Prospective multicentre DTA trial (cross-sectional design)
Trisomies 21, 18 and 13
1390 Index test trademark: NA (Illumina HiSeq2500 platform or Life Technologies 5500 W SOLID) Comparator: no intervention
Reference standard: CVS or amniocentesis, ultrasound data, genetic testing in products of conception and neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Persico et al.
[49], 2016
Prospective, multicentre DTA trial (cross-sectional design)
Trisomies 21, 18 and 13
259 Index test trademark: NA (SNPs) Comparator: no intervention
Reference standard: CVS or amniocentesis and/or aCGH
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Zhang et al. [50], 2016
Prospective DTA trial (cross-sectional design)
Trisomies 21, 18 and 13 and Turner syndrome
87 Index test trademark: Verifi™ Prenatal Test Comparator: no intervention
Reference standard: amniocentesis, neonatal blood karyotyping and neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Benachi et al.
[51], 2015
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
900 Index test trademark: NA (Illumina HiSeq1500 platform)
Comparator: no intervention
Reference standard: CVS or amniocentesis
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
or study name women enrolled
Hernández-Gómez et al. [52], 2015
Prospective DTA trial (cross-sectional design) Trisomy 18 and monosomy X
42 Index test trademark: Harmony® prenatal test Comparator: no intervention
Reference standard: amniocentesis and neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Ke et al. [53], 2015
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
2340 Index test trademark: NA (Illumina HiSeq2000 platform)
Comparator: no intervention
Reference standard: amniocentesis and neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Lee et al. [54], 2015
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
93 Index test trademark: NA (MiSeq and NextSeq (Illumina))
Comparator: no intervention
Reference standard: amniocentesis, CVS cordocentesis, neonatal peripheral blood or products of conception
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Sago et al. [55], 2015
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
7740 Index test trademark: MaterniT PLUS Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Sánchez-Usabiaga et al.
[56], 2015
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
270 Index test trademark: NA (SNPs) Comparator: no intervention
Reference standard: CVS or amniocentesis and neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Song et al. [57], 2015
Prospective DTA trial (cross-sectional design)
Trisomies 21, 18 and 13 and SCA
213 Index test trademark: NA (Illumina HiSeq 2000 platform)
Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Wang et al. [58], 2015
Prospective DTA trial (cross-sectional design) Trisomies 21 and 18
917 Index test trademark: NA (Illumina HiSeq2000 platform)
Comparator: no intervention
Reference standard: standard karyotyping, FISH and neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Jeon et al. [59], 2014
Prospective DTA trial (cross-sectional design) Trisomies 21 and 18
155 Index test trademark: NA (Ion Proton™ system) Comparator: no intervention
Reference standard: amniocentesis
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Korostelev et al.
[60], 2014
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
1968 Index test trademark: NA (SNPs) Comparator: no intervention
Reference standard: amniocentesis, chromosomal microarray analysis or neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Porreco et al.
[61], 2014
Prospective DTA trial (cross-sectional design)
Trisomies 21, 18 and 13 and SCA (Turner syndrome, trisomy X, Klinefelter syndrome and 47,XYY syndrome)
4170 Index test trademark: NA (Illumina HiSeq2000 platform)
Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Stumm et al.
[62], 2014
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
522 Index test trademark: NA (Illumina HiSeq2000 platform)
Comparator: no intervention
Reference standard: amniocentesis, CVS or cordocentesis
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Willems et al.
[63], 2014
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
3000 Index test trademark: Harmony® prenatal test Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Zhou et al. [64], 2014
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
7705 Index test trademark: NA Comparator: no intervention
Reference standard: amniocentesis or neonatal follow-up
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
or study name women enrolled Liang et al. [65],
2013
Prospective DTA trial (cross-sectional design)
Trisomies 21, 18, 13 and 9 and SCA (Turner syndrome, XXX, XXY or XYY)
435 Index test trademark: NA (Illumina HiSeq2000 platform)
Comparator: no intervention Reference standard: amniocentesis
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Nicolaides et al.
