Diagnostic Approach to hemolytic anemia

Diagnosis of hereditary and acquired hemolytic anemia

1. Diagnostic Approach to hemolytic anemia Part I – hereditary Causes By- Dr. Amita Yadav MBBS, MD (Pathology)

2. Hemolytic Anemia Definition: Those anemias which result from an increase in RBC destruction coupled with increased erythropoiesis Classification: Congenital / Hereditary Acquired

3. CLASSIFICATION OF HEMOLYTIC ANEMIAS INTRACORPUSCULAR DEFECTS EXTRACORPUSCULAR FACTORS HEREDITARY HEMOGLOBINOPATHIES ENZYMOPATHIES MEMBRANE-CYTOSKELETAL DEFECTS FAMILIAL HEMOLYTIC UREMIC SYNDROME ACQUIRED PAROXYSMAL NOCTURNAL HEMOGLOBINURIA MECHANICAL DESTRUCTION [MICROANGIOPATHIC] TOXIC AGENTS DRUGS INFECTIOUS AUTOIMMUNE

4. Classification- MAHA Transfusion reaction PNH Infections Snake bite Hemoglobinopathies Enzymopathies Membrane defects AIHA Intravascular hemolysis Extravascular hemolysis

5. How is Hemolytic Anemia Diagnosed? Two main principles To confirm that it is hemolysis To determine the etiology

6. GENERAL FEATURES OF HEMOLYTIC DISORDERS GENERAL EXAMINATION - PALLOR,JAUNDICE,DARK COLOURED URINE , BOSSING OF SKULL PHYSICAL FINDINGS - SOMETIMES SPLENOMEGALY HEMOGLOBIN - FROM NORMAL TO SEVERELY REDUCED MCV - USUALLY INCREASED RETICULOCYTE COUNT - INCREASED BILIRUBIN - INCREASED[MOSTLY UNCONJUGATED] LDH - INCREASED HAPTOGLOBULIN - REDUCED TO ABSENT

7. Hemolytic facies - chipmunk facies

8. EXTRAVASCULAR HEMOLYSIS-

9. INTRAVASCULAR HEMOLYSIS-

10. Laboratory Evaluation of Hemolysis Extravascular Intravascular   HEMATOLOGIC Routine blood film Reticulocyte count Bone marrow examination Polychromatophilia Erythroid hyperplasia Polychromatophilia Erythroid hyperplasia   PLASMA OR SERUM Bilirubin Haptoglobin Plasma hemoglobin Lactate dehydrogenase Unconjugated , Absent N (Variable) Unconjugated Absent (Variable)   URINE Hemosiderin Hemoglobin + +  severe cases  

11. POLYCHROMATOPHILIC CELLS

12. Patient History Acute or chronic Medication/Drug precipitants G6PD AIHA Family history Concomitant medical illnesses Clinical presentation

13. CASE 1 6 yr old child presenting with severe pallor,jaundice growth retardation Abnormal facies,hepatosplenomegaly + h/o recurrent blood transfusions CBC- Hb -3gm%, MCV-58FL, -MCH- 19pg RDW- 14% P.S- Microcytic hypochromic RBCs with target cells +

14. Differential Diagnosis- Iron Deficiency Anemia Thalassemia

15. Thalassemia - Thalassemia syndromes result from defects in rate of synthesis of alpha or beta chains.

16. Beta thalassemias - Mutation in Beta globin gene expression M.C- deranged splicing of m-RNA Thalassemia Major (Cooley’s Anemia)- Homozygous or double heterozygous form Accumulation of free alpha chains

17. . Type Genotype Anemia RBC mor-phology Hb electro. Clinical features Beta thalassemia Minor B/B+ ,B/B0 Absent or mild(Hb-10-12gm/dl)MCV & MCH reduced Mil d anisopoikilocytosis with mild microcytosis and hypochromia,target cells , basophillic stippling HbA - 90-93% Hb A2-3.5-8% (Diagnostic ) HbF - N to minor inc. Asymptomatic Beta thalassemia intermedia B+/ B+,Interaction of alpha,B,HbE,HbD,HbQ,HbS Moderate(7-10gm/dl), MCV & MCH reduced) Moderate changes HbF-10-30% Late onset,Not transfusion dependent,mild splenomegaly,facial and skeletal change B Thalassemia Major B0/B0,B0/B+,B+/B+ Mod. To severe( Hb - 3-8gm%) Dec.MCV,MCH,MCHC Severe changes,many nRBCs HbF -30-90% Onset in infancy,transfusion dependent,severe changes

