Laboratory Diagonosis of Thalassemia

by Chirantan Mandal

Moderator: Dr Santosh Kumar Mondal Assoc.Professor, Pathology

Initial Approach to Suspect

Clinical Features

Compensated Haemolytic Anaemia

Extramedullary Haematopoesis leads to Splenomegaly upto 1500 gm, even Hepatomegaly

Iron Overload causing Hemosiderosis & Secondary Hemochromatosis damage to endocrine organs, Heart etc

Serum Bilirubin Unconjugated in Beta TM major

X-ray film of the skull (showing perpendicular radiations resembling a crewcut)

striking expansion of hematopoietically active marrow. In the bones of the face and skull the burgeoning marrow (erythroid hyperplasia) perforates/erodes existing cortical bone

characteristic “hair-on-end” appearance

Complete Blood CountBeta Major Beta Minor Alpha HbH Alpha Trait

Hb%M => 13.8-18.0 g/dLF => 12 - 15 g/d

2-4 g/dl 9-11g/dl 2 - 8g/dl 10-14g/dl

MCV (fl)Normal 80 - 100 fl

50-60 60-80 60-70 60-80

MCH(pg) normal 27-31 pg/cel

16-22 28-32 24-28 28-32

MCHC Decrease Normal or Slight Decrease

Decrease Normal or Slight Decrease

RDW Normal => 11 - 15%

Increased Normal <14% Normal Normal

Reticulocytosis Moderately high

mild mild normal

Alpha Hydrops FoetAlpha Hydrops Foetalisl<6g/dl Hb Very High Reticulocytosis phaalislpha

Peripheral Blood Smear Basophillic Stiplling

Microcytic HypochromicTear Drop Cell Target Cell

Anisopoikilocytosis

HbH

Incubation with brilliant cresyl blue stain causes Hemoglobin H to precipitate

appearance of multiple discrete inclusions -golf ball appearance of RBCs

Heinz bodies that are evenly distributed throughout cell.

Reticulocyte count (increased erythropoesis)

alpha HbHHeinz Bodies inclusions within RBC composed of denatured

Hemoglobin

Bone Marrow Exam

Markedly increased Iron Deposition

erythroid hyperplasia

morphologic abnormalities of the erythroblasts

Test for Hemolysis

• UC-Bilirubin• UBG • MetheAlbumin

ReticulocyteNucleated RBCBM cellularity

Hb UriaHemosiderosisMetHb Uria

Increased RBC destruction Increased RBC production

Increased excretion Through Urine

(Fe3+ )

albumin complex = albumin+heme

Hb Electrophoresis

differentiate among Hb A, Hb A2, and Hb F

Detects presence of abnormal HbDiagnosing and differentiating various forms of thalassemias

Principle : Comparing their mobility to those of a known control sample (mixture of HbA+F+S/D+A2)

Cellulose Acetate Agarose Gel Electrophoresis Alkaline pHHb molecule is –vely charged & migrate towards anode

HbD & HbS and HbA2/C/E/O have same mobility

Citrate Agar Gel Electrophoresis Acidic pHSeparation of HbD & HbS and HbA2/C/E/O from each other

High Performance Liquid Chromatography

Cation Exchange HPLCSeparate Hbs that have identical mobility in Citrate Agar Gel &

Cellulose Acetate Agarose Gel Electrophoresis

Separation of HbA2 & HbE not Possible

Anion Exchange HPLCPattern of elution obtained here is opposite of Cation Exchange

HPLC

Separation of HbA2 & HbE is possible here

IEF (IsoElectric Focussing)

Formation of pH gradient along the gel during passage of current through the separation of carrier ampholites with different pHs

Separation of Hbs whose pI (IsoElectric point) differ by as little as 0.01 pH units

Can separate those Hbs from each other, which have identical mobility in Electrophoretic system

Beta Th Major An increased level of Hb F ranging from less than 50-90%

Hb A2 normal or high

Beta Th Minor HbA2 often elevated > 3%, sometimes reaching 7-8%.

