Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for ß-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with ß-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present.

Analytical evaluation of the Tosoh HLC-723 G7 automated HPLC analyzer for hemoglobin A2 and F determination.

OBJECTIVES: The analytical performance of a new automated HPLC system (Tosoh HLV-723 G7) for Hb A(2) and Hb F quantification in blood was studied. DESIGN AND METHODS: Hb A(2) and Hb F measurements were studied for imprecision, linearity, carry-over, interferences and sample concentration effect. Method comparison study was performed with the Bio-Rad Variant II HPLC system. Hb F results were also compared with those obtained by the alkaline denaturation test. The detection of some common Hb variants was also studied. RESULTS: Hb A(2) within-run and between-run CVs were found between 0.8-2.2% and 2.9-7.2%, respectively, while CVs for Hb F were up to 10.0% in normal and between 1.9-5.3% at more clinically relevant Hb F concentrations (>1.5%). Comparison study with Bio-Rad Variant II for Hb A(2) determination showed good correlation but highlighted calibration differences. The following results were obtained in two different laboratories: y = 1.163x - 0.52, r = 0.9918, n = 144 (Lab A); y = 1.060x - 0.40, r = 0.9920, n = 93 (Lab B). With regard to the determination of Hb F, the measurements performed by the tested method was found to correlate well with the alkaline denaturation test (y = 1.0138x - 0.36, r = 0.9842, n = 20) and with the Variant II HPLC system (y = 0.812x + 0.52, r = 0.9835, n = 110). An excellent linearity (r = 0.999) was found for both Hb A(2) and Hb F in the range 0.8-19%. Hb S, Hb C and Hb D can be presumptively identified by the assigned retention time windows. CONCLUSION: The new analyzer Tosoh HLV-723 G7 was found to be a reliable method for Hb A(2) and Hb F quantification and for the presumptive identification of some common Hb variants.