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Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point
Helene Puehringer1, Hossein Najmabadi, Hai-Yang Law
1ViennaLab Diagnostics GmbH, Vienna, Austria. puehringer@viennalab.co.at
Background:
alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations.
Methods:
We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2).
Results:
Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods.
Conclusions:
The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.
