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

Background: Approximately 15% of pregnant women in the US are RhD-negative. To prevent alloimmunization, current national guidelines endorse the administration of prophylactic anti-D immune globulin (RhIG) at 28 weeks of gestation and in any other episodes where alloimmunization can occur, such as bleeding, pregnancy loss, trauma or invasive procedures. Alloimmunization only occurs if the fetus is RhD-positive; however, 40% of RhD-negative mothers carry an RhD-negative fetus, resulting in, under the current guidelines, the sometimes repeated, use of unnecessary RhIG. Objective: We aimed to evaluate the performance of a next generation sequencing (NGS) with quantitative counting template (QCT) technology prenatal cell free DNA (cfDNA) assay in detecting the fetal RhD genotype in a diverse RhD-negative pregnant population in the United States (US). Study Design: This retrospective 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. 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. Fetal RhD was detected in 261 cases (65%), whereas it was negative in 140 (35%). 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. Despite the fact that 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 based on cfDNA results. This decrease was greater at certain sites and at later study periods. If the cfDNA results were fully utilized during the entire study period, up to 147 RhIG doses (24% of administered doses) could have been avoided, indicating the importance of guideline changes to support the use of cfDNA for fetal RhD detection to conserve this resource. 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. 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.