Red blood cells

First MBBS Physiology Lecture- RBC,Blood Indices ESR,

1. Red blood cells

2. The student will be able to: ( MUST KNOW ) Give the dimensions of red cell and normal red cell count in different age groups in males and females. List the functions of red cells . Give the list of abnormal forms of red cells and the common condition in which these abnormalities are observed. Appreciate the importance of specialities of red cell membrane. Understand the meaning of red cell fragility and give the causes of increased and decreased fragility of red cells.

3. The student will be able to : ( MUST KNOW ) Give the values of hematocrit in males and females and common conditions of variations in hematocrit. Explain the mechanism of erythrocyte sedimentation rate (ESR), list the factors affecting ESR, give the values of ESR in males and females, and list the physiological and pathological variations in ESR

4. ERYTHROCYTES The red blood cells are named as erythrocytes as they appear red ( erythros means red) in a stained smear of peripheral blood. Red cells are the major cellular elements of blood and perform transport of oxygen from lungs to tissues, and carbon dioxide from tissues to the lungs .

5. Blood Components: Cells Erythrocytes Red Blood Cells (RBC) O 2 & CO 2 transport White Blood Cells (WBC) Immune defense Phagocytosis Platelets: clotting

6. FORMED ELEMENTS Red Blood cell Are disk-shaped and biconcave ; no nucleus ; contains hemoglobin Live for about 120 days . Main component is the hemoglobin which is responsible for 98.5% of the oxygen transported in the blood 6.9 – 7.5 µm in diameter(average=7.2) Function: transports oxygen and carbon dioxide

7. Why Hb Should be inside RBCs : When Hb is free in the plasma, 3 % of Hb will leak through capillaries into tissue spaces or glomerular filtrate each time the blood passes through the capillaries. So, Hb must be inside red blood cells to remain in the human blood. The major function of red blood cells : Transport hemoglobin , which carries oxygen from the lungs to the tissues.

8. Another function 1.RBCs contain carbonic anhydrase enzyme which catalyzes the reversible reaction between carbon dioxide (CO 2 ) and water to form carbonic acid (H 2 CO 3 ). This reaction makes it possible for the water of the blood to transport CO 2 in the form of bicarbonate ion (HCO 3 - ) from the tissues to the lungs, where it is reconverted to CO 2 and expelled into the atmosphere as a body waste product. 2. The Hb is an excellent acid-base buffer , so that RBCs are responsible for most of the acid-base buffering power of whole blood.

9. Abundance of RBCs Erythrocytes account for slightly less than half the blood volume, and 99.9% of the formed elements Hematocrit measures the percentage of whole blood occupied by formed elements Commonly referred to as the volume of packed red cells

10. Structure of RBCs Biconcave disc, providing a large surface to volume ration Shape allows RBCs to bend and flex RBCs lack organelles Typically degenerate in about 120 days.

11. Red blood cells specialisations 2) no nucleus  extra space inside 3) contain haemoglobin  the oxygen carrying molecule  250million molecules / cell 1) biconcave shape increases the surface area so more oxygen can be carried

12. Figure 19.2 Figure 19.2 The Anatomy of Red Blood Cells

13. Red Blood Cell Diameter of each RBC is 7.2 µm (range6.9-7.4µm) Thickness -in periphery is 2µm and in center is 1µm. Surface area of each RBC is 120-140µm 2 Volume is about 80µm 3

14. Advantages of Biconcave Shape It renders the cells quite flexible so that they can pass through capillaries(3.5µm) Biconcave shape increases surface area 20-30%. Thus it allows easy exchange of gases. Biconcavity allows considerable alteration in cell volume .So RBCs can withstand considerable changes of Osmotic Pressure & resist hemolysis .

15. The shapes of red blood cells can change as the cells squeeze through capillaries . Because the normal RBC has an excess of cell membrane for the quantity of material inside, deformation while passing through smallest capillaries does not affect the membrane, so the Red Cells do not rupture passing through capillaries.

16. Life Span of Red Cells Red cells usually live for 120 days. Aged red cells are destroyed by tissue macrophage system . Life span of red cells can be measured by injecting radioactive iron or RBCs tagged with radioactive chromium ( 51 Cr). The tagged red cells lose radioactivity as they are destroyed. Complete disappearance of radioactivity gives the life span of red cells.

