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THALASSEMIA
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.
PREVALENCE 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.
PREVALENCE 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.
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.
Each globin gene has 3 exons and 2 introns.
Classification of thalassemia
According to deficient globin chain Alpha thalassemia Beta thalassemia Delta-beta thalassemia Gamma delta beta thalassemia
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
α Thalassemia
α
α α
α
αα/αα
αα/-α
αα/--
Normal
--/-α
--/--
Molecular basis of beta thalassemias
Beta o thalassemias Complete absence of beta chain synthesis
Beta + thalassemias Reduced synthesis
α 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.
Mutations causing α thalassemia :
Most cases of α thalassemia result from gene deletion
Other – 1) Mutation which cause aberrant splicing 2) Mutation of chain terminator codon3) Mutation which cause instability of α
globin chain after translation.
Hb barts’ hydrops foetalis syndrome
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.
Hb barts’ hydrops foetalis syndroms
Severe anisopoikilocytosis Microcytosis Erythroblastosis
Peripheral smear
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
Hemoglobin H disease. This blood film demonstrates microcytosis, hypochromasia, and numerous morphologic abnormalities, including target cells, microspherocytes, and fragments. Basophilic stippling may occur.
19
Hb H inclusion body test
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.
Hb H disease
Inclusion bodies
Hb H disease
Hb elctrophoresis demonstrates fast moving HbH band in the range of 5-35 %.
HbH also demonstrate on HPLC.
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.
BETA THALASSEMIA
Mutations causing β thalassemia :
1) Mutations which affect transcription2) Mutation that affect splicing of RNA3) Mutations affecting consensus sequences4) Polyadenylation mutations5) Mutations which lead to the formation of
the chain termination codon6) Frame-shift mutations7) Deletions
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)
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.
Pathophysiology of β thalassemia major
Accumulation of free alpha chains Extravascular hemolysis Marrow and bone changes Extramedullary hemopoiesis Synthesis of HbF Iron overload
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)
FACE : frontal bossing ( cranial bone thickening ), overgrowth of zygomatic bone.
JAUNDICE: mild BONE CHANGES : X ray demonstrates-
expansion of diploe, hair on end appearance.
β-Thalassemia facial bone abnormalities. These changes include bossing of theskull; hypertrophy of the maxilla, exposing the upper teeth; depression of nasal bridge; andperiorbital puffiness
β-Thalassemia major. Note the pallor, short stature, massive hepatosplenomegaly,and wasted limbs in this undertransfused case of β-thalassemia major
Beta thalassemia major
β-Thalassemia bone abnormalities. Note the “hair on end” appearance of the corticalbone caused by expansion of the bone marrow (arrows). The subperiosteal bone grows in radiating striations, which appears as “hairs.”
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.
Beta thalassemia major
ENDOCRINE SYSTEM : 1) Growth hormone deficiency2) Hypothyrodism3) Hypoparathyrodism4) Diabetes mellitus
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
• Thalassemias• Smear
Characteristics– Hypochromia– Microcytosis– Target Cells– Tear Drops
Basophilic stippling in thalassemia. Peripheral blood film demonstratingmicrocytic hypochromic RBCs and basophilic stippling (arrows). Basophilic stippling occurs inthalassemia as well as in other hematologic disorders.
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
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
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
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)
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 : 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.
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.
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.
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.
At alkaline pH Hb C, E, A2 and O migrate together to form a single band, Hb S, D and G also co migrate.
Electrophoresis Principle (2).
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
Hemoglobin electrophoresis at alkali pH
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
52
Hemoglobin electrophoresis at alkali pH
Reagent :
Tris-EDTA-Borate (TBE) pH 8.4-8.6
53
Equipment
• 1. Power supply for 500 V• 2. Electrophoretic chamber • 3. Cellulose acetate plate• 4. Sample applicator• 5. Stain box• 6. Large filter paper or blotter
54
Equipment
Sample preparation well Aligning base
Sample applicator
55
Equipment
BlotterCellulose acetate plate
56
Equipment
Power supply
Electrophoreticchamber
Cellulose acetate plate
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 300 volts for 10-20 min.
