Approach to diagnosis of hemolytic anemia.pptx

Hemolytic anemia

1. APPROACH TO DIAGNOSIS OF HEMOLYTIC ANEMIA Presentor : Dr. Aesha Soni Moderator : Dr. Neeraj Kumari

2. HEMOLYTIC ANEMIA Hemolytic anemias are characterised by increased red cell destruction. Anemia is the result of premature destruction of red cells exceeding the erythropoietic capacity of the bone marrow. Mainly of two types: 1. Extravascular Destruction 2. Intravascular Destruction

3. Aetiological classification of HA Intra-corpuscular Defect Extra-corpuscular Defect Membrane Disorders 1 Haemoglobin Defects 2 Enzymopathies 3 Immune System Mechanisms 1 Non-Immune System Mechanisms 2 Abnormalities in iron metabolism 4 Hemolytic anemias of unknown origin 5

4. HA Aetiological Classification | Intra-corpuscular Anemia INTRA-CORPUSCULAR (INTRINSIC) MECHANISM Primary Mechanisms Aetiological Classifications Causal Factors & pertaining disorders Membrane Disorders Hereditary Spherocytosis Hereditary Elliptocytosis Hereditary Stomacytosis Enzymopathies G6PD deficiency or any Penrose Phosphate Pathway enzyme deficiency Pyruvate Kinase deficiency or any Embden-Meyerhof pathway enzyme deficiency Haemoglobin Defects Haemoglobinpathies Sickle – cell anemia Other homozygous disorders (Hb-C, Hb-E, Hb-D, etc.) Thalassaemia β Thalassaemia major Hb-H disease Double heterozygous disorders Sickle – cell β Thalassaemia Abnormalities in iron metabolism Sideroblastic anemia Aceruloplasminemia Hemolytic anemias of unknown origin Idiopathic autoimmune hemolytic anemia

5. HA Aetiological Classification | Extra-corpuscular Anemia Extra-corpuscular (Extrinsic) Mechanism Primary Mechanisms Aetiological Classifications Causal Factors & pertaining disorders Immune System Mechanisms Autoimmune hemolytic anemia (AIHA): Warm AIHA Cold AIHA Alloimmune hemolytic anemia : Hemolytic disease of Newborn Hemolytic transfusion reactions Drug-induced immune hemolytic anemia : Non-Immune System Mechanisms Mechanical hemolytic anemia / Fragmentation Syndromes Microangiopathic hemolytic anemia ( MAHA) H emolytic uremic syndrome (HUS ) H eart valve disorders Microbial infections I ntravascular hemolysis in malaria S epsis-induced hemolysis B acterial toxins causing hemolysis . Chemical agents E xposure to certain chemicals, drugs, or toxins causing hemolysis , such as in drug-induced hemolytic anemia , or from exposure to oxidant chemicals like naphthalene and benzene derivatives.

6. 1. Establishing the presence of hemolysis Look for the S igns and Symptoms of H emolysis such as: Fatigue Pallor Jaundice Dark urine Splenomegaly Gall stones Skeletal abnormalities Leg ulcers Tachycardia Dyspnea on exertion

7. 2 . Determining the cause of hemolysis PERIPHERAL SMEAR EXAMINATION RETICULOCYTE COUNT IF ANEMIC, GO FOR COMPLETE BLOOD COUNT CBC PHYSICAL EXAMINATION PATIENT IS SYMPTOMATIC

8. 3 . Determining whether the hemolysis is intra or extracorpuscular :

9. PARAMETERS EXTRAVASCULAR HEMOLYSIS INTRAVASCULAR HEMOLYSIS Site of hemolysis Reticuloendothelial cells – Spleen, bone marrow. In circulating blood S. Methaemalbumin Negative Positive Haemoglobinemia Absent Present S. Ferritin Normal or increased Decreased S. Bilirubin ( Unconjugated) Increased (↑↑) Increased (↑) S. LDH (↑) (↑↑) Haemoglobinuria Absent Present Hemosiderinuria Absent Present S. Haptoglobin Normal Decreased Disease states Thalassemia, Sickle cell anemia , Hereditary spherocytosis. PNH, G6PD Deficiency, Black water fever. Tissue iron in spleen, liver (↑) (↓) or Normal Reticulcyte count (↑) (↑)

