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 Banerjee!!!!!!!!!

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Laboratory Diagonosis thalassemia Chirantan

  1. 1. Laboratory Diagonosis of Thalassemiaby ChirantanMandal<br />Moderator: Dr Santosh Kumar Mondal<br />Assoc.Professor, Pathology <br />
  2. 2. Initial Approach to Suspect<br />
  3. 3. Clinical Features<br />Compensated HaemolyticAnaemia<br />ExtramedullaryHaematopoesis leads to Splenomegalyupto 1500 gm, even Hepatomegaly<br />Iron Overload causing Hemosiderosis & Secondary Hemochromatosis damage to endocrine organs, Heart etc<br />Serum BilirubinUnconjugated<br />in Beta TM major<br />
  4. 4. X-ray film of the skull <br />(showing perpendicular radiations resembling a crewcut)<br />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<br />
  5. 5. Complete Blood Count<br />Alpha HydropsFoetAlphaHydropsFoetalisl<6g/dl HbVery High Reticulocytosis<br />pha<br />alislpha<br />
  6. 6. Peripheral Blood Smear<br />Anisopoikilocytosis<br />BasophillicStiplling<br />MicrocyticHypochromic<br />Tear Drop Cell Target Cell<br />
  7. 7. HbH<br />Incubation with brilliant cresyl blue stain causes Hemoglobin H to precipitate<br />appearance of multiple discrete inclusions -golf ball appearance of RBCs<br />Heinz bodies that are evenly distributed throughout cell.<br />
  8. 8. alpha HbH<br />Heinz Bodies inclusions within RBC composed of denatured Hemoglobin<br />Reticulocyte count<br />(increased erythropoesis)<br />
  9. 9. Bone Marrow Exam<br />Markedly increased Iron Deposition<br />erythroid hyperplasia <br />morphologic abnormalities of the erythroblasts<br />
  10. 10. Test for Hemolysis<br />Increased RBC production <br />Increased RBC destruction <br />Reticulocyte<br />Nucleated RBC<br />BM cellularity<br />UC-Bilirubin<br />UBG <br />MetheAlbumin<br /> albumin complex = albumin+heme<br />Increased excretion Through Urine <br />HbUria<br />Hemosiderosis<br />MetHbUria<br /> (Fe3+ )<br />
  11. 11. Hb Electrophoresis<br />differentiate among Hb A, Hb A2, and Hb F<br />Detects presence of abnormal Hb<br />Diagnosing and differentiating various forms of thalassemias<br />Principle : Comparing their mobility to those of a known control sample (mixture of HbA+F+S/D+A2)<br />Cellulose Acetate Agarose Gel Electrophoresis Alkaline pH<br />Hb molecule is –vely charged & migrate towards anode<br />HbD & HbS and HbA2/C/E/O have same mobility<br />Citrate Agar Gel Electrophoresis Acidic pH<br />Separation of HbD & HbS and HbA2/C/E/O from each other<br />
  12. 12.
  13. 13.
  14. 14. High Performance Liquid Chromatography<br />Cation Exchange HPLC<br />Separate Hbs that have identical mobility in Citrate Agar Gel & Cellulose Acetate Agarose Gel Electrophoresis <br />Separation of HbA2 & HbE not Possible<br />Anion Exchange HPLC<br />Pattern of elution obtained here is opposite of Cation Exchange HPLC<br />Separation of HbA2 & HbE is possible here<br />
  15. 15.
  16. 16.
  17. 17. IEF (IsoElectricFocussing)<br />Formation of pH gradient along the gel during passage of current through the separation of carrier ampholites with different pHs<br />Separation of Hbs whose pI (IsoElectric point) differ by as little as 0.01 pH units<br />Can separate those Hbs from each other, which have identical mobility in Electrophoretic system<br />
  18. 18.
  19. 19. Beta Th Major <br />An increased level of Hb F ranging from less than 50-90%<br />Hb A2 normal or high<br />Beta Th Minor <br />HbA2 often elevated > 3%, sometimes reaching 7-8%.<br />Hb F 3%<br />Alpha Trait Th<br />HbA2 either normal or slightly decreased<br />Small amount of HbBarts in neonatal period 2 to 5%<br />Alpha ThHbH<br />HbF 10% , HbH 2-4%<br />HydropsFeotalisHb Barts100 %<br />
  20. 20. HbA2<br />Cellulose Acetate Agarose Gel Electrophoresis , HPLC<br />Useful to confirm Beta TM carrier state <br /> HbA2 >3.5% are considered to have thalassemic trait<br />Sharp rise in 1st 4 months of life<br />Slightly elevated for rest of life<br />HbF<br />Alkali Denaturation technique<br />Acid Elution technique <br /> (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<br />HPLC<br />Sharp decline in 1st 10 months of life<br />
  21. 21. Molecular Detection (Determine specific defect at molecular DNA level)<br />Majority of alpha TM results from gene deletion<br />Majority of betaTM results from single nucleotide substitution / frameshift mutation<br />Gene mapping based on Southern Blotting<br />PCR based procedures<br />PreNatalDiagonostic Importance<br />
  22. 22. free erythrocyte protoporphyria (FEP)<br />
  23. 23. Iron Study(To differentiate thalassemia from IDA<br />Thalassemia<br />Serum Ferritin 200 ng/mL in female<br />300 ng/mL in male<br />Serum Iron Level Increased , 69-135ug/dL<br />Transferritin Saturation >50%<br />TIBC normal <br />Marrow Iron Store Increased<br />Iron deficiency anemia<br />Serum Ferritin <12ng/L<br /> Serum Iron Level very Low<br />Transferritin Saturation <10%<br />TIBC Increased <br />Marrow Iron Store very low<br />
  24. 24. Globin Chain(alpha, beta gamma)<br />Prenatal Diagnostic importance<br />By Reverse phase HPLC<br />
