title: The importance of determining the limit of detection of non-invasive prenatal testing methods.
abstract: OBJECTIVE
  Several non-invasive prenatal testing (NIPT) methods, which analyze circulating fetal cell-free DNA (cfDNA) in maternal plasma, suggest a fetal fraction (FF) ≥ 4% for a reportable result, with the assumption that fetal aneuploidies may not be detectable at lower FF. This study determined the actual limit of detection (LOD) of a massively parallel sequencing-based NIPT method and evaluated its performance in testing samples with low FF.
  
  
  METHODS
  An experimental model, involving the creation of artificial plasma mixtures with a final aneuploid FF ranging from 1% to 4%, simulated samples at different proportions of fetal cfDNA. We then analyzed 7103 blood samples, from pregnant women undergoing NIPT, to assess the impact of low FF on the performance of cfDNA testing.
  
  
  RESULTS
  Detection of common aneuploidies in samples with an FF as low as 2% is well within the ability of this technology. Of 105 pregnancies confirmed chromosomally abnormal, 25 (23.8%) involving a 2% <  FF  <  4% were consistently detected. These high-risk pregnancies would have not been identified using the suggested 4% FF cut-off.
  
  
  CONCLUSIONS
  This study underscores the importance of determining the actual LOD for each specific NIPT methodology. It may reduce the incidence of test cancelations and shorten the time required for the diagnosis of aneuploidy. © 2016 John Wiley & Sons, Ltd.
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Fiorentino
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Bono
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Pizzuti
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Mariano
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Polverari
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Duca
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Sessa
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Baldi
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Diano
authors.affiliation: GENOMA Group, Molecular Genetics Laboratory, Via di Castel Giubileo, 11 00138, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Spinella
mesh_terms: D000328:Adult; D000782:Aneuploidy; D002474:Cell-Free System; D025063:Chromosome Disorders / Q000175:diagnosis; D002882:Chromosomes, Human, Pair 13; D002887:Chromosomes, Human, Pair 18; D004247:DNA / Q000097:blood; D004314:Down Syndrome / Q000175:diagnosis; D005260:Female; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057230:Limit of Detection; D062145:Maternal Serum Screening Tests / Q000379:methods* / Q000592:standards; D008875:Middle Aged; D011247:Pregnancy; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014314:Trisomy / Q000175:diagnosis; D000073839:Trisomy 13 Syndrome; D000073842:Trisomy 18 Syndrome
chemical_list: D004247:DNA