10. RED CELL MEMBRANE DISORDERS

11. HEREDITARY SPHEROCYTOSIS Hereditary haemolytic anemia Autosomal dominant Both sexes equally May present in childhood or adulthood Due to mutation in Ankyrin or Spectrin or Band 3 protein gene. Clinical presentations: Intermittent jaundice Mild to moderate anemia Splenomegaly Pigment Gallstones

12. Anemia , reticulocytosis Splenomegaly Phagocytosed by RE cells of the spleen Red cells become spherical and fragile To compensate for membrane loss, cells become spherical SPHEROCYTES Reduced deformability Reduced membrane plasticity Increased extravascular hemolysis Point mutations causing B eta Spectrin and Ankyrin deficiency Reduced membrane stability with loss of RBC membrane Difficulty in passing through splenic sinusoids

13. Peripheral smear findings in Hereditary S pehrocytosis Microspherocytes : Small dense RBC’S without any central pallor. Uniform in size and density Polychromatic cells ( due to reticulocytosis )

14. Osmotic Fragility Test Screening test for Hereditary Spherocytosis This test assesses the ability of RBC’s to withstand osmotic stress when suspended in decreasing concentrations of hypotonic saline solutions Spherocytes have reduced surface area to v olume ratio and are osmotically fragile. .

15. Osmotic Fragility curve Shift to right – Hereditary Spherocytosis Shift to Left - Thalassemia

16. Other investigations MCV, MCH – Usually normal or slightly reduced, MCHC - Increased Bone marrow – Erythroid hyperplasia with normoblastic reaction Increased unconjugated bilirubin S. LDH – raised Coomb’s test – Negative Eosin-5-maleimide (EMA) Binding via Flow cytometry – Reduced in HS Treatment – Folic Acid supplementation, Splenectomy.

17. HEREDITARY ELLIPTOCYTOSIS Autosomal dominant disorder B- Spectrin mutation Usually presents as mild haemolytic anemia Peripheral smear : Presence of > 25% elliptocytes (oval shaped RBC’S ) Osmotic fragility: Normal

18. HEREDITARY STOMATOCYTOSIS Autosomal dominant Deficiency of Stomatin protein , leading to increased permeability. MCV raised , MCHC reduced. Peripheral smear: Stomatocytes (>50%) – red cells with central slit like pallor. Stomatocytes are also seen in : Alcoholism, drug induced hemolysis and hepatobiliary disease.

19. ENZYMOPATHIES

20. GLUCOSE - 6 - PHOSPHATE DEHYDROGENASE DEFICIENCY X- linked recessive disorder India has common 4 variants: Mediterranean, Kerala- kalyan , Orissa and Chatran . On exposure to oxidative stress (Infection, drugs like naphthalene, antimalarials , sulpha drugs, nitrofurans ) , G6PD deficient red blood cells will have accumulation of toxic free radicals (H202) and accumulation of denatured hemoglobin, causing intra as well as extravascular hemolysis resulting in anemia, jaundice and reticulocytosis .

21. Peripheral smear: Bite cells, Blister cells, Spherocytes Supraavital stain: Heinz bodies , Reticulocytosis . Definitive diagnosis depends on demonstration of G6PD deficiency by fluorescent spot test, methemoglobin reduction test, or dye decolorization test

22. PYRUVATE KINASE DEFICIENCY Rare, Autosomal recessive condition Nonspherocytic haemolytic anemia . Due to PK enzyme deficiency, ATP synthesis is impaired leading to shortened lifespan of red blood cells. Peripheral smear: Prickle cells (red cells with sharp thorn like projections) Clinically, may present as neonatal jaundice to chronic compensated haemolytic anemia . Diagnosis: Autohemolysis test, quantitative enzyme assay and DNA PCR analysis.

23. HEMOGLOBIN DEFECTS

24. THALASSEMIA It results from inherited defect in the rate of synthesis of globin chains . Imbalance of globin chain production leads to : Ineffective erythropoiesis, Defective Hb production, Red cell Hb precipitates, Hemolysis and variable degree of anemia .