17. Normal Counts At birth RBC count-6-7million/cubic m.m . In Adult male = 5-6.5million/cubic m.m.of blood (average=5.5million/cubic m.m .) In females = 4.5-5.5 million/cubic mm (average=4.8 million/cubic mm ) Clinically a count of 5million/cubic mm is considered as 100%

18. Variation in size, shape & counts of RBCs Variation in size is called anisocytosis : Microcytosis i.e. In size of red blood cells occurs in - iron deficiency anaemia , thalassaemia (MCV<80) Macrocytosis i.e. In size of RBCs seen in Megaloblastic Aneamia (MCV>95 )

19. Hypochromic Microcytic RBC

20. Megaloblastic Anemia

21. Poikilocytes This means red cells are of different shapes . Poikilocytosis is usually seen when older cells are present in circulation . Spherocytes When red cells assume spherical shape , are called spherocytes . This is seen in conditions like hereditary spherocytosis . Elliptocyte When red cells assume oval shape are called elliptocytes . Elliptocytosis occurs in hereditary elliptocytosis, thalassemia and iron deficiency anemia. Variation in shape is called Poikilocytosis

22. Her. Spherocytosis:

23. Elliptocytes i.e. elliptical shape of RBCs seen in some aneamia

24. Sickle cell,i.e . crecent shape of RBCs due to presence of HB S

25. Sickle Cell Disease:

26. Echinocyte (Burr cells) A “burr cell” or crenated red cell is an echinocyte . Echinocytosis occurs in uremia and liver disease.  

27. Dacryocyte (Tear Drop ) When the red cell assumes the appearance of a tear drop, is called dacryocyte Dacryocytosis occurs in myelofibrosis with myeloid metaplasia , thalassemia

28. Keratocytes Keratocytes are red cells having a pair of spicule . Keratocytosis usually occurs by mechanical damage or by removal of a Hinz body by pitting action of the spleen. ‘Hamlet cells ’ and ‘bite cells ’ are also used to describe keratocytes

29. Schistocytes Schistocytes are fragments of red cells . They are smaller than red cells and they have sharp angles and spurs. Schistocytosis (red cell fragmentation) occurs in thalassemia , mechanical stress ( microangio­pathic hemolytic anemia, cardiac hemolytic anemia etc.), and thermal injury (severe burns).

30. Basophilic Stippling This means presence of numerous basophilic granules (coarse and dark-blue granules) in red cells . This condition is called punctate basophilia . It is typically seen in lead and other heavy metal poisoning . It also occurs in thalassemia , megaloblastic anemia , infections and liver disease .

31. Sometimes in lead poisoning, Cabot’s ring (ring shape or figure of 8) at the periphery of red cells or Howell Jolly bodies (small nuclear fragments appear in cytoplasm) are found in erythrocytes. Nucleated red cells are seen in severe hemolytic anemia.

32. Variations in counts Physiological factors Age- at birth 6-7 million/ cmm of blood Sex- adult females =4.8million/ cmm adult males =5.5million/ cmm High Altitude- persons residing at mountains (>10,000feet) have high RBC counts 7million/ cmm because of hypoxic stimulation of erythropoisis

33. Physiological in RBC count After sleep In pregnancy- due to haemodilution At high barometric pressure

34. Polycythaemia -pathological in RBC count above 7million/ cmm Primary polycythaemia or polycythaemia vera (PV)– malignancy of bone marrow Secondary polycythaemia -occurs due to state of chronic hypoxia in the body such as: -congenital heart diseases Chronic respiratory disorder like emphysema Phosphorus & arsenic poisoning

35. Polycythemia Description: an increase in the number of circulating erythrocytes and the concentration of hemoglobin in the blood; also known as polycythemia vera , PV, or myeloproliferative red cell disorder, polycythemia can be primary or secondary

36. Polycythemia Etiology and Pathophysiology a. Primary Neoplastic stem cell disorder characterized by increased production of RBCs, granulocytes, and platelets With the over production of erythrocytes, increased blood viscosity results in congestion of blood in tissues, the liver, and spleen Thrombi form, acidosis develops, and tissue infarction occurs as a result of the diminished circulatory flow of blood caused by the increased viscosity