[66], 2013
Prospective DTA trial (cross-sectional design)
Trisomies 21, 18 and 13 and Turner syndrome
242 Index test trademark: NA (SNPs) Comparator: no intervention Reference standard: CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Verweij et al.
[67], 2013
Prospective DTA trial (cross-sectional design) Trisomy 21
595 Index test trademark: NA Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Lau et al. [68], 2012
Prospective DTA trial (cross-sectional design)
Trisomies 21, 18 and 13 and SCA (Turner syndrome and Klinefelter syndrome)
108 Index test trademark: Verifi™ prenatal test Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Norton et al. [69], 2012
Prospective DTA trial (cross-sectional design) Trisomies 21 and 18
4002 Index test trademark: Harmony® prenatal test Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Ehrich et al. [70], 2011
Prospective DTA trial (cross-sectional design) Trisomy 21
480 Index test trademark: NA (GAIIx sequencer; Illumina) Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Gil et al. [71], 2016
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
4012
(460 classified as high risk and 3552 classified as intermediate risk)
Index test trademark: Harmony® prenatal test Comparator: no intervention
Reference standard: CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Fosler et al. [72], 2017
Prospective DTA trial (cross-sectional design) Trisomy 21
487 Index test trademark: Verifi™ prenatal test Comparator: no intervention
Reference standard: amniocentesis, CVS or ultrasound findings
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Prospective DTA trial (cross-sectional design) Trisomies 21, 18 and 13
467 Index test trademark: Harmony™ prenatal test Comparator: no intervention
Reference standard: fetal karyotype (not specified method used)
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Tan et al. [73], 2016
Prospective DTA trial (cross-sectional design) Trisomy 21
565 Index test trademark: NA (MPS) Comparator: no intervention
Reference standard: amniocentesis or CVS
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Bevilacqua et al.
[74], 2015
Prospective DTA trial (cross-sectional design) Trisomy 21
515 Index test trademark: Harmony™ prenatal test Comparator: no intervention
Reference standard: amniocentesis, CVS or neonatal blood examination
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Huang et al. [75], 2014
Prospective DTA trial (cross-sectional design) Trisomies 21 and 18
189 Index test trademark: NA Comparator: no intervention
Reference standard: amniocentesis, CVS or cordocentesis
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Lau et al. [76], 2013
Prospective DTA trial (cross-sectional design) Trisomy 21
12 Index test trademark: NA
Comparator: first- or second-trimester screening and/or first-trimester ultrasound marker screening (NT, fetal nasal bone and Doppler assessment of the tricuspid valve and ductus venosus)
Reference standard: amniocentesis, CVS or cordocentesis
Safety and effectiveness of NIPT: FN, FP, test failure rate, S, Sp, PPV, NPV
Abbreviations: aCGH=microarray-based comparative genomic hybridisation; CARE=Comparison of Aneuploidy Risk Evaluations; CRL=crown–rump length; CVS=chorionic villus sampling;
DTA=diagnostic test accuracy; FISH=fluorescence in situ hybridisation; FN=false negative; FP=false positive; hCG=human chorionic gonadotropin; β-hCG=β subunit of human chorionic gonadotropin;
MPS=massive parallel sequencing; NA=not available; NIPT=noninvasive prenatal test; NPV=negative predictive value; NT=nuchal translucency; PAPP-A=pregnancy-associated plasma protein A;
PPV=positive predictive value; S=sensitivity; SNP=single nucleotide polymorphism; Sp=specificity; TN=true negative; TP=true positive; SCA=sex chromosome aneuploidy.
Only four manufacturers who had peer-reviewed publications were asked for submission files for feasibility reasons. All were contacted to provide information regarding
reimbursement.
The search was restricted to 2010 given that the first noninvasive prenatal test was launched in 2011.
Studies were restricted to those published in English, Spanish, French, Italian and Portuguese.
Subgroup analysis could not be performed because of the lack of data and was deleted from the project plan.