25. Alpha Thalassemia- The alpha gene deleted, loss of one gene or both gene from each chromosome 16 may occur in association with production of some or no alpha globin chain . Hydrops fetalis / Hb Barts Hemoglobin H disease Alpha Thalassemia trait Silent carriers Beta Thalassemia- (Major, Intermedia, Minor, Minima) Defective production usually result from disabeling point mutation causing no or reduced ß chain production.

26. PATHOPHYSIOLOGY 27

27. BETA THALASSEMIA MAJOR Autosomal Recessive Homozygous disorder, Transfusion dependent Baby is well at birth till 3 months (due to HbF ) Afterwards, presents in childhood with growth delay and failure to thrive. Ineffective Erythropoiesis with Extravascular hemolysis Progressive pallor- Severe Anemia Extramedullary hematopoesis - Hepatosplenomegaly Iron overload - Endocrine dysfunction Bone changes- Hair on End appearance due to widening of diploe in skull Bone marrow – Hypercellular , Marked erythroid hyperplasia ( normoblastic ) with hemosiderin laden macropahges (Increased iron stores)

28. PS features - Severe Microcytosis , Target cells, Nucleated RBC’s (5 to 40/ hpf ) Hb electrophoresis: HbF - 90-96 % HbA2 - 3.5 %- 5.5% HbA - 0 %

29. BETA THALASSEMIA MINOR (TRAIT) Asymptomatic carriers Mild Anisopoikilocytosis , microcytosis , hypochromia Few Target cells Similar picture of Iron deficiency Anemia MCV & MCH reduced , MCHC near normal Low RDW, HCT < 30-33% Mentzer index < 13

30. Beta Thalassemia Trait HPLC Hb electrophoresis: HbA2 - 4.5 to 8 % (normal <3.9 %) HbA - 90 to 93 %, HbF - 1 to 4% HbA2 between 3.3 to 3.7% - further assess iron status and repeat Severe IDA – reduces HbA2 Vit B12 def. – Increases HbA2

31. BETA THALASSEMIA INTERMEDIA Clinical spectrum between Thalassemia Major & Minor Anemic but not Transfusion dependent Less severe than major Moderate APC & Anemia , Microcytosis , Hypochromia Few Target cells and basophilic stippling. Hb electrophoresis - HbF - 20-100 % HbA2 -3.5%-5.5% HbA – 0-30%

32. SICKLE CELL DISORDERS Presence of HbS imparts sickle shape to red cells in a state of reduced oxygen tension . Homozygous Sickle cell A nemia – Only HbS (SS) Heterozygous Sickle cell Trait – Both HbS and HbA (AS) Double Heterozygous Sickle cell Disease– HbS + B thal / Hb D/ Hb E HbS is prevalent in Africa, Mediterranean countries and in india . India –MP, Orissa ,AP, Kerala, Tamilnadu and in South Gujarat.

33. SICKLE CELL DISEASE Mutation in the 6 th position of Beta globin chain Glutamic acid Valine Deoxy HbS ( Polymerised ) Ca influx, K leakage Stiff Viscous sickle cell Venocclusion Decresed RBC Survival Microinfarctions Anemia , Jaundice , Gallstones, Leg ulcers 34

34. CLINICAL FEATURES Growth and Development Retardation. Recurrent leg ulcers. Dactylitis or Hand Foot Syndrome . Acute infections – Pneumonia ,Meningitis. Osteomyelitis due to Salmonella Jaundice, Pigmented stones, Acute abdominal pain Priapism Sickle retinopathy- Salmon patches -Intra retinal haemorrhage and proliferation. 35

35. CRISIS IN SICKLE CELL DISEASE Hemolytic crisis- Low Hb, R eticulocytosis , Jaundice Sickling crisis ( Vaso -occlusive crisis) – Splenic infarct, bone pain, chest pain, stroke Aplatic crisis Splenic Sequestration crisis – Rapid organ enlargement, hypovolemic shock. Hand foot syndrome Acute chest syndrome (chest pain ,fever ,leukocytosis ) 36

