Haemolytic anemia

hemolytic anemia ppt

1. HEMOLYTIC ANEMIA Presenter – Dr Preeti Utnal Moderator - Dr Manjula K

2. OBJECTIVES Definition Classification Etiology Evaluation of hemolytic anemia References

3. DEFINITION Hemolytic disorders are characterised by signs of accelerated erythrocyte destruction together with those of vigorous blood regeneration. Shortened life span of RBC’s – 15-20 days. Elevated Erythropoietin Accumulation of haemoglobin degradation products.

4. CLASSIFICATION OF HEMOLYTIC ANAEMIA INTRINSI C EXTRINSIC CONGENITAL ACQUIRED MEMBRANE DISORDER HEMOGLOBINOPATHIES ENZYME DISORDER S PNH IMMUNE DRUG INDUCED NON-IMMUNE AUTO Ab ALLO Ab

5. SITE OF RBC DESTRUCTION Intravascular Red cells rupture within the vasculature releasing free hemoglobin into circulation Extravascular Liver Spleen Bone marrow Splenomegaly is a feature of extravascular helmolysis

6. Causes of extravascular hemolysis

7. Pathogenesis of Extra vascular hemolysis Extreme changes in shape required for RBC to navigate the splenic sinusoids successfully This alterations result in to less deformable RBC RBC sequestration & phagocytosis by Macrophages located within splenic cords Globin Protein pool Haem Biliverdin Bilirubin Unconjugated bilirubin Conjugated bilrubin Urobilinogen Excretion in Faeces & urine and formation of gall stones

8. Causes of intravascular hemolysis Mechanical injury Complement fixation Intracellular parasite Exogenous toxic factors

9. General consideration in the diagnosis of hemolytic anemia Is the anemia hemolytic ? If so, is it Intravascular / Extravascular haemolysis ? What is the etiology? What is the severity of anemia?

10. IS THE ANEMIA HAEMOLYTIC? Evidence for increased red cell production- IN BLOOD - peripheral smear 1.Increased reticulocyte count 2 .Circulating nucleated RBC 3 .Marked polychromasia.

11. Polychromatophilic red cells(A--green arrows) Spherocytes (B--blue arrows ) Nucleated red blood cells (D-- orange arrow)

12. Fragmented cells, spherocytes , blister cells and punctate basophilia

13. Reticulocytosis

14. BONE MARROW BIOPSY ERYTHROID HYPERPLASIA

15. INVESTIGATION OF MEMBRANE DEFECT

16. HEREDITARY SHPEROCYTOSIS

17. PATHOPHYSIOLOGY OF HS Primary membrane skeletal defect Membrane stability Membrane loss Surface to volume ratio deformability Splenic trapping Erythrostasis Glucose pH phagocytosis

18. Lab Findings Minimal / no anemia Spherocytes Polychromasia MCV , MCHC Negative antiglobulin test Polychromatic cell S pherocyte

19. Special tests done OSMOTIC FRAGILITY TEST Measure of the erythrocytes resistance to hemolysis by osmotic stress.

20. OSMOTIC FRAGILITY TEST NORMAL ABNORMAL INCREASED IN H.Spherocytosis H.Elliptocytosis H.stomatocytosis AI Hemolytic anemia. DECREASED IN Thalassemia Iron deficiency anemia.

21. OSMOTIC FRAGILITY TEST Shift to left – increased OF Shift to right – decreased OF

22. INCUBATED OSMOTIC FRAGILITY TEST Blood incubated for 24hrs At 37°C HS cells lose membranes more readily then normal RBC’s when incubated Increased sensitivity Most reliable diagnostic test for HS

23. AUTOHEMOLYSIS TEST Measures spontaneous hemolysis of blood incubated at 37ºc for 48 hrs Measure readings colorimetrically at 540nm Normal – 0.2-2 % With added glucose – 0-0.9 %

24. Decreased rate with added Glucose Hereditary Spherocytosis PNH G6PD Deficiency No response to added glucose Pyruvate Kinase Deficiency

