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

Abstract Objective: To evaluate the accuracy of NGS based quantitative cfDNA analysis for fetal antigen genotyping in alloimmunized pregnancies undergoing clinical testing across the US practices. Timely identification of the fetal red blood cell antigen genotype for the antigen to which the pregnant person is alloimmunized is vital for determining fetal risk for HDFN and guiding management. Presently in the US, recommended care is to determine fetal antigen genotype with reproductive partner testing and/or amniocentesis. This approach has many limitations, including availability of reproductive partner testing, risk of nonpaternity, and low uptake of invasive testing such as amniocentesis. These barriers to obtaining fetal antigen genotype information lead to pregnancies not at risk for HDFN undergoing burdensome monitoring and, in some cases, unnecessary intervention. PCR based qualitative cfDNA analysis for fetal antigen genotyping is available in Europe however it is offered at later gestational ages, may require a repeat sample, has a higher frequency of inconclusive results for individuals of non-European ancestry, and has logistical challenges related to shipping and insurance coverage for patients in the US. The availability of a NGS based quantitative cfDNA analysis for fetal antigen genotyping in the US that is robust for diverse populations and is available as early as 10 weeks presents an opportunity to assess performance. Methods: Patients with alloimmunized pregnancies undergoing clinical fetal antigen cfDNA analysis were recruited to the study along with the neonates resulting from the pregnancies. Neonatal buccal swabs were sent to an outside laboratory, blinded to the fetal cfDNA testing results, for antigen genotyping 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: We observed complete concordance between the fetal antigen cfDNA analysis result and neonatal genotypes for the 206 calls made on antigens to which the pregnant person was alloimmunized, for 100% sensitivity, specificity, PPV, and NPV across a racial and ethnically diverse cohort. Concordance was 99.8% for all antigens to which the pregnant person was genotype negative. Conclusion: This study demonstrates that cfDNA analysis for determining fetal antigen genotype is more accurate than real-life use of the current recommendations, ie., partner testing and amniocentesis, in a diverse US population. In addition, this noninvasive approach reduces barriers to obtaining timely, accurate information about fetal antigen genotype. These results support the routine implementation of fetal antigen cfDNA analysis to guide care of alloimmunized pregnancies in the US.