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.
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.
13.
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
15.
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.
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)
30.
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
33.
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)
35.
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
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
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).
53.
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.
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.
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
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
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
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)
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)
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.
(greek word) Thalassa- Mediterranean sea
About 250 million people (1.5%) of the world population carry thalassemia gene.
About 250 million people (1.5%) of the world population carry thalassemia gene.
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.
In India β thalassemia is frequent and α thalassemia is rare.
Specially alpha thalassemia distribution parallel to malaria prevalaent zone.
Each globin gene has 3 exons and 2 introns.
Synthesis of alpha chains is controlled by 2 gene clusters on chromosome 16 and of beta chains on chromosome 11.
Baby is pale and bloated ; placenta is oedamatous ; moderate to massive hepatomegaly.
Zeta 2 gamma 2
Hydrops fetalis at autopsy in hemoglobin Bart disease. Hepatosplenomegaly in a
newborn with hemoglobin Bart disease. The loss of all four α-globin genes results in severe
anemia, high-output heart failure, splenomegaly, edema, and intrauterine or immediately
postpartum death for the affected fetus. Dystocia, eclampsia, and hemorrhage can occur in the
mother carrying the affected fetus.
Severe anisopoikilocytosis
Microcytosis
Fragmented cells
Target cells
Tear drop cells
Erythroblastosis
demonstrates microcytosis, hypochromasia, and numerous morphologic abnormalities, including target cells, microspherocytes, and fragments.
Peripheral blood stained
with supravital stain brilliant cresyl blue.
Mutations which affect transcription
Mutation that affect splicing of RNA
Mutations affecting consensus sequences
Polyadenylation mutations
Mutations which lead to the formation of the chain termination codon
Frame-shift mutations
Deletions
Progeny
FACE : frontal bossing ( cranial bone thickening ), overgrowth of zygomatic bone.
JAUNDICE: mild
BONE CHANGES : X ray demonstrates- expansion of diploe, hair on end appearance.
“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.” Widening ofcalvarium.
nucleated red blood cells, microcytosis, and hypochromia with multiple morphologic changes: target cells, teardrop
cells, fragments, basophilic stippling.
β-Thalassemia major.. This peripheral blood film demonstrates many nucleated red blood
cells, microcytosis, and hypochromia with multiple morphologic changes: target cells, teardrop
cells, fragments, basophilic stippling, and Pappenheimer bodies. The nucleated red blood cells
may be dysplastic or show abnormal hemoglobinization. Neutrophilia and thrombocytosis may
occur. This patient has undergone splenectomy for hypersplenism and increased transfusion
requirements. Howell-Jolly bodies are present.
The KB test is performed to quantitate the number of fetal cells present in the maternal circulation.
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
At alkaline pH Hb C, E, A2 and O migrate together to form a single band, Hb S, D and G also co migrate.
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.
