Thalassemia

1. THALASSEMIA
Moderator – Dr. Poonam Nanwani

3. • "Whipple and Bradford” proposed the name
Thalassemia.
• THALASSEMIA is a heterogenous group of disorders
characterized by genetically determined reduction in
the rate of synthesis of normal globin chain.
• Commonest form of haemoglobinopathy.
History

4. PREVALENCE
• The alpha thalassemia is prevalent in southeast Asia,
Malaysia and southern china.
• The beta thalassemia are seen primarily in the area
surrounding Mediterranean sea, Africa and southeast
Asia.
• Carrier frequency of thalassemia in India is about 3 %
and estimated frequency of thalassemia at birth is
1:2700.

5. PREVALENCE IN INDIA
• In India β thalassemia is frequent and α thalassemia is rare.
• β thalassemia is more common in certain
communities such as Sindhis, Punjabis,
Bengalis, Gujratis, Parsis, Bhansalis, Jain
and Lohanas.
• Thalassemia is prevalent in those parts of world where malaria has
been common.

6. GENETICS
• Thalassemia are autosomal recessive disorders.
• Globin of haemoglobin A is made up of 2 alpha and 2
beta chains, synthesis of alpha chains is controlled by 2
gene clusters on chromosome 16 and of beta chains on
chromosome 11.

7. Hb A - 97% HbA2 – 1.5-3.5% HbF - <1%

8. STEPS IN SYNTHESIS OF GLOBIN CHAIN

9. CLASSIFICATION OF THALASSEMIA
• According to the deficient globin chain
• Alpha thalassemia
• Beta thalassemia
• Delta-beta thalassemia
• Gamma delta beta thalassemia

10. According to clinical severity -
Alpha thalassemia
• Silent carrier
• Thalassemia trait
• HbH disease
• Hb Barts/Hydrops foetalis syndrome
Beta thalassemia
• Thalassemia major
• Thalassemia intermedia
• Thalassemia minor

11. ALPHA THALASSEMIA
α
α α
α
αα/αα
αα/-α
αα/--
Normal
--/-α
--/--
Silent carrier
Thalassemia trait/minor
HbH disease
Hb Barts
Hydrops foetalis syndrome

12. ΑLPHA THALASSEMIA
• α chains of globin are not/partly synthesized.
• It is required for both HbA and HbF .
• Majority of α thalassemia cases result from gene
deletions.
• Others –
1) Mutation which cause aberrant splicing
2) Mutation of chain terminator codon
3) Mutation which cause instability of α globin chain after translation.

14. Redused biosynthesis of alpha chain
Beta and gamma chain produced
ϒ tetramer, α absent
Unable to carry and deliver oxygen
Intra uterine
hypoxia
Foetal death
Still birth
Formation of beta tetramer, present in
developing normoblast
Moderatly ineffective
erythropoiesis
Hb H inclusion in red cells,
cannot dissociate oxygen in
tissue
Spleen trap this cells
Hemolytic anemia
Tissue
hypoxia
Pathophysiology in alpha
thalassemia

16. HB BARTS’ HYDROPS FOETALIS SYNDROME
• Deletion of all 4 genes.
• Intrauterine death of such a baby or if born, dies
wihin first 2 hour.
• Hb barts’ ( free ϒ 4 chains ) has high affinity for
oxygen and therefore , oxygen does not dissociate
from ϒ 4 resulting in sever tissue hypoxia and
foetal death.

18. HB BARTS’ HYDROPS FOETALIS
SYNDROME
Hepatosplenomegaly

19. HB BARTS’ HYDROPS FOETALIS SYNDROME
Peripheral smear

20. HB H DISEASE
• --/-alpha
• Anemia, Hb 6-10gm/dl
• Reticulocyte count 4 - 15 %
• Icterus and hepatosplenomegaly
• Lab findings
• Anisopoikilocytosis
• Hypochromia
• Microcytosis
• Target cells
• Inclusions bodies
• Hb elctrophoresis demonstrates fast moving HbH band in the
range of 5-35 %.
• HbH also demonstrate on HPLC.

22. HbH Inclusion Bodies

24. Α THALASSEMIA TRAIT
• Α heterozygous cases 1 or 2 gene deletions.
• Clinically normal
• Hb 9-12 g/dl
• MCV ↓
• MCH ↓
• Mild microcytosis and hypochromia
• HbH Hb bart : not demonstrable
• Confirmation by DNA analysis.

