Anemia Section - Journey to Beta Thalassemia

A Journey to Beta Thalassemia

1. THE NAMES OF GOD

2. Hemoglobin Review Heme 1) Porphyrin ring 2) Ferrous iron (Fe 2+ ) Globins 1) Alpha family on chromosome 16  ]--//--[  2) Non alpha family on chromosome 11  ]--//--[ γ G ]--[ γ A  [ 

3. Hemoglobin Synthesis : Majority synthesized at the polychromatophilic normoblast stage in the B.M. Processes necessary for normal synthesis : Adequate iron supply & delivery Adequate synthesis of protoporphyrins Adequate globin synthesis Regulation : Stimulated by tissue hypoxia. Hypoxia causes the kidneys to increase production of EPO, which increases RBC & Hb production . Heme

4. Heme Synthesis Iron delivery & supply Iron is delivered to the reticulocyte by transferrin Synthesis of protoporphyrins Occurs in the mitochondria of RBC precursors Mediated by EPO & vit B6 Protoporphyrin IX + Iron = Heme

5. Globin Synthesis The rate of globin synthesis is proportional to the rate of porphyrin synthesis. Proper globin synthesis depends on genes. The precise order of amino acids in the globin chains is critical to the structure & function of Hb. Chain designations ( α , ζ , β , δ , ε , γ )

6. Introduction To Thalassemia

7. Thalassemia : Diverse group of disorders which manifest as anemia of varying degrees. Result of defective production of globin portion of Hb molecule. Distribution is worldwide. Occurs predominantly in persons of Mediterranean (such as Iran), African and Asian ancestry. In Greece and Southern Italy, the prevalence of β-thalassemia is around 10%. Globin chains structurally normal (is how differentiated from Hemoglobinopathy ), but have imbalance in production of two different types of chains.

8. Thalassemia : Results in overall decrease in amount of Hb production. Defect results from abnormal rate of synthesis in one of the globin chains. Two major types of thalassemia: 1) Alpha ( α ) thal 1. Caused by defect in rate of synthesis of α chains. 2. Usually caused by gene deletion . 2) Beta ( β ) thal 1. Caused by defect in rate of synthesis in β chains. 2. Usually caused by point mutation . May be either Homozygous or Heterozygous defect that results in microcytic, hypochromic anemias of varying severity.

9. Beta Thalassemia

10. Beta ( β ) thalassemia It is mainly prevalent in the Mediterranean region countries , such as Greece, Cyprus, Italy, Palestine and Lebanon. β -thalassemia result from over 150 mutations of the β globin genes that result in the absence or a reduction of the β globin chains. Severity depends on where the hit(s) lie : b : no b -globin synthesis b + : reduced synthesis Disease results in an overproduction of a -globin chains, which precipitate in the cells and cause splenic sequestration of RBCs. Ineffective erythropoiesis increases, sometimes becomes extramedullary hematopoiesis.

11. Pathophysiology of β -Thalassemia

12. Mutations of the β globin genes

13. Examples Of Inheritance: Marriage between carrier & normal person Marriage between two carriers

14. Classical Syndromes of Beta Thalassemia β -Thalassemia Minor (carrier state) Minor point mutation Heterozygous disorder resulting in minimal anemia; no treatment indicated β -Thalassemia Intermedia Homozygous minor point mutation or more severe heterozygote Severity can be a spectrum between the minor and major Most often do not require chronic transfusions β -Thalassemia Major (Cooley’s Anemia) Severe gene mutations Homozygous disorder resulting in severe transfusion-dependent hemolytic anemia Need careful observation and intensive treatment

15. Beta Thalassemia Clinical Syndrome Genotype Hb (gr/dl) Hb Analysis Minor (Trait)  /  + or  /  ° 10-13   HbA 2 ,  HbF Intermedia  + /  + 7-10  HbA 2 ,  HbF Major ( Cooleys )  + /  ° or  °/  ° < 7  HbA 2 ,  HbF

16. Beta Thalassemia Minor (β- Thal Trait; Carrier ; Cooley’s Trait) Caused by heterogenous mutations that affect β -globin synthesis.  Usually presents as mild, asymptomatic hemolytic anemia  unless patient in under stress such as pregnancy, infection, or folic acid deficiency. Have one normal β -gene and one mutated beta gene. Hb level in 10-13 g/ dL , Elevated RBC count (5-7 × 10 6 ), Fe ( sl ↑ ) , TIBC ( N to ↓ ), OFT ( ↓ ) Such people usually practice normal life, but may suffer from a mild form of anemia.  Thalassemic RBCs offers protection against severe malaria caused by P.F .

