approach to hemolytic anemias various causes.pptx

Hemoytic anemia red cell membrane defects enzymatic defects hemiglobinopathies

1. Hemoglobinopathies Structural hemoglobinopathies —(QUALITATIVE) hemoglobins with altered amino acid sequences that result in deranged function or altered physical or chemical properties.- HbS Defective biosynthesis of globin chains (QUANTITATIVE) α Thalassemias β Thalassemias δβ , γδ , βαβ thalassemias Thalassemic hemoglobin variants —structurally abnormal Hb associated with co-inherited thalassemic phenotype HbC HbD Hb Lepore Hereditary persistence of fetal hemoglobin —persistence of high levels of HbF into adult life

2. Hemolytic anemias Hemolytic anemias are a group of disorders characterized by - increased erythrocyte destruction - bone marrow compensates for hemolysis by increased erythropoiesis . - shortening of life span upto 15-20 days. Due to defect in RBC (intrinsic) or outside RBC (extrinsic)

3. Hemolysis (red cell destruction) Extravascular(80-90%) Intravascular (10-20%)

4. PARAMETER INTRAVSCULAR HEMOLYSIS EXTRAVASCULAR HEMOLYSIS Site Within circulation Macrophages of spleen causes PNH, PCH, incompatible blood transfusions, Erythrocyte fragmentation disorders Black water fever in falciparum malaria Clostritrial sepsis Drugs and Chemicals Snake & spider venom Thermal injury, Burns Hemoglobinopathies , hereditary and autoimmune hemolytic anemia Splenomegaly absent Present Reticulocyte count increased increased Indirect bilirubin increased increased Plasma hb Markedly increased Mild-moderately increased hemoglobinuria present Absent Hemosiderinuria present absent methalbuminemia present absent Serum haptoglobin Decreased Decreased Serum LDH increased increased

5. CLASSIFICATION

6. Clinical signs 1-Less Hemoglobin - Pallor 2-Increased destruction of RBC –Jaundice, Cholelithiasis (pigmented stones) 3-Compensatory Mechanism- Skeletal abnormalities (chip- munk facies- Thalassemia) 4-Cause of Hemolytic anemia Extravascular hemolysis - Splenomegaly Sickle cell anemia- vasoocclusive : Leg ulcers , Hand foot syndrome, Priapism Thalassemia- Iron overload ( ineffective erythropoises , Hemolysis, Regular blood transfusion. Manifest as  Growth retardation. Hyperpigmentation of the skin Heaptocellular damage (cirrhosis) Diabetes mellitus Gonadal dysfunction Hyperthyroidism

7. Microcytic hypochromic cells, target cells  Thalassemia

8. LABORATORY INVESTIGATIONS

9. Complete blood count Hemoglobin Decreased Red cell indices MCV MCHC RDW Increased Increased Increased RBC count Leucocyte count Platelet count Decreased Increased Increased Reticulocyte count Increased Peripheral blood film examination Specific cell abnormalities

10. MORPHOLOGICAL ABNORMALITIES IN BLOOD

11. A) Normal RBCs B) Microspherocytes (H.S) C) Spherocytes (AIHA) D)Hereditary Elliptocytosis

12. Target cells Schistocytes Blister cells (G6PD Def) CAD

13. Hb H Cabot ring

14. GENERAL EVIDENCE OF HEMOLYSIS Evidence of increased haemoglobin breakdown Evidence of compensatory hyperplasia Evidence of damage to the RBCs Demonstration of shortened RBC lifespan

15. Evidence of increased haemoglobin breakdown Reduction in blood hemoglobin levels (>1g/dl/week) Jaundice and hyperbilirubinemia Reduced plasma haptoglobin and hemopexin levels Reduced levels of glycosylated hemoglobin Increased plasma lactate dehydrogenase (LDH)levels

16. intravascular hemolysis Free Hb in the plasma( hemoglobinemia ) Hb-Haptoglobin complex Low plasma haptoglobin Filtration by glomerulus- Hemoglobinuria hemosiderinuri a Haem Methhemoglobin + albumin Methhemoglobinuri a

17. HEMOGLOBINEMIA - normal values of <1 mg/dl of plasma HEMOGLOBINURIA When plasma Hb exceeds the haptoglobin -binding capacity, Hb dimers are excreted into the urine, resulting in hemoglobinuria. Urine that contains Hb ranges from faint pink to deeper red, or even to almost black. Detected by- benzidine or orthotoludiene tests. HEMOSIDERINURIA Hb in the glomerular filtrate is partially reabsorbed by the proximal tubular cells, and the Hb iron is incorporated into ferritin and hemosiderin. Subsequently, the iron-containing tubular cells are sloughed into the urine. Detected by- qualitative test based on the Prussian blue reaction.