36. DIAGNOSIS: FAMILY HISTORY, ETHNICITY PERIPHERAL SMEAR – Sickle cells SICKLING TEST - Positive SOLUBILITY TEST

37. Sickle Cell Homozygous (SS) Sickle Cell Disease Moderate to severe anemia with Hb between 4-8gm/dl. HbS (S window) - 70 to 90% HbA2- 2-3 % (N / Slightly raised) HbF - 5 to 25% 38

38. Sickle Cell Heterozygous (SA) Sickle cell Trait Normal Hb ( HbA ) : 60 % HbS : between 30-40 % - It has enough normal Hb to prevent most complications which develop in homozygous state. 39

39. IMMUNE HEMOLYTIC ANEMIAS

40. IMMUNE HEMOLYTIC ANEMIA HEMOLYTIC TRANSFUSION REACTIONS ALLOIMMUNE HEMOLYTIC ANEMIA AUTOIMMUNE HEMOLYTIC ANEMIA HEMOLYTIC DISEASE OF THE NEWBORN WARM ANTIBODY TYPE COLD ANTIBODY TYPE MIXED WARM AND COLD ANTIBODY TYPE IDIOPATHIC DRUG INDUCED FOLLOWING ORGAN TRANSPLANTATION FOLLOWING MULTIPLE DRUG TRANSFUSIONS SECONDARY AIHA PAROXYSMAL COLD HEMOGLOBINURIA COLD AGGLUTININ DISEASE INFECTIONS AUTOIMMUNE DISORDERS CHRONIC DISORDERS

41. AUTOIMMUNE HEMOLYTIC ANEMIA Result from RBC destruction due to RBC autoantibodies : Ig G, M, E, A Most commonly – idiopathic Classification Warm Autoimmune Hemolysis: Ab binds at 37degree Celsius Cold Autoimmune Hemolysis: Ab binds at 4 degree Celsius

42. WARM ANTIBODY TYPE AIHA Ig G directed RBC agglutination occurs when RBCs are incubated with Antiglobulin antisera at 37 degree for 2 hours Causes: 50% Idiopathic Rest - secondary causes: 1.Lymphoid Neoplasm: Lymphoma, Myeloma 2.Solid Tumors: Lung, Colon, Kidney, Ovary, Thymoma 3.Connective tissue disease: SLE,RA 4.Drugs: Penicillin , Quinine, Chloroquine

43. INVESTIGATIONS Hemolysis, MCV is raised PS Findings: Reduced RBC count, Microspherocytosis , increased polychromatic cells and RBC’S with Pappenheimer bodies. Confirmation: Direct Coomb’s Test / Antiglobulin test

44. COOMB’S TEST Direct Coomb’s test Indirect Coomb’s test

45. COLD ANTIBODY TYPE AIHA Usually Ig M directed RBC agglutination occurs at 4 - 5 degree celsius without use of Antiglobulin . Infection: Mycoplasma pneumonia, Infectious Mononucleosis Neoplasms : Lymphoma, CLL, Kaposi sarcoma, Myeloma C/F: Elderly patients Exacerbations in the winter Bluish discoloration of fingers, toes, ears, or nose ( Acrocyanosis )

46. COLD AGGLUTININ DISEASE Production of IgM antibodies against i antigen of RBC’S. Complement mediated (Antic3b) Presents as acrocyanosis of hands, feets and ears and attacks of hemolysis in December to January in India Agglutination of RBC’s is seen in peripheral smear made at room temperature in winter, reversed by warming the slide at 37 degree. RBC count is difficult with erroneous values Direct Coomb’s test is Positive Treatment : Rituximab / Rituximab- Fludarabine .

47. PAROXYSMAL COLD HEMOGLOBINURIA Recurrent attacks of hemolysis following exposure to cold Usually occurs after acute viral infections (molecular mimicry) Donath- Landsteiner antibody present against P ag on RBC Complement mediated Coomb’s test – anti C3d positive and anti IgG negative Demonstarte Hemoglobinemia and hemoglobinuria Confirmatory test : D-L Antibody test Self limited condition with prompt recovery.