17. Beta Thalassemia Minor Anemia usually hypochromic and microcytic with slight aniso and poik , including target cells and elliptocytes ; May see basophilic stippling. Rarely see hepatomegaly or splenomegaly. HbA 2 (3.5-8.0%) > HbF (1-3%). Hb, HCT, MCV & MCH ( ↓ ) but MCHC ( N ). Make sure are not diagnosed with iron deficiency anemia. Usually no signs or symptoms are apparent, except for a mild anemia. Carriers are usually initially detected through screening, or when performing routine CBC. Later it can be confirmed using Hb electrophoresis.

18. Beta Thalassemia Intermedia Expression of disorder falls between thalassemia minor and thalassemia major. May be either heterozygous for mutations causing mild decrease in beta chain production or may be homozygous causing a more serious reduction in beta chain production. Patients able to maintain minimum Hb (7 g/ dL or greater) without transfusions. If unable to maintain Hb levels between 6–7 g/dl, transfusion or splenectomy is recommended. See increase in both HbA 2 and HbF production ( HbF > HbA 2 ). Peripheral blood smear picture similar to thalassemia minor.

19. Beta Thalassemia Intermedia Have varying symptoms of anemia, jaundice, hepatosplenomegaly . Have significant increase in bilirubin levels.  Bony deformities due to B.M trying to make more blood cells to replace defective ones causes late development, exercise intolerance, and high levels of iron in blood due to reabsorption in the GI ( gastrointestinal ) tract. Anemia usually becomes worse with infections, pregnancy, or folic acid deficiencies. May become transfusion dependent as adults. Usually survive into adulthood.

20. Beta Thalassemia Major (Cooley’s Anemia) Complete absence of β -globin. Characterized by severe microcytic, hypochromic anemia after 6 month birth.  Detected early in childhood: Infants fail to thrive.  Have pallor, variable degree of jaundice, abdominal enlargement, and hepatosplenomegaly . Severe anemia causes marked bone changes due to expansion of marrow space for increased erythropoiesis. See characteristic changes in skull ( hair on end ) & long bones.

21. Beta Thalassemia Major Have protrusion upper teeth and Mongoloid facial features.  Signs such as paleness and growth retardation, are readily detectable since the first year of life. Those are mainly due to severe anemia. Peripheral blood shows markedly hypochromic, microcytic erythrocytes with extreme poikilocytosis , such as target cells, teardrop cells and elliptocytes . See marked basophilic stippling and numerous NRBCs.  MCV in range of 50-60 fL , Elevated Retic count (2-8%), Fe & Ferritin ( ↑ ), TIBC( ↓ ). Hb level between 4-7 gr/ dL . Most of Hb present is HbF with slight increase in HbA 2 .

22. Beta Thalassemia Major Regular transfusions usually begin around one year of age and continue throughout life. Chronic transfusions required, in conjunction with chelation therapy ( Desferoxamine , Deferpirone & Deferasirox )  Excessive number of transfusions results in hemosiderosis ; Without iron chelation, patient develops cardiac disease. Danger in continuous tranfusion therapy: Development of iron overload. Development of alloimmunization (developing antibodies to transfused RBCs). Risk of transfusion-transmitted diseases (HCV, HBC, HIV & Yersinia). B.M transplantation may be future treatment, along with gene therapy and new drug therapies.

23. Protuberant spleen Mongoloid facial Hair-on-End

24. Comparison of Beta Thalassemias GENOTYPE Hb A Hb A 2 Hb F NORMAL N N N MINOR ↓ ↑↑ N to ↑ INTERMEDIA ↓ N to ↑ Usually ↑ MAJOR ZERO or ↓ Usually ↑ Usually ↑

25. MINOR INTERMEDIA MAJOR

26. Laboratory Diagnosis of Beta Thalassemia

27. Laboratory Diagnosis of Thalassemia Need to start with patient's individual & family history. Ethnic background is important. Perform physical examination: Pallor indicating anemia. Jaundice indicating hemolysis. Splenomegaly due to pooling of abnormal cells. Skeletal deformity, especially in β- Thal major.

