1. Haemolytic anaemias Ahmad Sh. Silmi Msc Haematology, FIBMS

2. Haemolytic anaemias (HAs) HAs are defined as those anaemias which result from an increase in the rate of red cell destruction. any condition which leads to a reduction in the mean lifespan of the red cell is a haemolytic disorder. 2

3. Because of erythropoietic hyperplasia and anatomical extension of bone marrow, red cell destruction may be increased several fold before the patient become anaemic --- compensated haemolytic anaemia. The normal adult marrow, after full expansion, is able to produce red cells at 6-8 times the normal rate. Therefore HA may not be seen until the red cell lifespan is less than 30 days. 3

4. Classification of HA The HA can be classified in several different ways: 1- Site of haemolysis: Extravascular haemolytic disorders - macrophages of the RES Intravascular haemolytic disorders- within the circulatory system In many of the cases there is a combination of both extra and intravascular haemolysis. 4

5. Extravascular haemolysis Red cell destruction usually occurs in the cell of the RES. 5

6. Intravascular haemolysis Destruction of red cells occur inside the blood vessels. 6

7. Classification of HA 2- Site of defect: Intrinsic defect (intracorpuscular)- structural or functional defect within the red cell. Extrinsic defect (extracorpuscular)- an abnormality in the red cell environment. 7

8. Classification of HA 3- Inherited or acquired: Inherited HA are usually caused by intrinsic defect. While acquired HA are caused by an extrinsic defect. However there are some exceptions: Paroxysmal nocturnal haemoglobinuria (PNH) which is an acquired intrinsic defect, and severe hereditaryG6PD enz deficiency which requires the presence of an extrinsic trigger such as the antimalarial drug for the intrinsic defect to manifest. 8

9. Inherited & acquired HA Hereditary HA Membrane defects e.g hereditary spherocytosis Metabolic defect e.g G6PD deficiency. Haemoglobin defects e.g sickle cell disease. Acquired HA –Immune -Autoimmune eg AIHA -Alloimmune e.g HDN, HTR –Red cell fragmentation syndromes –March haemoglobinaemia –Infections –Chemical and physical agents. –PNH 9

10. Inherited Haemolytic Disorders Disorders of globin synthesis and / or structure (thalassaemia and haemoglobinopathies and these are previously described). Primary membrane disorders. Enzyme disorders. 10

11. Primary membrane disorders Primary disorders of the red cell membrane are associated with alteration of cell shape. Many of these disorders are classified according to the shape of the abnormal cells. This scheme is by far the most common employed, and so will be used. 11

12. Hereditary Spherocytosis Hereditary spherocytosis (HS), also known as congenital haemolytic anaemia and congenital haemolytic jaundice, is the most common of the inherited primary red cell membrane abnormalities. Incidence rate: at least 1 in 5000 in North European populations. Inheritance: autosomal (non-sex linked) dominant, although a less common autosomal recessive variant exists. To date, no homozygous for the autosomal dominant have been described, suggesting that this state is incompatible with life. 12

13. Pathophysiology The defective red cells in HS have an increased flux of Na+ ions into the cell, because of weakened membrane structure, leading to greatly increased activity of the cation pump and necessitating an increased rate of glycolysis. Normally, the osmotic balance of the red cell is maintained with sufficient glucose and ATP to expel sodium at a rate equal to its influx. However, spherocyte consume glucose at a higher rate. When the amount of glucose is low, as in splenic cords, there is an increased rate of destruction of red cells. The water content of red cells is increases, and as a result, swelling and haemolysis of the red blood cells occur. 13

14. Cause The exact nature of the red cell defect is unknown. A variety of cytoskeletal defects have been described. Evidence suggests the primary defect involve the structure of spectrin, a skeletal protein in the red cell membrane. A quantitative decreases in spectrin that, correlates with the degree of spherocytosis and the severity of the disorder. 14

15. Symptoms Congenital haemolytic anaemia with a variable degree of spherocytosis. Increased red cell osmotic fragility. Episodic jaundice. Variable splenomegally. Cholelithiasis (pigment gallstones// less common). 15

16. Laboratory findings Moderate anaemia. Peripheral blood shows variable spherocytic cell varies from few to many. Decrease RBC's diameter and increase MCHC. Increase reticulocyte count up to 20%. Few NRBC's present. Normal WBC & platelet, except during haemolysis, where there is leukocytosis and thrombocytosis. Osmotic Fragilty is increased. Increase bilirubin. Increase urine and stool urobilinogen. Decrease haptoglobin and may be undetectable. Direct combs' is negative. Bone marrow is hypercellular where 25-60% is erythroid. 16

