RHD/CE typing by polymerase chain reaction using sequence-specific primers.

Abstract

BACKGROUND: Current DNA-based Rh system typing strategies may detect the two RH genes and their prevalent alleles, but they are known to fail sometimes, when rare RH alleles (e.g., D category phenotypes) are encountered. It is almost impossible to find a single DNA-based method that can accommodate the great heterogeneity within the human Rh system. STUDY DESIGN AND METHODS: An easy-to-perform DNA-based method for the detection of the two RH genes and their alleles, including variant RHD alleles, was developed. By the use of one RHD/C-, seven RHD-, and four RHCE-specific polymerase chain reactions, all triggered to work at identical thermocycling conditions, the DNA of 77 blood donors carrying weak D and that of 200 random donors with common D phenotype was investigated. In addition, 77 selected samples of ccDee and rare Rh system phenotypes were examined. RESULTS: Among 77 samples of weak D, one Rh33 and six DVI categories were detected, one of which showed new RHD-specific nucleotide patterns. In DFR and CCee samples, novel variant RHD alleles were found. RHD DNA types of 200 random donors were found to be concordant with their D phenotype. For RHE and RHe genotyping, a full correlation with serologic phenotypes was found. Our method for genotyping RHC and RHc failed in some cases, because of an already published RHc allelic variation, which we have called RHc(cyt48). An estimate of the frequency of this RHc(cyt48) allele in a white population was made. CONCLUSION: The presented exon-scanning RHD/CE polymerase chain reaction using sequence-specific primers complements current DNA-based Rh system typing strategies and is superior in the detection of variant RHD alleles.