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

Abstract Objective We 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 Design This 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 assay’s sensitivity, specificity, and accuracy were calculated considering the neonatal RhD serology results. Results A total of 401 non-alloimunized RhD-negative pregnancies were included in the analysis. The D antigen cfDNA result was 100% concordant with the neonatal serology, resulting in 100% sensitivity and positive predictive value and (both 95% CI: 98.6%-100%) 100% specificity and negative predictive value (both 95% CI: 97.4%-100%). There were 10 pregnancies where the cfDNA analysis identified a non- RHD gene deletion, including RhDΨ (n=5) and RHD-CE-D hybrid variants (n=5). A total of 616 doses of RhIG were administered. Even though the study occurred prior to the current RhIG shortage and the recent American College (ACOG) advisory change, there was a marked decrease in the use of antenatal RhIG in pregnancies when cfDNA results indicated an RhD-negative fetus, indicating providers were using the results to guide pregnancy management. Conclusion This cfDNA analysis via NGS for detecting fetal RhD status is highly accurate with no false positive or false negative results in 401 racial and ethnically diverse pregnancies with 100% follow up of all live births. Our data support implementing this assay for the routine management of non-alloimmunized RhD-negative individuals. This approach will result in more efficient and targeted prenatal care with administration of RhIG only when medically indicated.