Papageorghiou 2016b.

Study characteristics
Patient sampling	Study design: blinded, case‐control study (1:9). Participants: pregnant women selected at high risk of fetal aneuploidy presenting for invasive testing. Inclusion criteria: at least 18 years of age, a singleton or twin pregnancies of at least 10 weeks’ gestation and a clinical indication for an invasive procedure. Exclusion criteria: higher‐order multiple pregnancies (triplets or more), known mosaicism, partial trisomy or translocations, fetal demise, disappearing twin, malignancy or known aneuploidy in the pregnancy.
Patient characteristics and setting	Number enrolled: 442 pregnant women. Number available for 2 x 2 table: 11 twin pregnancies (subgroup of 2%). Setting: 6 hospital centres in England, UK. Recruitment period: April 2008 to November 2014. Ethnicity: not reported. Median gestational age (range): 15.4 (11 to 36.6) weeks. Median maternal age (range): 35 (18 to 55) 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 MPSS on Ion Proton™ sequencer in 8‐plex. Fetal fraction DNA: amount measured but not reported. Blood samples for gNIPT were collected before reference standard. Cutpoint: positive if likelihood ratio > 1 and maternal age‐adjusted probability risk score. Commercial test: IONA® test by Premaitha Health (public limited company).
Target condition and reference standard(s)	Target conditions: T21, T18 and T13. Reference standards: fetal karyotype of chorionic villi or amniotic fluid or medical record from birth.
Flow and timing	Blood samples for gNIPT were obtained prior to the invasive procedure (reference standard). gNIPT was a second‐tier test. 5/442 samples failed during sequencing process including 3 samples for low fetal fraction DNA and 2 samples did not have sufficient DNA fragment counts (no gNIPT result). 426/437 singleton pregnancies were not selected. No repeated test reported.
Comparative
Aim to study	To investigate the accuracy of the IONA® test in the discrimination between euploid pregnancies and those affected by fetal trisomies 21, 18 and 13.
Funding source or sponsor of the study	Study funded by Premaitha Health (public limited company). Some authors are employees of Premaitha Health plc.
Informations about the authors contacted	Author was contacted on: 19 September 2016. Reply received on: 20 September 2016.
Notes	Data from twin pregnancies only reported here. Data from singleton pregnancies reported in Papageorghiou 2016a.
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	No
Was a case‐control design avoided?	No
Did the study avoid inappropriate exclusions?	No
		High	Low
DOMAIN 2: Index Test MPSS
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