18. 3. Evidence of compensatory hyperplasia

19. POLYCHROMATOPHILS BASOPHILIC STIPLING

20. ERYTHROID HYPERPLASIA

21. 3. Evidence of damage to the RBCs Spherocytosis Increased osmotic fragility Fragmented RBCs Heinz bodies 4. Demonstration of shortened RBC lifespan Radioisotope labelled RBCs- Chromium labelled

22. HEREDITARY SPHEROCYTOSIS MC – Spectrin deficiency f/b combined ankyrin and spectrin deficiency and least common band 3 and protein 4.2 deficiency.

23. TESTS: Osmotic fragility Acidified glycerol lysis test Cryohemolysis : specific for HS Autohemolysis Flow cytometric test (dye binding test) Membrane protein analysis

24. OSMOTIC FRAGILITY TEST measure of the resistance of erythrocytes to hemolysis by osmotic stress PRINCIPLE: The test consists of exposing RBC to decreasing strengths of hypotonic saline solutions and measuring the degree of hemolysis colorimetrically Hemolysis is determined by spectrophometre . More sensitive is the incubated OF test RBCs incubated for 18-24 hrs at 37 C. This procedure takes advantage of the observation that all erythrocytes lose membrane under these incubation conditions; however, the process is markedly accelerated in HS red cells.

25. Take heparinized venous blood Take stock solution of buffered saline 10gm/l Dissolve 45gm of Nacl 6.5gm of Na di hydrogen phosphate 1.25gm of di Na hydrogen phosphate From this make different conc of hypotonic sol (12 TEST TUBES) Add 50 l vol of blood Mix by inverting Allow them to stand for 30 min Then remix & centrifuge for 5 min at 1200-1500rpm Read at 540 nm SPECTROMETERE 100% lysis in tube 12 (water)

26. Fresh blood Initial lysis 5.0 Complete lysis 3.0 Median corpuscular fragility 4.0-4.45 Fresh blood Initial lysis 8.0 Complete lysis 5.0 Median corpuscular fragility 6.5

27. Factors affecting osmotic fragility tests In carrying out osmotic fragility tests by any method, three variables capable of markedly affecting the results must be controlled, These are as follows: The relative volumes of blood and saline The final pH of the blood in saline suspension The temperature at which the tests are carried out. The effect of pH : a shift of 0.1 of a pH unit is equivalent to altering the saline concentration by 0.1 g/l, the fragility of the red cells being increased by a decrease in pH. An increase in temperature decreases the fragility, an increase of 5 °C being equivalent to an increase in saline concentration of about 0.1 g/l.

28. Acidified glycerol lysis test Assessed for rate of lysis by glycerol and expressed as length of time for 50% lysis. Principle : glycerol present in hypotonic saline sol slows the rate of entry of water molecule, so that the time taken for lysis may be measured conviently with spectrometer Result : normal blood takes 30 min to reach 50 % of lysis. In spherocytosis, it takes 25-150 sec Cryohemolysis : specific for HS Principle : Whereas osmotic fragility may be abnormal in any condition where spherocytes occur, it has been suggested that cryohaemolysis is specific for HS. This appears to result from the fact that the latter is dependent on factors that are related to molecular defects of the red cell membrane rather than to changes in the surface area-to-volume ratio. % cryohemolysis is observed after transferring cells from 37 C to 0 C for 10 mins. In H. S cryohemolysis >20% than normal 3-15%.

29. Autohemolysis Blood incubated at 37 C for 48hrs without glucose Compensatory increase in Na extrusion To differentiate membrane & enzyme defects Principle : blood is incubated both with & with out sterile glucose sol for 48 hr. After this spontaneous hemolysis is measure Interpretation If abnormal haemolysis is fully corrected by glucose, a membrane abnormality is likely If abnormal haemolysis shows little or no correction by glucose, a metabolic abnormality is likely Exhaustion of ATP Failure of pumps Cell swelling Authemolysis assessed by colorimeter

30. Flow cytometric test (dye binding test) Principle : The flow cytometric (dye-binding) test measures the fluorescence intensity of intact red cells labelled with eosin-5-maleimide (EMA), which reacts covalently with Lys-430 on the first extracellular loop of Band 3 protein. Flow cytometric screening test with reduced fluorescence of red cells in H.S due to absence of Band -3 protein.

31. Membrane protein analysis Sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE ) of the membranes identify qualitative and quantitative alterations in the specific proteins. Densitometry of protein bands on the gel gives an overall profile showing spectrin , ankyrin , Band 3 (the anion transprt protein) and protein 4.2.

