Thalassemia

HAEMOGLOBINOPATHY INTRODUCTION, ALPHA AND BETA THALASSEMIA AND TYPES, PATHOLOGY, INVESTIGATIONS

1. HEMOGLOBINOPATHIES DR.ANAMIKA

2. INTRODUCTION Genetically determined abnormality of the structure or synthesis of the haemoglobin molecule are called haemoglobinopathies. The abnormality is associated with globin chains , heme portion will be normal. Range in severity is from asymptomatic laboratory anomalies to death in utero.

3. GLOBIN GLOBIN – Different types of Hbs are formed depending on composition of associated globin chain tetrads; responsible for different physical properties of Hbs . α like chains – α & ζ chains Β Like chains – β , γ , ε , δ chains Globin chains – Held together by non-covalent bonds in a tetrahedral array, giving the Hb , a spherical shape.

4. Hemoglobin Tetramer of globin polypeptide chains: a pair of α- like chains and a pair of β- like chains 6 Wks – Hb Portland( ζ 2 γ 2 ) , Hb Gower I( ζ 2 ε 2 ) and Hb Gower II( α 2 ε 2 ) . 10-11 Wks – Hb F( α 2 γ 2 ) 35 Wks – Hb A 2 ( α 2 δ 2 ) 38 Wks – Hb A( α 2 β 2 ) New born – Hb F-50-85% 1 Yr – Hb A is the major Hb, HbF < 2%, HbA 2 - 1.8-3.5%

5. Hemoglobin

6. Classification of Hemoglobinopathies 5 Major classes STRUCTURAL – Qualitative abnormality. Decreased solubility – Sickle cell anemia ( Hb S), Hb C, Hb SC, Hb D. Decreased stability – Congenital Heinz body anemia. Altered O 2 Affinity – High- Polycythemia. Low- Anemia, cyanosis. THALASSEMIA SYNDROMES – Deficient globin chain biosynthesis. α – Thalassemia. β – Thalassemia. δβ Thal , γδβ Thal , α β Thal .

7. THALASSEMIC Hb VARIANTS – HbS / β - Thal . HbS / α - Thal . Hb E. Hb Constant Spring. Hb Lepore. HEREDITARY PERSISTENCE OF FETAL Hb- ( HPFH) – High levels of Hb F in adult life. ACQUIRED HEMOGLOBINOPATHIES – Modifications of normal Hb by toxins.

8. THALASSEMIA

9. “ Thalassa” – (Gk.) Sea. (referring to Mediterranean) Grp of congenital anemias that have deficient synthesis of one or more of the globin subunits of the normal Hb. Thomas Cooley identified it. It is quantitative hemoglobinopathy since no structurally abnormal hemoglobin is synthesized.

10. CLASSIFICATION OF THALASSEMIAS Normally α : β - 1:1 Degree of imbalance is related to the clinical expression. Impaired α –chain synthesis – α - Thal Impaired β –chain synthesis – β - Thal Misc. Thalassemic syndromes – HbS Thal , HbD Thal , HbE Thal , δβ - Thal

11. Beta - thalassemia

12. ß thalassemia syndromes Thalassemia Major – Most severe form, homozygous state Thalassemia Intermedia – Double heterozygous state, milder form Thalassemia Minor/Trait(BTT) – Heterozygous state, asymptomatic with little or no anemia Thalassemia Minima – Clinically undetectable

13. GENETICS Autosomal Recessive disorders 2 β - Thalassemia genes ( β Thal )– β ˚ - No production of β -Chains. β + - Some but still subnormal production. Any combination of normal β -genes and β Thal genes are possible – various phenotypes. Point mutation on globin gene cluster; i.e , single nucleotide substitution Splicing mutation – Most common cause of β + Thalassemia Promoter region mutation - β + Thalassemia Chain terminator mutation - Thalassemia  