37. Polycythemia b. Secondary Most common form of polycythemia The disturbance is not in the development of red blood cells but in the abnormal increase of erythropoietin, causing excessive erythropoiesis The increase in red blood cell production caused by increased erythropoietin release is a physiologic response to hypoxia ; hypoxia stimulates the release of erythropoietin in the kidney

38. Polycythemia Chronic hypoxic states may be produced by prolonged exposure to high altitudes, pulmonary diseases, hypoventilation , and smoking The results of an increased RBC production include the increased viscosity of blood, which alters circulatory flow

39. Cell Membrane & Metabolism Red Cell Membrane   Red cell membrane is made up of three major structural elements: Lipid bilayer Integral proteins and Membrane skeleton.  

40. Hematocrit Percent of formed elements Hematocrit Normal Hematocrit is around 45%, depending on gender

41. Packed Cell Volume (PCV) Hematocrit represents the percentage of red blood cells in blood (called Packed Cell Volume (PCV)              1. A lower than normal hematocrit is representative of a condition known as anemia              2. An abnormally high hematocrit is representative of polycythemia Hematocrit “ for males: 40%-54% ( 47% ); Females: 38%-46% ( 42% )

42. True Haematocrit = is calculated by multiplying observed haematocrit by 0.98 . it is calculated because 2% of plasma is trapped in between the cells. Body Haematocrit = is calculated by multiplying the observed haematocrit with 0.87 . it is calculated because of the fact that haematocrit estimated from venous blood whose haematocrit is greater than the whole body.

43. MCV (Mean Corpuscular Volume) It refers to average volume of single red blood cell. Normal value=80-100µm 3 one cubic micro meter is equal to one famto letre (fl) When MCV is normal=RBCs are termed Normocytes . When MCV = Microcytes seen in IDA (<80µm 3 ) When MCV = Macrocytes seen in Megaloblastic Anaemia (>100µm 3 )

44. Blood indices: 2. Mean Corpuscular Haemoglobin (M.C.H.): Is an expression of average amount of Hb per cell in picograms (pg =10 -12 gm). - Normal M.C.H. = 27-32 pg. * [email protected]

45. MCH(Mean Corpuscular Haemoglobin ) It means average amount of Hb present in each red blood cell In IDA - low HS and Megaloblastic Anaemia -high <27 pg. Hypochromic anaemia (Iron deficiency). * >32 pg. (Vit.B 12 deficiency).

46. MCHC(Mean Corpuscular Haemoglobin Concentration) It refers to the amount of Hb expressed as percentage of the volume of a RBC Normal value= 33.3%(range 32-36%) RBCs with normal value of MCHC are called Normochromic . MCHC<30% = Hypochromic RBCs seen in IDA,Thalassemia MCHC can’t be more than(36)%

47. So RBCs can’t be Hyperchromic since RBC’s can’t hold the HB beyond the saturation point. Whole enzymatic machinery of RBC’s after full working can’t form HB more than 34% of the volume of a RBC MCHC has greater clinical importance as it is independent of RBC count & RBC’s size It is simply ratio of MCH/MCV ×100

48. C.B.C Haemoglobin - 15±2.5, 14 ±2.5 - g/dl PCV - 0.47 ±0.07, 0.42 ±0.05 - l/l (%) Haematocrit , effective RBC volume RBC count - 5.5 ±1, 4.8 ± 1 x10 12 /l MCHC - Hb /PCV - 32-36 - g/dl Hb synthesis within RBC MCH - Hb /RBC - 29.5 ± 2.5 pg/dl Average Hb in RBC MCV - PCV/RBC- 85 ± 8 - fl

49. Roulaeux Formation Is the tendency of RBC’s to pile one over the another like a pile of coins . Albumin decreases the rouleaux formation, while fibrinogen and globulin & other products of tissue destruction increases it. This is a reversible phenomina ,but it promotes sedimentation of RBC;s It does not occur in normal circulation ,however within a blood vessel in absence of significant flow & when the blood is taken out the red cells tend to form roulaeux .

