Abstract

Background/Objectives: Non-invasive prenatal testing (NIPT) for fetal aneuploidy requires accurate trisomy detection together with reliable fetal fraction assessment. This study evaluated the clinical feasibility of a 106-plex digital PCR (dPCR) NIPT assay for trisomies 13, 18, and 21 with internal fetal fraction quantification. Methods: We consecutively recruited 470 women with high-risk singleton pregnancies. Fetal trisomies were detected using dPCR-NIPT and confirmed by invasive prenatal diagnosis. Pregnancies with negative prenatal diagnostic results were followed to birth. Analytical performance and quality control were assessed using trisomic DNA. The euploid cut-off and diagnostic performance were evaluated in two independent maternal plasma sample sets, using invasive diagnosis and clinical outcome as the reference standard. Results: dPCR-NIPT measured fetal fraction irrespective of fetal sex and detected trisomies at fetal fractions ≥3% using 5 ng DNA. A total of 12 of 470 plasma samples failed cell-free DNA quality control and were excluded before dPCR testing. Of the remaining 458 samples, 5 had fetal fractions below 3% and were classified as failed tests, yielding a nonreportable rate of 1.1%. Using a cut-off of 6.9 established in 103 training samples, no false-positive or false-negative trisomy calls were observed in the 350-sample testing set, corresponding to 100% sensitivity (95% confidence interval [CI], 85.18–100%) and 100% specificity (95% CI, 98.88–100%) for 23 confirmed trisomies. Conclusions: This proof-of-principle study supports the feasibility of fetal fraction-informed dPCR-NIPT for trisomy detection in high-risk singleton pregnancies. Larger prospective studies in average-risk and earlier-gestation populations are required.

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