The applicability of different PCR-based methods for fetal RHD and K1 genotyping: a prospective study

The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.