title: Validation of combinatorial probe-anchor ligation-based sequencing as non-invasive prenatal test for trisomy at a central laboratory.
abstract: OBJECTIVE
  To evaluate the clinical validity of a new ultrahigh-throughput non-invasive prenatal test (NIPT) based on combinatorial probe-anchor ligation (cPAL) sequencing of cell-free fetal DNA (cffDNA) using centralized testing.
  
  
  METHODS
  Maternal plasma samples were obtained from 10 594 singleton pregnancies in high-risk populations at 20 centers in China, including 8155 that were collected retrospectively and 2439 prospectively. Fetal outcome data and karyotyping results were documented as gold standard and were double blinded during NIPT. The clinical performance of the ultrahigh-throughput sequencing method, cPAL, for NIPT was validated by evaluating its sensitivity, specificity and positive predictive value (PPV) in detecting trisomies 21, 18 and 13 as the centralized testing mode in the reference laboratory. To ensure stable and reproducible performance of centralized cPAL-based NIPT in detecting trisomies, a series of quality-control systems, including sequencing of two sets of artificial samples, were employed and evaluated.
  
  
  RESULTS
  Ten prospective cases were excluded from the study because of incomplete clinical data. Four prospective samples failed to generate a NIPT result due to assay failure, presenting a failure rate of 0.16% (4/2429). A total of 168 retrospective cases and 47 prospective cases had a positive NIPT result for trisomy, giving respective positive rates of 2.06% and 1.94%. Four false-positive and no false-positive cases were observed in the retrospective and prospective groups, respectively, resulting in PPV of 97.62% (95% CI, 94.02-99.35%) and 100% (95% CI, 92.45-100%), respectively. In the retrospective group, sensitivity and specificity were, respectively, 100% (95% CI, 97.07-100%) and 99.98% (95% CI, 99.94-100%) for trisomy 21, 100% (95% CI, 97.75-100%) and 99.98% (95% CI, 99.94-100%) for trisomy 18, and 100% (95% CI, 15.81-100%) and 100% (95% CI, 99.95-100%) for trisomy 13. In the prospective group, sensitivity and specificity were, respectively, 100% (95% CI, 90.75-100%) and 100% (95% CI, 99.85-100%) for trisomy 21, 100% (95% CI, 63.06-100%) and 100% (95% CI, 99.85-100%) for trisomy 18, and 100% (95% CI, 2.50-100%) and 100% (95% CI, 99.85-100%) for trisomy 13.
  
  
  CONCLUSIONS
  In this multicenter study with a full quality-control system, NIPT by centralized cPAL-based testing showed high stability and performance comparable to those of previous validation studies in high-risk populations. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.
authors.affiliation: Peking University First Hospital, Beijing, PR China.
authors.forename: J
authors.initials: J
authors.lastname: Ma
authors.affiliation: BGI-Shenzhen, Shenzhen, PR China.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Hubei Maternal and Child Health Hospital, Wuhan, PR China.
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.affiliation: Southwest Hospital, The Third Military Medical University, Chongqing, PR China.
authors.forename: Y
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Women's Hospital School of Medicine Zhejiang University, Hangzhou, PR China.
authors.forename: C
authors.initials: C
authors.lastname: Xu
authors.affiliation: Wuhan Medical Care Center for Women and Children, Wuhan, PR China.
authors.forename: A
authors.initials: A
authors.lastname: Zhou
authors.affiliation: Nanjing Maternal and Child Health Hospital, Nanjing, PR China.
authors.forename: Z
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Guiyang Maternal and Child Health Hospital, Guiyang, PR China.
authors.forename: Z
authors.initials: Z
authors.lastname: Wu
authors.affiliation: First People's Hospital of Yunnan Province, Kunming, PR China.
authors.forename: X
authors.initials: X
authors.lastname: Tang
authors.affiliation: BGI-Shenzhen, Shenzhen, PR China.
authors.forename: F
authors.initials: F
authors.lastname: Chen
authors.affiliation: BGI Genomics Co., Ltd, Shenzhen, PR China.
authors.forename: Y
authors.initials: Y
authors.lastname: Yin
authors.affiliation: BGI Genomics Co., Ltd, Shenzhen, PR China.
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.affiliation: BGI-Manufacture, Shenzhen, PR China.
authors.forename: M
authors.initials: M
authors.lastname: Yan
authors.affiliation: BGI-Manufacture, Shenzhen, PR China.
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.affiliation: BGI-Manufacture, Shenzhen, PR China.|BGI-Wuhan, Wuhan, PR China.
authors.forename: F
authors.initials: F
authors.lastname: Mu
authors.affiliation: Peking University First Hospital, Beijing, PR China.
authors.forename: H
authors.initials: H
authors.lastname: Yang
mesh_terms: D000293:Adolescent; D000328:Adult; D002681:China; D025063:Chromosome Disorders / Q000175:diagnosis*; D020650:Combinatorial Chemistry Techniques; D004314:Down Syndrome / Q000175:diagnosis; D005260:Female; D006801:Humans; D007753:Laboratories / Q000592:standards; D062145:Maternal Serum Screening Tests; D008875:Middle Aged; D011237:Predictive Value of Tests; D011247:Pregnancy; D011296:Prenatal Diagnosis; D015203:Reproducibility of Results; D012189:Retrospective Studies; D000073839:Trisomy 13 Syndrome / Q000175:diagnosis; D000073842:Trisomy 18 Syndrome / Q000175:diagnosis; D055815:Young Adult
keywords: NIPT; cPAL; centralized mode; combinatorial probe-anchor ligation-based sequencer; next generation sequencing; non-invasive prenatal testing; prenatal screening