Novel proof of concept assay for rapid, simplified non-invasive fetal RhD screening.

The RhD antigen is a key component of the Rh blood group system and is highly immunogenic, posing risks for RhD-negative pregnant women. While anti-D prophylaxis prevents maternal alloimmunization, concerns regarding availability, overuse, and universal effectiveness persist. Recent advances in non-invasive fetal RHD genotyping have shown potential to reduce unnecessary interventions. This study presents a new non-invasive fetal RhD screening method using cell-free DNA (cfDNA) and dry chemistry quantitative polymerase chain reaction (qPCR) with fluorescence measurements of three RHD exons, aimed at enhancing the accuracy and efficiency of RHD genotyping and improving clinical decision-making. We evaluated this new method using cfDNA extracted from plasma samples of 28 RhD-negative women carrying singleton pregnancies. The 20µL cfDNA samples correctly identified all 18 RhD-positive pregnancies and 9/10 RhD-negative pregnancy samples. Our study provides proof of concept that the cfDNA and dry chemistry qPCR method for fetal RHD genotyping is feasible, as it achieved 100% sensitivity. Despite the small sample size, these findings support the use of RHD genotyping as a precise and effective alternative or complement to anti-D prophylaxis, offering improved risk management and care for pregnant women worldwide.