28. CBC With Differential Hct , Hb , MCV , MCH ( ↓ ).  MCHC ( N to sl ↓) . Microcytic, hypochromic anemia pattern (except in carrier states). Have normal or elevated RBC count with a normal RDW. Elevated RBC count with markedly decreased MCV differentiates thalassemia from IDA. Mild to moderate poikilocytosis . In more severe cases, see marked number of target cells and elliptocytes . Polychromasia , basophilic stippling and NRBCs.

29. β- Thal minor Haematological Profile Hypochromasia 1+ Anisocytosis 2+ ( Microcytes ) Polikilocytosis 1+ (Target cells) Immature Forms Coarse basophilic stippling Blood smear shows hypochromia , microcytosis (similar to IDA). Blood indices: MCV< 75 fl , Hb usually> 10, HCT> 30%, RDW < 14%. HbA2 > 3%, sometimes reaching 7-8%. Hyperbilirubinemia, LFT abnormalities (late finding) TFT abnormalities, hyperglycemia (late endocrine findings)

30. Beta Thalassemia major Haematological Profile Hypochromasia ) 3+ Anisocytosis 3+ ( Macrocytes , Microcytes ) Poikilocytosis 3+ Target Cells, Tear Drops, Schistocytes , Acanthocytes Howell Jolly Bodies, Target Cells (post splenectomy ) Polychromasia 2+ Immature Forms 3+ Nucleated RBC (B.M response) Blood smear shows profound microcytic anemia, with extreme hypochromia , tear drop, target cells & NRBCs. Hb may be very low at 3-4 gr/dl.

31. What is basophilic stippling? How does it occur? Beta thalassemia is caused by mutations in the in haemoglobin beta gene which prevents the production of the β-chains in Hb. This causes an increased ( α / β ) ratio and precipitation of alpha ribosomal DNA resulting coarse basophilic stippling. The stippling effect is aggregations of the ribosomal DNA or protein granulations in the cytoplasm of erythrocytes

32. Reticulocyte Count : Usually elevated. Degree of elevation depends upon severity of thalassemia. Osmotic Fragility : Have decreased osmotic fragility. Is not very useful fact for diagnosing thalassemia. Is an inexpensive way of screening for carrier states.

33. Hemoglobin Electrophoresis Important role in diagnosing and differentiating various forms of thalassemias . Can differentiate among HbA , HbA 2 , and HbF , as well as detect presence of abnormal hemoglobins such as Hb Lepore , Hb Bart's or HbCS . Also aids in detecting combinations of thalassemia and hemoglobinopathies .

34. Routine Chemistry Tests Indirect bilirubin elevated in thalassemia major and intermedia . Assessment of iron status, TIBC, and ferritin level important in differentiating thalassemia from iron deficiency anemia . MCV < 80 fL Serum Iron TIBC BM Perls stain IDA ↓↓ ↑↑ Thalassemia ↑↑ N 4+

35. Other Special Procedures Globin Chain Testing - determines ratio of globin chains ( α / β ) being produced in Reticulocyte (not RBC). DNA Analysis - Determine specific defect at molecular DNA level.

36. Differential Diagnosis of Microcytic, Hypochromic Anemias Parameter Disease RDW Serum Iron TIBC Serum Ferritin FEP IDA ↑ ↓ ↑ ↓ ↑ Alpha- Thal N N N N N Beta- Thal N N N N N HbE Disease N N N N N Anemia of Chronic Disease N ↓ ↓ ↑ ↑ Sideroblastic Anemia ↑ ↑ N ↑ ↓ Lead Poisoning N N N N ↑

37. Differential Formula : Name Formula - Thal Minor Iron Deficiency Seri - Vastava MCH RBC <3.8 >3.8 Mentzner MCV RBC <13 >13 DF formula (ENGLAND) MCV -RBC -5( Hb ) -3.4 - + Green - King MCV 2 ×RDW Hb×100 <65 >65 Kerman 1(K1) MCV×MCH RBC <300 >300 Kerman 2 K1×10 MCHC <80 >80 Shein - Lal MCV 2 ×MCH RBC <1530 >1530 New Formula MCV-(10×RBC) <15 >15 Name Formula Iron Deficiency Seri - Vastava MCH RBC <3.8 >3.8 Mentzner MCV RBC <13 >13 DF formula (ENGLAND) MCV -RBC -5( Hb ) -3.4 - + Green - King MCV 2 ×RDW Hb×100 <65 >65 Kerman 1(K1) MCV×MCH RBC <300 >300 Kerman 2 K1×10 MCHC <80 >80 Shein - Lal MCV 2 ×MCH RBC <1530 >1530 New Formula MCV-(10×RBC) <15 >15

