Prenatal non-invasive foetal RHD genotyping: diagnostic accuracy of a test as a guide for appropriate administration of antenatal anti-D immunoprophylaxis

Background. Foetal RHD genotyping can be predicted by real-time polymerase chain reaction (qPCR) using cell-free foetal DNA extracted from maternal plasma. The object of this study was to determine the diagnostic accuracy and feasibility of non-invasive RHD foetal genotyping, using a commercial multiple-exon assay, as a guide to appropriate administration of targeted antenatal immunoprophylaxis. Material and methods. Cell-free foetal DNA was extracted from plasma of RhD-negative women between 11-30 weeks of pregnancy. The foetal RHD genotype was determined non-invasively by qPCR amplification of exons 5, 7 and 10 of the RHD gene using the Free DNA Fetal Kit (R) RhD. Results were compared with serological RhD cord blood typing at birth. The analysis of diagnostic accuracy was restricted to the period (24-28(+6) weeks) during which foetal genotyping is usually performed for targeted antenatal immunoprophylaxis. Results. RHD foetal genotyping was performed on 367 plasma samples (24-28(+6) weeks). Neonatal RhD phenotype results were available for 284 pregnancies. Foetal RHD status was inconclusive in 9/284 (3.2%) samples, including four cases with RhD maternal variants. Two false-positive results were registered. The sensitivity was 100% and the specificity was 97.5% (95% CI: 94.0-100). The diagnostic accuracy was 99.3% (95% CI: 98.3-100), decreasing to 96.1% (95% CI: 93.9-98.4) when the inconclusive results were included. The negative and positive predictive values were 100% (95% CI: 100-100) and 99.0% (95% CI: 97.6-100), respectively. There was one false-negative result in a sample collected at 18 weeks. After inclusion of samples at early gestational age (<23(+6) week), sensitivity and accuracy were 99.6% (95% CI: 98.7-100) and 95.5% (95% CI: 93.3-97.8), respectively. Discussion. This study demonstrates that foetal RHD detection on maternal plasma using a commercial multiple-exon assay is a reliable and accurate tool to predict foetal RhD phenotype. It can be a safe guide for the appropriate administration of targeted prenatal immunoprophylaxis.