2. Thalassemia was defined as a clinical entity in
1925 when Dr. Thomas B. Cooley and his
associate Pearl Lee, pediatricians at the Detroit
Children’s Hospital,
In the early it is called as the anaemia splenica
infantum.
Whipple and Bradford proposed the name
thalassemia.
3. The alpha thalassemia is prevalent in southeast
Asia, Malaysia and southern china. α +
thalassemia is relatively more common in India.
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.
4. 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, P
arsis, Bhansalis, Jain and Lohanas.
Thalassemia is prevalent in those parts of
world where malaria has been common.
5. 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.
Each globin gene has 3 exons and 2 introns.
10. Beta o thalassemias
Complete absence of beta chain synthesis
Beta + thalassemias
Reduced synthesis
11. 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.
12. Most cases of α thalassemia result from gene
deletion
Other –
1) Mutation which cause aberrant splicing
2) Mutation of chain terminator codon
3) Mutation which cause instability of α globin
chain after translation.
13.
14. Deletion of all 4 genes.
Intrauterine death of such a baby or if
born, dies wihin first 2 hour.
Baby is pale and bloated ; placenta is
oedamatous ; moderate to massive
hepatomegaly.
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. Hemoglobin H disease. This blood film demonstrates microcytosis, hypochromasia, and
numerous morphologic abnormalities, including target cells, microspherocytes, and
fragments. Basophilic stippling may occur.
19. Principle
Hb H (b4) is an unstable hemoglobin
commonly seen in a-thalassemia. On
incubation with some oxidative chemicals
such as brilliant cresyl blue (BCB), HbH is
oxidised, denatured and precipitated in the
erythrocytes and seen as small, evenly-
distributed, intra-erythrocytic blue dots
which termed HbH inclusion bodies.
25. 1) Mutations which affect transcription
2) Mutation that affect splicing of RNA
3) Mutations affecting consensus sequences
4) Polyadenylation mutations
5) Mutations which lead to the formation of the
chain termination codon
6) Frame-shift mutations
7) Deletions
26. 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)
27. 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.
28. Accumulation of free alpha chains
Extravascular hemolysis
Marrow and bone changes
Extramedullary hemopoiesis
Synthesis of HbF
Iron overload
29.
30. 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)
31. FACE : frontal bossing ( cranial bone thickening
), overgrowth of zygomatic bone.
JAUNDICE: mild
BONE CHANGES : X ray demonstrates- expansion
of diploe, hair on end appearance.
32. 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
33. β-Thalassemia bone
abnormalities. Note the “hair on
end” appearance of the cortical
bone caused by expansion of the
bone marrow (arrows). The
subperiosteal bone grows in radiating
striations, which appears as
“hairs.”
34. 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.
38. Basophilic stippling in thalassemia. Peripheral blood film demonstrating
microcytic hypochromic RBCs and basophilic stippling (arrows). Basophilic
stippling occurs in
thalassemia as well as in other hematologic disorders.
39. Hypercellular
Erythroid hyperplasia is marked
Erythropoisis is normoblastic
M:E ratio 1:5
Dyserythropoisis
Myelopoisis and megakaryopoisis are normal
Bone marrow iron increased
42. 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 2 ml of 0.36%
buffered saline is taken in a test tube, 20ml of
whole blood is added to it, and is allowed to stand
at room temperature. After 20 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)
43. 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 :
Principle : Fetal hemoglobin (HbF) is more
resistant to denaturation in alkaline 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.
44. 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.
45.
46. 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.
47. 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.
At alkaline pH Hb C, E, A2 and O migrate
together to form a single band, Hb S, D and G
also co migrate.
48. At acid pH Hb C separates from E and O and
Hb S separates from D and G.
Hb E and O cannot be separated by
electrophoresis neither can Hb D and G.
49. Hemolysate preparation
• Centrifuge EDTA blood at 3000-5000 rpm and
remove plasma
• Wash packed red cell with NSS for three time
and remove supernatant as much as possible
at the last washing round
• Add DW 1.5 time the volume of PRC and mix
vigorously
• Add CCl4 to the half of the volume of lysed
red cells and mix vigorously
• Centrifuge 3000 -5000 rpm and collect the
upper red portion which is “Hemolysate or
Hemoglobin solution)
50. Hemoglobin electrophoresis at alkali pH
Hb: Amphoteric molecule
• Molecular net charge depends on pH of the
medium.
• pH > pI (Iso-electric point) : Molecular net
charge is negative.
• pH < pI : Molecular net charge is positive.
• pI (Iso-electric point) is the pH where
molecular net charge of hemoglobin is zero.
51. Principle
• In alkali medium, Hbs will gain negative net
charge.
• Different Hbs have different molecular
negative net charge.
