Persico 2016.

Study characteristics
Patient sampling	Study design: blinded, prospective cohort study. Participants: pregnant women selected from a high‐risk population. Inclusion criteria: singleton pregnancies. Exclusion criteria: multifetal pregnancies.
Patient characteristics and setting	Number enrolled: 259 pregnant women. Number available for 2 x 2 table: 249 pregnant women (subgroup of 96%). Setting: 4 fetal medicine centres in Italy. Recruitment period: March to December 2014. Ethnicity: not reported. Gestational age: not reported. Median maternal age (range): 36 (20 to 46) years. Relevant tests carried out prior to index test: ultrasonography (nuchal translucency measurement) or biochemical screening or both. Language of the study: English.
Index tests	gNIPT by TMPS (SNP‐based method) on Illumina Genome Analyzer IIx or HiSeq sequencers, 19,488‐plex targeted PCR with NATUS algorithm. Fetal fraction DNA: amount measured but not reported (usually NATERA used quality control criteria > 4%). Blood samples for gNIPT were collected just before reference standard. Cut‐off value: positive if risk score > 1%. Commercial test: Natera's prenatal test.
Target condition and reference standard(s)	Target conditions: T21, T18, T13, 45,X, 47,XXY and 47,XXX. 47,XYY was also assessed but no case was found. Reference standard: fetal karyotype of chorionic villi or amniotic fluid.
Flow and timing	Blood samples for gNIPT were obtained prior to the invasive procedure (reference standard). gNIPT was a second‐tier test. 10/259 samples failed during sequencing process (no gNIPT result) including 2 samples failed internal quality control and 8 samples had low fetal fraction DNA. No repeated test reported.
Comparative
Aim to study	To investigate a strategy for clinical implementation of ccfDNA testing in high‐risk pregnancies after first‐trimester combined screening.
Funding source or sponsor of the study	Study not funded by industry but the cost of ccfDNA testing were covered by Natera, Inc.
Informations about the authors contacted	No need for further contact.
Notes
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	Yes
Was a case‐control design avoided?	Yes
Did the study avoid inappropriate exclusions?	No
		High	Low
DOMAIN 2: Index Test TMPS
Were the index test results interpreted without knowledge of the results of the reference standard?	Yes
If a threshold was used, was it pre‐specified?	Yes
		Low	Low
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?	Yes
Were the reference standard results interpreted without knowledge of the results of the index tests?	Yes
		Low	Low
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?	Yes
Did all analysed patients receive the reference standard?	Yes
Were all patients included in the analysis?	No
		High