  25. 25. Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT)<br />screening test for carrier states<br />principle : limit of hypotonicity which the red cell can withstand<br />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.<br />if line is not visible it is considered as positive. <br />Positive test is due to the reduced osmotic fragility of red cells <br /> 1<br />osmotic fragility = --------- , <br /> S/V ratio <br />S/V ratio => osmotic fragility<br />The red blood cells are so markedly resistant to hemolysis in hypotonic sodium chloride solution<br />
  26. 26. Prenatal Diagnosis<br /> if the lady is found to be NESTROFT and red cell indices positive, HbA2 is done to confirm the carrier status. <br />If her HbA2 is 3.5. per cent, husband's carrier status is tested. <br />If both partners are carriers we study their DNA for 5 common and 12 rare mutations. <br />Prenatal diagnosis is offered if mutations are identified.<br />
  27. 27. 1st Trimester<br />Known Mutation <br />ARMS (Amplification Refractory Mutation System)<br />Reverse Dot Blot Hybridization<br />Dot Blot Hybridization using ASO probes<br />Direct Electrophorersis for 619bp deletion <br />619bp deletion , IVS1-5(G->C), codons8/9(+G), IVS1-1(G->T),<br />codons 41/42(-TCTT), codons15A(G->A)<br />Unknown Mutation <br />DGGE (Dnaturation Gradient Gel Electrophoresis)<br />Single Strand Confirmational Polymorphism<br />Sequence analysis of Beta Globin Gene<br />Mismatch PCR<br />
  28. 28. 2nd Trimester<br />method of choice where DNA mutations are unidentified in parents<br />Cordocentesis<br />(transabdominal route by USG guide)<br />Globin chain synthesis Ratio in<br /> Cord Blood @ 17 to 23 Weeks Pregnancy<br />Hemoglobin Electrophoresis @ 6 months <br />of Delivery to cross check Diagonosis<br />extract DNA from amniotic fluid @ >15 weeks of gestation <br />chorionic villus samples 10-12 weeks (upto 20 weeks)<br />Fetal DNA analysis<br />
  29. 29.
  30. 30. Pre-Marriage Thalassemia Test is Imperative<br />Over four crore people in India are<br />diagnosed with this form<br />Patients need blood transfusions every three to eight weeks to maintain hemoglobin levels<br />Permanent cures like Bone Marrow Transplantation and stem cell transplants are very expensive and also very risky<br />. <br />
  31. 31. It is thus advised that people getting married should take a simple blood test <br />ensure that both the partners are not carrying the Thalassemia trait.<br /> If found to be diagnosed with Thalassemia, consult your doctor before planning your family together. <br />
  32. 32. THANK YOU<br />
  33. 33.
  34. 34.  DNA Mutation Analysis<br />Once the carrier status of the couple is confirmed<br />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.<br />Reverse dot blot probes are attached to the membrane and DNA hybridizes with dot corresponding to the mutation.<br />Amplifica- tion refractory mutation system (ARMS) technique in which specific primers against normal and mutant sequences are used.<br />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<br />DGGE is based on the resolution of DNA fragments differing by single nucleotide substitution<br /> 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.<br />
  35. 35. Management<br />Transfusion<br />chronic hypertransfusion therapy to maintain a hematocrit of at least 27–30% so that erythropoiesis is suppressed. <br />Splenectomy is required if the annual transfusion requirement (volume of RBCs per kilogram of body weight per year) increases by >50%. <br />Folic acid supplements may be useful. <br />SuperTransfusion<br />vigorous transfusion program <br />pretransfusionhematocrit was kept at ≥35% <br /> aimed at keeping hemoglobin levels above 12.0 g/dL.[68]<br />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<br /> reserved for patients with poor tolerance of lower hemoglobin levels<br />
  36. 36. Complications of Transfusions<br />Excessive iron stores lead to depletion of ascorbic acid and vitamin E<br />Haemosiderosis<br />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<br />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<br />
  37. 37. Experimental Therapies<br /><ul><li>Bone Marrow Transplantation (HLA-compatible donor )</li></ul>(provides stem cells able to express normal Hb, curative in 80–90% of patients, survival into adult life is possible with conventional therapy)<br /><ul><li>Cord Blood Transplantation </li></ul>HLA-identical siblings<br /><ul><li>Gene Therapy</li></ul>(Uptake of gene vectors into the nondividing hematopoietic stem cells. Lentiviral-type vectors that can transducenondividing cells )<br /><ul><li>Reestablishing high levels of HbF</li></ul>( using pulsed hydroxyurea, cytarabine, Butyrates that stimulates proliferation of the primitive HbF-producing progenitor cell population<br />
  38. 38. Differential Diagonosis<br />
  39. 39. 39<br />Differential Diagnosis of Microcytic, Hypochromic Anemias<br />
  40. 40. Differential diagnosis<br />

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