14. Beta -THALASSEMIA MAJOR Also K/A Cooleys anemia Homozygous form of / or β + / β + or double heterozygous / β + Infants well at birth Later by 6 months , develop moderate to severe anemia , failure to thrive, hepatosplenomegaly and bone changes Transfusion dependent  

15. CLINICAL FEATURES Irritable, pale infant, failure to thrive, diarrhea, fever and enlarged abdomen. Severe anemia → Cardiac failure. Chronic Hemolysis → Gout, Gall stones, Icterus Thrombotic complications. Massive Splenomegaly . Flattened nose, wide set eyes, frontal bossing of the skull, prominent cheek bones, and overgrowth of zygomatic bones – “Chipmunk Facies” / thalassemic facies/ mongoloid facies. Enlarged marrow cavities d/t hyperplastic marrow. Hair-on-end appearance on X-Ray skull.

16. Hematologic findings Anemia – moderate to severe when first diagnosed ; 3-8gm% MCV – 50-70fl MCH -12-20pg MCHC - 22-32% PERIPHERAL SMEAR- RBCs are microcytic and hypochromia is marked and red cells are thin Moderate to marked degree of aniso -poikilocytosis Many target cells . Central puddle of Hb may be eccentric Basophilic stippling – constant feature Nucleated RBCs (mainly late normoblasts )- 5 - 40/100 WBC Presence of tear drop , elliptical, fragmented red cells and occasional red cell with Howel Jolly body Aggregates of free α chains can be seen by phase contrast microscopy on supravital staining with methyl violet Reticulocyte count <2 % because of ineffective erythropoiesis & does not correspond to severity of anemia

17. Iron status S. ferritin >1000 µg/L ( 50-150) Transferrin saturation ↑’d 55-90% (30-35%) Total iron binding capacity ↓ ‘d 250-300 µg% (320-360) S. Iron is increased

18. Bone marrow Markedly hypercellular Erythroid hyperplasia is marked M:E ratio is reversed to 1:1 to 1:2 Erythropoiesis is normoblastic Some normoblasts demonstrates features of dyserythropoiesis like irregular nuclear and cytoplasmic borders. Normoblasts demonstrate pink inclusions of free α chains and basophilic stippling in intermediate and late normoblasts Few gaucher like cells may be seen Myelopoiesis and megakaryopoiesis - normal Abundance of iron ( Prussian blue stain)

19. Laboratory tests for diagnosis Acid elution test / kleihauer’s cytochemical method Hb F levels are high 30%-90%; being higher in Thalassemia than in β + Thalassemia In the red cells, Hb F can be demonstrated by acid elution test Procedure : blood smear fixed with ethyl alcohol and incubated in prewarmed citric acid phosphate buffer solution for 5 mins and then stained with hematoxylin and erythrosine B Result: Red cells containing Hb F  stained pink (resistant to acid elution) Red cells containing Hb A  appear as GHOST cells  

20. Hb Electrophoresis Done on starch agarose/ cellulose acetate membrane Shows bands of both Hb A and Hb F in β + Thalassemia In Thalassemia – Hb F >90% HPLC Globin Chain synthesis analysis Due to lack of synthesis of ß chains, α :ß ratio is altered to 2-30:1 (normal 1:1). In transfused patients – Globin chain synthesis analysis is done by separating peripheral blood reticulocytes Done by incubation of red cells with a radioactive tracer such as H-leucine Mutation studies by DNA analysis, DNA Scanning and DNA Se quencing  

21. hplc

22. Thalassemia intermedia Double heterozygote for mild β + Thalassemia alleles It’s a clinical spectrum intermediate between thalassemia major and minor Pts are anemic but not transfusion dependent Hematological features are of moderate severity Hb- 7-10gm/dl Hb F – 10-30%

23. Thalassemia minor/ ß thalassemia trait (BTT) Heterozygotes for thalassemia gene Carriers are asymptomatic Women during pregnancy are diagnosed as trait cases in thalassemia screening programmes. Hematologic findings Hb- 10-12gm/dl RBC count > 5.2 million / cumm MCV -65-80fl MCH – 20-25pg MCHC is nearly normal – 29-33% Hb A -90-93% HbA2 – 3.6-8% (normal < 3.5 %)