Hb F 3%

Alpha Trait ThHbA2 either normal or slightly decreased

Small amount of Hb Barts in neonatal period 2 to 5%

Alpha Th HbHHbF 10% , HbH 2-4%

Hydrops Feotalis Hb Barts100 %

HbA2• Cellulose Acetate Agarose Gel Electrophoresis , HPLC• Useful to confirm Beta TM carrier state HbA2 >3.5% are considered to have thalassemic trait

• Sharp rise in 1st 4 months of life• Slightly elevated for rest of life

HbF• Alkali Denaturation technique• Acid Elution technique (Acid pH dissolves HbA from RBC. HbF is resistant, so remains

in cell. Eosin Stained slide cells with Hb F stains varying shades of pink. Normal RBC`s appear as "ghost" cells

• HPLC• Sharp decline in 1st 10 months of life

Molecular Detection (Determine specific defect at molecular DNA level)

Majority of alpha TM results from gene deletionMajority of betaTM results from single nucleotide substitution /

frameshift mutation

Gene mapping based on Southern BlottingPCR based procedures

PreNatal Diagonostic Importance

free erythrocyte protoporphyria (FEP)

Iron Study(To differentiate thalassemia from IDAThalassemia

Serum Ferritin 200 ng/mL in female300 ng/mL in male

Serum Iron Level Increased , 69-135ug/dLTransferritin Saturation >50%

TIBC normal Marrow Iron Store Increased

Iron deficiency anemiaSerum Ferritin <12ng/L

Serum Iron Level very LowTransferritin Saturation <10%

TIBC Increased Marrow Iron Store very low

Globin Chain(alpha, beta gamma)

Prenatal Diagnostic importance

By Reverse phase HPLC

screening test for carrier states

principle : limit of hypotonicity which the red cell can withstand

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.

if line is not visible it is considered as positive.

Positive test is due to the reduced osmotic fragility of red cells 

Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT)

The red blood cells are so markedly resistant to hemolysis in hypotonic sodium chloride solution

1osmotic fragility = --------- , S/V ratio

S/V ratio => osmotic fragility

Prenatal Diagnosis

if the lady is found to be NESTROFT and red cell indices positive, HbA2 is done to confirm the carrier status.

If her HbA2 is 3.5. per cent, husband's carrier status is tested.

If both partners are carriers we study their DNA for 5 common and 12 rare mutations.

Prenatal diagnosis is offered if mutations are identified.

1st TrimesterKnown Mutation

• ARMS (Amplification Refractory Mutation System)• Reverse Dot Blot Hybridization• Dot Blot Hybridization using ASO probes• Direct Electrophorersis for 619bp deletion

619bp deletion , IVS1-5(G->C), codons8/9(+G), IVS1-1(G->T), codons 41/42(-TCTT), codons15A(G->A)

Unknown Mutation • DGGE (Dnaturation Gradient Gel Electrophoresis)• Single Strand Confirmational Polymorphism• Sequence analysis of Beta Globin Gene• Mismatch PCR

2nd TrimesterCordocentesis

(transabdominal route by USG guide)

Globin chain synthesis Ratio in Cord Blood @ 17 to 23 Weeks Pregnancy

Hemoglobin Electrophoresis @ 6 months of Delivery to cross check Diagonosis

method of choice where DNA mutations are unidentified in parents

extract DNA from amniotic fluid @ >15 weeks of gestation chorionic villus samples 10-12 weeks (upto 20 weeks)

Fetal DNA analysis

Pre-Marriage Thalassemia Test is Imperative

Over four crore people in India arediagnosed with this form

Patients need blood transfusions every three to eight weeks to maintain hemoglobin levels

Permanent cures like Bone Marrow Transplantation and stem cell transplants are very expensive and also very risky

.