17. Treatment Splenectomy, which leads to: Increase RBC's lifespan. Increase Hb, Hct, and RBC's. Decrease bilirubin. Decrease reticulocyte. Osmotic fragility will continue increased. 17

18. Hereditary Elliptocytosis 18

19. Hereditary Elliptocytosis Hereditary Elliptocytosis disorders are characterized by the presence of a large proportion of oval or elliptical red cells. The frequency of HE is difficult to estimate because most forms are clinically silent but may be as high as 1 in 1000. The condition typically is transmitted as an autosomal dominant characteristic. In contrast to HS, homozygous form is well recognized as a severe transfusion-dependent haemolytic anaemia. 19

20. Cause A wide range of cytoskeletal defects has been described in association with HE. The most common cytoskeletal defects are structural abnormalities of domain 1 of α spectrin, the region responsible for dimer-dimer self-association in cytoskeleton. Resulting in free unconnected dimers. 20

21. Pathophysiology Most workers currently believe that, because of the weakened forces holding the cytoskeleton together in HE red cells; repeated passage through the microvasculature resulting in red cell fragmentation, poikilocytosis and micro- elliptocytosis. 21

22. Symptoms Approximately 90% of the individuals showing elliptocytosis have no clinical symptoms other than the presence of elliptical red blood cells. The remaining patients display a haemolytic anaemia and splenomegally. 22

23. Laboratory findings :The characteristic feature is increased osmotic fragility test. Treatment: Splenectomy typically provides a functional cure 23

24. Hereditary Enzyme Deficiency 24

25. The pathways of glucose metabolism 25

26. G6PD function 26

27. Glucose 6-Phosphate Dehydrogenase Functions Regenerates NADPH, allowing regeneration of glutathione Protects against oxidative stress Lack of G6PD leads to hemolysis during oxidative stress –Infection –Medications –Fava beans Oxidative stress leads to Heinz body formation,  extravascular hemolysis 27

28. Glucose-6-phosphate dehdrogenase deficiency The reduction of oxidized glutathione by NADPH 28

29. Geographical distribution Deficiency of G-6-PD has been reported in most populations of the world but is most commonly seen in Western and Central Africa, the Mediterranean region, the Middle East and SE Asia. Normally active G6PD has been designated type Gd B. It is the most common form of the enzyme in all populations and exists in 99% of Whites. Among whites, G6PD Mediterranean is the most common variant, although the overall prevalence is low. Another variety of the G6PD enzyme that is commonly found in Africans also has normal activity but differs from the Gd B by a single amino acid substitution that alter its electrophoretic mobility. This variant is designated as Gd A. The Gd A variant is found in about 20% of American black men. 29

30. Glucose 6-Phosphate Dehydrogenase Different Isozymes 30

31. Mode of Inheritance The gene, which encodes G-6-PD, is located on the tip of the q arm of the X chromosome, close to the factor VIII gene. The disorder is fully expressed in men (hemizygote) who inherit the mutant gene. In women, full expression of the disorder occurs only when two mutant genes (homozygous) are inherited. The heterozygous woman has two populations of red blood cells, one population with normal enzyme activity and the other with deficient enzyme activity. 31

32. Pathophysiology Oxidants cause two kinds of damage to red cells: Damage to haemoglobin Damage to the membrane. 32

33. Tests which could be done in the diagnosis of glucose-6-phosphate dehydrogenase deficiency elevated bilirubin levels low serum haptoglobin haemoglobinuria elevated absolute reticulocyte count low red blood cell count and haemoglobinuria Heinz bodies present on examination of the peripheral blood smear using stains methylene blue test methemoglobin reduction test 33

34. Acquired Haemolytic Disorders The acquired haemolytic disorders can be sub- classified into five groups, according to the nature of the defect: Haemolysis secondary to immune mechanisms. Haemolysis secondary to the action of chemicals, drugs or toxins. Haemolysis secondary to infection. Haemolysis secondary to physical damage. Miscellaneous disorders 34

