Naked Eye Single Tube Osmotic Fragility Test

Naked Eye Single Tube osmotic Fragility TestThe effectiveness of one tube osmotic fragility viewing in notice BTT was root introduced by Kattamis C. in 1981.(56) NESTROFT is the rapid simple and cost effective screening test.2.15.1 PrincipleThe tenet of NESTROFT is based on the limit of hypotonicity that the red cell finish withstand. There is a sound out decrease in osmotic fragility of red cells in -thalassemia(57) Cells with a decreased surface/ gaudiness ratio, bedevil a limited capacity to expand in low osmolarity solutions and lyse (rupture) at a higher(prenominal) concentration of sodium chloride than do average biconcave red cells. Therefore, thalassaemic cells that atomic number 18 hypochromic and fLatter collapse a greater capacity to expand and thus give up decreased osmotic fragility. (58)2.15.2 Clinical ImplicationsThe different saline solution concentration is used in NESTROFT test to detect spherocytosis and BTT. Positive test is due to reduced osmotic fragi lity of red cells at 0.36% buffered saline. Manglani M et al in 1997 studied 165 cases (with MCVRecent produce data has appearancen that the NSTROFT can be a very useful screening tool for -thalassemia Trait. (5, 63-66) Different studies show that NESTROFT with 0.36% saline could detect 96-100% of heterozygotes with -thalassemia. Study published in Indian J Pathol Microbiol, 2002 concludes NSTROFT to be 92.5% sensitive and 95.2% particular(prenominal) for screening of red cell microcytosis.(67) The test proves to be simple, cheap, easy to perform and adaptable for mass screening coming close to an ideal screening test. harmonize to a recent study conducted at PNS Shifa infirmary Karachi, NESTROFT has a Positive Predictive Value of 85.38% and Negative Predictive Value of 97.66%, this correlates to international published data. The diagnostic accuracy was 94.6 % (63) NESTROFT done with 0.36 % buffered saline solution provides more(prenominal) holy results comp ard to the other concentrations tested.(5) Routine use of hematologic data from automated cell counters may complement the results of the NSTROFT.(64)2.16 Supravital StainsSupravital stains are a group of special stains for display of intracellular inclusions in the living tissues. Common supravital stains used are methylene vipers bugloss, new methylene blue, vivid cresyl blue (BCB), methyl violet, crystal violet and azure B.Supravital stains in thalassemia are done for the demonstration of reticulocytes and Hb H inclusions as and when indicated. In thalassemia flattop screening reticulocyte count does not have a diagnostic value. However in the detection of -thalassaemia, especially Hb H disease, the brilliant cresyl blue stain will detect the characteristic Hb H inclusion bodies. Supravital stains (brilliant cresyl blue or new-methylene blue) are able to stain residual mRNA in immature red blood cells. There are now several automated electronic cell counters able to perform a reticulocyte c ount development specific RNA staining.(68)Reticulocyte numbers and maturation levels have been studied in different haemoglobinopathies and the results have been match with the degree of ineffective erythropoiesis. Laura C. et al in 2003 studied 219 samples from patients with Sickle Beta-thalassemia (n=7), HbSC disease (n=11), BTT (n=33) and IDA (n=47) and non-anaemic individuals(n=60). They found patients with HbS trait (0.83%), IDA (1.18%) and BTT (1.53%) showed Reticulocyte parameters corresponding to non-anaemic group (1.18%). A non-responsive bone marrow does not release reticulocytes in sufficient numbers to cut through for the degree anaemia. The authors concluded that the absolute number and immaturity fraction were higher in BTT than normal individuals, merely without statistical significance.(69)2.17 Haemoglobin ElectrophoresisHemoglobin dielectrolysis (also called Hgb electrophoresis), is a test that measures the different types of hemoglobin in the blood. The ru le used is called electrophoresis, a process that causes movement of particles in an electric field, resulting in formation of bands that spot toward one end or the other in the field.2.17.1 Types of Electrophoresis2.17.1.1 SDS-PAGESDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a mutual type of electrophoresis used for analyzing proteins, which separates proteins according to their size. The SDS is a protein denaturing detergent that causes unfolding of protein molecule. The detergent binds to the polypeptide in a 11 ratio with each segment of the protein to give it a charge. The movement of protein polypeptides through the colloidal colloidal gel occurs at different rates depending on size.2.17.1.2 Agarose GelsAgarose gels electrophoresis is used for separation of RNA and desoxyribonucleic acid molecules. the like SDS-PAGE, this separates the molecules based on