Laboratory Diagonosis thalassemia Chirantan
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The document discusses the laboratory diagnosis of thalassemia, including clinical features, laboratory tests, and diagnostic techniques such as hemoglobin electrophoresis and DNA mutation analysis. It emphasizes the importance of prenatal diagnosis, management strategies like blood transfusions and therapies, and the necessity for carrier screening before marriage. The document also outlines differential diagnoses for microcytic, hypochromic anemias and possible complications associated with chronic transfusions.
![ManagementTransfusionchronic 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 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](https://image.slidesharecdn.com/labdaigonosisthalassemia240-111003041922-phpapp02/85/Laboratory-Diagonosis-thalassemia-Chirantan-35-320.jpg)
Laboratory Diagonosis thalassemia Chirantan
- 1. Laboratory Diagonosis of Thalassemiaby ChirantanMandalModerator: Dr Santosh Kumar MondalAssoc.Professor, Pathology
- 2. Initial Approach to Suspect
- 3. Clinical FeaturesCompensated HaemolyticAnaemiaExtramedullaryHaematopoesis leads to Splenomegalyupto 1500 gm, even HepatomegalyIron Overload causing Hemosiderosis & Secondary Hemochromatosis damage to endocrine organs, Heart etcSerum BilirubinUnconjugatedin 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 CountAlpha HydropsFoetAlphaHydropsFoetalisl<6g/dl HbVery High Reticulocytosisphaalislpha
- 6. Peripheral Blood SmearAnisopoikilocytosisBasophillicStipllingMicrocyticHypochromicTear Drop Cell Target Cell
- 7. HbHIncubation with brilliant cresyl blue stain causes Hemoglobin H to precipitateappearance of multiple discrete inclusions -golf ball appearance of RBCsHeinz bodies that are evenly distributed throughout cell.
- 8. alpha HbHHeinz Bodies inclusions within RBC composed of denatured HemoglobinReticulocyte count(increased erythropoesis)
- 9. Bone Marrow ExamMarkedly increased Iron Depositionerythroid hyperplasia morphologic abnormalities of the erythroblasts
- 10. Test for HemolysisIncreased RBC production Increased RBC destruction ReticulocyteNucleated RBCBM cellularityUC-BilirubinUBG MetheAlbumin albumin complex = albumin+hemeIncreased excretion Through Urine HbUriaHemosiderosisMetHbUria (Fe3+ )
- 11. Hb Electrophoresisdifferentiate among Hb A, Hb A2, and Hb FDetects presence of abnormal HbDiagnosing and differentiating various forms of thalassemiasPrinciple : 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 anodeHbD & HbS and HbA2/C/E/O have same mobilityCitrate Agar Gel Electrophoresis Acidic pHSeparation of HbD & HbS and HbA2/C/E/O from each other
- 14. High Performance Liquid ChromatographyCation Exchange HPLCSeparate Hbs that have identical mobility in Citrate Agar Gel & Cellulose Acetate Agarose Gel Electrophoresis Separation of HbA2 & HbE not PossibleAnion Exchange HPLCPattern of elution obtained here is opposite of Cation Exchange HPLCSeparation of HbA2 & HbE is possible here
- 17. IEF (IsoElectricFocussing)Formation of pH gradient along the gel during passage of current through the separation of carrier ampholites with different pHsSeparation of Hbs whose pI (IsoElectric point) differ by as little as 0.01 pH unitsCan separate those Hbs from each other, which have identical mobility in Electrophoretic system
- 19. Beta Th Major An increased level of Hb F ranging from less than 50-90%Hb A2 normal or highBeta Th Minor HbA2 often elevated > 3%, sometimes reaching 7-8%.Hb F 3%Alpha Trait ThHbA2 either normal or slightly decreasedSmall amount of HbBarts in neonatal period 2 to 5%Alpha ThHbHHbF 10% , HbH 2-4%HydropsFeotalisHb Barts100 %
- 20. HbA2Cellulose Acetate Agarose Gel Electrophoresis , HPLCUseful to confirm Beta TM carrier state HbA2 >3.5% are considered to have thalassemic traitSharp rise in 1st 4 months of lifeSlightly elevated for rest of lifeHbFAlkali Denaturation techniqueAcid 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" cellsHPLCSharp decline in 1st 10 months of life
- 21. 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 mutationGene mapping based on Southern BlottingPCR based proceduresPreNatalDiagonostic Importance
- 22. free erythrocyte protoporphyria (FEP)
- 23. Iron Study(To differentiate thalassemia from IDAThalassemiaSerum Ferritin 200 ng/mL in female300 ng/mL in maleSerum Iron Level Increased , 69-135ug/dLTransferritin Saturation >50%TIBC normal Marrow Iron Store IncreasedIron deficiency anemiaSerum Ferritin <12ng/L Serum Iron Level very LowTransferritin Saturation <10%TIBC Increased Marrow Iron Store very low
- 24. Globin Chain(alpha, beta gamma)Prenatal Diagnostic importanceBy Reverse phase HPLC
- 25. Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT)screening test for carrier statesprinciple : limit of hypotonicity which the red cell can withstand2 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 1osmotic fragility = --------- , S/V ratio S/V ratio => osmotic fragilityThe 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 TrimesterKnown Mutation ARMS (Amplification Refractory Mutation System)Reverse Dot Blot HybridizationDot Blot Hybridization using ASO probesDirect 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 PolymorphismSequence analysis of Beta Globin GeneMismatch PCR
- 28. 2nd Trimestermethod of choice where DNA mutations are unidentified in parentsCordocentesis(transabdominal route by USG guide)Globin chain synthesis Ratio in Cord Blood @ 17 to 23 Weeks PregnancyHemoglobin Electrophoresis @ 6 months of Delivery to cross check Diagonosisextract DNA from amniotic fluid @ >15 weeks of gestation chorionic villus samples 10-12 weeks (upto 20 weeks)Fetal DNA analysis
- 30. Pre-Marriage Thalassemia Test is ImperativeOver four crore people in India arediagnosed with this formPatients need blood transfusions every three to eight weeks to maintain hemoglobin levelsPermanent 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
- 34. DNA Mutation AnalysisOnce the carrier status of the couple is confirmedASO (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 fragmentDGGE 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. ManagementTransfusionchronic 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 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 TransfusionsExcessive iron stores lead to depletion of ascorbic acid and vitamin EHaemosiderosiseach 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 ironfully 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 TherapiesBone 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 siblingsGene 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
- 39. 39Differential Diagnosis of Microcytic, Hypochromic Anemias