Cell-free DNA analysis for the determination of fetal red blood cell antigen genotype in alloimmunized pregnancies

Abstract Objective The objective of the study is to evaluate the accuracy of NGS based quantitative cfDNA analysis for fetal antigen genotyping in alloimmunized pregnancies undergoing clinical testing in practices across the US as early as ten weeks gestational age. Timely identification of the fetal red blood cell antigen genotype for the antigen to which the pregnant person is alloimmunized is vital for determining risk for hemolytic disease of the fetus or newborn (HDFN) and guiding management. The currently recommended approach to assessing risk for HDFN in the US relies on determining the antigen status of the reproductive partner and/or amniocentesis–an approach with limitations such as low uptake and the potential for nonpaternity, which could be alleviated by the utilization of cfDNA testing to determine fetal antigen status. Methods Patients with alloimmunized pregnancies undergoing clinical fetal antigen cfDNA analysis were recruited to the study along with the neonates resulting from the pregnancies via outreach from the laboratory. The laboratory issued the results prospectively as a part of clinical care. After delivery, neonatal buccal swabs were sent to an outside, independent laboratory for antigen genotyping. The outside laboratory was blinded to the fetal cfDNA results, and the results were compared. Concordance was reported for the fetal antigen cfDNA analysis for antigens to which the pregnant person was alloimmunized as well as for all antigens for which the pregnant person was genotype negative. Results A total of 156 participants from 120 clinics who received clinically ordered cfDNA fetal antigen testing provided neonatal buccal swabs for genotyping following delivery. Concordance between cfDNA analysis results and neonatal genotype was determined for 465 antigen calls for the following antigens K1 (n=143), E (124), C (60), Fy a (50), c (47), and D(RhD) (41). These 465 calls included 145 where the fetus was antigen positive and 320 where the fetus was antigen negative. We observed complete concordance between prenatal fetal antigen cfDNA analysis results and neonatal genotypes for the 465 calls, resulting in 100% sensitivity, specificity, and accuracy across a racially and ethnically diverse cohort. Conclusion This study demonstrates that cfDNA analysis for determining fetal antigen genotype is more accurate than real life application of the current recommendations, i.e., partner testing and amniocentesis, in a diverse US population. In addition, this noninvasive approach reduces barriers to obtaining timely and accurate information about fetal antigen genotype. Taken together with previously published evidence, this study supports the implementation of cfDNA testing to manage alloimmunized pregnancies as a clinically useful and cost effective approach.