RhD genotyping by quantitative fluorescent polymerase chain reaction: a new approach

Objective To develop a new method of RhD/d genotype determination using a q uantitative fluorescent PCR (QF-PCR) assay. Methods Polymerase chain reaction amplification (PCR) of fragments of exon 7 of both the RHD and RHCE genes was performed from 32 amniotic fluid and 2 6 chorionic villus samples known to be heterozygous for the RHD gene, 74 pe ripheral blood samples of RhD-positive blood donors (homozygous or heterozy gous) estimated by serologic typing and 24 RhD-negative fetal samples. The number of copies of the RHD gene in RhD-positive samples was determined by comparing the fluorescent intensities of the amplification products specifi c for the RHD and the RHCE genes. Results A ratio of fluorescent intensities of 1:1 clearly indicated D/D hom ozygous individuals whereas a ratio of 1:2 was demonstrated in samples from D/d heterozygous individuals. The mean fluorescent intensity ratio of the peak areas of homozygous samples was 1.12 (SD 0.128), the mean ratio of the peak areas of heterozygous samples was 0.51 (SD 0.060). Complete agreement was obtained between RhD/d typing by QF-PCR and RhD genotypes assessed by family studies and serological methods. Conclusions The fluorescent PCR-based DNA test allows easy, rapid and accur ate determination of the zygosity for the RHD gene. This new technique prov ides useful information for the clinical management of pregnancies of sensi tised RhD-negative mothers.