Naked Eye Single Tube Osmotic Fragility Test
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The effectiveness of one tube osmotic fragility screening in detecting BTT was first introduced by Kattamis C. in 1981.(56) NESTROFT is the rapid simple and cost effective screening test.
The principle of NESTROFT is based on the limit of hypotonicity that the red cell can withstand. There is a pronounced decrease in osmotic fragility of red cells in Î²-thalassemia(57) Cells with a decreased surface/volume ratio, have a limited capacity to expand in low osmolarity solutions and lyse (rupture) at a higher concentration of sodium chloride than do normal biconcave red cells. Therefore, thalassaemic cells that are hypochromic and fLatter have a greater capacity to expand and thus have decreased osmotic fragility. (58)
2.15.2 Clinical Implications
The different saline concentration is used in NESTROFT test to detect spherocytosis and BTT. Positive test is due to reduced osmotic fragility of red cells at 0.36% buffered saline. Manglani M et al in 1997 studied 165 cases (with MCV<80fL) with NESTROFT and found sensitivity 94.4% and specificity 64.2%, NPV 97.6% and PPV 35.3%. Hence they suggested NESTROFT in combination with MCV<80fL had a sensitivity of 100% and they suggested that in areas with limited laboratory facilities and economic resources a single test of NESTROFT would cost as little as Rs.1.50. Hence, it is a sensitive, cost-effective, rapid and reliable screening test for detection of BTT in a population.(43) Mehta BC et al in1991 studied 131 cases and found sensitivity, specificity, PPV and NPV to be 99.2%, 75.8%, 69.3% and 99.5% respectively.(59) Raghavan et al in 1991 found sensitivity, specificity, PPV and NPV to be 95.5%, 87%, 70.5% and 98.3%.(60) Susanna et al in 1996 studied 137 cases and found sensitivity, specificity, PPV and NPV to be 98.7%, 66.6%, 87% and 96.5%.(61) Maheshwari et al in1999 studied 1048 cases and found sensitivity, specificity, PPV and NPV to be 91%, 95%, 55% and 99%.(62)
Recent published data has shown that the NSTROFT can be a very useful screening tool for Î²-thalassemia Trait. (5, 63-66) Different studies show that NESTROFT with 0.36% saline could detect 96-100% of heterozygotes with Î²-thalassemia. Study published in Indian J Pathol Microbiol, 2002 concludes NSTROFT to be 92.5% sensitive and 95.2% specific 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. According to a recent study conducted at PNS Shifa Hospital 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 accurate results compared to the other concentrations tested.(5) Routine use of haematological data from automated cell counters may complement the results of the NSTROFT.(64)
2.16 Supravital Stains
Supravital stains are a group of special stains for demonstration of intracellular inclusions in the living tissues. Common supravital stains used are methylene blue, new methylene blue, brilliant 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 thalassaemia carrier screening reticulocyte count does not have a diagnostic value. However in the detection of Î±-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 count using specific RNA staining.(68)
Reticulocyte numbers and maturation levels have been studied in different haemoglobinopathies and the results have been correlated 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 similar to non-anaemic group (1.18%). A non-responsive bone marrow does not release reticulocytes in sufficient numbers to compensate for the degree anaemia. The authors concluded that the absolute number and immaturity fraction were higher in BTT than normal individuals, but without statistical significance.(69)
2.17 Haemoglobin Electrophoresis
Hemoglobin electrophoresis (also called Hgb electrophoresis), is a test that measures the different types of hemoglobin in the blood. The method used is called electrophoresis, a process that causes movement of particles in an electric field, resulting in formation of "bands" that separate toward one end or the other in the field.
2.17.1 Types of Electrophoresis
SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) is a common 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 1:1 ratio with each segment of the protein to give it a charge. The movement of protein polypeptides through the gel occurs at different rates depending on size.
18.104.22.168 Agarose Gels
Agarose gels electrophoresis is used for separation of RNA and DNA molecules. Like SDS-PAGE, this separates the molecules based on charge and size. DNA molecules are negatively charged, so they move through the gel quickly depending on size. Smaller DNA fragments move more quickly than larger ones due to friction resistance.
Electrofocusing analyze the charge and pH values of proteins. A container is filled with a gel solution that has an increasing 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.
Capillary 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.
22.214.171.124 Native Gels
Native gels are similar to SDS-PAGE, except 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 variant Hbs. Electrophoretic methods have been developed that allow for separation at alkaline pH 8.4 on cellulose acetate 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 acetate electrophoresis may be used for qualitative identification of variants, but also with elution for quantitation of the haemoglobins, A2, A, S, D, Lepore, Î±-chain variants, Hb H and Hb Bart's. Agarose gel electrophoresis is not a satisfactory screening technique because it cannot distinguish 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 various 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 assay 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 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 small sample volumes that perform newborn screening. This electrophoretic method utilizes carrier ampholytes, small proteins that are able to carry 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 better 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 resolution of charge variants. The capillary focusing process is analogous to conventional isoelectric focusing in gels, while the requirement for zone mobilization is unique to the capillary format with on-tube detection. A variety of mobilization methods have been described, and the selection 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 good solubility in aqueous buffers, but may be unsatisfactory for large or hydrophobic proteins.(72)
2.20 Globin chain electrophoresis
It is an ancillary procedure in which haemoglobin lysate with mercaptoethanol and 8mol/L Urea to dissociate 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 Î± and a Î² 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 available for many years. Cation-exchange HPLC is emerging 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 off the column at a particular retention time. This method has a advantage that HbC does not coelute with HbA2, however HbE and HbO-Arab still coelute with HbA2 with this method.(71)
2.22 DNA Analysis
The DNA analysis is gold standard for detection of carrier state of Î²-thalassemia. The 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 sequence 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 most common 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 Diagnosis
The 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 Î±- and Î²-thalassemia was carried out for the first time in the 1970s using globin chains synthesis analysis in fetal 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 sampling, fetal DNA analysis is performed by the PCR-based methods mentioned for carrier detection procedures. In general, the mutation to be detected 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, maternal contamination, and sample exchange). However, the risk of misdiagnosis can be significantly reduced using a number of precautionary measures, such as fetal DNA analysis for selected polymorphic markers.(35)
Fetal cells, known to be present in the maternal circulation, represent an attractive, noninvasive approach to prenatal diagnosis. Fetal cells, immunological isolated for their low purity, can only be used for prenatal diagnosis of Î²-thalassemia in women whose partners carry a different mutation. Recently, this problem has been overcome by development of a technique able to isolate single 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 mother's 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 permit 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 Î²-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 thalassemia major. Effective preventive programs have also been established in countries such as United 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 Î± and /Î² thalassaemia traits. (Figure 2.8)
Figure 2.8. FLow chart for carrier detection and prenatal diagnosis of thalassemia.
Source: Adopted from Maheshwari M, Arora S, Kabra M, Menon PSN. 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 Î± and /Î²
thalassaemia traits, The Thalassaemia Working Party of the BCSH General Haematology Task Force J Clin Pathol 1994;47:289-295
Prevention 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 International Federation, 2003). The countries where prevention programmes are effective resulting in increased survival of thalassemia major patients in comparison to countries where preventive strategies do not exist. (Figure 2.6)
Figure 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, Thalassaemia International Federation, 2003.
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