Objective: In this prospective study, we assessed the feasibility of foetal RHD genotyping by analysis of DNA extracted from plasma samples of Rhesus (Rh) D-negative pregnant women using real-time PCR and primers and probes targeted toward exon 7 and 10 of RHD gene. Methods: We analysed 24 RhD-negative pregnant woman and 4 patients with weak D phenotypes at a gestational age ranging from 11th to 38th week of gestation and correlated the results with serological analysis of cord blood after the delivery. Results: Non-invasive prenatal foetal RHD exon 7 genotyping analyses of maternal plasma samples was in complete concordance with the serological analysis of cord blood in all 24 RhD-negative pregnant women delivering 12 RhD-positive and 12 RhD-negative newborns. RHD exon-10-specific PCR amplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhD-positive foetus, despite the positive amplification in RHD exon 7 region observed in all cases. In 1 case red cell serology of cord blood revealed that the mother had D–C–E–c+e+ C^w– and the infant D+C–E–c+e+ C^w+ phenotypes. RhD exon 10 real-time PCR analysis of cord blood was also negative. These findings may reflect that DC^w– paternally inherited haplotype probably possesses no RHD exon 10. In another case no cord blood sample has been available for additional studies. The specificity of both RHD exon 7 and 10 systems approached 100% since no RhD-positive signals were detected in women currently pregnant with RhD-negative foetus (n = 8). Using real-time PCR and DNA isolated from maternal plasma, we easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 4) from truly RhD-negative patients since the threshold cycle (C_T) for RHD exon 10 or 7 amplicons reached nearly the same value like C_T for control β-globin gene amplicons detecting the total DNA present in maternal plasma. However in these cases foetal RhD status cannot be determined.Conclusion:Prediction offoetal RhD status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal foetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD-specific products.

S U M M A R YWe assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.