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Haemolytic anemia
- 1. HEMOLYTIC ANEMIA PRESENTER – DRPREETI UTNAL MODERATOR - DR MANJULA K
- 2. OBJECTIVES Definition Classification Etiology Evaluation of hemolytic anemia References
- 3. DEFINITION Hemolytic disordersare 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 INTRINSICEXTRINSIC CONGENITAL ACQUIRED MEMBRANE DISORDER HEMOGLOBINOPATHIES ENZYME DISORDERS PNH IMMUNE DRUG INDUCED NON-IMMUNE AUTO Ab ALLO Ab
- 5. SITE OF RBC DESTRUCTIONIntravascular Red cells rupture within the vasculature releasing free hemoglobin into circulation Extravascular Liver Spleen Bone marrow Splenomegaly is a feature of extravascular helmolysis
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- 8. Pathogenesis of Extravascular 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
- 9. Causes of intravascularhemolysis Mechanical injury Complement fixation Intracellular parasite Exogenous toxic factors
- 12. General consideration inthe 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?
- 13. IS THE ANEMIAHAEMOLYTIC? Evidence for increased red cell production- IN BLOOD - peripheral smear 1.Increased reticulocyte count 2.Circulating nucleated RBC 3.Marked polychromasia.
- 14. Polychromatophilic redcells(A--green arrows) Spherocytes (B--blue arrows) Nucleated red blood cells (D-- orange arrow)
- 15. Fragmented cells, spherocytes,blister cells and punctate basophilia
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- 21. PATHOPHYSIOLOGY OF HS Primarymembrane skeletal defect Membrane stability Membrane loss Surface to volume ratio deformability Splenic trapping Erythrostasis Glucose pH phagocytosis
- 22. Lab Findings Minimal/ no anemia Spherocytes Polychromasia MCV , MCHC Negative antiglobulin test Polychromatic cell Spherocyte
- 23. Special tests done 1.OSMOTIC FRAGILITY TEST Measure of the erythrocytes resistance to hemolysis by osmotic stress.
- 24. OSMOTIC FRAGILITY TEST NORMAL ABNORMAL INCREASED IN •H.Spherocytosis • H.Elliptocytosis • H.stomatocytosis • AI Hemolytic anemia. DECREASED IN • Thalassemia • Iron deficiency anemia.
- 25. OSMOTIC FRAGILITY TEST Shiftto left – increased OF Shift to right – decreased OF
- 26. 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
- 27. AUTOHEMOLYSIS TEST Measuresspontaneous hemolysis of blood incubated at 37ºc for 48 hrs Measure readings colorimetrically at 540nm Normal – 0.2-2 % With added glucose – 0-0.9 %
- 28. Decreased rate withadded Glucose Hereditary Spherocytosis PNH G6PD Deficiency No response to added glucose Pyruvate Kinase Deficiency
- 29. ACIDIFIED GLYCEROL LYSIS TIMETime 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
- 30. CRYOHEMOLYSIS Specific forHereditary 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%
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- 33. THALASSEMIA Autosomal dominant Reduced synthesis of normal Hb polypeptide chain due to molecular defects. Hematologic consequences: Low intracellular Hb Relative excess of unpaired chain
- 34. Pathogenesis Imbalanced synthesisof α & β chains Decreased total RBC Hb production Ineffective erythropoiesis Chronic hemolytic process Systemic iron overload
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- 36. LAB FINDINGS MicrocyticHypochromic anemia(2-3g/dl) Decreased MCV , MCH , MCHC Decreased Osmotic Fragility. Increased serum uric acid Anisopoikilocytosis
- 37. Lab Findings- BM Normoblastic erythroid hyperplasia Increased macrophages Inclusion bodies in normoblasts
- 39. SICKLE CELL ANAEMIA Hereditary disorder – autosomal recessive RBC contains Hb S α2β2 6GLU ↔ VAL Hypoxia , acidosis, hypertonicity, ↑temp→ deoxygenation → Hb S polymerisation → sickle cells
- 40. SICKLE CELL ANAEMIA •Normocytic normochromic anemia • Sickle cells • Reticulocytosis ( 10 – 20 %) • Anisopoikilocytsis • Howell jolly bodies • Normoblast • ↑RDW • BM – Erythroid hyperplasia
- 41. SOLUBILITY TEST Sicklecell 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
- 42. SICKLING TEST Blooddeoxygenated with reducing substances (sodium metabisulphite) Place on slide Seal coverslip Immediate sickling – Disease Sickling in 1 hr – Trait 1. Hb electrophoresis 2. High performance liquid Chromatography (HPCL) 3. Isoelectric focusing – agar gel 4. Prenatal diagnosis - PCR OTHER DIAGNOSTIC TESTS
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- 44. G6PD DEFICIENCY INTRODUCTION 1.Its an enzyme of HMP pathway 2. Protects against oxidative stress 3. X linked disorder 4. Deficiency result in to, Impaired NADPH production Accumulation of oxidants in cell Oxidative stress leads to Heinz body formation & extra- vascular hemolysis
- 45. BLOOD SMEAR ING6PD DEFICIENCY
- 46. 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
- 47. 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
- 48. OTHER TESTS ASCORBATECYANIDE 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)
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- 50. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA AcquiredClonal 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.
