Multiplexed Analysis of Circulating Cell-Free Fetal Nucleic Acids for Noninvasive Prenatal Diagnostic RHD Testing

The fetuses of RhD-negative pregnant women that are positive for the Rh blood group D-antigen gene (RHD) are at risk for hemolytic disease of the fetus and newborn. Prophylactic anti-D therapy is administered to Rh-negative pregnant women carrying RHD-positive fetuses. However, a substantial number of Rh-negative women carry Rh-negative fetuses, and are therefore subjected to unnecessary injections of anti-D therapy. Noninvasive molecular tests that have been shown to accurately identify RHD-positive fetuses through detection of fetal RHD sequences in serum DNA are already being used routinely in several European countries. Use of these tests would restrict administration of prophylactic anti-D therapy to women with RHD-positive fetuses.The aim of this study was to evaluate the efficacy of a novel noninvasive multiplex assay to detect fetal RHD loci in the maternal plasma of pregnant women negative for RhD. This genotyping assay used the MassARRAY system to detect RHD exons 4, 5, 7, and 10, and RHD (pseudogene) of the RHD gene along with a Y chromosome-specific marker. A total of 150 plasma samples from RHD-negative pregnant women were analyzed with the multiplex assay for fetal RHD genotype. All 150 samples had been previously characterized for fetal RHD status using a real-time polymerase chain reaction amplification control.The assay correctly identified fetal RHD status in 98.7% (148/150) of the samples based on comparison with the real-time polymerase chain reaction control results. Overall, 86 (57.3%) and 62 (41.3%) of the samples were correctly classified as positive and negative, respectively.These findings show that the multiplex assay using the MassARRAY system has potential as a noninvasive prenatal diagnostic test for RHD typing.