1. Thalassemias are genetic blood disorders caused by mutations in the globin genes that result in reduced or absent globin chain production and imbalanced hemoglobin synthesis. 2. There are two main types: alpha-thalassemia affects alpha chain production and beta-thalassemia affects beta chain production. 3. The clinical severity of thalassemias depends on the number of defective globin genes and ranges from asymptomatic carriers to severe anemias requiring lifelong blood transfusions. Laboratory tests can identify the type and severity through hemoglobin analysis and peripheral blood smears.

1. THALASSEMIAS
Muhammad Asif Zeb

2. HEMOGLOBIN DISORDERS
 Genetic disorders of hemoglobin are the most
common genetic disorders worldwide
 Mutations in the globin genes are the most
prevalent monogenic disorders worldwide &
affect approx. 7% of the world population

3. Α AND Β THALASSEMIA
• The α gene is duplicated: 2
genes per chromosome,
therefore 4 α genes in a normal
adult.
• The β gene is single therefore
the normal adult will have two β
genes
α and β globin gene clusters
Chromosomes 11 and 16

4. NORMAL HUMAN HEMOGLOBIN
Embryonic Hemoglobin
• Hgb Gower 1 (ζ2,ε2)
• Hgh portland (ζ2, γ2)
• Hgb Gower 2 (α2, ε2)
Fetal Hemoglobin
• Hgb F (α2,γ2)
Adult Hemoglobin
• Hgb A (α2,β2) 98%
• Hgb A2 (α2,δ2) 1.5- 3.2%
• Hgb F (α2,γ2) <1%
Early embryogenesis
Yolk sac erythroblasts
Major
Hemoglobin of
Intra-uterine life
Adult Hemoglobin

6. THALASSEMIA
 Are characterized by a reduced rate of
production of normal hemoglobin due to
absent or decreased synthesis of one or
more types of globin polypeptide chain

7. TYPES OF THALASSEMIA
• Inherited anemia characterized by a decrease in synthesis
one or more of the globin chains. The defect may affect the
α chains
β chains
γ chains
δ chains
• The clinical syndromes arise from a combination of
Inadequate production of hemoglobin
Unbalanced accumulation of globin chains

9. Α-THALASSEMIA SYNDROMES
• Group of disorders characterized by decreased
synthesis of α-chains usually due to gene
deletions
• The mutation is a functionally abnormal α
gene
• The clinical severity can be classified according
to the number of genes that are missing or
inactive

10. Α-THALASSEMIA SYNDROMES
Four possible α thalassemia syndromes:
1. α-thalassemia (silent carrier): One of the four α-
globin gene fails to function.
2. α-thalssemia-trait (α Thal trait minor): Two of the
four α-globin gene fails to functions.
3. Hgb H disease: Three of the four α-globin gene
fails to functions.
4. Bart’s Hydops Fetalis: Four of the four α-globin
gene fails to functions.

11. CLASSIFICATION

13. Syndrome
# alpha genes
deleted
Newborn Hb
Barts (γ4) %
Clinical Picture
Silent Carrier 1 1-2 Silent
Alpha
thalassemia trait
2 3-10
Mild hypochromic,
microcytic anemia
Hb H Disease 3 25
Hb H (β4) mild
hemolytic anemia
Hydrops fetalis 4 80-100
Death in utero or
shortly after birth
α Thalassemia

14. Α-THALASSEMIA MAJOR
• --/--( α0 -thal) (Hydrops fetalis)
• Most severe form of α-thalassemia, involving
the deletion of all 4 genes resulting the
absence of α chains.
• In the absence of α chains, erythrocytes
assemble hemoglobin using the δ,ϒ & β
chains available.
• Therefore abnormal hemoglobin tetramer
involving gamma chains (Hb Bart’s, ϒ4) &
beta chains(HbH, β4 )is produced.

15. Α-THALASSEMIA MAJOR
 Hb Bart’s has high affinity for oxygen so this
Hb can’t supply tissue with sufficient O2 to
sustain life & developing infant dies of
hypoxia & congestive heart failure.

16. CLINICAL FEATURES
• Infants that survive until birth exhibit significant
physical changes upon routine exam
• The babies are
- Underweight, edematous with distend
abdomen,
- Hepatosplenomegaly due to extramedulary
hematopoiesis
- Massive bone marrow hyperplasia
• Hemolysis is severe, as there is extensive
deposition of hemosiderin

17. LAB DIAGNOSIS
• There is severe anemia
• hemoglobin 3-10 g/dl
• Microcytic hypochromic RBCs
• marked anisocytosis & poikilocytosis
• Increased NRBCs
• Hb electrophoresis on cellulose acetate
membrane at alkaline PH shows:
Hb Bart’s 80-90%
Hb Portland 10-20%
Hb H sometimes detectable
Hb A, HbA2, Hb F absent

18. HEMOGLOBIN H DISEASE
 --/-α (HETEROZYGOUS)
 Occurs when 3 of 4 α genes are deleted
 This disorder usually results when 2
heterozygous parents, one with --/αα & the
other expressing –α/αα genotype, bear
children.

