Hemolytic anemia sandip

approach to hemolytic anemia with emphasis on thalassemia

1. HEMOLYTIC ANEMIA DR SANDIP GUPTA PGT,PEDIATRICS B.S.M.C.H.

2. CLASSIFICA TION 1.RED CELL ABNORMALITY ( INTRACORPUSCULAR DEFECT ) A.HEREDITARY 1.Membrane defect( spherocytosis,ellipsocytosis ) 2.Enzyme defect 3.Hemoglobinopathies( Thalassemia,SCD , other) B.ACQUIRED 1.Paroxysmal nocturnal hemoglobinuria

3. 2.Extra corpuscular defect A.IMMUNE HEMOLYTIC ANEMIA 1.AUTOIMMUNE HEMOLYTIC ANEMIA Warm antibodies Cold anti bodies 2.ALLOIMMMUNE HEMOLYTIC ANEMIA Hemolytic disease of newborn Incompatible blood transfusion B.NONIMMUNE HEMOLYTIC ANEMIA Microangiopathic hemolytic anemias (DIC ,TTP,HUS) Trauma:prosthetic cardiac valve Burns,ECMO,snake bite Infection: malaria, babesia Chemical injury : lead,wilson disease, Hypersplenism

4. Evaluation of anemia Low Hgb/Hct Corr. Retic Ct >2% Corr. Retic Ct <2% Acute Blood Loss MCV>100 MCV 80-100 MCV<80 Evaluate & treat appro - priately Evaluate for Hemolytic Anemias Evaluate for microcytic anemias Evaluate for macrocytic anemias Evaluate for normocytic anemias NO YES

5. Step by step approach 1. Calculate for Corrected Reticulocyte Count Retic count: 10% Pt’s Hct 29 Control Hct 40 Corrected Retic Count = % Retic x Pt’s Hct Control Hct = 10% x 29/ 40 = 7.73 % > 2% if no blood loss Indicates hemolysis

6. Hemolytic Anemia Premature destruction of RBCs Confirm (+) hemolysis : Corrected retic count > 2% Inc indirect bilirubins Inc LDH Low/absent haptoglobin Look for cause of hemolysis - occult blood in urine, urine hemosiderin - peripheral blood smear - direct antiglobulin test, Hgb electrophoresis, RBC enzyme analysis

7. Role of pbs 1 Sickled cells Bite cells Schisto- cytes Acantho- cytes Sphero - cytes Target cells parasite inclusions DAT (+) DAT (-) Hgb electro- phoresis G6PD level PT/PTT Crea platelets Auto- Immune Hemo- lytic Anemia Heredi- tary Sphero- cytosis Sickle Cell Ds G6PD Deficient Vs Unstable Hgbs Thalas- semias Hemo- globino- pathy Liver Ds Liver Ds Malaria Babe- siosis Barto- nella TTP-HUS DIC Prosthe- tic Valve Malignant HTN Hemolytic Anemia (CRC>2% + no blood loss)

8. HEMOLYTIC ANEMIA Intravascular Causes in Red WITHIN THE RED CELL 1. Membrane defects - HS - HE - Hereditary pyropoikilocytosis - Hereditary stomatocytosis 2. Enzyme defects -G6PD - Pyruvate kinase 3. Hemoglobin defects - SCA - Thalassemias - Unstable hemoglobin NON-IMMUNE 1. Hypersplenism 2. Fragmentation syndromes - grafts / valves / AS - HTN / Pre- eclampsia - March hemoglobinuria - MAHA - TTP/HUS - DIC - hemangioma 2. Infections/Toxins (Malaria, Babeosis , Bartonella , Clostridium welchii , snakes, spiders) 3. Drugs 4. Liver dz (Spur cell) 5. PNH AUTO-IMMUNE 1. Warm 2. Cold 3. Transfusion reactions 4. Drug associated OUTSIDE THE RED CELL

9. Intravascular Hemolysis RBC LYSIS HBG HAPTOGLOBIN REMOVED BY LIVER HEMOGLOBINEMIA HEMOGLOBINURIA HBG TAKEN UP BY RENAL TUBULAR CELLS HEMOSIDERIN CELLS SLOUGHED IN URINE 1 WEEK LATER

10. Features specific to intravascular haemolysis: Haemoglobinaemia ( haptoglobin and haemopexin exhausted). Methaemoglobinaemia . Haemoglobinuria . Haemosiderinuria .