48. COLD AGGLUTININ DISEASE PAROXYSMAL COLD HEMOGLOBINURIA D-L Antibody Test

49. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Acquired Clonal / Stem cell Non Malignant disorder, Young adults PIG-A gene mutation leading to loss of GPI- linked anchor proteins . Intravascular Hemolysis , Thrombosis and/or Bone marrow failure. Normocytic normochromic anemia Reticulocytosis – Not proportional to anemia Passage of Dark/red colored urine early morning , subsequent samples normal. Thrombotic episodes.

50. HAM’S acidified serum test – Diagnostic of PNH. Flow cytometry (FLAER) test Gold standard for diagnosis of PNH Reduced/ N o expression of CD55 and CD59.

51. FRAGMENTATION SYNDROMES

52. Schistocytes are the hallmark of this group of disorders

53. MICROANGIOPATHIC HEMOLYTIC ANEMIA Small vessel disease of capillaries and arterioles with thrombotic lesions As red cells pass through these platelet fibrin microthrombi , part of RBC gets fragmented by high shear forces. PS: Schistocytes (>1%) Helmet cells, thrombocytopenia

54. Hemolytic Uremic Syndrome - Thrombotic Thrombocytopenic Purpura (TTP-HUS) HUS Children Localised to kidney Microangiopathy of kidney MAHA Thrombocytopenia Following Enterohemorrhagic . Coli or Shigella Dysentriae infection TTP Adults ADATS13 deificency – vWF multimers – Platelet hyaline thrombi throughout body End organ damage to CNS, kidney and heart, often lethal Secondary TTP ( Pregnancy, HELLP syndrome, APLA, SLE, MALIGNANCY)

55. PERIPHERAL SMEAR FINDINGS Blood smear Sickle cells G6PD Deficiency MAHA Hereditary Spherocytosis AIHA Hemolytic disease of newborn Sickle cell anemia Bite cells Schistocytes Spherocytes Thalassemia Microcytic hypochromic cells Target cells Pancytopenia Paroxysmal Nocturnal Hemoglobinuria

56. COMPENSATORY MECHANISMS TO HEMOLYSIS This indicates increased red cell production by bone marrow in response to hemolysis Peripheral blood findings: Polychromatophilia , Nucleated RBC’S, Schistocytes , Acanthocytes , s pherocytes , sickle cells, target cells, thrombocytosis, neutrophilia with shift to left. Reticulocytosis : Raised reticulocyte count. Bone marrow: Erythroid hyperplasia Reversal of M:E ratio adults . adults.

57. 58 Routine Ix Suggestive of Hemolysis Evaluate for Hemolysi s CBC, PS, RC, LDH, bilirubin, Haptoglobin Micro-Hypo With Target Cells Thalessemia Sickle shape RBC Sickle cell anemia Sickling test HPLC Electrophoresis Family study Spherocytes DCT + ve - ve Immune Hemolysis Family h/o Spherocytosis AIHA Cold Ab (Igc3 + ve ) Warm Ab (Igc3 - ve ) Hereditary Spherocytosis Schistocytes MAHA HUS DIC TTP ECLAMPSIA Anemia, Increase RC, Hyperbilirubinemia Bite/Blister Pickle Cell Enzymopathy Ie . G6PD, PK Enzymopathy H/o Drugs, Inf Eliptocytes Severe Poikilocytes Stomatocytes Positive Family history Hereditary elliptocytosis Hereditary Stomatocytosis NC:NC Mild Micro-Hypo History Hemoglobinuriaria at night Paroxysmal Nocturnal Hemoglobinuria Flowcyto FLAER Test Infection Fever Malaria Babesia

58. References ATLAS and TEXT of HEMATOLOGY 4 th edition – Dr.Tejinder Singh. ROBBINS AND COTRAN –Pathologic basis of disease - 10 th Edition. Wintrobe’s Clinical Hematology 12th Edition © 2009 Lippincott Williams & Wilkins . Dacie and lewise 12 th edition- Pracical hematology . Atlas of Peripheral Blood : The Primary Diagnostic Tool - Lippincott Williams & Wilkins . 59