Haemopoesis Erythropoesis

Stages of erythropoesis, it's regulation, factors affecting it.

1. ERYTHROPOIESIS AND ITS REGULATION AND FUNCTIONS DR.PRIYANKA VERMA MBBS,MD PHYSIOLOGY DEPARTMENT OF PHYSIOLOGY LNCT MEDICAL COLLEGE INDORE

2. Red Blood Cells (Mature Erythrocytes) Each red blood cell (RBC) - bounded by a cell membrane N on-nucleated Has no nucleus , no mitochondria and no ribosome . The cytoplasm of the RBC contains a special pigmented protein called the haemoglobin. ( Hb) - 90 % of the weight of the erythrocytes. The red color of the RBCs and blood is due to - Hb .

3. Normal size Diameter - 7.2 μm (range 6.9–7.4 μm ), Thickness - Periphery - 2 μm - Centre - 1 μ m, Surface area - 120–140 μm² Volume - 80 μm³ (range 78–86 μm³) NORMAL SIZE, SHAPE AND COUNTS OF RBCS

4. Normal shape The RBCs are circular, biconcave discs Advantages of biconcave shape are : Allows considerable change in cell volume. Thus, can withstand considerable changes of osmotic pressure and resist hemolysis. Allows easy folding of RBC on itself when it passes through capillaries . Haemoglobin remains distributed in the center of the RBC which facilitates optimal and quick exchange of gases (ie. oxygen and carbon dioxide).

5. Composition 62.5 % water 35 % Haemoglobin (29.5 ± 2.5 pg / RBC) Sugar - glucose Lipids - cephalin, cholesterol and lecithin Protein - Glutathione , albumin like insoluble protein , acts as a reducing agent, thus prevents damage of haemoglobin. Enzymes of glycolytic system ; carbonic - anhydrase and catalase. I ons - Na ⁺ , K ⁺ , Ca²⁺, PO ₄³⁻ and SO ₄ ²⁻

6. RED CELL MEMBRANE Structure Erythrocytes are covered by cell membrane. Below the cell membrane structural proteins - Spectrin , Ankyrin , Actin and other proteins are present. These proteins are arranged in a mesh like pattern.

7. Schematic diagram showing ultra structure of red cell membrane.

8. They are responsible for Flexibility of RBC membrane Maintain the biconcave shape of RBC provides membrane stability and durability to the red cells Contain specific blood group antigen like A or B.

9. Common membrane defects: Therefore , deficiency of ankyrin and spectrin results in membrane defects like spherocytosis, elliptocytosis and poikilocytosis , etc. These defects also make the cells rigid so that the cells are destroyed prematurely.

10. Permeability Semi-permeable membrane Impermeable to sodium, calcium and barium ions, fats and sugars, Slightly impermeable to amino acids Freely permeable to all anions like Cl ⁻ , SO ₄⁻ and HCO₃⁻ , and to urea, ammonia, aldehyde, alcohol and bile salts.

11. Energy Sources Energy comes from Glycolysis (80%) and the HMP shunt pathway (20%). It is required for the RBC to maintain Ionic gradient across its membrane, Volume and To keep iron in fe ⁺⁺ state. Since mitochondria are absent, there is no citric acid cycle and electron carrier system in the RBC.

12. PROPERTIES Suspension stability Rouleux formation Sedimentation Fragility

13. Normal counts At birth : 6-7 million/ μ L Adults - Male: 5-6 million/ μ L (average 5.5 million/ μ L) Females: 4.5-5.5 million/ μ L (average 4.8 million/ μ L) Clinically , a count of 5 million/ μL is considered as 100 %.

14. VARIATION IN RBC COUNT Physiological Increase High altitude: RBC count increases to more than 7 million/mm³ due to hypoxia. Newborn and infant: High RBC counts due to ↑ sed activity of the bone marrow which continues after birth.

15. Muscular exercise: RBC increases due to: Stored pool entering the circulating pool. Decreased plasma volume (haemoconcentration). Sex: - Male - androgens increase EP from the kidney. - Female- oestrogen does not stimulate EP production , hence the sex variation .

16. 2. Physiological Decrease Children - RBC counts are low, compared to newborn and young adults due to low production . Pregnancy- less RBC count due to haemodilution . Old age – production of RBC is less and so the RBC count is 4.5 million/mm³.

17. 3. Pathological Increase Polycythaemia vera- RBC count increases to 8 million/ mm³ or above. malignancies of the bone marrow. Congenital heart disease - RBC increase due to hypoxia as there is a mixing of venous blood with arterial blood. Chronic lung disease - RBC increase due to hypoxia .

18. Leukemia - Increased RBC due to an increase activity of bone marrow. Cushing syndrome - Production is high. EP production increases due to cortisol. Hyperthyroidism - Production is high. EP production increases due to higher levels of T₃ and T ₄.

19. VARIATION IN SIZE Normocytes:- When the size is 7.2 μm . Microcyte:- The size of the RBC is smaller than normal. Microcytes are seen in : Iron deficiency anaemia Chronic infections Thalassemia Increased osmotic pressure in blood .