25. MOLECULAR BASIS OF BETA
THALASSEMIAS
• Beta0 thalassemias
• Complete absence of beta chain synthesis
• Beta+ thalassemias
• Reduced synthesis

26. Β THALASSEMIA
β
β
βN/βN
β0/βN
β+/βN
Normal
β+/β+
Thalassemia minor
Thalassemia minor
Thalassemia intermedia

27. Β THALASSAEMIA
β
β
βN/βN
β0/β0
β0/β+
Normal
Thalassemia Major
Thalassemia Major

28. MUTATIONS CAUSING Β THALASSEMIA

29. MUTATIONS FREQUENTLY OBSERVED
IN INDIANS IN Β THALASSAEMIA
• Intron 1 position 5 (G-C)
• 619 base pair deletion
• Intron 1 position 1 (G-T)
• Frame shift mutation in codon 41 – 42 (-CTTT)
• Codon 15 (G-A)

31. THALASSEMIA MAJOR
• Beta thalassemia major was first described by a Detroit
pediatrician, Thomas Cooley, in 1925.
• Also known as Cooley's anemia
• It is the homozygous form of β 0 / β 0 or β + /β + or
double heterozygous β 0 / β +.
• Infant are well at birth but develop moderate to sever
anemia, failure to thrive, hepatosplenomegaly and bone
changes which are prominent in face.

32. PATHOPHYSIOLOGY OF Β THALASSEMIA
MAJOR
• Accumulation of free alpha chains
• Extravascular hemolysis
• Marrow and bone changes
• Extramedullary hemopoiesis
• Synthesis of HbF
• Iron overload

34. CLINICAL FEATURES
• AGE :
1) Present within first year of life, at birth asymptomatic and after
3 month anemia develops.
2) Infant may present with failure to thrive, intermittent infections
and poor feeding.
• PALLOR ( progressive increase )
• SPLENOMEGALY ( Hemosiderosis and hyperfunction of
spleen)

36. β-Thalassemia facial bone abnormalities. These
changes include bossing of the
skull; hypertrophy of the maxilla, exposing the
upper teeth; depression of nasal bridge; and
periorbital puffiness
β-Thalassemia major. Note the pallor, short
stature, massive hepatosplenomegaly,
and wasted limbs in this undertransfused case
of β-thalassemia major

37. BETA THALASSEMIA MAJOR

38. BETA THALASSEMIA MAJOR
• Growth is retarded and delayed puberty.
• Increase susceptibility to infections.
• CARDIAC CHANGES : Myocardial hemosiderosis develops
especially in transfused patients. Arrhythmias and congestive
cardiac failure supervene.

39. BETA THALASSEMIA MAJOR
• HEPATOMEGALY : Mainly first 3 to 4 year..
• ENDOCRINE SYSTEM :
1) Growth hormone deficiency
2) Hypothyrodism
3) Hypoparathyrodism
4) Diabetes mellitus

41. PERIPHERAL SMEAR INDICES
• Microcytic hypochromic
anemia , basophilic stippling ,
marked anisopoikilocytosis ,
Target cells
• Reticulocyte count;mildly
increased
• Leucocyte ;increased ,
Platelet ;normal
• Hb 3- 8 g/dl
• MCV= <70fl
• MCHC=(22to 30g/dl)
• MCH=(20 -28pg)
• S.iron( >200µg/dl), s.ferritin
–markedly increased
• Transferrin saturation
increased, TIBC –Normal or
redused

43. • Thalassemias
• Smear Characteristics
– Hypochromia
– Microcytosis
– Target Cells
– Tear Drops

45. BETA THALASSEMIA MAJOR
Target cells
Tear drop cells

46. BONE MARROW
• Hypercellular
• Erythroid hyperplasia is marked
• Erythropoisis is normoblastic
• M:E ratio 1:5
• Dyserythropoisis
• Myelopoisis and megakaryopoisis are normal
• Bone marrow iron increased

47. THE BONE MARROW HAS INCREASED NUMBERS OF
ERYTHROID PRECURSORS (A LOW MYELOID TO ERYTHROID
RATIO) RELATED TO THE INCREASED PERIPHERAL RBC
DESTRUCTION IN THIS DISEASE.
Bone marrow Aspirate

48. THE BONE MARROW HAS INCREASED NUMBERS OF ERYTHROID
PRECURSORS (A LOW MYELOID TO ERYTHROID RATIO) RELATED TO THE
INCREASED PERIPHERAL RBC DESTRUCTION IN THIS DISEASE.
Bone marrow Biopsy

49. SPECIAL LABORATORY TEST FOR
DIAGNOSIS
• Hb F ↑ : the levels are higher in β zero then in β plus thalassemia.
There are various method method for estimation of HbF.
• The commonly used method is Betke method : a. Principle : Fetal
hemoglobin (HbF) is more resistant to denaturation in acidic
solution than adult hemoglobin (HbA). Alkali converts HbA to
alkaline hematin. Alkaline hematin is insoluble and precipitates.
• HbF is quantitated by measuring the hemoglobin concentration
before and after denaturation.

