Validation of a Non-invasive Prenatal Test for Fetal RhD, C, c, E, Kell and FyA Antigens

Abstract

We developed and validated a next generation sequencing (NGS) based NIPT assay using quantitative counting template (QCT) technology to detect RhD, C, c, E, Kell, and Fya fetal antigen genotypes in the diverse U.S. population. The assay quantifies paternally derived fetal antigen cell free DNA molecules after calibration to fetal fraction and a reference gene. The assay correctly determined fetal antigen status for all 1087 preclinical samples; analytical sensitivity 100% (95% CI: 99-100%) and analytical specificity 100% (95% CI: 99-100%). The assay showed a clear separation between antigen detected and not detected for 15,939 clinical samples and a fetal antigen determination was made for all samples with RHD psi, a variant more common among RhD-negative Black individuals. The NIPT results were 100% concordant with neonatal antigen genotype/ serology for 27 clinical samples. This NGS-based fetal antigen NIPT assay had excellent performance in a validation study of samples from a diverse U.S. population for fetal fractions as low as 1.1% and as early as 10 weeks of gestation, without the need of a sample from the biological partner. Implementation of NIPT for the detection of fetal antigen in RhD-negative and alloimmunized pregnant individuals will streamline care and reduce unnecessary treatment, monitoring and patient anxiety.