Extended blood grouping of blood donors with automatable PCR-ELISA genotyping

BACKGROUND: In the past 10 years, PCR-based methods have been described to allow the detection of gene polymorphisms responsible for many blood group antigens. These methods are routinely used to test samples of fetal origin and to resolve serologic discrepancies. Another interesting application of blood group genotyping could be the extended typing of blood donors for minor antigens to facilitate the procurement of compatible blood for alloimmunized patients. STUDY DESIGN AND METHODS: PCR-based tests have been modified to allow multiplex amplification of specific fragments of blood group genes and the convenient detection of hybridized amplicons by ELISA in a microplate format. RESULTS: The results obtained show that fragments of the Rh (D, c, C, e, E), Kell (K, k), Duffy (Fya, Fyb), and Kidd (Jka, Jkb) genes could be amplified along with controls in multiplex PCR reactions. Labeling of amplicons with digoxigenin allowed their solid-phase detection in microplate wells previously coated with individual blood group-specific oligonucleotides. A comparative study performed with 100 individuals showed a 99.7 percent concordance between genotypes and phenotypes for the 11 antigens assayed, with only three discrepant Fyb genotypes. CONCLUSION: Extended genotyping could be performed once on regular donors and confirmed when needed by standard serologic RBC assays. The format of these tests will allow easy automation of the procedure including the interpretation and downloading of the results with existing ELISA software.