32. G-6-PD DEFICIENCY

33. VARIANTS CLASS ENZYME ACTIVITY C/F I <5% of normal Rare, cong non- spherocytic H.A II <10% of normal Episodic, drug induced hemolysis III 10-60% of normal Acute self limited hemolysis IV 60-100% of normal - V Inc. -

34. PATHOGENESIS Exposure to oxidants Heinz bodies Cross linking of sulph-hydryl groups Denatured protein- Heinz bodies Intravascular hemolysis Red cell deformability (IN SPLEEN) BITE CELL

35. DIAGNOSIS- PBF-half ghost cells , Bite cells and Heinz bodies. Qualitative or screening tests- fluorescent spot test Methemoglobin reduction test G6PD assay: confirmatory Cytochemical tests

36. FLUORESCENT SPOT TEST- Principle NADPH, generated by G6PD present in a lysate of blood cells, fluoresces under long-wave ultraviolet (UV) light. In G6PD deficiency, there is an inability to produce sufficient NADPH; this results in a lack of fluorescence Reagent mixture- G-6-PO4 NADP Saponin GSSG G-6PD present-NADPH produced Fluorescence (>20%) Red cells with <20% of normal G6PD activity do not cause detectable fluorescence False-normal . If there is reticulocytosis , a vivid fluorescence may be seen with a genetically G6PD-deficient blood sample because young red cells have more G6PD activity. If the test is carried out during an acute haemolytic episode, the patient’s blood should be retested when the reticulocyte count has returned to normal.

37. METHEMOGLOBIN REDUCTION TEST- Principle: Na nitrite reduces oxyhb to methb . Methylene blue( redox dye) Reduces methb to hb in presence of G-6PD. Red coloured blood Absent G6PD –inc. methb thus brown coloured blood

38. Hemoglobin Methemoglobin (Hi) Add methylene blue Normal subject Patient Hi Hi Hb Hi G6PD - G6PD + Methemoglobin Reduction Test Na Nitrite Interpretation: Cherry red color: G6PD within normal limit Brown color: G6PD deficient

39. QUANTITATIVE ASSAYS- G6PD assay measured at 340nm by spectrophotometer .

40. CYTOCHEMICAL TESTS FOR DEMONSTRATING DEFECTS OF RED CELL METABOLISM Cytochemical methods have been developed by means of which red cells deficient in G6PD can be demonstrated. Principle : red cells are treated with sodium nitrite to convert their oxyhaemoglobin (HbO2) to methaemoglobin (Hi). In the presence of G6PD, Hi reconverts to HbO2, but in G6PD deficiency, Hi persists. The blood is then incubated with a soluble tetrazolium compound (MTT), which will be reduced by HbO2 (but not by Hi) to an insoluble formazan form .

41. Interpretation Normnal G6PD activity: all the red cells are stained. In hemizygotes for G6PD deficiency:majority of the red cells are unstained. In heterozygotes, mosaicism is usually seen; usually 40–60% of the cells are unstained

42. HEMOGLOBINOPATHIES

43. SICKLE CELL ANEMIA PATHOGENESIS Non-polar valine is substituted for polar glutamic acid at 6 th position of A 3 helix of β -chain [ β 6 (A 3 ) Glu →Val ] Solubility markedly reduced → In deoxygenated states → form long microtubular arrays called tactoids → Erythrocytes stretch around the tactoids → Long, pointed, slightly curved cells called “Sickle Cells”. Life span of RBC – 14 Days. Polymerisation – Begins when O 2 saturation < 85% and is complete at 38%.

44. Tests to confirm Sickling test Hb S solubility test Hb electrophoresis on cellulose acetate & citrate agar HPLC is a sensitive method for confirmation

45. SICKLING TEST Priniciple : if oxygen is removed from red cells of sickle cell disease, red cells become sickle shaped resulting from the presence of abn HbS which in deoxidized state undergoes a hydrophobic bond dependent polymerization. Procedure A thin wet film preparation Reagents: Sodium meta bisulphite : 2% Add anticoagulated blood with reducing reagent and seal between cover slip & glass slide with a petroleum jelly/paraffin wax mixture The process takes 12 hr for sickle cell trait and evident immediately for sickle cell anemia

46. Sickling test Sodium metabisulphite Add 1 drop of reagent to 1 drop of anticoagulated blood Sodium dithionite ( freshly prepared) Add 5 drops of reagent to 1 drop of blood Seal between slide and coverglass with a petroleum jelly/paraffin wax Immediate sickling : sickle cell anemia A min of 1 hr in sickle cell trait SICKLING TEST

47. Solubility test Rapid confirmatory test. Principle : Sickle cell haemoglobin is insoluble in the deoxygenated state in a high molarity phosphate buffer. The crystals that form refract light and cause the solution to be turbid A mixture of a lysing agent and a reducing agent is added to blood HbS liberated from lysed RBCs is deoxygenated with dithionite → HbS polymerises → Opacity of solution.