11. Hereditary spherocytosis Incidence:1/5000 in North European population Autosomal dominant Defect in RBC cytoskeleton( spectrin,ankyrin ) Pathophysiology:A deficiency in spectrin , ankyrin,protein 3, leads to weakening of the “vertical” interaction of the lipid bilayer & loss of membrane microvescicle . Loss of surface area,↑cation permeability, ATP use,& glycolysis leading to premature destruction in spleen.

12. Clinical features Neonatal period: anemia+ jaundice, more severe. Infancy&childhood:variable severity. Mild: asymptomatic Moderate: intermittent jaundice,spleenomegaly,anemia . Severe:tranfusion dependeant,bone expansion,gall stone LAB. DIAGNOSIS:Anemia (Hb:6-10g%),PBS : Spherocytes lacking central pallor , reticulocytes,MCV -N MCH ↑, MCHC >35 ,RDW>14.5,DCT: Negative Osmatic fragility& Incubated osmotic fragility test. Differential diagnosis: autoimmune hemolytic anemia, G6PD def, Clostridial sepsis, wilsons disease.

13. treatment Splenectomy was routine in past. Anemia,reticulocytosis , hyperbilirubinemia resolve. Transfusion requirement↓,risk of gall stone falls. Current approach is to spenectomize pts with severe hemolytic anemia &those with s/s of anemia, growth failure, skeletal changes, leg ulcer, etramedullary hematopoiesis , aplastic crises,cardiomegaly . Pt’s with Hb >10%& retic count<10% may not need splenectomy . Partial splenectomy in infants & young children with severe hemolysis & transfusion dependent anemia has been recently advocated. Suppoertive therapy : 1mg folic acid daily. Laparoscopic splenectomy . Lifelong antibiotic prophylaxis.

14. 2.Hereditary Elliptocytosis Equatorial Africa, SE Asia AD / AR Functional abnormality in one or more anchor proteins in RBC membrane- Alpha & beta spectrin & defective spectrin heterodimer self association , Protein 4.1& glycophorinC . Usually asymptomatic Mx : Similar to H. spherocytosis Variant: 3.SE-Asian ovalocytosis : Common in Malaysia , Indonesia… Asymptomatic-usually Cells oval , rigid ,resist invasion by malarial parasites SAO is associated with protein3 abnormality.

15. Red Cell Enzymopathies Physiology: EM pathway: ATP production HMP shunt pathway: NADPH & Glutathione production

16. Glucose-6-Phosphate Dehydrogenase ( G6PD ) Deficiency Pivotal enzyme in HMP Shunt & produces NADPH to protect RBC against oxidative stress Most common enzymopathy -10% world’s population 1% of indian males have G6PD deficiency Protection against Malaria X-linked recessive Clinical Features: Acute drug induced hemolysis : Aspirin, primaquine , quinine, chloroquine , dapsone …. Chronic compensated hemolysis Infection/acute illness Neonatal jaundice Favism

17. (Oxidised form) (Reduced form)

18. Inv: e/o non- spherocytic intravascular hemolyis P. Smear: Bite cells, blister cells, irregular small cells, Heinz bodies, polychromasia G-6-PD level Treatment: Stop the precipitating drug or treat the infection Acute transfusions if required

19. 2. Pyruvate Kinase Deficiency AR Deficient ATP production, Chronic hemolytic anemia Inv; P. Smear: Prickle cells Decreased enzyme activity Treatment: Transfusion may be required

20. Paroxysmal Nocturnal Hemoglobinuria Clonal cell disorder Ongoing Intra- & Extravascular hemolysis ; classically at night Testing Acid hemolysis (Ham test) Sucrose hemolysis CD-59 negative (Product of PIG-A gene) Acquired deficit of GPI-Associated proteins (including Decay Activating Factor)

21. 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.Misc: UC, HIV

22. Inv: e/o hemolysis , MCV P Smear: Microspherocytosis , n-RBC Confirmation: Coomb’s Test / Antiglobulin test Treatment Correct the underlying cause Prednisolone 1mg/kg po until Hb reaches 10mg/dl then taper slowly and stop Transfusion: for life threatening problems If no response to steroids  Spleenectomy or, Immunosuppressive: Azathioprine , Cyclophosphamide

23. 2. Cold AI Hemolysis Usually Ig M Acute or Chronic form Chronic: C/F: Elderly patients Cold , painful & often blue fingers, toes, ears, or nose ( Acrocyanosis ) Inv: e/o hemolysis P Smear: Microspherocytosis Ig M