25. ACIDIFIED GLYCEROL LYSIS TIME Time taken for 50% hemolysis of a blood sample in a buffered hypotonic saline glycerol mixture. Glycerol retards the osmotic swelling of red cells Rate of hemolysis is measured by rate of fall of turbidity Half time for AGLT > 30min for normal RBC’s HS cells 25-150 sec

26. CRYOHEMOLYSIS Specific for Hereditary Spherocytosis Dependent on molecular defects of RBC membrane. HS cells are particularly sensitive to cooling at 0 °C in hypertonic saline. Normal: 3 – 15% HS - >20%

27. HERIDITARY ELLIPTOCYTOSIS

28. INVESTIGATIONS OF HEMOGLOBINOPATHIES

29. THALASSEMIA Autosomal dominant Reduced synthesis of normal Hb polypeptide chain due to molecular defects. Hematologic consequences: Low intracellular Hb Relative excess of unpaired chain

30. Pathogenesis Imbalanced synthesis of α & β chains Decreased total RBC Hb production Ineffective erythropoiesis Chronic hemolytic process Systemic iron overload

31. THALASSAEMIA

32. LAB FINDINGS Microcytic Hypochromic anemia(2-3g/dl) Decreased MCV , MCH , MCHC Decreased Osmotic Fragility. Increased serum uric acid Anisopoikilocytosis

33. Lab Findings- BM Normoblastic erythroid hyperplasia Increased macrophages Inclusion bodies in normoblasts

34. SICKLE CELL ANAEMIA Hereditary disorder – autosomal recessive RBC contains Hb S α 2 β 2 6GLU ↔ VAL Hypoxia , acidosis, hypertonicity, ↑temp→ deoxygenation → Hb S polymerisation → sickle cells

35. SICKLE CELL ANAEMIA Normocytic normochromic anemia Sickle cells Reticulocytosis ( 10 – 20 %) Anisopoikilocytsis Howell jolly bodies Normoblast ↑RDW BM – Erythroid hyperplasia

36. SOLUBILITY TEST Sickle cell Hb is insoluble in deoxygenated state in a high molality phosphate buffer Crystals formed refract light, cause solution to be turbid. Positive test – sickling Hb Doesn’t differentiate b/w homozygous & heterozygous POSITIVE TEST

37. SICKLING TEST Blood deoxygenated with reducing substances (sodium metabisulphite ) Place on slide Seal coverslip Immediate sickling – Disease Sickling in 1 hr – Trait Hb electrophoresis High performance liquid Chromatography (HPCL) Isoelectric focusing – agar gel 4. Prenatal diagnosis - PCR OTHER DIAGNOSTIC TESTS

38. INVESTIGATION OF ENZYME DEFICIENCY

39. G6PD DEFICIENCY INTRODUCTION Its an enzyme of HMP pathway Protects against oxidative stress X linked disorder Deficiency result in to, Impaired NADPH production Accumulation of oxidants in cell Oxidative stress leads to Heinz body formation & extra-vascular hemolysis

40. BLOOD SMEAR IN G6PD DEFICIENCY

41. FLORESCENT SPOT TEST Sensitive screening test Whole blood + G6P + NADP + SAPONIN Kept on a filter paper Examine under UV light Examine the Fluorescence G6P + NADP 6-Phosphogluconate + NADPH (Fluoresces) Lack of fluorescence ------- G6PD Deficiency

42. DYE REDUCTION TEST Pts blood hemolysate + G6P + NADP + brilliant cresyl blue ------ incubation If G6PD present ----- NADP → NADPH BLUE → COLOURLESS Time taken is inversely proportional to the amt. of G6PD present Controls- normal blood Specific test

43. OTHER TESTS ASCORBATE CYANIDE TEST METHEMOGLOBIN REDUCTION TEST G6PD ASSAY Glutathione stability test-Decrease sensitivity in G6PD deficiency. PCR-To reveal genetic abnormality (Florescent labelled probes are used to detect mutant G6PD alleles)

44. ACQUIRED HEMOLYTIC ANEMIA

45. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Acquired Clonal cell disorder Somatic mutation in hematopoietic stem cell. Defect in glycosyl – phosphatidyl inositol (GPI) molecule embedded in cell membrane GPI linked proteins – decay accelerating factor (CD 55), Inhibitor of reactive lysis(CD 59)-Prevents activation of complement.