20. Macrocytes: These are bigger RBC: Megaloblastic anemia Decreased osmotic pressure in blood Anisocytosis: RBC with unequal size are called anisocytes . These are seen in pernicious anemia.

21. VARIATION IN STRUCTURE Punctate basophilia: Small dots of basophilic material in the cytoplasm. Cabot's ring: Ring shape or figure of '8' material appears in the cell. Howell Jolly bodies: Small nuclear fragments are present in the cytoplasm. All the above are seen in lead poisoning . Siderocyte : RBC containing nonhaem iron granules in the cytoplasm are called siderocytes. seen in lead poisoning and splenectomized persons .

22. VARIATION IN SHAPE Crenation : [hypertonic solution]. Spherocytosis : [hypotonic solution]. Elliptocytosis : elliptical . Sickle cell: Crescentic shape Poikilocytosis : Presence of different shaped cells [sickle cell anaemia].

23. FUNCTIONS OF RBC O ₂ transport . RBC transports CO ₂ . RBC transport nitric oxide (NO ) and release in the tissue where NO causes vasodilatation. RBC participate in acid-base balance. RBC contribute to the viscosity of blood . RBC influence erythrocyte sedimention rate . inverse relationship. Antigens of the RBC membrane are useful for identification of blood groups .

24. Haemopoesis

25. Blood cell production is referred to as haemopoiesis. This includes : a ) Erythropoiesis (RBC production ) b ) Leucopoiesis (WBC production ) c ) Thrombopoiesis ( Platelet production ) Two theories have been proposed: Monophyletic theory Polyphyletic theory

26. Monophyletic theory: According to this theory all the blood cells are produced from a single cell called pluripotent haemopoietic stem cell or totipotent stem cell . Polyphyletic theory: According to this theory different types of blood cells take origin from different stem cells. Monophyletic theory has been accepted because of the evidences available in favour of it.

27. SITES OF HAEMOPOIESIS First 2 months of gestation - yolk sac - (Mesoblastic Stage). From 3 rd months of gestation - till birth , liver and spleen . (Hepatic Stage). From 20 th week of gestation, - bone marrow and by 7 th or 8 th month- main site (Myeloid Stage).

28. Red Bone Marrow - active haemopoietic bone marrow - due to marked cellularity Yellow Bone Marrow – P rogressive fatty replacement throughout the long bones converting red bone marrow into yellow BM.

29. Extra medullary hemopoiesis after birth is abnormal: Hemopoiesis in liver and spleen is physiological during intrauterine life. However, hemopoiesis in these organs or in any other organ ( Extramedullary hemopoiesis ) after birth is considered abnormal.

30. BLOOD CELL PRECURSORS Stem cells The monophyletic theory - all blood cells originate from the pluripotent or multipotent stem cell. Stem cells possess 2 fundamental properties: Self-replication - capable of cell division Differentiation and commitment - ability to differentiate into specialized cells called progenitor cells .

31. The mother hematopoietic stem cell is the Pluripotent Hematopoietic Stem Cells ( PHSC). Producing 2 important groups of stem cells. Lymphoid (immune system) stem cells which ultimately develop into lymphocytes Myeloid (trilineage) stem cells which later differentiate into 3 types of cell lines

32. Myeloid (trilineage) stem cells Granulocyte–monocyte progenitors which produce all leucocytes except the lymphocytes. Erythroid progenitors - produce RBCs. Megakaryocyte progenitors - produce platelets.

33. There are 4 major cell-stages or steps of erythropoiesis: Stem cells, Progenitor cells Precursor cells and Mature cells

34. Progenitor Cells Have ability to give rise to clones (group of cells), so they are also called colony forming cells or colony forming units (CFU).

35. CFU-GEMM (colony forming unit–granulocyte, erythroid, megakaryocyte and macrophage ) BFU-E ( burst forming unit– erythroid ) - large colonies of erythroid series . CFU-E (colony forming unit– erythroid ) - erythrocytes. Ba–CFU - basophil colony forming units. Eo –CFU - eosinophil colony forming units. M–CFU - monocyte colony forming units. G–CFU -neutrophil forming units

36. Formation of the multiple different blood cells from the original pluripotent hematopoietic stem cell in the bone marrow

37. CONTROL OF HAEMOPOIESIS Growth of all haemop. Cell - by the Haemopoietic Growth Factors = cytokines = colony stimulating factors (CSF) : Cytokine - the proteins released by the cells that act as intercellular mediators . Also called - G-CSF stimulates the  granulocytic precursors , M-CSF  monocytic precursors , GM-CSF  granulocytic and monocytic precursors . Interleukins (IL)  lymphocytic precursor , for ex- IL-1, IL-3 , etc. Erythropoietin  erythroid series of cells .

38. Functions of Haemopoietic Growth Factors Causes differentiation and maturation ex- G- CSF, GM-CSF, CSF-I, IL-3, and IL-6. Multiplication of the progenitors . Maturation of a single lineage progenitor cells , e.g., EP for erythrocytes, TPO for thrombocytes. Some growth factors like IL-1, IL-2, IL6 and TNF participate in immune responses.