48. Hb electrophoresis Principle of Hb electrophoresis Hb is (-) charged protein and when subjected to electrophoresis will migrate toward the anode (+) Diverse Hbs have different charges, and it is on the basis of these charges and amount, that the hbs are found to operate at different speeds both in the alkaline and acid gel

49. Hb electrophoresis Cellulose acetate pH 8.4-8.6 Satisfactory separation of Hbs C, S, F, A, Hbs S, D, and G migrate together as do Hbs C, E, and O Arab Citrate agar pH 6.0 Satisfactory separation of Hbs S & C with others Hbs A, D, lepore migrate together as do Hbs F & barts

50. HPLC

51. Hb E – elutes with A 2 Hb D – Unknown peak at 3.8 +/- 0.1 min Hb S – S- window Hb C – C-window 22-40 % - heterozygous 70 –90 % - homozygous Compound conditions also present (S+ Beta thal , Hb SD , E+ Beta thal etc )

52. Neonatal diagnosis – Isoelectric focussing and High performance liquid chromatography (HPLC) Because HbS in infants is low. Prenatal Screening – DNA based analysis (PCR) – from amniotic fluid cells (15-20 wks) or by CVS (10-12 wks) Multiparameter flow cytometry – non-invasive

53. THALASSEMIA SYNDROMES Grp of congenital anemias that have deficient synthesis of one or more of the globin subunits of the normal Hb . Thomas Cooley in Detroit in 1925. MC genetic disorders worldwide. PATHOPHYSIOLOGY – - Results from an alteration in the rate of globin chain production. - Impedes Hb synthesis and creates an imbalance with the other, normally produced globin chains. - An excess of the abnormally produced type is present and accumulates in the cell as an unstable product - Cell Destruction.

54.  Thalassemia major HbF levels are high How to demonstrate HbF Acid elution test Alkali denaturation method Electrophoresis

55. NESTROF test Naked Eye Single Tube Rapid Osmotic Fragility Test Screening test for thalasemia trait Reduced osmotic fragility of RBCs. 2 ml of 0.36% buffered saline sol. is taken in 2 test tubes Control : Add 0.02 ml of normal blood Test : Add 0.02 ml of patient blood Mix well. After ½ hr put a white paper with a dark line behind both the tubes Interpret: in Thalessemia trait cases black lines are not seen

56. Acid elution test HbF containing RBCs are resistant to elution at acidic pH Interpret: HbF cells stain red, adult ghost cells are pale pink

57. Automated high-performance liquid chromatography (HPLC) is increasingly replacing haemoglobin electrophoresis as the initial investigative procedure Isoelectric focusing (IEF) is used only to a limited extent, mainly for neonatal screening Molecular techniques demonstrating specific gene mutations

58. DNA based analysis (PCR)

59. FRAGMENTATION HEMOLYSIS

60. Paroxysmal nocturnal hemoglobinuria HAM’s test (Acidified Serum Test ) Sucrose lysis test Gel card method Flow cytometry for GPI linked proteins

61. Acidified Serum Test (Ham Test ) Definitive diagnostic test Principle : Acidified serum activates alternative complement pathway resulting in lysis of patient’s rbcs Method - red cells incubated in separate tubes to fresh acidified serum (0.5mL). Lysis determined by optical density of the supernatant fluid after 1hr incubation. 10-50% Lysis -Positive test Acidified-serum lysis test with additional magnesium (modified Ham test) Principle : The sensitivity of the Ham test can be improved by the addition of magnesium to the test to enhance the activation of complement. 76

62. Sucrose Hemolysis Test Screening test 10% sucrose provides low ionic strength which promotes complement binding resulting in lysis of patient’s rbcs . Amount of hemolysis quantified- spectrophotometer Interpretation The sucrose lysis test is based on the fact that red cells absorb complement components from serum at low ionic concentrations. PNH cells, because of their great sensitivity, undergo lysis but normal red cells do not Less specific than Ham test >10% hemolysis –positive test 77

63. PNH Diagnosis by Flow Cytometry Considered method of choice / gold standard for diagnosis of PNH Detects actual PNH clones lacking GPI anchored proteins For Granulocytes-CD14 & Cd66b RBC & Platelet- CD55 &CD59 More sensitive and specific than Ham and sucrose hemolysis test

64. Gel Based Method (Column agglutination technique ) Normal PNH

65. IMMUNE-HEMOLYTIC ANEMIAS

66. Autoimmune hemolytic anemia

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