35. IMMUNE HEMOLYTIC ANEMIA General Principles All require antigen-antibody reactions Types of reactions dependent on: –Class of Antibody –Number & Spacing of antigenic sites on cell –Availability of complement –Environmental Temperature –Functional status of reticuloendothelial system Manifestations –Intravascular hemolysis –Extravascular hemolysis 35

36. IMMUNE HEMOLYTIC ANEMIA General Principles - 2 Antibodies combine with RBC, & either: 1.Activate complement cascade, &/or 2.Opsonize RBC for immune system If 1, if all of complement cascade is fixed to red cell, intravascular cell lysis occurs If 2, &/or if complement is only partially fixed, macrophages recognize Fc receptor of Ig &/or C3b of complement & phagocytize RBC, causing extravascular RBC destruction 36

37. IMMUNE HEMOLYTIC ANEMIA Coombs Test - Direct Looks for immunoglobulin &/or complement of surface of red blood cell (normally neither found on RBC surface) Coombs reagent - combination of anti-human immunoglobulin & anti-human complement Mixed with patient’s red cells; if immunoglobulin or complement are on surface, Coombs reagent will link cells together and cause agglutination of RBCs 37

38. Direct Antiglobulin Test 38

39. IMMUNE HEMOLYTIC ANEMIA Coombs Test - Indirect Looks for anti-red blood cell antibodies in the patient’s serum, using a panel of red cells with known surface antigens Combine patient’s serum with cells from a panel of RBC’s with known antigens Add Coombs’ reagent to this mixture If anti-RBC antigens are in serum, agglutination occurs 39

40. Indirect Antiglobulin Test 40

41. HEMOLYTIC ANEMIA - IMMUNE Drug-Related Hemolysis Alloimmune Hemolysis –Hemolytic Transfusion Reaction –Hemolytic Disease of the Newborn Autoimmune Hemolysis –Warm autoimmune hemolysis –Cold autoimmune hemolysis 41

42. IMMUNE HEMOLYSIS Drug-Related Immune Complex Mechanism –Quinidine, Quinine, Isoniazid “Haptenic” Immune Mechanism –Penicillins, Cephalosporins True Autoimmune Mechanism –Methyldopa, L-DOPA, Procaineamide, Ibuprofen 42

43. DRUG-INDUCED HEMOLYSIS Immune Complex Mechanism Drug & antibody bind in the plasma Immune complexes either –Activate complement in the plasma, or –Sit on red blood cell Antigen-antibody complex recognized by RE system Red cells lysed as “innocent bystander” of destruction of immune complex REQUIRES DRUG IN SYSTEM 43

44. DRUG-INDUCED HEMOLYSIS Haptenic Mechanism Drug binds to & reacts with red cell surface proteins Antibodies recognize altered protein, ± drug, as foreign Antibodies bind to altered protein & initiate process leading to hemolysis 44

45. DRUG-INDUCED HEMOLYSIS True Autoantibody Formation Certain drugs appear to cause antibodies that react with antigens normally found on RBC surface, and do so even in the absence of the drug 45

46. ALLOIMUNE HEMOLYSIS Hemolytic Transfusion Reaction Caused by recognition of foreign antigens on transfused blood cells Several types –Immediate Intravascular Hemolysis (Minutes) - Due to preformed antibodies; life-threatening –Slow extravascular hemolysis (Days) - Usually due to repeat exposure to a foreign antigen to which there was a previous exposure; usually only mild symptoms –Delayed sensitization - (Weeks) - Usually due to 1st exposure to foreign antigen; asymptomatic 46

47. INCOMPATIBLE RBC TRANSFUSION Rate of Hemolysis 47

48. ALLOIMMUNE HEMOLYSIS Testing Pre-transfusion ABO & Rh Type of both donor & recipient Antibody Screen of Donor & Recipient, including indirect Coombs Major cross-match by same procedure (recipient serum & donor red cells) 48

49. ALLOIMMUNE HEMOLYSIS Hemolytic Disease of the Newborn Due to incompatibility between mother negative for an antigen & fetus/father positive for that antigen. Rh incompatibility, ABO incompatibility most common causes. Usually occurs with 2nd or later pregnancies Requires maternal IgG antibodies vs. RBC antigens in fetus 49

50. Erythroblastosis Fetalis 50

51. ALLOIMMUNE HEMOLYSIS Hemolytic Disease of the Newborn - #2 Can cause severe anemia in fetus, with erythroblastosis and heart failure Hyperbilirubinemia can lead to severe brain damage (kernicterus) if not promptly treated HDN due to Rh incompatibility can be almost totally prevented by administration of anti-Rh D to Rh negative mothers after each pregnancy 51