18. Type Genotype anaemia RBC Changes Hb Electro. Clinical Features Silent Carrier aa /a- absent None Hb Bart not demon. Asymptomatic a Thalassemia trait a-/a-, aa /-- Absent or mild Microtic hypochromic Hb Bart not demon. Clinically normal HbH Disease ( beta tetramer) a-/-- Progressive anemia Hb-6-10 gm/dl Mod. anisopoikilocytosis with mild microcytosis and hypochromia,target cells Retic . Prep. HbH inclusion HbH Band- 5-35% Splenomegaly,moderate skeletal deformity Hydrops foetalis (gamma tetramer) --/-- Severe Severe changes,numerou erythroblasts 80-100% Hb Barts Fatal, pale and bloated baby,oedematous placenta

19. Reticulocyte preparation showing Golf ball HbH inclusions attached to red cell membrane.

20. Investigations- Peripheral blood examination Test for inclusion bodies Hb Electrophoresis Acid elution Test Bone marrow examination Radiograph

21. Peripheral blood films in Thalassemia major

22. Nestrof Test-

23. Acid Elution Test for Hbf ( Kleihauer’s Test) – A.red cells containing hbf are stained pink. B.Ghost cells indicating presence of HbA

24. a.Giemsa stained BM –macrophages laden with golden brown granules of hemosiderin pigment B.perl’s stain demonstrates iron stores

25. Bone changes in THALASSEMIA MAJOR- Hair on end appearance due to new bone formation and widening and thickening of diploe

26. Case 2 3 yr old male child presenting with pallor,jaundice,Severe pain of long bones, fever, Growth retardation CBC- Anemia , leucocytosis , reticulocytosis , LAB - S.bilirubin - 5mg% S.LDH -600 IU/L (normal upto 200)

27. Peripheral smear- Anisopoikilocytosis,hypochromia.Presence of drapanocytes,target cells Few red cells showing Howell Jolly bodies and nRBC

28. What is the diagnosis ? SICKLE CELL ANEMIA- Characterised by presensce of HbS which imparts sickle shape to red cells in a state of reduced oxygen tension. Substitution of valine in place of glutamic acid at 6 th position of beta globin chain. Altered solubility Polymerisation of HbS forming tactoids .

29. Sickle cell disease Mutation beta globin-6 Glu Val. Deoxy HbS ( polymerised ) Ca influx, K leakage stiff,viscous sickle cell venocclusion decreased RBC survival microinfarctions,ischemic pain anemia,jaundice , autoinfarct,leg ulcers gallstones

30. Sickling disorders- Type Genotype Clinical manifestation RBC morphology Hb Electrophoresis Sickle Cell Anemia β s/ β s Moderate to severe anemia.Hb5-10gm/dl. Mod.to severe anisopoikilocytosis.sickle cells,target cells,polychromatic cells ,Howell jolly bodies,reticulocytosis Hbs Predominant(70-90%0, HbA(0-10%), HbF (10-30%),HbA2 normal Sickle cell disease β s/ β 0, β s/ β + Moderate to severe anemia Moderate to severe changes Depending upon hemoglobinopathy present Sickle cell trait β s/ β Mild or no anemia. Hb 11-13%, Mild changes HbS 25-40%,HbA 60-75%,HbF<1%,

31. Clinical manifestations Vasoocclusive crisis Hemolytic crisis Aplastic crisis Splenic sequestration crises Hand foot syndrome( Dactylitis ) Acute chest syndrome

32. DIAGNOSIS – Peripheral blood examination ESR-Low Sickling Test Solubility test Hb electrophoresis- HbS is slow moving as compared to HbA and HbF HPLC Estimation of HbF by alkali denaturation test