charge and size. DNA molecules are negatively charged, so they move through the gel quickly d epending on size. Smaller DNA break ups move more quickly than larger ones due to clangor resistance.2.17.1.3 ElectrofocusingElectrofocusing analyze the charge and pH values of proteins. A container is change with a gel solution that has an increase pH gradient. The amino acids that form polypeptides have different acidic or basic charges. The protein travels through the gel, obtaining or losing protons depending on its charge. As the protein particle moves through the gel, it eventually becomes neutral and gets stuck in an isoelectric position.2.17.1.4 CapillaryCapillary electrophoresis is a method similar to SDS-PAGE. It separates molecules based on their charge and mass. Molecules are placed in rows called capillaries filled with conductive, electrolyte fLuid. The analytes move in a speed relative to their charge and mass. This method is an older technique introduced in the 1960s. SDS-PAGE is usually preferred in labs.2.17.1.5 Native GelsNative gels are similar to SDS-PAGE, ex cept the detergent (SDS) is not used to denature proteins. Native gels are only able to separate proteins up to 2,000 kDa in size. Because the proteins are left folded, the dyes used are also different than SDS-PAGE.Hemoglobin electrophoresis is the method for identification and quantification of strain Hbs. Electrophoretic methods have been developed that allow for separation at alkaline pH 8.4 on cellulose ethanoate and at acidic pH 6.2 on agarose gels. These provide a clear background, allowing for quantification of the Hb present by densitometric scanning.(47) Cellulose ethanoate electrophoresis may be used for qualitative identification of variants, besides also with elution for quantitation of the haemoglobins, A2, A, S, D, Lepore, - string variants, Hb H and Hb Barts. Agarose gel electrophoresis is not a satisfactory screening technique because it cannot get laid many abnormal haemoglobins from Hb A. However it can separate the C group into three fractions HbC, O-Arab, and Hb E plus HbA2. The method can also distinguish Hb S from Hb D, Hb F from Hb A, Hbs Little Rock, Rainier and Bethesda from Hb A, and Hb H from Hb I. (68) The diagnosis of BTT relies on an accurate estimation of HbA2 levels.(69) Raised HbA2 level (3.5%) is the gold standard for the diagnosis of BTT. Subjects found to be positive in preliminary screening tests by Red cell indices, DFs and NESTROFT are confirmed for thalassemic carrier status by unhomogeneous methods such as cellulose acetate electrophoresis, microcolumn chromatography, capillary isoelectrofocussing and HPLC (high performance liquid chromatography). Subjects with HbA2 levels of 3.5% and above are considered to have BTT. However precautions have to be taken when HbA2 levels fall between 3.3 and 3.7%. In such cases it is recommended to repeat the tick to rule out technical error or treat the patient for IDA before the analysis is repeated.(60) According to the Thalassemia working party of BCSH General haematology Task force both(prenominal) electrophoresis and elution from cellulose acetate or microcolumn chromatography are recommended. They suggested that precision and accuracy of automated scanning densitometry was inadequate for HbA2 estimation.(70)2.18 Isoelectric focusing (IEF)IEF is another popular method used by laboratories that have a large number of specimens or very niggling sample volumes that perform newborn screening. This electrophoretic method utilizes carrier ampholytes, small proteins that are able to guard both current and pH (Zwitterions). When the current is applied to the support medium, the ampholytes will gradually establish a pH gradient throughout the gel (for example, a pH range of 6 to 8 for Hb analysis). IEF gives develop separation of Hb variants that show similar mobilities on alkaline electrophoresis, which are much sharper. Hb variants such as Hb-Malmo, show separation from HbA which is not seen on alkaline electrophoresis. Minor bands such as HbH, Hb-Barts and Delta chain variants are easily seen.(71)Figure 2.6 Examples of many hemoglobin variants and their migration patterns on Isoelectric focusing.2.19 Capillary isoelectric focusing (CIF)CIF is a useful analytical technique for characterization of protein mixtures and determination of protein isoelectric points. It is particularly useful in separation of protein glycoforms, characterizing protein microheterogeneity, and issue of charge variants. The capillary focusing process is analogous to conventional isoelectric focusing in gels, while the prerequisite for zone mobilization is unique to the capillary format with on-tube detection. A variety of mobilization methods have been described, and the extract of the mobilization method for a particular application depends on the capillary type, the instrument configuration, and the type of proteins to be analyzed. Capillary IEF is generally successful for proteins with a molecular weight up to about 150,000 that exhibit erect solubil ity in aqueous buffers, but may be unsatisfactory for large or hydrophobic proteins.