor and erythropoietic stimulating cofactor and corticosteroids </li></ul>
  66. 66. <ul><li>Cytoplasm of reticulum cell contains hemosiderin granules and interdigitates between erythroid cells, thus nourishing them (nurse cell) providing ferritin for use in heme synthesis and may be a stimulant for further rubriblasts </li></ul><ul><li>Maturing erythroid cell move away from reticulum cell to become reticulocytes. Reticulum cells also are phagocytic, engulfing free nuclei of metarubricytes and aged red cells </li></ul>
  67. 67. <ul><li>Rubriblast (proerythroblast, pronormoblast) </li></ul><ul><li>Largest of erythroid series , round to oval, with narrow rim of cytoplasm, 15-20  m in diameter </li></ul><ul><li>Non granular deep blue cytoplasm with or without lightly stained peri or paranuclear zone of Golgi complex </li></ul><ul><li>Central nucleus is round, 1-2 nucleoli or nuclear rings </li></ul>
  68. 68. <ul><li>Nuclear chromatin finely stippled, staining slightly more purple than immature granulocytes, with reddish tinge </li></ul><ul><li>Rubriblasts, circular, cytoplasm and nuclei stain deeper than myeloblasts because of higher DNA and RNA content </li></ul>
  69. 69. <ul><li>Prorubicyte (basophilic erythroblast, early erythroblast, early normoblast) </li></ul><ul><li>Morphology similar to rubriblast except its lack of nucleoli </li></ul><ul><li>May be larger than rubriblast, highly basophilic cytoplasm, light chromatin condensation. </li></ul>
  70. 70. <ul><li>Basophilic rubricyte (basophilic erythroblast, basophilic normoblast) </li></ul><ul><li>Smaller than prorubricyte </li></ul><ul><li>Condensed coarse granular nuclear chromatin and moderate to intense blue cytoplasm. Nuclear chromatin separated by light streaks (cart wheel appearance) </li></ul><ul><li>Nucleoli absent in this and subsequent stages </li></ul><ul><li>Cytoplasmic colour changes from narrow rim of deep blue to gray orange, low RNA, increase of haemoglobin </li></ul>
  71. 71. <ul><li>Polychromatic rubricyte (early polychromatic erythroblast, normoblast) </li></ul><ul><li>Smaller than basophilic rubricyte </li></ul><ul><li>Grayish cytoplasm </li></ul><ul><li>Increasing chromatin clumping </li></ul><ul><li>Small nucleus than previous stage </li></ul><ul><li>Synthesis of hemoglobin is advanced </li></ul><ul><li>Nucleus has dark blobs of chromatin separated by light streaks. </li></ul>
  72. 72. <ul><li>Normochromic (Orthochromic) rubricyte </li></ul><ul><li>Stage in cats and horses </li></ul><ul><li>Nucleus remains viable </li></ul><ul><li>Cytoplasm stains similar to mature erythrocyte </li></ul>
  73. 73. <ul><li>Metarubricyte (Late polychromatic erythroblast or normoblast) </li></ul><ul><li>Smallest of nucleated erythrocytic series </li></ul><ul><li>Nucleus highly condensed, non viable, solid black pycnotic, or fragmented, partially extruded or partially autolyzed </li></ul><ul><li>Nuclear staining is homogenous deep purple, with few light streaks and spots </li></ul><ul><li>Cytoplasm grayish, polychromatic or normochromic depending on hemoglobin content. </li></ul>
  74. 74. <ul><li>Reticulocyte </li></ul><ul><li>Polychromatic red cell maturing to an erythrocyte. When metarubricyte expels its nucleus it skips reticulocyte stage, giving rise to giant red cell (macrocyte) </li></ul><ul><li>Reticulocytes are non-nucleated </li></ul><ul><li>When stained with new methylene blue they present some granules or diffuse network of fibrils </li></ul>
  75. 75. <ul><li>With Romanowsky stains the reticulocyte is polychromatophilic and may contain nuclear remnants called Howell-Jolly bodies. </li></ul><ul><li>Erythrocyte (red blood cell) </li></ul><ul><li>Erythrocyte is non-nucleated (mammals) or nucleated (birds, reptiles, fish, amphibians) definitive stage of erythropoietic series </li></ul>
  76. 76. Megakaryocytopoiesis and thrombopoiesis <ul><li>Mammalian platelets are cytoplasmic fragments of megakaryocytes not nucleated </li></ul><ul><li>Thrombocytyes of lower vertebrates are nucleated platelets, originate from successive divisions of precursor cells called thromboblasts within bone marrow sinusoids. </li></ul>
