The performance of grey zone in common foetal aneuploidy screening by semiconductor sequencing

Abstract

A total of 15,267 pregnancies were tested by NIPT in this study. Grey zone (z score: 2.58 ∼ 4 and −4∼−2.58) was set for screening out aneuploidy 21, 18 and 13. Cases with z score located in the grey zone were retested starting from DNA extraction. The chi-squared test and/or the Fisher’s exact test were used to compare variables. One hundred and eight screening-positive samples in the first run of NIPT were common trisomies 21 (N = 83), trisomies18 (N = 13) or trisomies 13 (N = 12), with PPVs of 87.18%, 76.92%, and 30% respectively. For the cases in the grey zone, most of them (67.15%, 184/274) were reported with Z score of Chromosome 21 in the grey zone and 176 were reclassified as negative by the second run of NIPT; while 3 cases reclassified as trisomy 21 and 1 case reclassified as trisomy 13 were finally confirmed by karyotyping analysis, with PPV 25% and 20% respectively. The grey zone and the second run of NIPT in this study showed that the grey zone and second run NIPT approach was able to accurately help categorise cases as negative and positive. Invasive diagnosis is recommended to prevent false negative aneuploidies for samples located in the special z score scope of 2.58–3 for two runs of NIPT. IMPACT STATEMENT What is already known on this subject? Grey zone was widely used in NIPT. The performance of grey zone of clinical samples on Illumina HiSeq 2000 instrument has been reported, and the performance of grey zone on some mainstream sequencers with simulated samples has also been summarised. Reported treatments for samples located in the grey zone in NIPT usually included being classified into ‘unclassified’ or ‘no call’ followed by following up and/or karyotyping analysis. What do the results of this study add? This study investigated the performance of the grey zone on Ion Proton DA8600 with clinical samples; and it present an alternative treatment for samples in grey zone that reclassified them as negative or positive by the second run of sequencing. What are the implications of these findings for clinical practice and/or further research? Our own data for the performance of the grey zone in the cfDNA assay on the semiconductor sequencing platform might provide raw materials for other researchers’ meta-analysis, cohort study, or other studies. Details of Z score distributions of chromosomes in grey zone, results of the second run of NIPT for samples in the grey zone, and false negative samples in the grey zone would help lab technicians better analyse the NIPT results and help doctors to improve genetic counselling.