(72)2.20 Globin chain electrophoresisIt is an ancillary subroutine in which haemoglobin lysate with mercaptoethanol and 8mol/L Urea to disjoint the globin chain is used. It is run both at alkaline and acid pH. It gives additional information on haemoglobin variants that have similar mobilities by other methods.(71)Globin chain electrophoresis is run at both alkaline and acid pH because some hemoglobin variants show slight differences in mobility at the two pHs. This method often gives additional information on hemoglobin variants that have similar mobilities by other methods. In confusing cases, this method may be useful to document the presence of both an and a chain variant Examples of different hemoglobin variants on globin chain electrophoresis are shown in Figure 2.6. (www.cap.org/apps/docs/cap_press/hemoglobinatlas_intro.pdf)Figure 2.7.Examples of hemoglobin variants on both acid (pH 6.2) and alkaline (pH 8.9) globin chain electrophoresis.Source Adopted from hemoglobin atlas. (www.cap.org/apps/docs/cap_press/hemoglobinatlas_intro.pdf)2.21 High-Performance Liquid Chromatography (HPLC)HPLC is a method that has been acquirable for many years. Cation-exchange HPLC is appear as the method of choice for the initial screening of Hb variants.(56) Run lengths have been shortened from more than 20mins to 6 to 7mins. These instruments are approved by U. S. Food and Drug Administration (FDA) for the measurements of HbS, A2 and F. These instruments generally utilize a weak cation exchange column. Gradually increasing the ionic strength of the eluting solution causes the Hb protein to come finish up the column at a particular retention time. This method has a advantage that HbC does not coelute with HbA2, heretofore HbE and HbO-Arab still coelute with HbA2 with this method.(71)2.22 DNA AnalysisThe DNA analysis is gold standard for detection of carrier area of -thalassemia. Th e prenatal diagnosis of affected couple should be carried out to prevent the birth of thalassemic child by selective abortion of affected foetuses.It is essential to characterize the DNA mutations of the parents for prenatal diagnosis of affected couple. The methods available to study DNA mutations are allele specific oligonucleotide (ASO) screening, (73) reverse dot blot, and restriction endonuclease allele recognition.(74) The ASO method is for detection point mutations, nucleotide insertion or deletion in genomic DNA. In this method ASO probes of 18-20 per order are used. DNA is denatured and dot blotted on to a nylon membrane and then hybridized to different probes. In reverse dot blot probes are attached to the membrane and DNA hybridizes with dot corresponding to the mutation. A recent method is amplification refractory mutation system (ARMS) technique in which specific primers against normal and mutant sequences are used.(60)More than 150 mutations causing beta-thalassemia have been reported from different parts of the world.(74) Studies conducted in Pakistan show the five some mutual mutations are IVS1-5 (G-C), IVS1-1 (G-T), Fr 41-42 (-TTCT) Fr 8-9 (+G) and deletion 619 bp.(75) Ahmed et al found that there are important ethnic and regional differences in the prevalence of mutations. The five most common mutations, IVSI-5 (G-C) (37.3%), Fr 8-9 (+G) (25.9%), del 619 (7.0%), Fr 41-42 (-TTCT) (6.7%) and IVSI-1 (G-T) (5.4%), constitute 82.3% of the total. Fr 8-9 (+G) is the most common mutation in Northern Pakistan (41.3%), whereas IVSI-5 (G-C) is the most frequent mutation in Southern Pakistan (52.2%). (76)2.23 Prenatal DiagnosisThe availability of prenatal diagnosis added a new option to couples at risk for major haemoglobinopathy, leading to a significant change in the effectiveness of screening and counseling in hemoglobinopathy prevention. Prenatal diagnosis of both - and -thalassemia was carried out for the first time in the 1970s using globin ch ains synthesis analysis in foetal blood, obtained by fetoscopy or placental aspiration around the nineteenth week of gestation. The advent of DNA analysis and the introduction of chorionic villi sampling resulted in a notable improvement in prenatal diagnosis because it could be performed generally at 10 to 12 weeks of gestation. Fetal DNA can be obtained also from aminocytes at 15 to 17 weeks of pregnancy. The reported risk of fetal loss with this procedure ranges from 0.5 to 4.5%. after(prenominal) sampling, fetal DNA analysis is performed by the PCR-based methods mentioned for carrier detection procedures. In general, the mutation to be detect in the fetus is first identified in the parents. The results of DNA analysis are very accurate, but misdiagnosis may occur for several reasons (failure to amplify the target DNA fragment, mispaternity, parental contamination, and sample exchange). However, the risk of misdiagnosis can be significantly reduced using a number of precaution ary measures, such as fetal DNA analysis for selected polymorphic markers.