- 51. LAB FINDINGS Anemia Thrombocytopenia Hemosiderinuria Positive sucrose hemolysis test Positive Ham’s test Normal Osmotic fragility
- 52. SUCROSE HEMOLYSIS TEST Screeningtest Patient’s blood incubated in sucrose solution Sucrose promotes binding of complement to RBC Hemolysis
- 53. HAM’S TEST(Acidified serum lysistest) 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
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- 56. PATHOPHYSIOLOGY RBC’s + IgGAb 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
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- 60. Positive test 1. Isoimmunisation 2.Presence of free auto Ab in patient’s serum
- 61. WARM AIHA Individualsproduce Ab against their own erythrocyte Ag (autoantibodies) Ab react with red cell Ag best at 37oc. Lab findings PBS – Normocytic normochromic anemia - Reticulocytosis - Spherocytes, - Schistocytes, Polychromasia, NRBC’s - Neutrophilia - Platelet - normal or decreased
- 62. Bone marrow -Normoblastic erythroid hyperplasia - Erythrophagocytosis Other tests - Direct Coombs’ test (DAT)- positive
- 63. COLD AIHA Associatedwith IgM Ab which fixes complement & is reactive below 32o c First indication of the presence of unsuspected cold agglutinins is blood counts. 1. RBC count is inappropriately decreased for Hb% 2. MCV is falsely elevated (due to agglutination) 3. PCV is falsely low 4. MCH & MCHC are falsely elevated • Visible autoagglutination can be observed in tubes of anticoagulated blood as the blood cools to room temperature.
- 64. When RBC indicesgo haywire think of the possibility of cold agglutinin disease
- 65. PAROXYSMAL COLD HEMOGLOBINURIA Labfindings 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
- 66. DONATH LANDSTEINER(D-L) Test PATIENT’S WHOLE BLOOD CONTROLTEST INCUBATE FOR 30 MIN AT 370 C 40 C INCUBATE FOR 30 MIN AT 370 C 370 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
- 67. AUTOIMMUNE HEMOLYTIC ANEMIA Hemolyticanemia induced by immunization of an individual with RBC Ag’s from another individual . Eg: 1. Hemolytic transfusion reactions 2. Hemolytic disease of new born
- 68. DRUG INDUCED AIHA It is the result of an immune mediated hemolysis precipitated by ingestion of certain drugs. MECHANISM: 1. Drug adsorption (hapten type) 2. Immune complex formation 3. Autoantibody induction 4. Membrane modification
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- 71. Other conditions where hemolysisis seen Disseminated malignancy Leukemia Malignant lymphomas Renal failure Liver disease Rheumatoid arthritis Megaloblastic anemia
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- 74. REFERENCE Henry’s Clinicaldiagnosis & Management by Lab. Methods Shirlyn B. Mc Kenzie, Text book of Haemotology Wintrobe Dacie & Lewis, Practical Haematology de Gruchy’s clinical Haematology 5th edition, pages 137-210.
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Editor's Notes
- #16 Fragmented cells, spherocytes, blister cells and punctate basophilia blood film of a patient with Pyruvate Kinase Deficiency? Thorny Apple sputnik cells, polychromasia
- #17 Reticulocytosis
- #46 Blister cells (arrows) Heinz bodies 9new methylene blue stain) (Inset)
- #47 False normal-Reticulocytosis,False deficient-anemia
- #59 Differential diagnosis for causes of intravascular hemolysis resulting in a positive DAT. DAT = direct antiglobulin test; PCH = paroxysmal cold hemoglobinuria; WAHA = warm autoimmune hemolytic anemia; MT-AIHA = mixed-type autoimmune hemolytic anemia; CAS = cold agglutinin syndrome.
- #65 When RBC indices go haywire think of the possibility of cold agglutinin disease
- #71 Falciparum malaria Pl.vivax