Fetal RHD genotyping by analysis of maternal plasma in a mixed population

Abstract

Maternal plasma analysis for the determination of the fetal RHD status is an exciting tool for the management of RhD-negative pregnant women, specially sensitized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi-ethnic population. We analyzed plasma samples from 88 RhD-negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic background. Fourteen patients (16%) had anti-D alloantibody. We used Taqman primers and probes to detect by real-time PCR, exons 4, 5, and 10 of RHD. As internal controls we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective neonates were collected during delivery and hemagglutination was performed. Fifty-eight samples (66%) were genotyped as RHD+, 27 samples (31%) showed complete absence of RHD and 3 samples (3 %) presented a D variant (RHDψ). All the results agreed with the neonatal typing, including the three fetuses with the RHDψ, phenotyped as RhD-negative. Thus, the accuracy of the fetal RHD genotyping in this mixed population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. Our findings indicate that the accuracy of RHD gene using three regions (exons 4, 5, and 10) can be sufficient for clinical application in a multi-ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma for our population.