19. PATHOPHYSIOLOGY
• The reduction of α-chain synthesis results in the
decrease in the assembly of HbA, HbA2, and HbF.
• A decrease in α-chain creates a relative excess
of beta chain which unite to form a tetrad of 4
beta chains called HbH.
• HbH is unstable & tend to
precipitate inside erythrocytes trigering chronic
hemolytic anemia.
• It has an O2 affinity 10 times that of
HbA, which reduces oxygen delivery to tissues
causes tissue hypoxia.

20. LAB DIAGNOSIS
• Hemoglobin level 8-10 g/dl
• Reticulocyte count moderately raised ranging
5-10%
• Microcytic hypochromic red cells
• Marked poikilocytosis
• Target cells
• Basophilic stippling
• NRBCs
Peripheral Blood Smear: stained
With brilliant cresyl blue, showing
Precipitated Hgb H

21. LAB DIAGNOSIS
• Hemoglobin electrophoresis of affected neonates
shows
- Hb Bart’s 25% with decreased levels of
HbA, HbA2,HbF
• After birth β chains begin to replace gamma
chains & Hb H replaces the Hb Bart’s.
- Hb H in Adults 2-40%
- HbA2 Decreased (1.5%)
- HbF Normal

23. DEMONSTRATION OF HB H INCLUSION BODIES
 “In alpha thalssemia, red cell containing Hb H
are incubated with a solution of a redox
dye(brilliant cresyl blue), HbH which is relatively
unstable, precipitates & red cells are pitted by
numerous inclusions, an appearance likened to
the surface of a golf ball”

25. THALASSEMIA TRAIT
 --/αα and -α/-α
 Also called Thalassemia minor, occurs when
two of the four α gene, either on same or
opposite chromosomes, are missing
 There is a measureable decrease in the
production of α- containing hemoglobins, the
unaffected hemoglobin genes are able to direct
synthesis of globin

26. THALASSEMIA TRAIT
chains faster than normal & compensate
for effected genes
• Patients with α-thalassemia trait are
asymptomatic with mild anemia and are
often diagnosed incidentally or when being
evaluated for family studies
• These patients have normal life span & do
not need medical intervention for their
thalassemia

27. LAB DIAGNOSIS
 MCV 60-70 fl
 Slight anemia
 In peripheral blood film, red cells with
- Hypocromia & Microcytosis
- Poikilocytosis
- Target Cells
- Basophilic Stippling
 In Hb electrophoresis, Hb Bart’s 5-6%

28. BETA THALASSEMIA
• occurs when one or both of the two genes
needed for making the beta globin chain of
hemoglobin are variant.
• The severity of illness depends on whether one
or both genes are affected and the nature of
the abnormality.
• If both genes are affected, anemia can range
from moderate to severe.
• The severe form of beta thalassemia is also
known as Cooley’s anemia.

29.  THALASSEMIA
 Greek letter used to designate globin chain:

 +: Indicates diminished, but some production of globin
chain by gene:
+
 0 :Indicates no production of globin chain by gene:
0

30. CLASSIFICATION
Genotype Genetic
description
Phenotype
βο/βο Homozygous Major
βο/β+ Heterozygous Major or
Intermedia
β+/β+ Homozygous Major or
Intermedia
βο/β Heterozygous Intermedia or
minor
β+/β Heterozygous Minor
β /β Homozygous Normal

31. Β-THALASSEMIA MAJOR
• βο/βο, βο/ β+, β+/ β+
• Is a severe, transfusion dependent, inherited
anemia
• Also referred to as COOLEY’S ANEMIA
• Caused by a homozygous or double
heterozygous inheritance of abnormal β gene
resulting in marked reduction or absence in β-
chain synthesis.
• Presents early in life

32. PATHOPHYSIOLOGY
• The molecular defects in - β thalassemia result in
absent or reduced β-chain production. α-Chain
synthesis is unaffected leading to an excess of α-
chains
• Lack of beta chain production can be classified
into four categories
– Reduced HbA
– Compensatory production of abnormal Hb
– Ineffective erythropoiesis
– Erythroid hyperplasia