33. CASE 3 45 yr old male came to opd in a remote PHC with burning micturition Urine R/M shows numerous pus cells++++ UTI diagnosed & medical officer gave cotrimoxazole 2 bd X 5days 1 wk later,pt developed severe pallor,palpitation,jaundice Lab- increased LDH, S.BILIRUBIN,RETIC COUNT P.S- shows polychromasia and irregular cells like bite cells and blister cells

34. Bite cells and blister cells in peripheral smear

35. Differential Diagnosis- G-6PD Deficiency Unstable hemoglobinopathy (Positive for Heat instability Test and Isopropanol precipitation Test)

36. G-6PD deficiency- First enzyme in HMP shunt pathway. Protects red cells from oxidant injury. Deficiency results in episodes of hemolysis .

37. Definitive risk Possible risk Doubtful risk antimalarials Primaquine Dapsone cholrproguanil chloroquine quinine Sulphonamides / sulphones Sulphametoxazole Dapsone Sulfasalazine Sulfadimidine Sulfisoxazole Sulfadiazine Antibacterials / Antibiotics Cotrimoxazole Nalidixic acid Nitrofurantoin Ciprofloxacin Norfloxacin Cholramphenicol p - Aminosalicylic acid Antipyretic/ Analgesics Acetanilide Phenazopyridine [ pyridium ] Acetylsalicylic acid High dose[>3g/d] Acetylsalicylic acid [<3g/d] Acetaminophen

38. Hematologic findings- Abrupt fall in Hb to 6-10 gm/dl Reticulocyte count increases to 20-50% Heinz bodies are seen in reticulocyte preparation Peripheral Smear- Moderate degree of anisopoikilocytosis , polychromatophilia , microspherocytes , bite cells, blister cells Hemoglobinuria Uribilinogen increased.

39. Bite cells and blister cells in peripheral smear

40. Diagnosis- History Peripheral blood smear Screening tests- 1.Methemoglobin Reduction Test 2.Fluorescent spot Test Quantitative assay of G-6PD ( assess a few weeks after the acute hemolysis ) DNA analysis by PCR

41. 2. Pyruvate Kinase Deficiency AR Deficient ATP production, Accumulation of G-3-p,2,3-DPG and glucose Chronic hemolytic anemia Clinical features- Pallor,jaundice Uncommonly gall stones and/or splenomegaly

42. Hematologic Finding- Moderate anemia Peripheral smear-Moderate degree of anisocytosis,Prickle cells( red cells with sharp thorn like projections,few echinocytes . Autohemolysis Test Quantitative assay

43. CASE 4 14 YR old female present with anemia, jaundice Rt hypochondrial pain O/E- vitals stable.pallor+,icterus+,splenomegaly + Usg - cholilithiasis Lab investigation- elevated LDH, S.Bilirubin Peripheral smear shows- Spherocytes

44. Differential diagnosis for spherocytosis - Hereditary spherocytosis Autoimmune hemolytic anemia Burn, G-6-PD deficiency, transfusion reaction, Clostridial sepsis, Snake/spider bite, copper sulphate poisoning, microangiopathic hemolytic anemia

45. Red Cell Membrane Defects 1.Hereditary Spherocytosis Usually inherited as AD disorder Defect: Deficiency of Beta Spectrin or Ankyrin  Loss of membrane surface area becomes more spherical Destruction in Spleen

46. C/F: Mild to moderate anemia Intermittent jaundice Splenomegaly Pigmented gall stones- in 50-75% of patients

47. Complications Clinical course may be complicated with Crisis: Hemolytic Crisis : associated with infection Aplastic crisis : associated with Parvovirus infection

48. Investigations- Mild to moderate anemia Peripheral smear- microspherocytes,polychromatophilia S.Bilirubin increased mainly unconjugated type Fecal stercobilinogen increased S. haptoglobin reduced. Osmotic fragility Test

49. Peripheral Blood Picture-

50. Osmotic Fragility test Curve-

51. Glycerol Lysis Test Flow Cytometry based on EMA ( Eosin-5-Maleimide)-mean fluorescence intesity of EMA tagged red cells is lower in HS.

