2018 ISPD Conference

Restricting NIPT for fetal fraction only, does it still make sense?
Vincenzo Cirigliano1, Elena Ordoñez1, Laura Rueda2, Sara Nicolas2, Isabel Castilla2, Manuel Grau2, Cristina Puertollano2, Mireia Lechuga2

1Synlab, Barcelona, Spain
2Synlab, Esplugues de Llobregat, Spain
Objectives
One common approach to improve NIPT performance is limiting analysis to samples above predefined FF (mostly 4%) to not compromise detection (DR) and false positive rates (FPR). However, being NIPT sensitivity mostly dependent on sequencing depth, detection limits should be evaluated at different depths instead of only being set to an arbitrary FF.
Paired-end MPSS allows discriminating fetal cfDNA by fragment sizes distribution. This provides FF estimates and allows performing counting statistics on fetal cfDNA, thus delivering more evident differences in case of fetal aneuploidy.
We evaluated the clinical utility of this approach in screening a large cohort of consecutive average-risk pregnancies.
Methods
A total of 23668 samples were collected above 10w of gestation (728 twins) regardless their risk category. Trisomies 13, 18 and 21 were screened using the NeoBona test, generating trisomy likelihood ratios (Tscore) for each chromosome of interest based on estimated FF, inter-chromosome statistics from fragments size distributions and the total sequencing counts on each chromosome. Chromosome specific cut-offs were applied at Tscores to classify normal and aneuploid cases. High-risk results were followed up by invasive diagnostic procedures in 98% of cases, low risk results were assumed to be concordant with pregnancy outcome unless otherwise advised by the referral centre.
Results
A total of 98.2% of cases were reported at first draw, 472 as high risk (2%). Ten FP were observed (cumulative FPR 0,04%), 1 T21 was classified as normal male with 7% FF (overall DR 99.8%). 
FF below 4% was observed in 1361 samples (5.7%), 84% yielded valid results, at high risk for trisomies in 34 cases, including 2 FP results.
T18 and T13 were detected even below 1% FF, accounting for 20% of all cases in this study, the lower FF for T21 was 2%. Including low FF cases didn’t impact DR while only increasing overall FPR by 0.005%.
Conclusions
The new analysis algorithm exploiting paired-end MPSS output of NeoBona test, enabled additional counting statistics to be performed on cfDNA of fetal origin while also monitoring sequencing counts reached for the estimated FF in each sample.
In the course of this study, this approach provided valid results in the vast majority of cases independently from the FF, allowing detecting all trisomies in samples reported with less than 4% fetal cfDNA at reduced FPR. The increased proportion of T18 and T13 observed in this subset of samples confirms the clinical utility of extending NIPT benefits to a wider population of pregnancies