39. ERYTHROPOIESIS

40. ERYTHROPOIESIS Haemocytoblast (CFU-S ) -a pluripotent stem cell  BFU (E ) burst forming unit ( erythrocytic)  CFU (E ) colony forming unit ( erythrocytic) Proerythroblast Basophilic Normoblast (Early Normoblast ). Intermediate normoblast (polychromatic normoblast) Late normoblast ( orthochromatic normoblast) Reticulocyte Erythrocytes

41. From pro-erythroblast to mature RBC it takes 7 days. From reticulocyte to matured RBC: 1-2 days.

42. Characteristic features of cells at different stages of erythropoiesis Stage Size (mm) Nucleus Cytoplasm Mitosis Hb Staining Homocytoblast (stem cell) 19–23 Very large (almost occupying whole of the cell), deep basophilic containing 4–5 nucleoli Absent Deep basophilic Present++ Pronormoblast (proerythroblast ) 15–20 Large (central) Deep basophilic Fine reticular chromatin 2–3 nucleoli Absent Scanty and deep basophilic ++

43. Stage Size (mm) Nucleus Cytoplasm Mitosis Hb Staining Early normoblast 12–16 Large Chromatin strand becomes thicker and coarser Nucleoli disappears Absent Still basophilic ++ Intermediate normoblast (polychromatic normoblast) 10–14 Nucleus becomes condense, coarse and basophilic Nucleoli absent Appears Acidophilic with basophilic hue (polychromatic) +

44. Stage Size (mm) Nucleus Cytoplasm Mitosis Hb Staining Late normoblast (orthochromatic normoblast) 8–10 Nucleus small, pyknotic with dark chromatin ( cart-wheel appearance) Nucleoli absent Increased in amount Acidophilic Absent Reticulocyte 7–7.5 Nucleus absent With supravital stain ( brilliant cresyl blue ) remnants of RNA appears in the form of reticulum in the cytoplasm Increased in amount Acidophilic Absent Erythrocyte 7.2 – 7.4 Nil Increased in amount Acidophilic Absent

45. Mature Cells - Reticulocytes Immediate precursors of red cells. Therefore = juvenile red cells . Slightly larger than red cells. They have a network of reticular nuclear material - remnants of disintegrated organelles , nuclear fragments. Therefore called reticulocytes . Maturation of reticulocyte into RBC takes about 1-2 days. Increase in the reticulocyte count is called reticulocytosis .

46. The increase in the reticulocyte count in response to iron and other nutrient therapy is called reticulocyte response . An increase in reticulocyte count is an index to the bone marrow activity. Reticulocyte index (RI) is calculated from Reticulocyte count and PCV a ) Normal RI = > 2 b ) < 2 indicates depressed bone marrow activity.

47. REGULATION OF ERYTHROPOIESIS General factors – Erythropoietin , Hypoxia Special maturation factors Dietary factors – Vitamins , Minerals Castle’s intrinsic factors Hormones

48. Erythropoietin Glycoprotein having molecular weight of 34,000. 85% - Produced by the juxta glomerular apparatus of kidney . 15% - by extra renal sources like liver and tissue macrophage system. Stimulant  hypoxia or ↓se in the number of RBCs (e.g. after haemorrhage or in haemolytic anaemia ).

49. Actions EP  multiplication of BFU (E), CFU ( E), proerythroblasts, basophilic and polychromatophilic normoblasts. Stimulates Hb synthesis by increasing globulin synthesis. Causes maturation of all stages of erythropoiesis. Promotes release of RBC from bone marrow into the peripheral circulation.

50. Function of the erythropoietin mechanism to increase production of red blood cells when tissue oxygenation decreases

51. Special Maturation Factors (Factors responsible for final maturation of RBCs ) Proteins help in 'globin' formation. Iron , manganese, copper, cobalt, nickel help in ' haem ‘ formation . Calcium increases iron absorption from GIT. Vitamins C - helps in absorption of iron. B₆  cofactor in the formation of haem . B12 and folic acid help in synthesis of nucleic acid . Castle’s Intrinsic Factor

52. Vitamin B12, intrinsic factor of Castle and folic acid Maturation of a RBC -  essential for the synthesis of DNA Required for the formation of thymidine triphosphate , one of the essential building blocks of DNA. Intrinsic factor  helps in absorption of vitamin B₁₂ from ileum.

53. Androgens - stimulate erythropoiesis. [Males - higher red cell count] Oestrogens - inhibits erythropoiesis by inhibiting erythropoietin production and also by ↓ ing the response of stem cells to erythropoietin. Thyroxin, Cortisol and Growth Hormone Thyroxin stimulates erythropoiesis , it stimulates erythropoietin production. Growth hormone increases the mitosis and maturation of erythroid precursors. Cortisol produces mild erythrocytosis .

54. FACTORS THAT REGULATE ERYTHROPOIESIS

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