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

Abstract

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 1061 preclinical samples with an analytical sensitivity of 100% (95% CI: 99–100%) and analytical specificity of 100% (95% CI: 99–100%) with only a 3.8% no-call rate, including challenging samples at 1.5% fetal fraction. The assay showed a clear separation between antigen detected and not detected for 15,939 clinical plasma samples in a general population setting, with an estimated clinical sensitivity of 99.6%-100%. The precision of the assay in which two replicate plasma samples were independently analyzed was 99.9% for 1683 clinical samples. Moreover, a fetal antigen determination could be made for samples with RHDΨ, a variant more common among RhD-negative Black individuals. The NIPT results were 100% concordant with neonatal antigen genotype/serology for 23 RhD negative pregnant individuals and 12 other antigen evaluations in 4 alloimmunized pregnant individuals. 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 for 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.