24. Other causes of Cold Agglutination: Infection: Mycoplasma pneumonia, Infec Mononucleosis PCH : Rare cause seen in children in association with viral infection. Demonstrable DONATH LANDSTEINER ANTIBODY Treatment: Treatment of the underlying cause Keep extremities warm Steroids treatment Blood transfusion

25. 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

26. Acquired hemolysis 2.Infection F. malaria: intravascular hemolysis : severe called ‘ Blackwater fever’ Cl. perfringens septicemia 3.Chemical/Drugs: oxidant denaturation of hemoglobin Eg : Dapsone , sulphasalazine , Arsenic gas, Cu, Nitrates & Nitrobenzene

27. Thalassemias / Thalassemia Syndrome Epidimiology : Most Common genetic disorder in Pediatric ward 7% of the world population is carriers of hemoglobin disorder 1.5% of world population is carriers of ß Thalassemia gene (20 millions in India alone) 8 to 10 thousand children born in India with homozygous state for the Thalassemia in every year. There are around 65 to 67 thousand Thalassemia patients in our country. In India, Prevalence of defective ß gene varies from 1 to 17 %.

28. Hallmark of Thalassemia is decreased or absent synthesis of Globin chains of Hemoglobin i.e. it is quantitative disorder of Hb Synthesis. Based on the chain affected Thalassemias are classified as α and ß Thalassemia. If ß gene is absent, it is term as ß Thalassemia. If partially affected, it is called ß + Thalassemia. The genetic classification does not necessarily define the phenotype and the degree of Anemia does not always predict the genetic classification. Thus for the management, the Thalassemias are classified into four groups, each for α & ß depending on clinical severity . Salient Features

29. α Thalassemia Syndromes : Syndrome Clinical Features Hemoglobin Pattern α- globin genes affected and genotype Silent carrier No Anemia, normal red cells 1-2% Hb Bart’s(γ 4 ) at birth 1 - α/ αα Thalassemia Trait Mild anemia, hypochromic 5-10 % Hb Bart’s(γ4) at birth, microcytic red cells 2 - α/ -α, --/ αα HbH Disease moderate anemia, Hepatosplenomegaly, malar prominence etc. 5-30 % HbH (ß4) red cells 20-30% Hb Bart’s(γ4) at birth 3 --/ -α Hydrops Fetalis / Hb Bart’s Syndrome Severe anemia, Hepatosplenomegaly, Cardiac defect, Genito-Urinary Systems abnormality, PET in mother Death in Utero Mainly Hb Bart’s 90 %, small amount of HbH, gower 1, gower 2 and portland 4 --/--

30. Classification , Clinical & Hematological features of ß Thalassemia : Syndrome Clinical Features Hemoglobin Pattern ß- globin genes affected and genotype Heterozygous State Silent Carrier Thalassemia trait No Anemia, normal Mild anemia, hypochromic , microcytic red cells Hb > 10 gm% RBC > 5.5 x 10 12 per liter Normal, HbF < 5% Elevated HbA2 (3.6-8 %) 1 ß + / A 1 ß / A, ß + / A Homozygous State Thalassemia Intermedia Thalassemia Major or Cooley’s Anemia Moderate anemia, requires some transfusion Hb > 7-10 gm% RBC < 5.5 x 10 12 per liter Severe anemia, transfusion dependent Hb < 7 gm% RBC < 4 x 10 12 per liter HbF elevated(20 -100 %) HbA2 < 3.5 % HbF elevated (90%) HbA2 = 2% HbE = 30-40% 2 ß + / ß + 2 ß / ß , ß / ß +, E / ß

31. Principles of Management Confirmation of the Diagnosis By HPLC Diagnose of Complication Correction of Anemia Packed Red Blood Cell (PRBC) transfusion Management of Complications Iron Overload and Chelation Therapy Anemia/ Hypoxia Arrest of Growth Infections Hypersplenism Pharmacological Methods Increase gamma chain Synthesis ( HbF ) Curative Treatment Stem cell transplantation Future Treatment Gene Replacement therapy Prevention of Disease

32. Diagnosis of Complication: It is not sufficient to diagnose the case as Thalassemia ONLY. For complete management of the case, it is necessary to think about its genetic classification, clinical and patho-physiological stage in which it now belongs to. THALASSEMIA Spenomegaly Skeletal Deformity & Arrest of Growth Iron Overload & Chelation Therapy Anaemia Recurrent Blood BormeInfection DEATH

