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.

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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.

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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)

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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.

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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

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Hemoglobin electrophoresis at alkali pH

Reagent :

Tris-EDTA-Borate (TBE) pH 8.4-8.6

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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

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Equipment

Sample preparation well Aligning base

Sample applicator

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Equipment

BlotterCellulose acetate plate

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Equipment

Power supply

Electrophoreticchamber

Cellulose acetate plate

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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

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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

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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

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Normal or a-thal trait b-thal trait

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Homo EHbE trait

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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

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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 +++ ++ +