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Laboratory Diagonosis  thalassemia Chirantan
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Laboratory Diagonosis thalassemia Chirantan


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22september 2011, GLT medical College Kolkata88, India..............under Pathology Dept!!!!!!!!1Dr.S.K.Mandal.........chief speakers aVIK bASU, Chirantan Mnadal, Urmimala Bhattacharya, Soumaditya …

22september 2011, GLT medical College Kolkata88, India..............under Pathology Dept!!!!!!!!1Dr.S.K.Mandal.........chief speakers aVIK bASU, Chirantan Mnadal, Urmimala Bhattacharya, Soumaditya Banerjee!!!!!!!!!

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  • 1. Laboratory Diagonosis of Thalassemiaby ChirantanMandal
    Moderator: Dr Santosh Kumar Mondal
    Assoc.Professor, Pathology
  • 2. Initial Approach to Suspect
  • 3. Clinical Features
    Compensated HaemolyticAnaemia
    ExtramedullaryHaematopoesis leads to Splenomegalyupto 1500 gm, even Hepatomegaly
    Iron Overload causing Hemosiderosis & Secondary Hemochromatosis damage to endocrine organs, Heart etc
    Serum BilirubinUnconjugated
    in Beta TM major
  • 4. 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
  • 5. Complete Blood Count
    Alpha HydropsFoetAlphaHydropsFoetalisl<6g/dl HbVery High Reticulocytosis
  • 6. Peripheral Blood Smear
    Tear Drop Cell Target Cell
  • 7. 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.
  • 8. alpha HbH
    Heinz Bodies inclusions within RBC composed of denatured Hemoglobin
    Reticulocyte count
    (increased erythropoesis)
  • 9. Bone Marrow Exam
    Markedly increased Iron Deposition
    erythroid hyperplasia
    morphologic abnormalities of the erythroblasts
  • 10. Test for Hemolysis
    Increased RBC production
    Increased RBC destruction
    Nucleated RBC
    BM cellularity
     albumin complex = albumin+heme
    Increased excretion Through Urine
     (Fe3+ )
  • 11. Hb Electrophoresis
    differentiate among Hb A, Hb A2, and Hb F
    Detects presence of abnormal Hb
    Diagnosing 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 pH
    Hb molecule is –vely charged & migrate towards anode
    HbD & HbS and HbA2/C/E/O have same mobility
    Citrate Agar Gel Electrophoresis Acidic pH
    Separation of HbD & HbS and HbA2/C/E/O from each other
  • 12.
  • 13.
  • 14. High Performance Liquid Chromatography
    Cation Exchange HPLC
    Separate Hbs that have identical mobility in Citrate Agar Gel & Cellulose Acetate Agarose Gel Electrophoresis
    Separation of HbA2 & HbE not Possible
    Anion Exchange HPLC
    Pattern of elution obtained here is opposite of Cation Exchange HPLC
    Separation of HbA2 & HbE is possible here
  • 15.
  • 16.
  • 17. IEF (IsoElectricFocussing)
    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
  • 18.
  • 19. 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 Th
    HbA2 either normal or slightly decreased
    Small amount of HbBarts in neonatal period 2 to 5%
    Alpha ThHbH
    HbF 10% , HbH 2-4%
    HydropsFeotalisHb Barts100 %
  • 20. 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
    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
    Sharp decline in 1st 10 months of life
  • 21. Molecular Detection (Determine specific defect at molecular DNA level)
    Majority of alpha TM results from gene deletion
    Majority of betaTM results from single nucleotide substitution / frameshift mutation
    Gene mapping based on Southern Blotting
    PCR based procedures
    PreNatalDiagonostic Importance
  • 22. free erythrocyte protoporphyria (FEP)
  • 23. Iron Study(To differentiate thalassemia from IDA
    Serum Ferritin 200 ng/mL in female
    300 ng/mL in male
    Serum Iron Level Increased , 69-135ug/dL
    Transferritin Saturation >50%
    TIBC normal
    Marrow Iron Store Increased
    Iron deficiency anemia
    Serum Ferritin <12ng/L
    Serum Iron Level very Low
    Transferritin Saturation <10%
    TIBC Increased
    Marrow Iron Store very low
  • 24. Globin Chain(alpha, beta gamma)
    Prenatal Diagnostic importance
    By Reverse phase HPLC
  • 25. Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT)
    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 
    osmotic fragility = --------- ,
    S/V ratio
    S/V ratio => osmotic fragility
    The red blood cells are so markedly resistant to hemolysis in hypotonic sodium chloride solution
  • 26. 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.
  • 27. 1st Trimester
    Known 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
  • 28. 2nd Trimester
    method of choice where DNA mutations are unidentified in parents
    (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
    extract DNA from amniotic fluid @ >15 weeks of gestation
    chorionic villus samples 10-12 weeks (upto 20 weeks)
    Fetal DNA analysis
  • 29.
  • 30. Pre-Marriage Thalassemia Test is Imperative
    Over four crore people in India are
    diagnosed 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
  • 31. 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.
  • 32. THANK YOU
  • 33.
  • 34.  DNA Mutation Analysis
    Once 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.
  • 35. Management
    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.
    vigorous transfusion program
    pretransfusionhematocrit 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
  • 36. Complications of Transfusions
    Excessive iron stores lead to depletion of ascorbic acid and vitamin E
    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
  • 37. 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 transducenondividing cells )
    • Reestablishing high levels of HbF
    ( using pulsed hydroxyurea, cytarabine, Butyrates that stimulates proliferation of the primitive HbF-producing progenitor cell population
  • 38. Differential Diagonosis
  • 39. 39
    Differential Diagnosis of Microcytic, Hypochromic Anemias
  • 40. Differential diagnosis