33. Why need a Transfusion? Correct Anemia &prevention of hypoxia Reduce Hepatosplenomegaly & Hypersplenism Reducing ineffective erythropoesis & GI absorption Reduce hemolytic facies & skeletal deformities. Improve growth BT is Mandatory For All children with Thalassemia Major Thalassemia Intermedia , Hb < 7 gm % Evidence of growth retardation Types of Transfusion Palliative(8.5g%) Hyper Transfusion(10g%) Super Transfusion(>12g%) Moderate Transfusion(9-10.5g%) Transfusion Therapy in Thalassemia

34. Transfusion Therapy ( contd …) Frequency of Transfusion Every 3-4 weeks Shorter interval of 2-3 weeks is more physiological avg. time taken 3-4 hrs(@5mi/kg/hr). Amount of Transfusion 180 ml / kg. / yr in non spenctomised , non- sensitised pt. 130 ml / kg /yr in spenctomised , sensitised pt. ( 30 % less) Efficacy of Transfusion Rate of fall of Hb should not exceed 1 gm / dl /week with spleen Rate of fall of Hb should not exceed 1.5 gm / dl /week without spleen Allo immunisation of RBC Hyperspenism Drag induced hemolysis Infection

35. Transfusion Therapy (Contd…) Adequacy of Transfusion First decade : normal growth No. of Normoblast < 5 / 100 WBC Complication of Transfusion Non hemolytic febrile Transfusion reaction NHFTR Allo Immunisation Plasma Borne Infection Steps to prevent those infections Allergic reaction INDICATION OF SPLENECTOMY A nnual PRBCc >200-250ml/kg > 1.5times bas al requirement Massive spleenomegaly hypersplenism

36. Iron Overload Causes of Iron Overload Treatment with multiple transfusion One bottle blood increases iron store by 200 – 250 mg iron. Ineffective erythropoesis Excessive dietary absorption of Iron Consequence of Iron overload Iron overload in Liver Hepatomegaly , Fibrosis & cirrhosis Iron overload in Spleen Splenomegaly , Hypersplenisim Cardiac complication Failure & Arrhythmia Endocrinal Dysfunction Thyroid, Para-Thyroid, Pituitary, Pancreas, Gonads Iron overload in Bones Osteoporosis, Osteopenia

37. Iron Chelation Therapy Iron Chelation Therapy Goal Reduce the Iron store & sub sequently maintain it at low level ( < 1000 µg/ml ) When to start Start after 15-20 transfusion or S.Ferritin > 1000 µg/ml (approx. 3 yrs of age) Needle Biopsy of Liver : 3.2Mg iron per gm of Liver tissue ( Drugs presently used Inj. Deferrioxamine (SC/IV) : DFO/ Desferal Oral Deferiprone Oral Deferasirox Newer Iron Chelator Desferrithiocin ( DFT) Hydroxy Benzyl Ethilene Diamine Diacetic acid (HBED) Pyridoxal iso nicotinyl Hydrazone (PIH) GT 56-252 40 SD02 (CHF 1540)

38. Iron Chelation Therapy ( contd …) Inj. Deferrioxamine (SC/IV) : DFO/ Desferal Dose <2000ug/l →25mg/kg/d,2000-3000ug/kg/d →35mg/kg/d Mode of Delivery s/c:over 8-10 hrs as 10% sol Dipot DFO is more effective & latest . Recently I.V. is used in severe cardiac involvement. Toxicity/ adverse effect Local reaction Visual abnormality ( 4-10 % of pt.) Sensori -neural hearing loss ( 4-38 % of pt.) Delayed linear growth Pulmonary Infarction Auditory & Visual Toxicity is reversible Yersinia sp. Infection Vit -c in a dose of 50-200mg/d

39. Curative Treatment Stem Cell Transplantation This is the only curative therapy available today. Though expensive, it is cost effective as compared to yearly cost of regular BT & chelation therapy Sources Bone Marrow Cord Blood Fetal Liver Peripheral Blood

40. Future Treatment Gene Therapy Aim : Insertion of a normal copy of gene along with key regulatory sequences(LOCUS CONTROL REGION) in the stem cells of recipients. Two main approaches Somatic gene therapy in which non-germ line cells are involved. Transgenic approach in which transfuse gene can be expressed in subsequent generations Need high titre vectors for sustained expression Lentiviral vector from HIV is a hope.

41. SCREENING & PREVENTION Premarital screening programmes Alternative is to screen pregnant woman in early pregnancy. PRENATAL DIAGNOSIS: BY CVS AT 9-11WK Recently there has been attempt to isolate fetal cells from maternal blood. PARENTERAL COUNSELLING

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