87
THALASSEMIA Moderator – Dr. Poonam Nanwani

Thalassemia gs

Embed Size (px)

Citation preview

Page 1: Thalassemia gs

THALASSEMIA

Moderator – Dr. Poonam Nanwani

Page 2: Thalassemia gs
Page 3: Thalassemia gs

• "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

Page 4: Thalassemia gs

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.

Page 5: Thalassemia gs

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.

Page 6: Thalassemia gs

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.

Page 7: Thalassemia gs

Hb A - 97% HbA2 – 1.5-3.5% HbF - <1%

Page 8: Thalassemia gs

STEPS IN SYNTHESIS OF GLOBIN CHAIN

Page 9: Thalassemia gs

CLASSIFICATION OF THALASSEMIA

• According to the deficient globin chain• Alpha thalassemia• Beta thalassemia• Delta-beta thalassemia• Gamma delta beta thalassemia

Page 10: Thalassemia gs

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

Page 11: Thalassemia gs

ALPHA THALASSEMIA

α

α α

ααα/αα

αα/-α

αα/--

Normal

--/-α

--/--

Silent carrier

Thalassemia trait/minor

HbH disease

Hb BartsHydrops foetalis syndrome

Page 12: Thalassemia gs

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.

Page 13: Thalassemia gs
Page 14: Thalassemia gs

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

Page 15: Thalassemia gs
Page 16: Thalassemia gs

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.

Page 17: Thalassemia gs
Page 18: Thalassemia gs

HB BARTS’ HYDROPS FOETALIS SYNDROME

Hepatosplenomegaly

Page 19: Thalassemia gs

HB BARTS’ HYDROPS FOETALIS SYNDROME

Peripheral smear

Page 20: Thalassemia gs

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.

Page 21: Thalassemia gs
Page 22: Thalassemia gs

HbH Inclusion Bodies

Page 23: Thalassemia gs
Page 24: Thalassemia gs

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.

Page 25: Thalassemia gs

MOLECULAR BASIS OF BETA THALASSEMIAS

• Beta0 thalassemias • Complete absence of beta chain synthesis

• Beta+ thalassemias• Reduced synthesis

Page 26: Thalassemia gs

Β THALASSEMIA

β

β

βN/βN

β0/βN

β+/βN

Normal

β+/β+

Thalassemia minor

Thalassemia minor

Thalassemia intermedia

Page 27: Thalassemia gs

Β THALASSAEMIA

β

β

βN/βN

β0/β0

β0/β+

Normal

Thalassemia Major

Thalassemia Major

Page 28: Thalassemia gs

MUTATIONS CAUSING Β THALASSEMIA

Page 29: Thalassemia gs

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)

Page 30: Thalassemia gs
Page 31: Thalassemia gs

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.

Page 32: Thalassemia gs

PATHOPHYSIOLOGY OF Β THALASSEMIA MAJOR

• Accumulation of free alpha chains

• Extravascular hemolysis

• Marrow and bone changes

• Extramedullary hemopoiesis

• Synthesis of HbF

• Iron overload

Page 33: Thalassemia gs
Page 34: Thalassemia gs

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)

Page 35: Thalassemia gs
Page 36: Thalassemia gs

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

Page 37: Thalassemia gs

BETA THALASSEMIA MAJOR

Page 38: Thalassemia gs

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.

Page 39: Thalassemia gs

BETA THALASSEMIA MAJOR

• HEPATOMEGALY : Mainly first 3 to 4 year..

• ENDOCRINE SYSTEM :

1) Growth hormone deficiency

2) Hypothyrodism

3) Hypoparathyrodism

4) Diabetes mellitus

Page 40: Thalassemia gs
Page 41: Thalassemia gs

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

Page 42: Thalassemia gs
Page 43: Thalassemia gs

• Thalassemias• Smear Characteristics

– Hypochromia– Microcytosis– Target Cells– Tear Drops

Page 44: Thalassemia gs
Page 45: Thalassemia gs

BETA THALASSEMIA MAJOR

Target cells

Tear drop cells

Page 46: Thalassemia gs

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

Page 47: Thalassemia gs

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

Page 48: Thalassemia gs

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

Page 49: Thalassemia gs

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.

Page 50: Thalassemia gs

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.

Page 51: Thalassemia gs

• 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

Page 52: Thalassemia gs

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).

Page 53: Thalassemia gs
Page 54: Thalassemia gs

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.

Page 55: Thalassemia gs

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.

Page 56: Thalassemia gs

GEL ELECTROPHORESIS

Alkaline pH

Acidic pH

Page 57: Thalassemia gs

(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.

Page 58: Thalassemia gs

Gel electrophoresis instrument

Page 59: Thalassemia gs
Page 60: Thalassemia gs

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.

Page 61: Thalassemia gs

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.

Page 62: Thalassemia gs

HPLC INSTRUMENT

Page 63: Thalassemia gs

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.

Page 64: Thalassemia gs

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

Page 65: Thalassemia gs

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 )

Page 66: Thalassemia gs

CLINICAL FEATURES

• Mild to moderate anemia

• Mild to moderate splenomegaly

• Mild skeletal and facial changes.

• Iron overload

• Recurrent leg ulcer

• Repeated infection

Thalassemia Intermedia

Page 67: Thalassemia gs

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

Page 68: Thalassemia gs

MODERATE DEGREE OF ANISOPOIKILOCYTOSIS,

MICROCYTIC HYPOCHROMIC,TARGET CELLS,

Page 69: Thalassemia gs

THALASSEMIA MINOR

• Heterozygous carrier state characterized by little or no anemia but prominent morphological changes of red cells

Page 70: Thalassemia gs

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

Page 71: Thalassemia gs

BETA THALASSEMIA MINOR

MICROCYTOSIS

HYPOCHROMIA

ANISOPOIKILOCYTO-SIS

TEAR DROP CELL

TARGET CELL

Page 72: Thalassemia gs

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

Page 73: Thalassemia gs

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 +++ ++ +

Page 74: Thalassemia gs
Page 75: Thalassemia gs

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

Page 76: Thalassemia gs

• Minor thalassemia :

Alpha beta

delta-beta

• Anemia of chronic disease (in late stages specially

in renal disease )

Anemia with Normal RDW

Page 77: Thalassemia gs

• Iron deficiency anemia

• Beta thalassemia major & intermedia

• Sickle thalassemia (high Hb S & F )

• Hb H disease

• Red cell Fragmentation syndrome

Anemia with high RDW

Page 78: Thalassemia gs

MENTZER INDEX(M.I)

M.I =

<13 SEEN IN THALASSEMIA

AND

>13 IN IRON DEFICIENCY ANEMIA

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

Page 79: Thalassemia gs

MISCELLENEOUS THALASSEMIC SYNDROME

• Hb S – Thalassaemia

• Hb E – Thalassaemia

• Hb D – Thalassaemia

• HPFH – Hereditary persistence of foetal hemoglobin

Page 80: Thalassemia gs

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.

Page 81: Thalassemia gs

SICKLE CELL BETA THALASSEMIA

• Also k/a Micro drepanocytic disease

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

Page 82: Thalassemia gs

HB D THALASSEMIA

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

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

Page 83: Thalassemia gs

HPFH

• Increase Hb F production in adult life.

• Heterozygote have 20-30 % Hb F and in homozygous 90 – 95 %.

Page 84: Thalassemia gs

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.

Page 85: Thalassemia gs

• 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)

Page 86: Thalassemia gs

When this picture will disappear?

Page 87: Thalassemia gs

THANK YOU

Presented by – Dr. Gaurav Shelgaonkar