Clinical performance of cell free DNA for fetal RhD detection in RhD-negative pregnant individuals from the US population.

ObjectiveWe aimed to evaluate the performance of a cell free DNA (cfDNA) assay that uses next generation sequencing (NGS) with quantitative counting templates (QCT) for the clinical detection of the fetal RHD genotype in a diverse RhD-negative pregnant population in the United States (US). Study DesignThis retrospective cohort study was conducted in four US healthcare centers. The same NGS QCT cfDNA fetal RhD assay was offered to non-alloimmunized, RhD-negative pregnant individuals as part of clinical care. Rh immune globulin (RhIG) was administered at the discretion of the provider. The assays sensitivity, specificity, and accuracy were calculated considering the neonatal RhD serology results. ResultsA total of 401 non-alloimunized RhD-negative pregnancies who received clinical care in the period from August 2020 to November 2023 were included in the analysis. The D antigen cfDNA result was 100% concordant with the neonatal serology, resulting in 100% sensitivity, 100% positive predictive value (both 95% CI: 98.6%-100%), 100% specificity, and 100% negative predictive value (both 95% CI: 97.4%-100%). There were 10 pregnancies where the cfDNA analysis identified a non-RHD gene deletion, including RhD{Psi} (n=5) and RHD-CE-D hybrid variants (n=5). RhIG was administered to 93% of pregnant individuals with cfDNA results indicating an RhD-positive fetus compared to 75% of pregnant individuals with cfDNA results indicating an RhD-negative fetus, signifying providers were using the results to guide pregnancy management. ConclusionThis cfDNA analysis via NGS for detecting fetal RhD status is highly accurate with no false-positive or false-negative results in 401 racially and ethnically diverse pregnancies with 100% follow up of all live births. This study and prior studies of this assay support a recommendation to offer cfDNA screening for fetal Rh status as an alternative option to prophylactic RhIG for all non-alloimmunized RhD-negative individuals, which will result in more efficient and targeted prenatal care with administration of RhIG only when medically indicated.