High-throughput red blood cell antigen genotyping using a nanofluidic real-time polymerase chain reaction platform

Abstract

Background: Serologic testing of donors to obtain antigen-negative blood for transfusion is limited by availability and quality of reagents. Where sequence variant information is available, molecular typing platforms can be used to determine the presence of a variant allele and offer a high-throughput format correlated to the blood group antigen. We have investigated a flexible high-throughput platform to screen blood donors for antigen genotypes in the African American population.

Study design and methods: Genomic DNA from 427 African American donors was analyzed for single-nucleotide polymorphisms responsible for red blood cell (RBC) antigens E/e, Fy(a)/Fy(b), Fy gene promoter, Jk(a)/Jk(b), Lu(a)/Lu(b), K/k, Js(a)/Js(b), Do(a)/Do(b), Jo(a), and Hy using primer/probe sets (Taqman, Applied Biosystems) on a high-throughput genotyping platform (OpenArray, BioTrove). Where available, the phenotype obtained by serologic testing was compared to genotype data.

Results: Serologic antigen types were available for 2037 of the 4270 genotypes generated. There were five discordant results. Three resolved with repeat serologic typing, one resolved after repeat genotyping, and one discordance was clarified by confirmation of the BioTrove genotype by Sanger sequencing. Triplicate determinations were made for each sample genotype and the results were identical more than 99% of the time.

Conclusions: The nanofluidic genotyping platform described here provides an accurate method for predicting blood group phenotypes. The user-specified array layout provides flexibility of target selection and number of replicate determinations and is suitable for screening antigen types.