  77. 77. <ul><li>Megakaryocytes are giant cells in bone marrow which give rise to platelets </li></ul><ul><li>Megakaryocytes arise from self replicating pluripotential stem cell (PPSC) in bone marrow </li></ul><ul><li>PPSC gives rise to megakaryocyte progenitor cell, Colony Forming Unit Megakaryocyte (CFU-Meg) </li></ul><ul><li>CFU-Meg divides to give rise to (1) self perpetuating CFU-Meg and (2) Megakaryocyte precursors, with several intermediates </li></ul>
  78. 78. <ul><li>Factors that influence divisional differentiation of PPSC to CFU-Meg </li></ul><ul><li>Haematopoietic microenvironment </li></ul><ul><li>Cell-cell interactions </li></ul><ul><li>Short range humoral factors </li></ul>
  79. 79. <ul><li>Regulation of differentiation of CFU-Meg into megakaryoblast </li></ul><ul><li>Colony Stimulating Factor (Meg-CSF) </li></ul><ul><li>Thrombopoietin (required for development of megakaryocytes) </li></ul><ul><li>  Megakarycytopoiesis involves polyploidization of precursor cells due to endoreduplication (nuclear division without cytoplasmic division). </li></ul>
  80. 80. <ul><li>Megakaryocytopoiesis passes six stages to final platelet productive form </li></ul><ul><li>Pluripotential Stem Cell </li></ul><ul><li>Progenitor cell (CFU-Meg) </li></ul><ul><li>Megakaryoblast </li></ul><ul><li>Promegakaryocyte </li></ul><ul><li>Mature megakaryocyte </li></ul><ul><li>Productive megakaryocyte </li></ul>
  81. 81. Progenitor cell Megakaryoblasts Promegakaryocyte
  82. 82. <ul><li>Mature megakaryocyte </li></ul><ul><li>Productive megakaryocyte </li></ul>
  83. 83. <ul><li>  Cytomorphology of megakaryocytopoiesis </li></ul><ul><li>Largest haematopoietic cells (4.7x10 -13 l or 4,700 fl) volume, 10-65 μm diameter </li></ul><ul><li>Megakaryocytes classified into 3 types, stages or groups based cytoplasm and nucleus; megakaryoblast (Stage I or Group I) cell, promegakaryocyte (Stage II or Group II) cell and megakaryocyte (Stage III) cell. Megakaryocytes are categorized as granular (Stage III) and mature or productive (Stage IV). </li></ul>
  84. 84. Ancestors and progenies of haematopoietic cell series Recognizable cell series 1. Pluripotential Stem Cell 2. Unipotential stem cell 2. Unipotential stem cell 3. Myeloblast 4. Promyelocyte 5. Neutrophil myelocyte 6. Neutrophil metamyelocyte 7. Neutrophil stab cell (band) 8. NEUTROPHIL 3. Myeloblast 5. Eosinophil/Basophil myelocyte 8. EOSINOPHIL/BASOPHIL - 6. Eosinophil/Basophi metamyelocyte 7. Eosinophil/Basophi stab cell/band
  85. 85. <ul><li>Generation </li></ul>1 3 4 5 6 7 8 2 Recognizable cell series Pluripotential Stem Cell Unipotential stem cell Unipotential stem cell Proerythroblast Early normoblast Intermediate normoblast Late normoblast Metarubricyte Reticulocyte ERYTHROCYTE Lymphoblast Prolymphocyte LYMPHOCYTE
  86. 86. Recognizable cell series 1. Pluripotential Stem Cell 2. Unipotential stem cell 2. Unipotential stem cell 3. Megakaryoblast 4. Promegakaryocyte 5. Early megakaryocyte 6. Mature megakaryocyte 7. Productive megakaryocyte 8. PLATELET (Thrombocyte) 3. Monoblast 5. - 8. MONOCYTE 4.- 6. - 7. -
  87. 87. Terminology in erythrocytic series <ul><li>Pluripotential stem cell (CFU-S) </li></ul><ul><li>Burst forming unit-erythroid (BFU-E) </li></ul><ul><li>Colony forming unit –erythroid (CFU-E) </li></ul><ul><li>Rubriblast (proerythroblast, pronormoblast) </li></ul><ul><li>Prorubicyte (basophilic erythroblast, early erythroblast, early normoblast) </li></ul>
  88. 88. <ul><li>Basophilic rubricyte (basophilic erythroblast, basophilic normoblast) </li></ul><ul><li>Polychromatic rubricyte (early polychromatic erythroblast, normoblast) </li></ul><ul><li>Normochromic (Orthochromic) rubricyte </li></ul><ul><li>Metarubricyte (Late polychromatic erythroblast or normoblast) </li></ul><ul><li>Reticulocyte </li></ul>
  89. 89. <ul><li>Fig. 2: Human bone marrow: Red bone marrow shows great cellularity, developing erythrocytic and myelocytic cells and large round clear fat cells. A megakaryocyte (a), larger venous sinuses (d), bone (b), periosteum (c). HE x26 </li></ul>
  90. 90. <ul><li>Fig. 3: Rabbit bone marrow: Red bone marrow showing great cellularity, developing erythrocytic and myelocytic cells, sinusoids (b), large clear fat cells. Megakaryocytes are largest (except for fat cells) in bone marrow with abundant cytoplasm and multilobulated nuclei (a). HE x 52 </li></ul>
