Haemolytic anaemias

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.

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.

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.

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

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

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.

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.

9. 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 Inherited & acquired HA

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

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.

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.

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.

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.

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).

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.

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.

18. Hereditary Elliptocytosis

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.

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.

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.

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.

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

24. Hereditary Enzyme Deficiency

25. The pathways of glucose metabolism

26. G6PD function

27. Glucose 6-Phosphate DehydrogenaseFunctions • 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

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

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.

30. Glucose 6-Phosphate DehydrogenaseDifferent Isozymes

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.

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

33. Tests which could be done in the diagnosis ofglucose-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

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

35. IMMUNE HEMOLYTIC ANEMIAGeneral 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

36. IMMUNE HEMOLYTIC ANEMIAGeneral Principles - 2 • Antibodies combine with RBC, & either: • Activate complement cascade, &/or • 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

37. IMMUNE HEMOLYTIC ANEMIACoombs 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

38. Direct Antiglobulin Test

39. IMMUNE HEMOLYTIC ANEMIACoombs 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

40. Indirect Antiglobulin Test

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

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

43. DRUG-INDUCED HEMOLYSISImmune 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

44. DRUG-INDUCED HEMOLYSISHaptenic 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

45. DRUG-INDUCED HEMOLYSISTrue 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

46. ALLOIMUNE HEMOLYSISHemolytic 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

47. INCOMPATIBLE RBC TRANSFUSIONRate of Hemolysis

48. ALLOIMMUNE HEMOLYSISTesting 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)

49. ALLOIMMUNE HEMOLYSISHemolytic 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

50. Erythroblastosis Fetalis