38. Treatment of Beta Thalassemia

39. TREATMENT Treatments for thalassemias depend on the type & severity of the disorder. Thalassemia minor (trait) : No need for any treatment, since the carriers are usually symptomless. Beta-thalassemia intermedia : Modulation of gamma-globulin chain production with hydroxyurea or other drugs, transfusion, splenectomy , and stem cell transplantation[1]. [1];(Guidelines for diagnosis and management of Beta-thalassemia intermedia . Karimi M, Cohan N, De Sanctis V, Mallat NS, Taher A. Pediatr Hematol Oncol . 2014 Oct;31(7):583-96. doi : 10.3109/08880018.2014.937884)

40. Thalassemia major includes : Chronic blood transfusion therapy + Iron Chelation Therapy Splenectomy Folic Acid Supplements (5mgr) Vit C (200mg/day) plus Deferoxamine Stem Cell Allogenic Transplantation Cell therapy ( Neocyte ) Gene therapy (is also an option still researched)

41. Iron Chelation Therapy: Because the Hb in RBCs is an iron-rich protein, regular blood transfusions can lead to a build up of iron in the blood (Iron overload). It damages the liver, heart, and other parts of the body. To prevent this damage, iron chelation therapy is needed to remove excess iron from the body. Two medicines are used for iron chelation therapy: Deferoxamine : is a liquid medicine that's given slowly under the skin, usually with a small portable pump used overnight. This therapy takes time and can be mildly painful. Side effects include loss of vision and hearing. Deferpirone : 3 dose/day , very effective than Deferoxamine. Deferasirox (ICL670,Exjade) : 1 pill/day. Side effects include headache, nausea, vomiting, diarrhea, joint pain, and fatigue.

42. TREATING COMPLICATIONS Heart and Liver Disease ( Sidrosis ) Sepsis Osteoporosis Enlarged spleen Iron overload

43. Prevention efforts Pre-marital screening to make sure that the couple are not both carriers. Provision of counseling and health education for the thalassemics , their families and the public . Provision of prenatal testing for thalassemia. Reduction of marriages between relatives.

44. Problems commonly faced by thalassemia major patients in developing countries Reduced availability of blood for transfusion. High cost of treatment. Limited services that blood banks are able to give. Unavailability of counseling services. Lack of experience & appropriate training among the health providers to handle thalassemia cases.

45. Other Thalassemias Caused by Defects in the Beta-Cluster Genes Delta Beta Thalassemia 1) F-Thalassemia (δβ ) 2) Lepore Hemoglobinopathy (δβ + ) Hereditary Persistence of Fetal Hemoglobin (HPFH) 1) Pancellular 2) Heterocellular (or Swiss)

46. δβ - Thalassemia (F-thalassemia) β- and δ-chains are not produced; this is nearly, although not completely, compensated for by increased output of γ-chains. Thalassemic indices plus significantly increased levels of Hb F. Types : 1) Heterozygous 2) Homozygous

47. Heterozygous : Is similar to a β-thalassemia trait, except that HbA2 is not increased or is even slightly reduced (mean level is 2.4%) Without clinical symptoms Hb is normal or slightly reduced, HbF is 5.4–20% , MCH is 21-26 pgr , MCV is 65–79 fL. Homozygous : Mi ld form of thalassemia intermedia with a Hb level of 10–13 g/ dL , mildly thalassemic red cell indices. Minimal hepatosplenomegaly . It is most common in the Mediterranean population.

48. Hemoglobin Lepore ( δβ + - Thalassemia) Rare class of δβ thalassemia. Caused by gene crossing-overs between δ locus on one chromosome & β locus on second chromosome. [ α 2( δβ )2]

49. Hemoglobin Lepore (δβ + -Thalassemia) Because HbF production is only slightly increased and the composite δβ-chain is synthesized at a very slow rate, a severe thalassemic phenotype is produced. Hematologic abnormalities are similar to those seen in β0-thalassemia. Heterozygote : Hb Lepore 10%, HbA2 2%, and HbF is 2–3%. Homozygotes : Hb Lepore is 10–15%, and the rest is HbF .

