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
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
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.
alpha HbH Heinz Bodies inclusions within RBC composed of denatured Hemoglobin Reticulocyte count (increased erythropoesis)
Bone Marrow Exam Markedly increased Iron Deposition erythroid hyperplasia morphologic abnormalities of the erythroblasts
Test for Hemolysis Increased RBC production Increased RBC destruction Reticulocyte Nucleated RBC BM cellularity UC-Bilirubin UBG MetheAlbumin albumin complex = albumin+heme Increased excretion Through Urine HbUria Hemosiderosis MetHbUria (Fe3+ )
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
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
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
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 %
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 deletion Majority of betaTM results from single nucleotide substitution / frameshift mutation Gene mapping based on Southern Blotting PCR based procedures PreNatalDiagonostic Importance
Iron Study(To differentiate thalassemia from IDA Thalassemia 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
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 1 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
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 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
2nd Trimester method of choice where DNA mutations are unidentified in parents Cordocentesis (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
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 .
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.
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.
Management Transfusion 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. SuperTransfusion vigorous transfusion program pretransfusionhematocrit was kept at ≥35% aimed at keeping hemoglobin levels above 12.0 g/dL. 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 Excessive iron stores lead to depletion of ascorbic acid and vitamin E 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