33. PATHOPHYSIOLOGY

34. CLINICAL FEATURES
• Symptoms 1st observed in infants are;
- Irritability, pallor,
- Diarrhoea, fever, enlarged abdomen
- Growth retardation
- Brown pigmentation of skin
- Chronic hemolysis often produces
gallstones
- Gout & icterus

35. CLINICAL FEATURES
- Bone changes accompany the hyperplastic marrow,
marrow cavities enlarge in every bone,expanding the
bone & producing characteristic bossing of the skull
- Facial deformities & “hair-on-end” appearance of the
skull on X-ray
- Massive splenomegaly
• Severe infections, septicemia,pericarditis
• Iron deposition leads to organ dysfunction, diabetes
& cirrhosis

36. Marked bony changes from bone
marrow proliferation

37. Bone marrow hyper expansion gives
the classic “hair on end” appearance
on X-ray of the skull

38. LAB DIAGNOSIS
 On CBC
 TLC Normal
- Hb low (2-3 g/dl)
- MCV, MCH, low
PLT: Normal to Decrease
- Reticulocyte count increases to the degree
expected for the severity of anemia
because of the increase degree of
ineffective erythropoises

39. • On peripheral blood picture
- Marked anemia
- Marked microcytosis & hypochromia
- Anisocytosis & poikilocytosis
- Target cells, tear drop cells, fragmented
RBC, polychromasia
- NRBCs
- Basophilic stippling
• Osmotic fragility test reveals increased
resistance to hemolysis

41. • Bone Marrow:
- is not usually necessary for diagnosis
but, when performed, show marked
erythroid hyperplasia
• Bone Marrow iron stores increased

42. BIOCHEMISTRY
- Serum iron raised
- TIBC saturated
- Serum transferrin receptor variable
- Serum ferritin raised
- Serum unconjugated bilirubin raised
- Serum uric acid raised
- Urine dipyrrols (hB degradation products)
are present
serum & red cell folate reduced

43. HB ELECTROPHORESIS
 In adults, the absence of HbA, 90% HbF, & low,
normal, or increase HbA2 is characteristic of
βο/βο thalassemia

45. Β-THALASSEMIA MINOR
 βο/ β or β+/ β
 Results from the heterozygous inheritance of
either a β+ or βο gene with one normal β gene
 The normal β gene directs synthesis of
sufficient amount β-chains to synthesize
enough HbA for near normal oxygen delivrey &
erythrocyte survival

46. Β-THALASSEMIA MINOR
 In case of a heterozygous β+ patient, the
thalassemic gene can also contribute to β-
chain production
 They are asymptomatic except in periods of
stress as can occur during pregnancy,
infections & folic acid deficiency
 Under these conditions,a moderate microcytic
anemia develops

47. LAB DIAGNOSIS
 Erythrocyte count is doubled (>5×10/L) for
what is expected at the given Hb conc.
 MCV 55-70fl
 MCH less than 22pg
 MCHC 29-33 g/dl
 Mild anemia with hemoglobin ranges 9-14 g/dl
 Reticulocyte count mild elevated

48. LAB DIAGNOSIS
• Peripheral blood picture shows mild anemia,
Anisocytosis & poikilocytosis with
- Target cells
- Basophilic stippling
- NRBCs are not usually found
• On Hb electrophoresis
- HbF normal
- HbA > 3.5%

50. Β-THALASSEMIA INTERMEDIA
• βο/ β+, β+/ β+ or βο/ β+
• All three patterns of inheritance, homozygous,
heterozygous, double heterozygous can
produce β-Thalassemia intermedia
• The homozygous & double heterozygous forms
represent a mutation in both β alleles resulting
in a moderate degree of β chain synthesis
• Symptoms intense during pregnancy &
infection

51. LAB DIAGNOSIS
 CBC reflects a moderate microcytic
hypochromic anemia with Hb value 6-10 g/dl
 Erythrocyte count raised
 Target cells are prominent poikilocytes
observed, basophilic stippling & NRBCs are
present
 HbA2 5-10%
 HbF 30-75%

52. SECOND LINE IDENTIFICATION OF THALASSEMIA
• Second line identification tests based on
results of Hb electrophoresis on cellulose
acetate membrane are;
- Estimation of HbA2
- Estimation HbF by
– Alkali denaturation by modified betke
method
– Acid elution method

53. OTHER TESTS
 PCR for identification of mutation
 Hb Electrophoresis

55. DIFFERENTIAL DIAGNOSIS
• Iron deficiency anemia
• Anemia of chronic disease
• Sideroblastic anemia

57. THANX