In this prospective study, we assessed the feasibility of fetal RHD genotyping by analysis of DNA extracted from maternal plasma samples collected from RhD negative pregnant women using RQ-PCR and primers and probes targeted toward exon 7 and exon 10 of RHD gene.We analysed 46 samples from 39 RhD negative pregnant women including 5 sensitised (1x anti-D, 2x anti-D and anti-C, 2x non-specific antibodies in enzymatic assay) and correlated the results with serological analysis of cord blood after the delivery. Non-invasive prenatal fetal RHD exon 7 genotyping analysis of maternal plasma samples was in complete concordance with the analysis of cord blood in all 20 RhD negative pregnant women delivering 12RhDpositive and8RhDnegativenewbornsuntilnow.RHDexon 10 specificPCRamplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhDpositive fetus, despite the positive amplification in RHD exon 7 region observed in all cases. We supposed that false negative results might be caused by a partly deletion or aberrant structure of the 3´ untranslated exon 10 region of RHD gene where specific primers and probe should anneal. The specificity of both RHD exon 7 and exon 10 systems approached 100% since no RHD positive signals were detected in women currently pregnant with RhD negative fetus (n = 8). Prediction of fetal Rhesus D status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal fetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD specific products. Fetal RHD genotyping may allow the identification of fetuses at risk of haemolytic disease of newborn. However,we suggest to apply a non-invasive prenatal RHD genotyping assay in the routine testing of allRhDnegativewomen involving both sensitised as well as nonsensitized pregnancies, which is the majority.

        Key words: fetal DNA, haemolytic disease of newborn, real-time PCR, maternal plasma, RHD gene