46. LAB FINDINGS Anemia Thrombocytopenia Hemosiderinuria Positive sucrose hemolysis test Positive Ham’s test Normal Osmotic fragility

47. SUCROSE HEMOLYSIS TEST Screening test Patient’s blood incubated in sucrose solution Sucrose promotes binding of complement to RBC Hemolysis

48. HAM’S TEST(Acidified serum lysis test) Patient’s RBCs are exposed at 37°C to action of normal / patient own serum suitably acidified to optimal pH for lysis( activate alternate pathway) 10 – 50 % of total RBC ---- Lysis

49. FLOW CYTOMETRY

50. IMMUNE MEDIATED HEMOLYTIC ANEMIA

51. PATHOPHYSIOLOGY RBC’s + IgG Ab Pass through the spleen Fc receptor of spleen macrophages attracts Fc portion of Ab – RBC complex Phagocytosis of RBC by macrophage Fragmentation of RBC membrane Membrane reseals and forms spherocytes Extravascular hemolysis

52. Indications

53. INDIRECT ANTIGLOBULIN TEST

54. Positive test Isoimmunisation Presence of free auto Ab in patient’s serum

55. WARM AIHA Individuals produce Ab against their own erythrocyte Ag (autoantibodies) Ab react with red cell Ag best at 37 o c. Lab findings PBS – Normocytic normochromic anemia - Reticulocytosis - Spherocytes , - Schistocytes , Polychromasia , NRBC’s - Neutrophilia - Platelet - normal or decreased

56. Bone marrow - Normoblastic erythroid hyperplasia - Erythrophagocytosis Other tests - Direct Coombs’ test (DAT)- positive

57. COLD AIHA Associated with IgM Ab which fixes complement & is reactive below 32 o c First indication of the presence of unsuspected cold agglutinins is blood counts. RBC count is inappropriately decreased for Hb % MCV is falsely elevated (due to agglutination) PCV is falsely low MCH & MCHC are falsely elevated Visible autoagglutination can be observed in tubes of anticoagulated blood as the blood cools to room temperature .

58. When RBC indices go haywire think of the possibility of cold agglutinin disease

59. PAROXYSMAL COLD HEMOGLOBINURIA Lab findings a) Between the attacks - peripheral blood is normal except for anemia b) During the attack – sharp drop in Hb c)DAT – weakly + ve with anticomplement antisera - Ab are not detected d) Indirect coomb’s test may be + ve ,if performed in cold e) Donath – Landsteiner test

60. DONATH LANDSTEINER(D-L) Test PATIENT’S WHOLE BLOOD CONTROL TEST INCUBATE FOR 30 MIN AT 37 C 4 C INCUBATE FOR 30 MIN AT 37 C 37 C Centrifuge: Observe plasma for presence of hemolysis Interpretation D-L antibodies present No hemolysis Hemolysis NO D-L antibodies present No hemolysis No hemolysis

61. AUTOIMMUNE HEMOLYTIC ANEMIA Hemolytic anemia induced by immunization of an individual with RBC Ag’s from another individual . Eg : 1. Hemolytic transfusion reactions 2. Hemolytic disease of new born

62. DRUG INDUCED AIHA It is the result of an immune mediated hemolysis precipitated by ingestion of certain drugs. MECHANISM: Drug adsorption ( hapten type) Immune complex formation Autoantibody induction Membrane modification

63. Haemolytic uremic syndrome

64. MALARIA

65. Other conditions where hemolysis is seen Disseminated malignancy Leukemia Malignant lymphomas Renal failure Liver disease Rheumatoid arthritis Megaloblastic anemia

66. Diagnostic approach

67. REFERENCE Henry’s Clinical diagnosis & Management by Lab. Methods Shirlyn B. Mc Kenzie , Text book of Haemotology Wintrobe Dacie & Lewis, Practical Haematology de Gruchy’s clinical Haematology 5 th edition, pages 137-210 .

68. THANK YOU 