50. ESR(Erythrocyte Sedimentation Rate) Is the rate at which the red blood cells sediment(settle-down) when the blood containing an anti coagulant is allowed to stand in a vertically placed tube. It is expressed to m.m . at the end of 1 st hour. Westergren’s Method normal value 0-15 mm 1 st hour(M) 0-20mm1st hour (F) Wintrobe’s Method : 0-9mm 1 st hr(M), 0-20(increase with age)

51. Put anti-coagulated blood in vertical tube, then RBC will sink slowly for its larger density. ESR is expressed by RBC sinking distance during the first hour. Measurement of ESR

52. ESR Has no specific diagnostic value . However ,raised level of ESR do suggest presence of some chronic inflammatory condition in body Estimation of ESR is more useful as a prognostic test i.e. to judge the progress of the disease in patients under treatment.

53. Factors affecting ESR Rouleaux formation- = ESR ,fibrinogen & proteins which enters in plasma in inflammatory and neoplastic disease favours RF In size of RBC’s= ESR When no of RBC’s increased the ESR is decreased & when the no of RBC’s decreased(as in aneamia ) the ESR is increased ESR increases when the viscosity of blood is & vice versa. Males =ESR is higher then females Pregnancy=ESR is High New Born=ESR is low

54. The most important factor that determines ESR is the extent of rouleaux formation by erythrocytes Rouleaux formation is determined mainly by the nature of plasma. Increased rouleaux formation occurs when plasma contains increased amounts of fibrinogen (as in pregnancy) and serum globulin (as in inflammatory diseases). Red cell characteristics also affect rouleaux formation and therefore affect the ESR too. Red cells with higher MCHC (mean corpuscular hemoglobin concentration) tend to fall faster in plasma than those with normal or low MCHC. Poikilocytosis (excessive variation in shape of the red cells) or anisocytosis (excessive variation in size of the red cells) reduces ESR

55. Two well-known pathological causes of elevated ESR are tuberculosis and rheumatoid arthritis . However, since ESR is elevated in almost all inflammatory disorders and collagen diseases, it has little diagnostic utility. Its main utility is in prognosis , that is, prediction of the probable course of a disease in an individual and the chances of recovery. Thus, during a 6-month course of tuberculosis treatment, serial measurements of ESR will indicate if the patient is improving. It has similar use in certain malignancies , especially Hodgkin disease.

56. Pathological variation of ESR ESR ESR Tuberculosis Malignancy Chronic infections All anemia's except Sickle cell Collagen diseases. Polycythemia Decreased fibrinogen level Sickle Cell Anemia Allergic Conditions

57. APPLIED ASPECTS Red Cell Fragility: The tendency of the cells to hemolyse is called fragility of the cells. There are 2 types of fragility: Mechanical Osmotic.

58. Mechanical Fragility Lysis of red cells due to mechanical stress and strain is called mechanical fragility . Therefore, when red cells pass through capillaries and splenic pulp, their membrane undergoes mechanical stress. On average, a red cell passes about three lakh times though capillaries during its life span, which makes the cell more fragile. Also, when red cells become older, the membrane becomes rigid. Increased membrane stiffness and mechanical stress make cell vulnerable to rupture. Red cell membrane defects increase mechanical fragility.

59. Osmotic Fragility Lysis of red cells on exposure to different osmotic solutions is called osmotic fragility . It assesses the integrity of red cellmembrane . The osmotic fragility test helps in the diagnosis of anemia in which the physical properties of the red cells are altered. This test detects whether or not the red cells can easily be hemolyzed . .

60. In an isotonic solution , the solution of equal concentration as that of red cell content, the red cells remain intact. Such a solution has same tonicity with that of plasma. Examples are 0.9% NaCl, 5% glucose, 10% mannitol and 20% urea. When suspended in hypertonic solution , a solution with more tonicity (> 0.9% NaCl), red cells shrink due to loss of water from them by exosmosis .

