Determination of Fetal RHD RHD RHD Genotype Including the RHD RHD RHD Pseudogene in Maternal Plasma

摘要

Objective To examine the accuracy of fetal RHD genotype and RHD pseudogene determination in a multiethnical population. Methods Prospective study involving D‐negative pregnant women. Cell‐free DNA was extracted from 1 ml of maternal plasma by an automated system (Mag NA Pure Compact, Roche) and real‐time PCR was performed in triplicate targeting the RHD gene exons 5 and 7. Inconclusive samples underwent RHD pseudogene testing by real‐time PCR analysis employing novel primers and probe. Results A positive result was observed in 128/185 (69.2%) samples and negative in 50 (27.0%). Umbilical cord blood phenotype confirmed all cases with a positive or negative PCR result. Seven (3.8%) cases were found inconclusive (exon 7 amplification only) and RHD pseudogene testing with both conventional and real‐time PCR demonstrated a positive result in five of them, while two samples were also RHD pseudogene negative. Conclusion Real‐time PCR targeting RHD exons 5 and 7 simultaneously in maternal plasma is an accurate method for the diagnosis of fetal D genotype in our population. The RHD pseudogene real‐time PCR assay is feasible and is particularly useful in populations with a high prevalence of this allele.