It is thus advised that people getting married should take a simple blood test

ensure that both the partners are not carrying the Thalassemia trait.

If found to be diagnosed with Thalassemia, consult your doctor before planning your family together.

THANK YOU

 DNA Mutation AnalysisOnce the carrier status of the couple is confirmed

ASO (allele specific oligonucleotide) method detects point mutations, nucleotide insertion or deletion in genomic DNA. In this method ASO probes of 18-20 mer sequence are used. DNA is

denatured and dot blotted on to a nylon membrane and then hybridized to different probes.

Reverse dot blot probes are attached to the membrane and DNA hybridizes with dot corresponding to the mutation.

Amplifica- tion refractory mutation system (ARMS) technique in which specific primers

against normal and mutant sequences are used.

SSCP is based on the mobility shift in a neutral polyacrylamide gel due to conformational change caused by substitution of a base in a single stranded DNA fragment

DGGE is based on the resolution of DNA fragments differing by single nucleotide substitution Both the methods could be used for detection of rare mutations. This can be followed by

sequencing using automated sequencers which are available now. We are using ARMS technique for character-isation of mutations in our laboratory. Using this technique we are able to detect

five common mutations, namely, IVS 1-5, IVS 1-1, 619 bp del, Fr41-42 and Fr8-9 (Fig. 2) in 90-95% of the subject and 12 rare mutations in 1-2% of the subjects. The families where mutations

were not characterized could be helped by doing linkage studies.

ManagementTransfusion

chronic hypertransfusion therapy to maintain a hematocrit of at least 27–30% so that erythropoiesis is suppressed.

Splenectomy is required if the annual transfusion requirement (volume of RBCs per kilogram of body weight per year) increases by >50%.

Folic acid supplements may be useful.

SuperTransfusionvigorous transfusion program

pretransfusion hematocrit was kept at ≥35%

aimed at keeping hemoglobin levels above 12.0 g/dL.[68] This approach rests on the assumption that the benefits of further suppression of erythropoiesis and gastrointestinal iron absorption will offset the increased need for

red blood cells generally

reserved for patients with poor tolerance of lower hemoglobin levels

Complications of Transfusions

Haemosiderosis each unit of blood contains approximately 200 mg of iron, a patient who receives 25 to 30 units of blood a year, by the

third decade of life, in the absence of chelation, will accumulate over 70 g of iron

fully saturated transferrin, a significant fraction of the total iron in plasma circulates in the form of low-molecular-weight

complexes not bound to transferrin, iron-induced peroxidative injury to the phospholipids of lysosomes and mitochondria,

produced by free hydroxyl radicals

Excessive iron stores lead to depletion of ascorbic acid and vitamin E

Experimental Therapies•Bone Marrow Transplantation (HLA-compatible donor )(provides stem cells able to express normal Hb, curative in 80–90% of patients, survival into adult life is possible with conventional therapy) •Cord Blood Transplantation HLA-identical siblings

•Gene Therapy(Uptake of gene vectors into the nondividing hematopoietic stem cells. Lentiviral-type vectors that can transduce nondividing cells )

•Reestablishing high levels of HbF ( using pulsed hydroxyurea, cytarabine, Butyrates that stimulates proliferation of the primitive HbF-producing progenitor cell population

Differential Diagonosis

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Differential Diagnosis of Microcytic, Hypochromic Anemias

RDW Serum Iron

TIBC Serum Ferritin

FEP

Iron Deficiency

Inc Dec Inc Dec Inc

Alpha Thal Norm Norm Norm Norm Norm

Beta Thal Norm Norm Norm Norm Norm

Hgb E Disease Norm Norm Norm Norm Norm

Anemia of Chronic Disease

Norm Dec Dec Inc Inc

Sideroblastic Anemia

Inc Inc Norm Inc Dec

Lead Poisoning

Norm Norm Norm Norm Inc

Differential diagnosis