24. If Hb A2 is 3.3 -3.7%  iron status to be studied because it can be BTT with associated IDA Peripheral smear – mild degree of aniso -poikilocytosis with microcytosis, hypochromic , few target cells and low RDW compared to IDA Serum iron and serum ferritin are normal to increased NESTROF test Naked eye single tube red cell osmotic fragility test Method : 5 ml of 0.35% saline solution is taken in two test tube. To the tubes is added 0.02 ml of blood of a normal person( control) and patients blood ( test ) After half an hour put a white paper with dark black line behind both tubes In control tube , black line is clearly seen. In test tube, line is not clearly seen in positive cases since microcytic hypochromic red cells of thalassemia are more resistant to lysis than normal normocytic normochromic red cells Used in screening programmes If + ve  do HbA2 estimation to detect BTT False positives : IDA, Hb E thalassemia , Hb D thalassemia

25. Thalassemia Vs Iron Deficiency Differential diagnosis of BTT – iron deficiency anemia BTT – high RBC count , target cells, and stippled cells, HbA2 > 3.5% IDA – RDW increased and MCHC is low, Hb A2 – 1-3.5% Thal Minor Fe Def Anemia RDW N ↑ RBC N or ↑ ↓↓ MCV ↓↓ ↓ MCH ↓↓ ↓ MCHC ↓ ↓↓ HbA 2 ↑ N FEP N ↑

26. Management of Thalassemia Major Regular blood transfusions at an early age – to prevent hyperstimulation of the bone marrow. “ Supertransfusion Regimen” Bone marrow transplantation Cord blood transplantation In-utero Stem cell transplantation Gene Therapy – to correct defects at molecular level. HbF reactivation - 5-Azacytidine, Hydroxyurea , Cytarabine , Vinblastine, Butyrates . Splenectomy – when excessive destruction of RBCs.

27. Complications of Treatment Transfusion Related – Infections – HBV, HCV, HGV. Hemosiderosis –Each unit BT contains 200mg Iron. PATHOGENESIS –tissue damage d/t- Iron-induced peroxidative injury to the phospholipids of lysosomes and mitochondria; free radical formation. Myocardial Iron Toxicity Hepatic fibrosis and cirrhosis. Endocrinal Pulmonary insufficiency CHELATION – Desferrioxamine (DFO) , Deferiprone(DPO) and Deferasirox

28. BM Transplantation Related Chronic Graft-Vs-Host Disease. Short stature and fertility related problems.

29. Prevention of thalassemia maJOR All mothers during first antenatal check up  screened for Hb, MCV, MCH, MCHC, PS, and NESTROF test. Pts with MCV<70, MCH <23 and + ve NESTROF test  Assess HbA2 level of mother ,if 3.6-8% , then evaluate father also. If positive for trait  if < 12 weeks – chorionic villous biopsy sampling 12-20 weeks – amniotic fluid sampling Then PCR analysis on fetal DNA is done to detect point mutations

30. Misc. Thalassemic syndromes HbS / β -Thalassemia( β S / β Thal ) Double heterozygous state of HbS & β thalassemia . Severity depends upon inheritance of β - Thal gene i.e β˚ or β + CLINICAL FEATURES Hepatosplenomegaly. Lymphadenopathy. Vaso -occlusive crisis Mild episodes of skeletal pain and fever

31. LABORATORY FEATURES – Similar to SCA to Heterozygous Thal Microcytic hypochromic anemia (Clue to the presence of Thalassemia ) Reduced MCV, MCH, MCHC Hb – 5-10g/ dL PS – Anisocytosis, Poikilocytosis, Target cells, basophilic stippling Increase in HbF and Hb S levels Hb Electrophoresis – - In HbS / β ˚ Thal – HbA – Absent HbS – 70% HbA 2 & HbF – Increased - In HbS / β + Thal – HbA – 50% HbS – 50% HbA 2 & HbF - Increased