61. Red cells absorb water by endosmosis , when kept in hypotonic solutions , a solution with less tonicity (< 0.9% NaCl). Endosmosis results in hemolysis due to swelling and rupture of the cells

62. Cell shrinkage or swelling ： Isotonic: cell neither shrinks nor swells Hypertonic: cell shrinks ( crenation ) Hypotonic: cell swells ( lysis )

63. Properties of RBC 3. Osmotic Fragility ： The resistance of RBC to h ypotonic solution. 0.8% 0.46% 0.34% 0.9%  Normally, osmotic fragility begins at 0.45 to 0.50 and completes at 0.30 to 0.33

64. Interpretation: When the rate of hemolysis of red cells is increased, the osmotic fragility is said to be increased, and when the rate of hemolysis is decreased, the osmotic fragility is said to be decreased

65. Normal Value & Variations Normally, osmotic fragility begins at 0.45 to 0.50 and completes at 0.30 to 0.33 . Conditions of Diminished Fragility : - Iron deficiency anemia - Thalassemia - Sickle cell anemia - Obstructive jaundice - Post-splenectomy Conditions of Increased Fragility: - Hereditary spherocytosis - Congenital hemolytic anemia - Other conditions in which spherocytes are found in the blood.

66. Metabolism of Red Cells Red cells have no nuclei, mitochondria and ribosomes . Therefore, adequate synthesis of proteins and lipids does not occur in red cells. Glucose is the primary fuel for red cells . Though enzymes for glycolysis are present, enzymes for TCA cycle are absent. ATP is formed by Embden- Mayerhoff pathway (EM pathway). The HMP shunt provides NADPH . Glucose entry into the red cells occurs easily by facilitated diffusion, which is independent of insulin action . Red cells depend mostly on glucose metabolism for their energy supply. 90% of glucose is oxidized by EM pathway and 10% by HMP shunt .

67. Glucose Metabolism in RBCs 1- Glycolysis 2- Hexose mono-phosphate shunt (HMPS)

68. Glycolysis in RBCs Glucose 2 NAD 2 NADH+H 2 ATP 2 Lactate Glucose 1,3Disphosphoglycerate 2,3Disphosphoglycerate

69. EM Pathway Red cells metabolize glucose, usually by anerobic glycolysis using EM pathway. Two ATP molecules are generated by glycolysis through EM pathway. 2, 3- DPG is produced in red cells. 2, 3- DPG influences oxygen affinity of hemoglobin and therefore, plays an important role in red cell function.

70. Importance of glycolysis in red cells: a) Energy production : it is the only pathway that supplies the red cells with ATP. haemolytic anaemia may occur due to an inherited deficiency of glycolytic enzymes mainly pyruvate kinase deficiency . b) Reduction of methaemoglobin : glycolysis provides NADH for reduction of met Hb by NADH-Cyto.b5 reductase c) In red cells 1,3 Disphosphoglycerate is converted to 2,3 Disphosphoglycerate which binds to oxy Hb and helps release of O 2 to tissues.

71. HMP Shunt The enzyme in the red cell, glucose-6-phosphate dehydrogenase (G-6-PD) is the main enzyme for HMP shunt. HMP shunt generates NADPH , keeps glutathione in reduced state , which is a strong reducing agent and prevents damage to the red cell. Therefore, G-6-PD deficiency interferes with red cell functions and produce Hemolytic anemia

72. Importance of HMPS in Red cells: Red cells are liable for oxidative damage by H 2 O 2 due to their role in O 2 transport. In RBCs, H 2 O 2 can cause both oxidation of iron in haemoglobin (to form methaemoglobin ) and lipid peroxidation ( increases the cell membrane fragility ). The major role of HMS in red cells is the production of NADPH , which protect these cells from oxidative damage by reduction of glutathione that helps removal of H 2 O 2 .

73. Non- Oxidative Phase Oxidative Phase Reversible Non-Regulatory Irreversible Regulatory 6 moles of Pentose-P 5 moles of G-6-P 6 moles of G-6-P 6 moles of Pentose-P 12 NADP 12 NADPH +12 H 6 H2O 6 CO2

74. Role of NADPH+H in reduction of glutathione G-S-S-G 2-GSH 2 GSH +H2O2 G-S-S-G + 2 H2O Glutathione Reductase Glutathione peroxidase NADPH+H NADP