Thalassemia gs

THALASSEMIA

Moderator – Dr. Poonam Nanwani

• "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

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.

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.

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.

Hb A - 97% HbA2 – 1.5-3.5% HbF - <1%

STEPS IN SYNTHESIS OF GLOBIN CHAIN

CLASSIFICATION OF THALASSEMIA

• According to the 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

ALPHA THALASSEMIA

α

α α

ααα/αα

αα/-α

αα/--

Normal

--/-α

--/--

Silent carrier

Thalassemia trait/minor

HbH disease

Hb BartsHydrops foetalis syndrome

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.

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

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.

HB BARTS’ HYDROPS FOETALIS SYNDROME

Hepatosplenomegaly

HB BARTS’ HYDROPS FOETALIS SYNDROME

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

• Hb elctrophoresis demonstrates fast moving HbH band in the range of 5-35 %.

• HbH also demonstrate on HPLC.

HbH Inclusion Bodies

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.

MOLECULAR BASIS OF BETA THALASSEMIAS

• Beta0 thalassemias • Complete absence of beta chain synthesis

• Beta+ thalassemias• Reduced synthesis

Β THALASSEMIA

β

β

βN/βN

β0/βN

β+/βN

Normal

β+/β+

Thalassemia minor

Thalassemia minor

Thalassemia intermedia

Β THALASSAEMIA

β

β

βN/βN

β0/β0

β0/β+

Normal

Thalassemia Major

Thalassemia Major

MUTATIONS CAUSING Β THALASSEMIA

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)

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

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

• HEPATOMEGALY : Mainly first 3 to 4 year..

• ENDOCRINE SYSTEM :

1) Growth hormone deficiency

2) Hypothyrodism

3) Hypoparathyrodism

4) 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

BETA THALASSEMIA MAJOR

Target cells

Tear drop cells

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

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.

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.

• 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

Kleihauer Betke 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).

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.

GEL ELECTROPHORESIS

Alkaline pH

Acidic pH

(1) Normal (2) New born (3) Hb C trait [A-C] (4) Hb SC disease [S-C] (5) Sickle cell disease [S-S], (6) Sickle cell trait [A – S] (7) New born (8) Normal.

Gel electrophoresis instrument

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.

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

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 reduced 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

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

CLINICAL FEATURE

T.MAJOR T.INTERMEDIA T.MINOR

GROWTH,DEVELOPMENT

impaired

SPLENOMEGALY ++++ ++SKELETAL CHANGE,THALASSEMIC FACIES

++++++++

++

Hb <7 7-10 >10RED 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 0-5HbA2 <4 <4 4-8

MICROCYTOSIS +++ ++ +HYPOCROMIA +++ ++ +

Serum iron decrease normal Decrease

ironStorage

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 - HbFHbA2

-

IRON DEFICIENCY ANEMIA

THALASSEMIA ANEMIA OF CHRONIC DISEASE

• Minor thalassemia :

Alpha 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 (high Hb S & F )

• Hb H disease

• Red cell Fragmentation syndrome

Anemia with high RDW

MENTZER INDEX(M.I)

M.I =

<13 SEEN IN THALASSEMIA

AND

>13 IN IRON DEFICIENCY ANEMIA

MCV (fl)RED CELL COUNT (millions/ul)

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

• Also k/a Micro drepanocytic disease

• Two forms• Sickle cell Beta 0 thalassemia• Sickle cell Beta + thalassemia

HB D THALASSEMIA

• There is interaction of Hb D and β – thalassemia genes.

• Electrophoresis demonstrates Hb A, Hb F and Hb D.

HPFH

• Increase 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.

• 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)

When this picture will disappear?

THANK YOU

Presented by – Dr. Gaurav Shelgaonkar