() SLR I 24 studies 146,940 tests Bivariate meta-analysis produced a summary sensitivity of 0.977 (95% and specificity of 0.999 (95% CI a positive likelihood of 1569 (95% CI 810–3149) and negative likelihood ratio of 0.023 (95% CI 0.011–0.048). Of 12/ 24 studies reporting inconclusive results, seven documented an explanation (in order of frequency): low fetal fraction; test failure; no reason given; mosaicism. The most common reasons given for false results were: confirmed low fetal fraction; confirmed mosaicism; presumed low fetal fraction/human error; maternal CNV; no reason given. Neither test technique nor population risk had a significant effect. () SLR I 33 studies The pooled sensitivity was 97.4% (95.8% to 98.4%) and specificity was 99.9% (99.9% to 100%). Including test failures in an intention to diagnose analysis in the meta-analysis decreased sensitivity estimates by 1.6% and specificity estimates by nearly 2%. SLR included case-control studies (Level Trisomy 13 Study ref Design LoE N Aim, setting, population, methods Results Comments () SLR I 18 studies Studies reported on the performance of screening by cfDNA analysis for trisomy 13, in a combined total of 139 trisomy-13 and 18,059 non-trisomy-13 singleton pregnancies. In individual studies, the detection rate varied between 40.0% and 100% and the false positive rate varied between 0% and 1.14%. The pooled weighted detection and false positive rates were 91.0% (95% CI, and 0.13% (95% CI, 0.05–0.26%), respectively. SLR includes case-control studies (Level and does not include the two largest studies.
() SLR I 16 studies 134,691 tests There was a summary sensitivity of 0.906 (95% CI 0.823–0.958) and specificity of 1.00 (95% CI 0.999–1.00). The positive likelihood ratio was 453 (95% CI 26–7864) and negative likelihood ratio was 0.188 (95% CI with a diagnostic odds ratio of 2788 (95%CI 285–27252). Of 6/16 studies reporting inconclusive results, 4 documented an explanation for inconclusive results: low fetal fraction; different fragmentation rate; contamination; assay failure; and human error. The only reason given for false results was confirmed low fetal fraction. () SLR I 24 studies The pooled sensitivity was 97.4% (86.1% to 99.6%) and specificity was >99.9% (99.9% to 100%). Including test failures in an intention to diagnose analysis in the meta-analysis decreased sensitivity estimates by 7.1% and specificity estimates by nearly 2%. SLR included case-control studies (Level Monsomy X ref Design LoE N Aim, setting, population, methods Results Comments () SLR I 16 studies Studies reported on the detection of monosomy X by cfDNA analysis, for a combined total of 177 singleton pregnancies with fetal monosomy X and 9,079 with no monosomy X. In individual studies, the detection rate varied between 66.7% and 100% and the positive rate varied between 0% and 0.52%. The pooled weighted detection and false positive rates were 90.3% (95% CI, and 0.23% (95% CI, 0.14–0.34%), respectively. SLR includes case-control studies (Level and does not include the two largest studies.
() Cohort III-2 4,736 Aim: to report the clinical experience and performance of massively parallel sequencing-based cDNA as a screening method in detecting trisomy 21, 18, and 13 in a mixed-risk population in Thailand. Setting: Thailand Population: high-risk pregnancies either with advanced maternal age or positive serum biochemical tests (n=2,840) and low-risk pregnancies without conventional indications (n=1,889). Methods: In a 30-month period, 121 medical centers in Thailand offered cfDNA as clinical screening tests for fetal T21, T18, and T13 in the mixed-risk population. All cfDNA-positive cases were recommended to undergo invasive prenatal diagnosis. Outcomes: Detection of trisomies 21, 18 and 13 99.9% (4732/4736) of the participants with a median maternal age of 35 years old received reports, and 1.3% (63/4732) were classified as test positive, including 36 T21, 19 T18, and 8 T13; 82.5% (52/63) took prenatal diagnosis, and 11.5% (6/52) false-positive cases were observed. There were 31 T21, 16 T18, 5 T13, and 4669 euploid cases. We observed one FP of T21, four FPs of T18 and one FP of T13 in our test, giving the sensitivities of 100.00%, 100.00%, and 100.00% and specificities of 99.98%, 99.92%, and 99.98% for detecting T21, T18, and T13, respectively. The PPV for T21, T18, and T13 were 96.77% (n=31; 83.3 to 99.9%), 75.00% (n=16; 47.6 to 92.7%), and 80.00% (n=5; 28.4 to 99.5%), respectively. The overall specificity for detecting these three chromosomal anomalies combined was 99.87%, and the overall sensitivity and PPV were 100.00% and 88.46%, respectively. The incidence of T21, T18, and T13 were 0.64%, 0.25%, and 0.08%, respectively.