• Being placed between cathode and
anode, Hbs will move away from the anode.
• The velocity of the movement depends solely
on the molecular net charge.
• Pattern from cathode to anode is :
A2/E, F, A, Bart’s, H
57. Procedure
• Hemolysate in wells
• Serum applicator dipped and applied
on soaked cellulose acetate plate
• Place cellulose acetate, face-down, in
electrophoretic chamber.
• Run elctophoresis at 3 volts for 1 -2
min.
• Stained with Ponceau S
58. Dip cellulose acetate plate in the stain and
leave for 5 min
Wash with destaining solution (5% HOAc)
twice and 5 min each time or until
background becomes white
Read Hb bands
60. 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.
61.
62. Principle
Hb is amphoteric molecule and changes net
charge according to pH of medium.
If pH < PI, net charge becomes positive (cation
) and different Hbs have different positive
charge.
HPLC separation of Hbs is based on cation
exchange chromatography
Stationary phase is negatively charged by
functional group, e.g. polyaspatic acid.
Mobile phase is buffer with pH lower than pI
of Hbs
Order of Hbs : Bart’s, H, F, A, A2/E according
to RT
66. 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.
67. 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 redused in alpha thalassemia and increased
in beta thalassemia
68. 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 )
69. Mild to moderate anemia
Mild to moderate splenomegaly
Mild skeletal and facial changes.
Iron overload
Recurrent leg ulcer
Repeated infection
Thalassemia intermedia
70. 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
71.
72. Heterozygous carrier state characterized by
little or no anemia but prominent
morphological changes of red cells
73. 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
75. Bone marrow is cellular with erythroid
hyperplasia.
Osmotic fragility test shows resistance to
hemolysis.
Elevation of HbA2.
HbF may be mildly increased
77. Serum iron decrease normal Decrease
iron
Storage
decrease N/increase Increase/N
TIBC increase normal Decrease
Osmotic fragility decrease decrease _
Bone marrow Decrease iron staining Erythriod
hyperplasia
Normal morphology
electrophoresis - HbF
HbA2
-
IRON DEFICIENCY
ANEMIA
THALASSEMIA ANEMIA OF CHRONIC
DISEASE
78. Minor thalassemia :
Alpha (Hb electrophoresis ) beta
delta-beta
Anemia of chronic disease (in late stages
specially in renal disease )
Anemia with normal RDW
79. Iron deficiency anemia
Beta thalassemia major & intermedia
Sickle thalassemia
Hb H disease
Red cell Fragmentation syndrome
Anemia with high RDW
80. MENTZER INDEX(M.I)=
<13 SEEN IN THALASSEMIA AND >13 IN IRON
DEFICIENCY ANEMIA
M.I=
MCV
RED CELL COUNT
81. KERMAN INDEX 1:(MCV*MCH/RBC )
<250 : Minor thalassemia =>check Hb elect.
251-320: Mixed iron def. & minor thalassemia
=> trial of iron & folate then check CBC & Hb elect
321-370: iron def.=> trial of iron for 1 mo.
>371: normal
Sensitivity =99% , Specificity=86%
82. KERMAN INDEX 2: MCV*MCH/RBC*MCHC
<8 : Minor thalassemia
8-10.5: Mixed iron def & minor thal.
10.5-13: Iron deficiency
>13: Normal
Note : Sensitivity=99% , Specificity=93%
83. Hb S – Thalassaemia
Hb E – Thalassaemia
Hb D – Thalassaemia
HPFH – Hereditary persistence of foetal
hemoglobin
84. 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.
85. Two forms
Sickle cell Beta 0 thalassemia
Sickle cell Beta + thalassemia
86. There is interection of Hb D and β –
thalassemia genes.
Electrophoresis demonstrates Hb A, Hb F and
Hb D.
87. Incrase Hb F production in adult life.
Heterozygote have 20-30 % Hb F and in
homozygous 90 – 95 %.
88. 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.
89.
90.
91.
92. Red Cell Studies : CBC, One- Tube OF
Test, DCIP Test
Hb Studies : Electrophoresis, Microcolumn
chromatography, Alkali Denaturation
Test, HPLC/LPLC, Imnunologic
Detection, Acid elution test
DNA studies : Gene mapping, PCR, Nt
sequencing, RFLP analysis
KleihauerBetke test. This peripheral blood from a postpartum womanwith fetomaternal hemorrhage demonstrates HbF containing fetal cells (dark red) in a background of maternal cells (ghost-like cells). it can be used to detect HbF–containing cells in β-thalassemia, hereditary persistence of hemoglobin F (some types have homogeneous distribution of HbF in the cells), sickle cell disease, δβ- thalassemia, and myelodysplastic syndrome