• Stained with Ponceau S
58
Ponceau S staining
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
Gel electrophoresis
Alkaline pH
Acidic pH
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.
HPLC instrument
62
Hb identification by HPLC
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
63
Normal or a-thal trait b-thal trait
64
Homo EHbE trait
65
Hb H disease in newborn HbE/bO-thalassemia
DNA Analysis. 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.
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 redused in alpha thalassemia and increased in beta thalassemia
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 )
Clinical features
Mild to moderate anemia
Mild to moderate splenomegaly
Mild skeletal and facial changes.
Iron overload
Recurrent leg ulcer
Repeated infection
Thalassemia intermedia
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
Moderate degree of anisopoikilocytosis,
microcytic hypochromic,target cells,
Thalassemia minor
Heterozygous carrier state characterized by little or no anemia but prominent morphological changes of red cells
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
Beta Thalassemia minor
MICROCYTOSIS
HYPOCHROMIA
ANISOPOIKILOCYTO-SIS
TEAR DROP CELL
BASOPHILIC STIPPLING
TARGET CELL
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
THE BLOOD SMEAR IN ANEMIAANEMIAS ANISOCYTOSIS
POIKILOCYTOSISBASOPHILIC STIPPLING
TARGET CELL DIMORPHISM
IRON DEFICIENCY ANEMIA
1-3+ 0 ± ±
ANEMIA OF CHRONIC DISORDER
± 0 ± ±
THALASSEMIAMINORMAJOR
±3+
2+3+
5%3+
00
Hb C OR E 2+ ± 50% 0
Serum iron decrease normal Decrease
ironStorage
decrease N/increase Increase/N
TIBC increase normal Decrease
Osmotic fragility decrease decrease _
Bone marrow Decrease iron staining
Erythriod hyperplasia
Normal morphology
electrophoresis - HbFHbA2
-
IRON DEFICIENCY ANEMIA
THALASSEMIA ANEMIA OF CHRONIC DISEASE
Minor thalassemia :
Alpha (Hb electrophoresis ) beta
delta-beta
Anemia of chronic disease (in late stages
specially in renal disease )
Anemia with normal RDW
Iron deficiency anemia
Beta thalassemia major & intermedia
Sickle thalassemia
Hb H disease
Red cell Fragmentation syndrome
Anemia with high RDW
MENTZER INDEX(M.I)= <13 SEEN IN THALASSEMIA AND >13 IN IRON
DEFICIENCY ANEMIA
M.I=MCV
RED CELL COUNT
DDX OF MINOR THALASSEMIA & IRON DEF.
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%
DDX OF MINOR THALASSEMIA & IRON DEF.
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%
Miscelleneous thalassemic syndrome
Hb S – Thalassaemia
Hb E – Thalassaemia
Hb D – Thalassaemia
HPFH – Hereditary persistence of foetal
hemoglobin
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.
Sickle cell Beta Thalassemia
Two forms Sickle cell Beta 0 thalassemia Sickle cell Beta + thalassemia
Hb D thalassemia
There is interection of Hb D and β – thalassemia genes.
Electrophoresis demonstrates Hb A, Hb F and Hb D.
HPFH
Incrase Hb F production in adult life. Heterozygote have 20-30 % Hb F and
in homozygous 90 – 95 %.
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.
THANK YOU
92
Laboratory Thalassemia Diagnosis
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
CLINICAL FEATURE
T.MAJOR T.INTERMEDIA T.MINOR
GROWTH,DEVELOPMENT
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 +++ ++ +
ANISOPOIKILOCYTOSIS
+++ ++ ±
B.M.IRON ++++ ++ ±HbF 30-90 10-30 0-5
HbA2 <4 <4 4-8
MICROCYTOSIS +++ ++ +HYPOCROMIA +++ ++ +