One additional assessment element was identified for the description and technical characteristics of technology domain (B0018).
Element ID Research question
B0001 What are noninvasive prenatal tests and the comparators?
B0002 What is the claimed benefit of NIPT in relation to the comparators?
B0003 What is the phase of development and implementation of NIPT and the comparators?
B0004 Who administers NIPT and the comparators and in what context and at what level of care are they provided?
B0008 What kind of special premises are needed to use NIPT and the comparators?
B0009 What equipment and supplies are needed to use NIPT and the comparators?
B0018 Are the reference values or cut-off points clearly established?
A0020 For which indications has NIPT received marketing authorisation or CE marking?
A0021 What is the reimbursement status of NIPT in prenatal screening?
3.2 Results
Features of the technology and comparators
[B0001] – What are noninvasive prenatal tests and the comparators?
General characteristics of NIPT
Noninvasive prenatal tests are in vitro diagnostic products that use cfDNA from maternal blood of pregnant women. Though commonly referred to as cell fetal free DNA, the DNA does not derive from the fetus but originates from the cytotrophoblast layer of the chorionic villi (the outer placental cell layer). It comprises around 10%–15% of the total cfDNA in the maternal circulation during the late first and early second trimester [2]. Testing can be performed between 8–10 weeks’ gestation.
Since the development of the first commercialised NIPT in 2011, which was designed for the de-tection of T21, T18 and T13, different laboratories have developed different assays, expanding the panel of chromosomal anomalies detected to other common chromosomal anomalies. Table 3 illustrates the features of the commercialised tests which were identified by the search, although many laboratory-developed tests with local redesigned workflows also exist given the transfer of existing technology to many laboratories worldwide. According to an international survey from 28 countries performed in 2015, the most common providers of noninvasive prenatal tests are currently Ariosa Diagnostics Inc./Roche Sequencing Solutions Inc. (Harmony®), BGI Diagnostics Technology Co. Ltd., (NIFTY™ test), Illumina Inc. (Verifi™, VeriSeq NIPT Solution and Serenity (Verifi™ and VeriSeq NIPT Solution)), Igenomix (NACE®), LifeCodexx AG (PrenaTest®), Natera (Panorama®) and Sequenom (MaterniT 21 PLUS test). Of these, the MaterniT 21 PLUS test is a laboratory-based test which is commercialised only in the USA [3].
ploidy risk analysis. Regardless of the tests, all require a sufficient proportion of cfDNA in the maternal plasma to be able to cfDNA-differentiate between the status of the mother and the fetus.
Initially many manufacturers established a minimum fetal fraction of 4% for NIPT evaluation, alt-hough with further development of NIPT technologies and analysis methods, this limit has been lowered. It has been highlighted that specimens containing excessive amounts might also affect the performance of the test (Harmony submission file) [77]. However, not all laboratories quantify fetal fraction in individual samples. Fetal fraction can be influenced by factors such as fetal aneu-ploidy, gestational age and maternal body mass index and weight, although failure to obtain an NIPT result could also be due to different technical/statistical reasons [5, 6]. In cases of a low fetal fraction, a redraw can be requested.
The results of NIPT can be confounded by several biological factors such as confined placental mosaicism, maternal copy number variations, maternal mosaicism, fetal partial trisomy or translo-cations, fetal mosaicism, fetal structural chromosomal anomalies other than trisomy, intrauterine fetal demise and disappearing twin [4]. Other factors which are liable to confound results are recent maternal transfusions, maternal organ or bone marrow transplants, maternal immunotherapy or stem cell therapy or maternal malignancy [77-79].
