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

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.