Non-invasive prenatal rhesus D genotyping using cell-free foetal DNA

BACKGROUND & OBJECTIVES: Non-invasive prenatal diagnosis (NIPD) of rhesus D (RHD) genotype using cell-free foetal DNA is extensively used in many developed countries. Studies on NIPD from India are scarce. The aim of the present study was to evaluate the performance of non-invasive foetal RHD genotyping by targeting exon 10 of the RHD gene using cell-free DNA. METHODS: DNA was extracted from the maternal plasma of alloimmunized and non-alloimmunized women between 7 and 34 wk of gestation. RHD sequence was determined by quantitative real time polymerase chain reaction (PCR). Results were compared with RhD phenotype obtained from cord blood samples of neonates. RESULTS: A total of 135 samples from RhD-negative pregnant women were collected. The foetal RHD status was conclusive in all 135 (100%) cases. The highest number of cases reported for RHD genotyping were from Punjab (38.5%) followed by Haryana (24.4%), Himachal Pradesh (17.0%) and Chandigarh Union Territory (13.3%). The non-invasive test correctly predicted the foetal RhD phenotype in 133 of 135 cases, making the accuracy of the test as 98.51 per cent [95% confidence interval (CI): 97.90-99.50%]. The overall sensitivity and specificity of the test were 99.18 per cent (95% CI: 95.52-99.98%) and 92.31 per cent (95% CI: 63.97-99.81%), respectively, with negative and positive predictive values of 99.80 per cent (95% CI: 94.85-99.87%) and 96.31 per cent (95% CI: 62.87-98.84%), respectively. INTERPRETATION & CONCLUSIONS: Non-invasive foetal RHD determination by single-exon quantitative PCR exhibited high accuracy and could be used in routine clinical practice after confirmatory studies are done.