Genotyping of 19 red cell antigens, including RHD, using liquid bead arrays

Traditionally, the determination of blood group has been performed using serological techniques. Due to the drawbacks of using antisera, molecular typing of blood groups has been introduced. However, the commonly used genotyping assays in blood banks have limitations because the panels for these were based on genetic variations in Caucasians.

We developed a multiplex human erythrocyte antigen genotyping assay using allele-specific primer extensions and hybridization with bead microarrays. Single nucleotide polymorphisms (SNP) were genotyped for the 18 red cell antigens and wild-type RHD, DEL, and complete deletion were examined for RHD. The cut-off of median fluorescence intensity (MFI) for each allele was optimized. In the evaluation stage, the results of 87 samples were compared with those from other genotyping methods, and 26 serologic results were also compared.

The cut-off values were determined using -1 and -2 standard deviation (SD) of the minimum adjusted MFI for SNP detection. Complete deletion was determined with raw MFI+2SD. In comparison with the other methods, the kappa values were 0.984 overall. Compared with serologic methods, our assay showed discrepancy in 2 S/s, 3 C/c, and 3 Di^a/Di^b antigens.

Our method reliably predicts the presence or the absence of the 19 antigens.