50. SPECIAL LABORATORY TEST FOR
DIAGNOSIS
• For higher level of HbF, method of Jonxis and visser can be used.
In this method rate of alkali denaturation is measured in
spectrophotometer and extraploated back to zero time to get the
amount of HbF.
• Other method are radioimmunoassay and high performance liquid
chromatography.

51. • Used to detect the presence of Hb F (fetal hemoglobin).
• RBCS on a slide are stained to detect the presence of Hb F.
• Can distinguish heterocellular HbF from pancellular HbF seen in
HPFH.
• Rarely done and difficult to interpret and standardize due to
significant variability between observers.
• Confirms maternal blood contamination with fetal blood in cases
of fetomaternal hemorrhage, with D mismatch.
• Flow cytometry is now the primary tool for investigation of fetal
haemoglobins in Australia.
Kleihauer Betke test For Hb F

52. Kleihauer Betke test. This peripheral blood from a postpartum woman
with fetomaternal hemorrhage demonstrates HbF containing fetal cells (dark red) in a
background of maternal cells (ghost-like cells).

54. ELECTROPHORESIS
•Principle-The term electrophoresis describes the
migration of a charged particle under the influence of
an electric field. Different haemoglobin have different
net charge because of variation in their structure.
• Under the influence of an electric field these charged
particles will migrate either to the cathode or to the
anode, depending on the nature of their net charge.

55. ELECTROPHORESIS PRINCIPLE.
• Separation of haemoglobins with electrophoresis at
pH 8.4 (alkaline) and pH 6.2 (acid).
• Scanning allows quantification of the hemoglobin
present, bands are seen by staining.

56. GEL ELECTROPHORESIS
Alkaline pH
Acidic pH

57. (1) Normal (2) New born (3) Hb C trait [A-C] (4) Hb SC disease [S-C] (5) Sickle cell disease [S-
S], (6) Sickle cell trait [A – S] (7) New born (8) Normal.

58. Gel electrophoresis instrument

60. HPLC PRINCIPLE
• In this automated technique , blood sample is introduced into
column packed with silica gel. different Hb get absorbed onto
the resin
• Cation-exchange HPLC can be preformed on an automated
instrument that can quantify Hb A2, Hb F, Hb A, Hb S, and Hb
C.
• Studies show equivalence or superiority over electrophoresis in
terms of identification of variant hemoglobins and quantification
of HbA2 level.
• Negatively charged carboxyl molecules bound to silica make up
the cartridge matrix.

61. HPLC PRINCIPLE
• Positively charge molecules (salt and hemoglobin) bind to
the carboxyl groups.
• Haemoglobin molecules are bound and displaced by
increasing salt concentration.
• Haemoglobin variants separate out due to variation in
charge.

62. HPLC INSTRUMENT

63. DNAANALYSIS.
• Indicated when the hemoglobinopathy not confirmed
by other methods or when the underlying mutation
important to management.
• These are of value in predicting the severity of
disease..
• For genetic counseling defining the particular
mutation or deletion is often required – this is
achieved by a variety of molecular techniques.

64. GLOBIN CHAIN SYNTHESIS
• It is helpful when electrophoretic and other usual
haematological studies fail to diagnose.
• It demonstrate α : β ratio. Normal ratio is about 1.0.
• It is reduced in alpha thalassemia and increased in beta
thalassemia

65. THALASSEMIA INTERMEDIA
• Clinical spectrum between thalassemia trait and
thalassemia major.
• This include cases of interaction of β,α, Hb E, Hb
D and Hb S genes.
• Present in the later age ( 2-5 yr )

66. CLINICAL FEATURES
• Mild to moderate anemia
• Mild to moderate splenomegaly
• Mild skeletal and facial changes.
• Iron overload
• Recurrent leg ulcer
• Repeated infection
Thalassemia Intermedia

67. Thalassemia intermedia
• Mild degree of anemia
• Red cell count is
increased
• MCV<70 fl
• MCH<25 pg
• MCHC is reduced
• Hb 6- 9 gm/dl
• Reticulocyte count ( 2-5%) and S.
bilirubin are slightly raised
• HbF 10-30%, H bA2 < 4%
• Moderate degree of
anisopoikilocytosis,
microcytic hypochromic,
target cells,
basophilic stippling

68. MODERATE DEGREE OF
ANISOPOIKILOCYTOSIS,
MICROCYTIC
HYPOCHROMIC,
TARGET CELLS,

69. THALASSEMIA MINOR
• Heterozygous carrier state characterized by little or
no anemia but prominent morphological changes
of red cells

