laboratory diagonosis thalassemia chirantan
DESCRIPTION
22september 2011, GLT medical College Kolkata88, India..............under Pathology Dept!!!!!!!!1Dr.S.K.Mandal.........chief speakers aVIK bASU, Chirantan Mnadal, Urmimala Bhattacharya, Soumaditya Banerjee!!!!!!!!!TRANSCRIPT
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
39
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