52. Diagnostic Approach to hemolytic anemia Part II – Acquired Causes By- Dr. Amita Yadav

53. Immune Hemolytic Anemia Result from RBC destruction due to RBC autoantibodies : Ig G, M, E, A Most commonly-idiopathic Classification 1.Autoimmune Hemolytic Anemia Warm AI hemolysis:Ab binds at 37degree Celsius Cold AI Hemolysis : Ab binds at 0-10 degree Celsius 2.Alloimmmune Hemolytic Anemia Hemolytic transfusion reaction Hemolytic disease of new born

54. Classification-

55. Warm and Cold antibodies-

56. 1.Warm AI Hemolysis : Can occurs at all age groups F > M Causes: 50% Idiopathic Rest - secondary causes: 1.Lymphoid neoplasm: CLL, Lymphoma, Myeloma 2.Solid Tumors: Lung, Colon, Kidney, Ovary, Thymoma 3.CTD: SLE,RA 4.Drugs: Alpha methyl DOPA, Penicillin , Quinine, Chloroquine 5. UC, HIV

57. 2. Cold AI Hemolysis Usually Ig M directed at the RBC I antigen CAUSES- Infection: Mycoplasma pneumonia, Infec Mononucleosis Neoplasms : waldenstrom macroglobulinemia , lymphoma,CLL,kaposi sarcoma, myeloma. C/F: Elderly patients Exacerbations in the winter Cold , painful & often blue fingers, toes, ears, or nose ( Acrocyanosis )

58. Mechanism of hemolysis -

59. Mechanism of hemolysis due to drugs-

60. Clinical features- Progressively increasing pallor Mild to moderate jaundice Cola coloured urine

61. Investigations- History of drug intake Hb - low Reticulocytes - 4-30% Peripheral smear- Anisocytosis with polychromatophilia Microspherocytes S.Haptoglobin -decreased S.LDH-elevated S.Bilirubin - >4mg/dl Urine urobilinogen -increased Coomb’s Test

62. Case 5 32 yr old presented 4 days history of distention of abdomen and rt hypochondrial pain and has h/o passage of dark colored urine early morning On USG- hepatomegaly,gross ascites,hepatic vein thrombosis Lab : Hb – 7gm%. WBC- 2200, PLC- 80,000 LDH- 600, S.Bilirubin - 4 mg% urine bile pigment +, heme dip stick++ What is the diagnosis?

63. Paroxysmal nocturnal hemoglobinuria Acquired chronic H.A Persistent intra vascular hemolysis H/0 passing dark coloured urine early morning. Pancytopenia Lab : hemoglobinburia,hemosiderinuria,increased LDH,bilirubin Risk of venous thrombosis P.S – polychromatophilia , normocytic normochromic anemia B.M – normoblastic hyperplasia

64. Diagnosis- -flow cytometry CD59-,CD55- -Hams’ acidified serum test - Sephacryl microtyping gel card test - FLAER(Flow cytometry based assay utilises Fluorescent labelled inactive variant of protein aerolysin .

65. Urine samples from a typical PNH patient-

66. Case 6 25 yr old male with RHD – severe MR done MVR,after 10 days presented with pallor, palpitation,jaundice CBC shows Hb – 7.5 gm %, Hct -22 % Lab : S.bilirubin -4.5mg% LDH -600 Retic count 10% Peripheral smear – Fragmented red cells,schistocytes (helmet cells,triangular cells),few spherocytes,polychromasia

67. Microangiopathic hemolytic anemia

68. Non-Immune Acquired Hemolytic Anemia 1. Mechanical Trauma A). Mechanical heart valves, Arterial grafts: cause shear stress damage B).March hemoglobinuria: Red cell damage in capillaries of feet C). Thermal injury: burns D). Microangiopathic hemolytic anemia (MAHA): by passage of RBC through fibrin strands deposited in small vessels  disruption of RBC eg: DIC,PIH, Malignant HTN,TTP,HUS

69. Microangiopathic hemolytic anemia( maha ) Other findings - leukocytosis - thrombocytopenia(DIC,TTP) - hemoglobinuria - deranged RFT - PT,APTT prolonged (DIC,TTP)

70. Hemolysis due to infections- P.falciparum malaria P.Vivax Trypanosomiasis Clostridial Sepsis Bartonellosis E.coli (HUS) Leptospirosis