32. Hb E THALASSEMIA Patient inherits ßthal gene from one parent and HbE gene from another parent HPLC and Hb electrophoresis  HbA , HbF and HbE in ß +/E No HbA in ß 0 /E Hb D THALASSEMIA HbA , HbF and HbD δß THALASSEMIA Reduced or absent production of both δ and ß chains and an increase in γ chain synthesis 2 types – δß + & ( δß ) 0 Δ ß +  production of Hb Lepore  has normal α chains and δß chains instead of ß chain HbF 10-20% HPLC – shows humps on downward slope

33. 5. HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN Group of heterogenous disorders in which the absence of δ and β -chains synthesis is compensated for by increased γ -chain production into adult life. Deletion / inactivity of the β and δ -structural gene complex. No β and δ -Chains production → Excess of α –chains → Combine with γ – chains to form HbF . Typical finding – PANCELLULAR distribution Hb F HPLC- near total absence of HbA and HbF >90%

34. Alpha Thalassemia Normal Hb α -Chain Production α chains of globin are not / partly synthesized. most frequently by deletions of DNA that involve one or more α -genes. Less common causes are point mutations and presence of an abnormal α -gene.

35. Hydrops fetalis/ hb barts Deletion of all the four α genes Intra-uterine death; if born , dies in 2 hours Baby is pale and bloated Placenta is edematous Moderate to massive hepatomegaly Barts has high affinity for oxygen, therefore oxygen does not dissociate from γ 4  severe tissue hypoxia and fetal death Hb Electrophoresis Hb Bart’s – 80-90% Hb H & Hb Portland – 10-20% TREATMENT No effective therapy. IU transfusions, In-utero stem cell transplants attempted unsuccessfully. Early termination of at-risk pregnancy.

36. Hb H DISEASE–( α -Thal-1/ α -Thal-2 )Or (–,–/–, α ) Most frequent in South-East Asia. Excess of β -Chains form tetramers( β 4 ) – Hb H. β β β β β β β β Hb H PATHOPHYSIOLOGY HbH – An unstable thermolabile protein with high O 2 affinity. Form intracellular inclusions Membrane Damage

37. Shortened RBC life span → Chronic Hemolytic Anemia. Ineffective Erythropoesis – Not very severe. CLINICAL FEATURES – Mild to severe anemia. Worsening of anemia during pregnancy, infections, oxidant drugs intake. Splenomegaly. Respiratory infections, leg ulcers, gall stones, Jaundice Moderate skeletal changes. LABORATORY FEATURES Hb – 6-10 g/dL. Reticulocytosis – 5-10% MCV, MCH, MCHC – Reduced Erythrocytosis .

38. PS – Microcytic Hypochromic BP. – Variable poikilocytosis , anisocytosis . HbH inclusions when stained with supravital stain . Brilliant cresyl blue;40X – Blue globules, Many per cell, require time for formation – Gives a golf ball appearance BM – Erythroid hyperplasia with normoblasts having scant supply of Hb . Hb Electrophoresis – HbH – Up to 40% HbA 2 – Decreased • HbF - Normal

39. α -THALASSEMIA TRAIT Common in Mediterranean Area, West Africa and in South-East Asia. Asymptomatic or mild anemia. Hb – 10-12 g/dL. MCV – 60-70 fL , MCH – 20-25 pg. PS – Microcytosis, Hypochromia – Occasionally HbH inclusions – Target cells, Basophilic stippling

40. SILENT CARRIER – ( α -Thal-2/Normal) One α -gene is deleted. Adequate normal Hb synthesis. Definitive diagnosis by Gene Mapping. Asymptomatic

41. references Tejinder singh . Atlas and text of hematology . 3 rd edition. Kumar, Abbas, Fausto. Robbins and Cotran Pathologic Basis of Disease.9th ed. Internet sources