(35)Fetal cells, known to be present in the maternal circulation, comprise an attractive, noninvasive approach to prenatal diagnosis. Fetal cells, immunological isolated for their low purity, can only be used for prenatal diagnosis of -thalassemia in women whose partners carry a different mutation. Recently, this problem has been overcome by development of a technique able to isolate hit fetal erythroblasts from maternal blood by microscopic micromanipulation, making possible the analysis of both fetal genes in a single cell. However, this procedure is associated with several technical and biological problems and it is not widely applicable.(35) The discovery of free fetal DNA in maternal plasma provided the basis for developing another method for noninvasive prenatal diagnosis. However, because free maternal DNA is also present, the application to prenatal diagnosis of thalassemias would be possible only to exclude paternally derived pathologic alleles different from the mothers mutation.(35)The advent of DNA amplification has made it possible to define the geneotype of a single cell biopsied from cleaving embryos (preimplantation diagnosis) and to analyze the polar body obtained during the maturation of the oocyte (preconceptional diagnosis). These procedures avoid the need to terminate affected pregnancies and leave the transfer of only healthy embryos established from in vitro fertilization. Successful experiences in many couples with this approach have been reported in hemoglobinopathies. However, preimplantation genetic diagnosis is a technically challenging, intensive procedure, which requires the close collaboration of a team of specialists. (35)To date, programmes for -thalassemia prevention based on carrier screening, genetic counseling, and prenatal diagnosis are on-going in several areas at risk in Mediterranean countries, with a marked decline in the incidence of thalasse mia major. Effective preventive programs have also been established in countries such as get together Kingdom, where thalassemia is a rare disorder that affects diverse minority ethnic groups.Special attention should be given in these programmes to the different religious and social issues and to the different attitude towards prenatal diagnosis of the various ethnic minorities. In case the mutations are not identified linkage studies using restriction fragment length polymorphisms (RFLP) or globin chain synthesis by cord blood sampling are the other options used for prenatal diagnosis. (60)In 1999, Maheshwari M and colleagues suggested fLow chart for carrier detection and prenatal diagnosis of thalassemia. (Figure 2.7) In 1994 the thalassemia working party of British Society of Hematology suggested guidelines for investigation of the and / thalassaemia traits. (Figure 2.8)Figure 2.8. FLow chart for carrier detection and prenatal diagnosis of thalassemia.Source Adopted from Mahesh wari M, Arora S, Kabra M, Menon PSN. mail carrier screening and prenatal Diagnosis of Beta-thalassemia. Indian Pediatr 1999 36 1119-1125. Figure 2.9. FLow chart for thalassemia carrier detection suspected on red cell indices Source adopted from Guidelines for investigation of the and /thalassaemia traits, The thalassaemia Working Party of the BCSH General Haematology Task Force J Clin Pathol 199447289-295Prevention is better than cure. It is important to develop prevention programmes for thalassemia prevention where there is high frequency, to avoid fatalities from untreated thalassaemia cases, the expense and difficulty of providing optimum treatment for patients which creates a burden on patients, families and national health services.thalassaemia patients may be left untreated (indeed, they often die without a diagnosis) or grossly under-treated. At the same time, quality of treatment is firmly linked to both survival rates and quality of life. (Thalassemia external Federation, 2003). The countries where prevention programmes are effective resulting in increased survival of thalassemia major patients in likeness to countries where preventive strategies do not exist. (Figure 2.6)Graph AGraph BFigure 2.10. Graph A Age distribution of thalassemics in a country without prevention Patients are mostly infants (non-prevention) and children (early deaths) Graph B Age distribution of thalassemics in a country with full prevention treatment. There is gap in early years with patients mostly in their mid-twenties. Source Adopted from Prevention of thalassemia other hemoglobinopathies, thalassemia International Federation, 2003.