52. AUTOIMMUNE HEMOLYSIS Due to formation of autoantibodies that attack patient’s own RBC’s Type characterized by ability of autoantibodies to fix complement & site of RBC destruction Often associated with either lymphoproliferative disease or collagen vascular disease 52

53. AUTOIMMUNE HEMOLYSIS Warm Type Usually IgG antibodies Fix complement only to level of C3, if at all Immunoglobulin binding occurs at all temps Fc receptors/C3b recognized by macrophages. Hemolysis primarily extravascular 70% associated with other illnesses Responsive to steroids/splenectomy 53

54. AUTOIMMUNE HEMOLYSIS Cold Type Most commonly IgM mediated Antibodies bind best at 30º or lower Fix entire complement cascade Leads to formation of membrane attack complex, which leads to RBC lysis in vasculature Typically only complement found on cells 90% associated with other illnesses Poorly responsive to steroids, splenectomy; responsive to plasmapheresis 54

55. HEMOLYTIC ANEMIA Summary Myriad causes of increased RBC destruction Marrow function usually normal Often requires extra folic acid to maintain hematopoiesis Anything that turns off the bone marrow can result in acute, life-threatening anemia 55

56. Paroxysmal Nocturnal Haemoglobinuria

57. 57 Sudden At Night Blood in Urine Nocturnal haemolysis was once thought to be associated with a slight drop in plasma pH during sleep, but this is now known to be untrue

58. Paroxysmal Nocturnal Haemoglobinuria Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder, which arises following a somatic mutation in a multipotential stem cell 58

59. Characteristics RBC’s are susceptible to the lytic action of complement. Chronic but episodic intravascular haemolysis. Occurs mainly during sleep. Moderately to severe panhypoplasia. Neutrophil dysfunction. Renal insufficiency. Dysphagia. Tendency to venous thrombosis. 59

60. Classification Broadly, the red cells in cases of PNH can be divided into three groups: 1- PNH I red cells are normal with regard to their sensitivity to complement-mediated lysis. 2- PNH II red cells show a moderately increased sensitivity. 3- PNH III red cells show a markedly increased sensitivity. 60

61. The size of hemolysis The major severity of haemolysis is the size of the PNH III red cell population. Where more than 50% of the circulating red cells are PNH III cells haemolysis is severe and relatively constant. Typical episodic or sleep-related hamolysis is seen when 20-50% of the circulating red cells is a PNH III cell. 61

62. Pathogenesis Deficiency of several red cell membrane proteins has been noted in PNH, at least some of which contribute to the pathogenesis of the haemolysis: 62

63. Acetylcholesterase Acetylcholesterase deficiency is relatively constant finding in PNH red cells and was originally thought to be implicated in the pathogenesis. However, this is untrue because artificially-induced inhibition to this enzyme has no effect on red cell life span in vitro or in vivo. 63

64. Decay Accelerating Factor (DAF) Red cell membrane integral protein which binds to complement components C 3 b and C 4 b on the membrane surface, thereby inhibiting the construction of C 3. DAF therefore protects the red cell from complement-mediated haemolysis. Deficiency of DAF causes a relatively mild increase in susceptibility to complement- mediated haemolysis. 64

65. Membrane inhibitor of reactive lysis (MIRL) This is a red cell membrane protein which protect normal red cell from complement- mediated haemolysis by inhibiting the assembly of the complement membrane attack complex, C5b-9. 65

66. Homologous Restriction Factor (HRF) This is a red cell membrane integral protein, which, in normal cells, limits the assembly of the complement membrane attack complex by binding C 8. HRF has been shown to be absent in PNH III. 66

67. Clinical picture Episodes of intravascular haemolysis. Haemoglobinuria. Haemorrhage. Infection. Thrombotic complications. 67

68. Triggering factors for haemolysis Mostly is unknown. Infection. Vaccination. Blood transfusion. Menstrual cycle. 68

69. Causes of death The excess of bacterial and fungal infection secondary to leukopenia and neutrophil dysfunction is a common cause of death. The leading cause of death in PNH, however, is venous thrombosis. Thrombosis is explained by the release of thromboplastin-like substances from lysed red cells and platelet aggregation. 69