Specific features of the process and tests
NGS technologies can be broadly categorised as whole genome sequencing (WGS) or targeted sequencing (chromosome-specific sequencing and single nucleotide polymorphism (SNP) analy-sis) [80]:
WGS analyses the whole genome and generates DNA sequence reads from all chromo-somes nonspecifically. This method analyses random sequences and can allow millions of short DNA fragments to be sequenced rapidly in a single run. It can allow screening of more conditions than T21, T18 and T13 in extended screening programmes. The depth of sequencing or coverage (number of reads giving information about a base present at a set position in the reference sequence, or the number of times a base is represented within all the reads) will determine the resolution achievable; hence more sequencing will increase performance and allow the test to be more specific and sensitive.
Targeted sequencing as the name indicates targets a subset of genomic regions. This ap-proach differs from the whole genome apap-proach by selectively amplifying and sequencing specific genomic regions, significantly reducing the total number of analysed reads. The re-gion can include certain chromosomal loci (chromosome-specific sequencing) or SNPs of interest. The genome region will be predetermined by the specific test used, and the test would need to be modified and validated to provide information about other regions of the genome.
Most existing tests use first-generation quantitative WGS approaches or counting methods (Table 3) to determine the percentage of cfDNA compared with cell-free maternal DNA. Practically all of these use the Illumina platforms (Illumina Inc., San Diego, CA, USA) for multiple parallel sequenc-ing. Accessing results commonly takes from around 3 to 10 days. The IONA® test (Premaitha Health plc, Manchester, UK) uses another platform, the Ion Proton™ ion semiconductor sequencing platform (Thermo Fisher Scientific), which is categorised by a total turnaround time, from the start of sample processing to a result, of 3 days.
removes the need for presequencing PCR. The turnaround time from sample accessing to results for the VeriSeq NIPT Solution is 26 hours. Natera has commercialised an NGS approach which specifically targets SNPs to determine ploidy (Panorama®). This second-generation approach, used also by other companies such as Imegen-Instituto de Medicina Genómica- (Spain), in comparison with other first-generation tests, has the ability to differentiate between cell-free maternal DNA and cfDNA, and in addition to identifying T21, T18, T13, monosomy X, and sex chromosome trisomies, it can identify the presence of a vanishing twin and maternal duplications. Currently it is being vali-dated for common microdeletion conditions, including 22q11.2 deletion syndrome, 1p36 deletion syndrome, cri du chat syndrome, Prader-Willi syndrome and Angelman syndrome. Several other tests such as TrisoNIM and Prendia also target other trisomies and microdeletions.
The Harmony® test, initially commercialised as a laboratory NGS test based on Illumina technology, changed in 2014 to use an alternative microarray sequencing process that operates on the Affy-metrix technology platform. This targeted amplification process, termed digital analysis of selected regions amplifies a set of universal PCR products from genomic intervals on chromosomes 1–13, 18, 21, X and Y and determines the fetal fraction by also measuring SNPs. These can be analysed to distinguish between cfDNA and cell-free maternal DNA. No data have been published regarding the Vanadis™ NIPT system, which is an innovative approach based on fluorescence. This system has still not been placed on the market in the European Union (EU).
Existing noninvasive prenatal tests have different software for analysis and interpretation of screening results with differentiating features, algorithms and quality standards. For example, not all tests measure fetal fraction to ensure adequate DNA analysis, or if done, include it in the analysis of fetal risk. VeriSeq NIPT Solution tests use a quality control metric to ensure that samples have sufficient coverage to make a confident call, eliminating the need for a set fetal fraction percentage cut-off point. The neoBona®, for example, integrates sequencing depth on each chromosome, the percent-age of fetal fraction and size of the fragments to calculate risks [81]. The Harmony® prenatal test establishes standards thresholds for all quality control metrics. This test uses an analysis algorithm termed FORTE to compute the probability of trisomies and probability of fetal sex chromosomes.
The algorithm takes into account the total cfDNA quantification from chromosomes measured with digital analysis of selected regions, the amount of fetal DNA in a sample as measured by NIPT, maternal age and gestational age-related risk for trisomy in calculating the probability score [82].