  91. 91. <ul><li>Fig. 4: Rabbit bone marrow: R abbit injected intraperitoneal with lithium carmine (red). Reticular cells around sinuses are phagocytic, have actively ingested red dye. Phagocytized dye in their cytoplasm follows elongated cytoplasmic processes which form sinusoidal lining (arrows). HE x 100 </li></ul>
  92. 92. <ul><li>Fig. 5: Bone marrow smear shows developing cells differing in sizes, nuclear shapes, contour and chromatin pattern, cytoplasm for basophilia and presence or absence of granules. Wrights – Giemsa x 200 </li></ul>
  93. 93. <ul><li>Fig. 6: Human bone marrow: Reticular cell (f) makes basic cellular stroma of bone marrow. Macrophage (a) contains phagocytized cellular debris in cytoplasm . </li></ul><ul><li>Others are lymphocyte (b), early normoblast or polychromatophilic erythroblast (e), mature neutrophil (d), damaged cell (c) with distorted nucleus, no cytoplasm. Wrights-Giemsa x 400 </li></ul>
  94. 94. <ul><li>Fig. 7a: Human bone marrow: Early neutrophilic promyelocytes (a), late promyelocyte (b) </li></ul>
  95. 95. <ul><li>Fig. 7b: Human bone marrow: neutrophilic myelocytes (d, e), neutrophilic metamyelocytes (c, h, i), band neutrophil (g), lymphocyte (f) with few azurophil granules. Wrights-Giemsa x 400 </li></ul>
  96. 96. <ul><li>Fig. 8: Human bone marrow: Small neutrophilic promyelocyte (g), neutrophilic metamyelocyte (a, b, c, e), Wrights-Giemsa x 400 </li></ul>
  97. 97. <ul><li>Fig. 8: Human bone marrow: Neutrophilic metamyelocyte (f) band neutrophil (d) and two lobed mature neutrophil (h). Wrights-Giemsa x 400 </li></ul>
  98. 98. <ul><li>Fig. 9: Human bone marrow: Mature neutrophils (b, c), mature eosinophil (d), an eosinophilic myelocyte (f) and basophilic myelocyte (a) Wrights-Giemsa x 400 </li></ul>
  99. 99. <ul><li>Fig. 9: Human bone marrow: Late promyelocyte or early neutrophilic myelocyte (g), two metamyelocytes (e, h) and early normoblast (i). Wrights-Giemsa x 400 </li></ul>
  100. 100. <ul><li>Fig. 10: Human bone marrow: Very early neutrophilic promyelocyte (a) is very basophilic & chromatophilic, proerythroblast (c). Wrights-Giemsa x 400 </li></ul>
  101. 101. <ul><li>Fig. 10: Human bone marrow: Band neutrophil (b), neutrophilic metamyelocytes (e), late neutrophilic myelocyte (d), normoblast (f). Wrights-Giemsa x 400. </li></ul>
  102. 102. Fig. 11: Human bone marrow: Lymphocyte (a), mature neutrophil (b), normoblast (c), band neutrophil (d), normoblast (e), W-Giemsa x 400
  103. 103. Fig. 11: Human bone marrow: Neutrophilic, metamyelocytes (f, g), mature eosinophil (h), neutrophlic myelocyte (i), eosionophilic myelocyte (j), lymphocyte (k). W-Giemsa x 400
  104. 104. Fig. 11: Human bone marrow: Neutrophilic myelocyte (m, n), lymphocytes (o), late neutrophilic metamyelocyte (p), neutrophilic myelocyte (q), monocyte (r). W-Giemsa x 400
  105. 105. <ul><li>Fig. 12: Human bone marrow: Early erythroblasts (a), late erythroblast (c), and normoblast (b). Wrights-Giemsa x 400. </li></ul>
  106. 106. <ul><li>Fig. 13: Human bone marrow: Normoblasts (b, d, f) differing in sizes and degree of chromatin clumping, late erythroblasts ( c). W-G x 400. </li></ul>
  107. 107. <ul><li>Fig. 13: Human bone marrow: Neutrophilic myelocyte (h), metamyelocyte (a), and mature (e) and band (g) neutrophil. W-G x 400. </li></ul>
  108. 108. <ul><li>Fig. 14: Human bone marrow: A megakaryocyte shedding its cytoplasm to form platelets (arrow). W-G x 400. </li></ul>
  109. 109. <ul><li>Blood supply to bone marrow </li></ul><ul><li>A nutrient artery system extends through the long axis, giving off radial branches, which terminate peripherally to form vascular sinuses. </li></ul><ul><li>Sinuses carry blood back to a central vein. </li></ul>
  110. 110. <ul><li>The thin walled vascular sinuses originate at periphery from terminal branches of nutrient artery, running transversely towards center. </li></ul><ul><li>Adventitial processes project into haemopoietic spaces, producing partial compartmentalization . </li></ul>
  111. 111. <ul><li>In mammals haemopoietic marrow lies between the broad radial sinuses. </li></ul><ul><li>Total bone marrow in the widely distributed bones constitutes an organ two-thirds the size of the liver </li></ul>