| In this assay, we established and evaluated two kinds of thalassemia diagnosis methods. One method is to diagnosis thalassemia with capollary electrophoresis technology. The other method is to genotype the five most common mutations of beta thalassemia in Chinses with primer-extension in combination with denaturing high-performance liquid chromatography techology. Here are the two motheds:To establishe and evaluate methods based on capillary electrophoresis used in thalassemia diagnosisObjective To evaluate the significance of capillary isoelectric focusing (CIEF) for the estimation of blood hemoglobin A2 (Hb A2) concentrations in routine thalassemia screening. Methods For this study, we collected 105 samples from healthy adults and 93 samples with positive phenotypes by routine thalassemia screening. CIEF was compared with Helena Spife combo Electrophoresis System for Hb A2 measurement and its precision and reproducibility was tested by analyzing intra-assay or inter-assay coefficient of variations(CPs'). The reliability and veracity of Hb A2 measurement by CIEF for the detection of a- and p-thalassemia including Hb E was evaluated by genotyping of 93 consecutive samples for routine thalassemia screening. Results By use of CIEF for measurement of Hb A2, reference ranges were obtained to be 3.59%-5.23% for a local healthy adult population. The results of CIEF showed good linearity relation to that of conventional Hb electrophoresis assay. All thalassemia (43 of a-thaLs and 44 of p-thaLs) or HbE (6 cases) carriers presumptively identified by the present CIEF for the quantification of Hb Aj, combined by routine RBC parameters for indicating microcytosis and hypochromia were confirmed to be the heterozygous or compound heterozygous defects of a- or p-globin gene by molecular diagnosis, without any false positive or false negative results. Conclusion The measurement of Hb A2 by CIEF method is rapid, precise and reproducible and could be as a useful tool in routine screening for a- or P-thalassemia.Key wordscapillary isoelectric focusing (CIEF); thalassemia; hemoglobin A.2To establishe and evaluate methods based on denaturing high-performance liquid chromatography used in thalassemia diagnosisObejective p-thalassemia, one of the most-common single-gene inherited diseases in South China, results from one or more of a total of more than 29 different Chinese mutations in the human p-globin gene (HBB). We have developed a primer-extension in combination with denaturing high-performance liquid chromatography (PE/DHPLC)-based assay for molecular diagnosis of the five most common p-thalassemia mutations in Chinese. Methods In this assay, the human p-globin gene fragment was amplified by PCR followed by detection of a multiple PE reaction specific for each five mutations with the purified PCR product as template. Then the five PE product mixtures were separated for genotyping of P-globin gene mutations on the Transgenomic Wave DNA fragment analysis system using fully-denaturing DHPLC analysis. Results In a blind study, On one hand, 120 unrelated samples with the five most common p-thalassaemia mutation in Chinese or a normal P-globin gene sequence that had been previously identified by RDB assay or direct sequencing were recruited to test the specificity. On the other hand, prenatal diagnosis was performed on thirty-six at-risk families for P-thalassemia major. Reverse dot blot (RDB) analysis was used to validate each result. Results show an accuracy rate of 100% for PE-DHPLC in total of 120 unrelated samples and 108 prenatal diagnosis samples tested. Overall, by PE-DHPLC analysis, we were able to unambiguously identify the genotypes involving these five common P-thalassemia mutations and normal alleles corresponding to 94.4% (102/108)of our prenatal testing samples and actually make final decision for prenatal diagnosis covering 97.2% (35/36) of all 36 at-risk families including those results by prenatal exclusion of p-thalassaemia major in three families. Conclusion The PE/DHPLC pro...
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