70. BETA THALASSEMIA MINOR
• Mild degree of anemia
• Red cell count is incrased
• MCV<70 fl
• MCH<25 pg
• MCHC is normal
• Hb >9.0 gm/dl
• Reticulocyte count and S. bilirubin are slightly raised

71. BETA THALASSEMIA MINOR
MICROCYTOSIS
HYPOCHROMIA
ANISOPOIKILOCYTO
-SIS
TEAR DROP CELL
TARGET CELL

72. BETA THALASSEMIA MINOR
• Bone marrow is cellular with erythroid hyperplasia.
• Osmotic fragility test shows resistance to hemolysis.
• Elevation of HbA2.
• HbF may be mildly increased

73. CLINICAL
FEATURE
T.MAJOR T.INTERMEDIA T.MINOR
GROWTH,DEVELOPM
ENT
impaired
SPLENOMEGALY ++++ ++
SKELETAL CHANGE,
THALASSEMIC
FACIES
++++
++++
+
+
Hb <7 7-10 >10
RED CELL COUNT 2-4 X 10¹² 3-4.5 X10¹² >5 x 10¹²
BASOPHILIC
STIPPLING
++ + +
TARGET CELL +++ ++ +
ANISOPOIKILOCYTOS
IS
+++ ++ ±
B.M.IRON ++++ ++ ±
HbF 30-90 10-30 0-5
HbA2 <4 <4 4-8
MICROCYTOSIS +++ ++ +
HYPOCROMIA +++ ++ +

75. Serum iron decrease normal Decrease
iron
Storage
Decrease N/increase Increase/N
TIBC increase normal Decrease
Osmotic fragility Decrease(mild to
moderate)
Decrease(marked) _
Bone marrow Decrease iron staining Erythriod hyperplasia Normal morphology
electrophoresis - HbF
HbA2
-
IRON DEFICIENCY
ANEMIA
THALASSEMIA ANEMIA OF CHRONIC
DISEASE

76. • Minor thalassemia :
Alpha beta
delta-beta
• Anemia of chronic disease (in late stages specially
in renal disease )
Anemia with Normal RDW

77. • Iron deficiency anemia
• Beta thalassemia major & intermedia
• Sickle thalassemia (high Hb S & F )
• Hb H disease
• Red cell Fragmentation syndrome
Anemia with high RDW

78. MENTZER INDEX(M.I)
M.I =
<13 SEEN IN THALASSEMIA
AND
>13 IN IRON DEFICIENCY ANEMIA
MCV (fl)
RED CELL COUNT
(millions/ul)

79. MISCELLENEOUS THALASSEMIC
SYNDROME
• Hb S – Thalassaemia
• Hb E – Thalassaemia
• Hb D – Thalassaemia
• HPFH – Hereditary persistence of foetal hemoglobin

80. HB S THALASSEMIA SYNDROME
• Double heterozygote state of Hb S and β thalassemia.
• Clinical feature - Mild growth retardation , pallor and
splenomegaly .
• Hematological feature – microcytic hypochromic red cells,
basophilic stippling and target cells are present.
• MCV and MCH ↓
• Hb F ↑
• Hb A, Hb F and Hb S are demonstrated by Hb electrophoresis,
Sickling and HPLC.

81. SICKLE CELL BETA THALASSEMIA
• Also k/a Micro drepanocytic disease
• Two forms
• Sickle cell Beta 0 thalassemia
• Sickle cell Beta + thalassemia

82. HB D THALASSEMIA
• There is interaction of Hb D and β – thalassemia
genes.
• Electrophoresis demonstrates Hb A, Hb F and Hb D.

83. HPFH
• Increase Hb F production in adult life.
• Heterozygote have 20-30 % Hb F and in
homozygous 90 – 95 %.

84. PREVENTION
• Health education
• Carrier screening and genetic counselling
• Prenatal diagnosis.
Commonly employed method for screening :
• Red cell indices
• Single tube osmotic fragility test
• Estimation of Hb A2
• Haemoglobin electrophoresis at alkaline pH
• Estimation of Hb F and Hb H inclusion.

85. • NESTROFT, a rapid, simple and
cost effective screening test. The
principle of NESTROFT is based on
the limit of hypotonicity which the
red cell can withstand. In this
procedure 4 ml of 0.36% buffered
saline is taken in a test tube, 0.02ml
of whole blood is added to it, and is
allowed to stand at room
temperature. After 0 minutes reading
is taken on a NESTROFT stand on
which a thin black line is marked.
Positive test is due to the reduced
osmotic fragility of red cells.
Naked Eye Single Tube Red Cell Osmotic Fragility Test
(NESTROFT)

86. When this picture will disappear?

87. Presented by – Dr. Gaurav Shelgaonkar