50. Hereditary Persistence of Fetal Hemoglobin (HPFH) Rare condition characterized by continued synthesis of HbF in adult life.  Do not have usual clinical symptoms of thalassemia. Little significance except when combined with other forms of thalassemia or hemoglobinopathies . If combined with sickle cell anemia, produces milder form of disease due to presence of HbF . Classified into two groups according to distribution of HbF among red cells: Pancellular HPFH : HbF uniformly distributed throughout red cells. Heterocellular HPFH : HbF found in only small number of cells.

51. Beta Thal / HbS This is the third most common sickling disorder after Hb SS and Hb SC in African Americans, and the most common one in people from the Mediterranean. It usually runs a milder course in black people (usually S/β+-thalassemia) but causes a severe sickling disorder with manifestations similar to those of sickle cell anemia in people of Italian, Turkish, or Greek descent. Types : 1. HbS/ β+- thalassemia 2. HbS/ β0- thalassemia

52. HbS/ β0- thalassemia : Hb A is absent; HbS is 75%–90%, HbF is 5%–20%, and HbA2 is 4%–6%. This disorder clinically & hematologically resembles sickle cell disease, except for the spleen, which remains enlarged after childhood and into adult life. The main difference is that in Hb S/β0-thalassemia, the MCV and MCH are decreased, and the HbA2 might be significantly increased. Family study is often necessary for a clear distinction. Microcytosis , variable hypochromia & many target cells are present. HbS/ β+- thalassemia : HbA is 15–30%; HbS >50%, HbF is 1–2%, and HbA2 is 4–6%. Although these individuals clinically may resemble sickle cell trait ( HbAS ) The amount of HbS always exceeds HbA .

53. Beta Thal / HbE

54. Beta Thal / HbC Hb C/β+-Thalassemia : This occurs mainly in black people, in whom it tends to result in little disability, except for anemia in pregnancy. Red cell indices are typical of β- thal trait, but there is anisocytosis and 20–50% target cells. Usual values are 65–80% for HbC , 16–30% for HbA and 2–5% for HbF . Hb C/β0-thalassemia : People of Mediterranean extraction usually have a moderately severe hemolytic anemia with β0 or a severe β+ genotype. This combination is extremely rare in the African population. May be difficult to distinguish from HbC disease in that HbA is absent in both, and there is an overlap in HbA2 and F levels. HbA2 is elevated to about 2% and HbF is elevated at 3–10%.

55. Beta Thal / HbE Is unusual because results in more severe disorder than homozygous E disease. Very severe anemia developing in childhood.  Transfusion therapy required. It is one of the most important thalassemia syndromes and is most common in Southeast Asia. Few symptoms, but the usual picture is that of thalassemia intermedia or thalassemia major. Ineffective erythropoiesis (consequence of excess α- globins ), RBC indices & morphology, and clinical manifestations are similar to those in homozygous β- thal . HbF is 5–85% (mean : 42%), and HbE is 58%.

56. Current Clinical Research on Thalassemia Stem Cell Transplant in Patients With β - Thal Gene therapy in β - Thal Patients

57. References : Beta Thal : Epidemiology and Diagnostic & Treatment Approaches in Iran; Najmaldin Saki, Ali dehghani Fard , Saeid Kaviani , Mohammad Ali Jalali Far, Seyed Hadi Mousavi , Kheirollah Al –Ali, Fakher Rahim; Submission Date: 7. Mar. 2012 Acceptance Date: 14. May. 2012. Postgraduate Haematology ; A Victor Hoffbrand , Daniel Catovsky , Edward GD Tuddenham , Anthony R Green; 6 th ed , 2011. Hematology: Basic Principles and Practice ; Ronald Hoffman, etc ; 5 ed , 2009. Clinical Diagnosis & Management by Laboratory Methods; Richard A. McPherson, Matthew R. Pincus , 22 nd edition , 2011. www.pubmed.gov www.royaninstitute.org www.iranianthalassemia.ir

58. THANKS FOR YOUR ATTENTION