Company Brand name Platform provider/
technology
Mechanism of action
Chromosomal anomalies detected Sample and reporting
T21, T18, T13 and sex chromosome aneuploidies in singleton pregnancies
T21, T18, T13 and presence of Y for women with twins through natural or reproductive methods Additional indications: T9, T16 and microdeletions ( Di George, Prader-Willi/ Angelman, Cri-du-Chat, Wolf-Hirschhorn and 1p36 deletion)
3–5 days All pregnant women ≥ 10 weeks gestation who have chosen to have T13, T18 and T13 prenatal screening Not intended to be used in isolation from other clinical findings and tests results
Single, twin or egg donor pregnancies Illumina Inc.
(San Diego, CA, USA)
VeriSeq NIPT Solution Includes: the VeriSeq NIPT Workflow Manager for the VeriSeq NIPT Microlab STAR, the VeriSeq NIPT Sample Prep Kits, and the VeriSeq Onsite Server with the VeriSeq NIPT Assay Software.
T21, T18, T13 and sex chromosome aneuploidies
1 day (26 hours)
Intended for use in pregnant women of at least 10 weeks gestation The product must not be used as the sole basis for diagnosis or other pregnancy management decisions
T21, T18, T13 and sex chromosome aneuploidies
— Single or twin pregnancies
technology time (days)
NeoBona® Advanced NeoBona® Advanced+
(http://www.neobona.es/)
NeoBona Advanced: T21, T18, T13 and sex chromosome aneuploidies (singleton
pregnancy only)
NeoBona Advanced+: T21, T18, T13, T16, T9, sex chromosome aneuploidies and microdeletions (singleton pregnancy only) Prenatal Test Extended Panel: T21, T18, T13, sex chromosome aneuplodies and microdeletions (DiGeorge, Angelman/Prader -Willi, 1p36 deletion, Wolf -Hirschhorn y Cri-du-chat) (singleton pregnancy only)
Prenatal Test Extended Panel + All chromosomes: T21, T18, T13, sex chromosome aneuplodies, microdeletions (DiGeorge, Angelman/Prader -Willi, 1p36 deletion, Wolf -Hirschhorn y Cri-du-chat) and all chromosome aneuploidies. (singleton pregnancy only)
10 days Can be used in pregnancies
≥ 10 weeks of gestation Single, twin, IVF and egg donor pregnancies
VisibiliT™: T21 and T18
MaterniT® 21 PLUS:T21, T18, T13, sex chromosome aneuploidies and 7 microdeletions (T21, T18, T13, sex chromosome aneuplodies, T16, T22 and microdeletions (Di George, Prader-Willi/ Angelman, Cri-du-Chat, Wolf-Hirschhorn, Jacobsen, Langer-Giedion and 1p36 deletion)
MaterniT® GENOME: All chromosomes and deletions or duplications of chromosome material 7 Mb or larger, as well as seven clinically micro-deletion regions less than 7 Mb in size (Di George, Prader-Willi, Cri-du-Chat, Wolf-Hirschhorn, Jacobsen, Langer-Giedion and 1p36 deletion)
5 days Can be utilized in pregnant women
≥ 10 weeks gestation
MaterniT21 Plus is relevant for pregnancies at increased risk of fetal anomalies
provider/
technology
of action reporting
time (days)
T21, T18, T13, sex chromosome aneuploidies and most common microdeletions, including 22q11.2 deletion syndrome, 1p36 deletion syndrome, cri du chat, Prader-Willi and Angelman
9 days Panorama could be useful for the general pregnant population ≥ 9 weeks gestation
T21, T18 and T13 3–5 days Suitable for all pregnant women
≥ 10 weeks of gestation
Intended to be used by a clinician in combination with other risk factors to estimate the risk of affected pregnancies Single, twin, surrogate or in-vitro fertilization pregnancies
Harmony® prenatal test Affymetrixa F.
T21, T18, T13, Monosomy X*, sex chromosome aneuploidies* and 22q11.2 deletion syndrome
*singleton pregnancy only
≤7 days Intended for use in pregnant women
≤7 days Intended for use in pregnant women