() Cohort III-2 5,267 Aim: To assess the implementation of CFDNA into clinical practice utilising both first- and second-line screening models. Setting: Metropolitan private practices in Australia Population: Singleton pregnancies in a mixed risk population Methods: cfDNA testing was offered as a first-line screen, ideally followed by combined first-trimester screening (cFTS), or as a second-line test following cFTS, particularly in those with a calculated risk between 1:50 and 1:1000. Outcomes: Detection of trisomies 21, 18 13, sex chromosome aneuploidies and other anomalies screening was performed in 5,267 women and as a first-line screening method in 3,359 (63.8%). Detection rates were 100% for trisomies 21 and 13 and 88% for trisomy 18. Of cases with known karyotypes, the positive predictive value (PPV) of the test was highest for trisomy 21 (97.7%) and lowest for monosomy X (25%). Ultrasound detection of structural anomaly resulted in the detection of five additional chromosome anomalies, two of which had high-risk cFTS results. For all chromosomal anomalies, cfDNA alone detected 93.4%, cfDNA in a model detected 81.8% and cFTS alone detected 65.9% (P < 0.005). If had only had cFTS, the rate of invasive procedures would have been 5.5% higher (p<0.0001). Given the false-positive rate for all aneuploidies, cfDNA is an advanced screening test, rather than a diagnostic test. Study population may not be generalisable to the Australian obstetric community as women were predominantly older, Caucasian and from higher socioeconomic groups
() Cohort III-2 216 Aim: to determine the positive predictive (PPV) of noninvasive prenatal screening (NIPS) for various aneuploidies based on cases referred for follow-up cytogenetic testing. Secondarily, to determine the (FN) rate for those cases with a negative NIPS result. Setting: United States Population: Pregnancies at high-risk of fetal aneuploidy Methods: We compared the cytogenetic findings (primarily from chromosome analysis) from 216 cases referred to our laboratories with either a positive or negative NIPS result, and classified NIPS results as true positive, false positive, true negative, or FN. Diagnostic cytogenetic testing was performed on the following tissue types: amniotic fluid (n = 137), chorionic villi (n = 69), neonatal blood (n=6), and products of conception (n = 4). Outcomes: Detection of trisomies 21, 18, 13, monosomy X, XXY The PPV for cfDNA were as follows: 93% for trisomy (T)21 (n=99; 95%CI 86-97.1%), 58% for T18 (n=24; 95%CI 36.6-77.9%), 45% for T13 (n=11; 95% CI 16.7-76.6%), 23% for monosomy X (n=26; 95% CI 9-43.6%), and 67% for XXY (n=6; 95% CI, 22.3-95.7%). Of the 26 cases referred for follow-up cytogenetics after a negative cfDNA result, 1 (4%) was false negative (T13). Two cases of triploidy, a very serious condition but one not claimed to be detectable by the test providers, were among those classified as true negatives. T21, has the highest prevalence of all aneuploidies, demonstrated a high rate, resulting in a high PPV. However, the other aneuploidies, with their lower prevalence, displayed relatively high false-positive rates and, therefore, lower PPV. Patients and physicians must fully understand the limitations of this screening test and the need in many cases to follow up with appropriate diagnostic testing to obtain an accurate diagnosis. trial
() Cohort III-2 5,267 Aim: To assess the implementation of CFDNA TESTING into clinical practice utilising both first- and second-line screening models. Setting: Metropolitan private practices in Australia Population: Singleton pregnancies in a mixed risk population Methods: cfDNA testing was offered as a first-line screen, ideally followed by combined first-trimester screening (cFTS), or as a second-line test following cFTS, particularly in those with a calculated risk between 1:50 and 1:1000. Outcomes: Detection of trisomies 21, 18 13, sex chromosome aneuploidies and other anomalies screening was performed in 5,267 women and as a first-line screening method in 3,359 (63.8%). Detection rates were 100% for trisomies 21 and 13 and 88% for trisomy 18. Of cases with known karyotypes, the positive predictive value (PPV) of the test was highest for trisomy 21 (97.7%) and lowest for monosomy X (25%). Ultrasound detection of structural anomaly resulted in the detection of five additional chromosome anomalies, two of which had high-risk cFTS results. For all chromosomal anomalies, cfDNA alone detected 93.4%, cfDNA in a model detected 81.8% and cFTS alone detected 65.9% (P < 0.005). If women had only had cFTS, the rate of invasive procedures would have been 5.5% higher (p<0.0001). Given the false-positive rate for all aneuploidies, cfDNA is an advanced screening test, rather than a diagnostic test. Study population may not be generalisable to the Australian obstetric community as women were predominantly older, Caucasian and from higher socioeconomic groups Retrospective cohort studies Trisomy 21 only Study ref Design LoE N Aim, setting, population, methods Results Comments
() Cohort III-2 675,332 Aim: to examine the screening performance of a trisomy 21 screening strategy based on maternal age, cFTS and cfDNA testing as well as the combinations maternal age and cfDNA and FTS and cfDNA. Setting: Germany Population: all births in 2012 Methods: a model-based approach was used to evaluate all births together with the percentage of euploid and trisomic pregnancies. Detection rates (DR), false positive rates (FPR), the costs of different screening strategies for trisomy 21 and combinations of these strategies were compared. The number of fetuses with trisomy 21 at 12+0 weeks of gestation was estimated based on maternal age distribution. Outcomes: Detection of trisomy 21 and Screening based on FTS (1:250) resulted in a detection rate (DR) of 92.2% and a false positive rate (FPR) of 8.0%. Screening based on cfDNA resulted in a DR of 99.9% and a FPR of 0.1%. If a 3% fail rate requiring subsequent invasive testing was postulated, the total FPR was 3.1% and the DR remained the same. When maternal age was combined with cfDNA (ie testing only offered to women over 30 years), this resulted in a DR of 85.2% and a FPR of 1.7%. Estimates were based on a detection rate of 99.9% and a false positive rate of 0.1% Trisomy 21 and other chromosomal anomalies ref Design LoE N Aim, setting, population, methods Results Comments
() Cohort study III-2 193,638 Aim: to determine the risk of missing other abnormal karyotypes of probable phenotypic significance by cfDNA testing. Setting: Denmark Population: singleton pregnancies booked for combined first trimester screening over a 4-year period Methods: Data concerning maternal demographics, cFTS and prenatal or postnatal karyotypes were collected from the Danish Medicine database. Karyotypes were classified according to whether the chromosomal anomaly would have been detected by cfDNA testing and whether it was likely to affect phenotype. 10,205 (5.3%) pregnancies had cytogenetic or molecular analysis performed. Of 1,122 (11.0%) had an abnormal karyotype, of which 262 (23.4%) would have been missed by CFDNA TESTING, but would probably have been clinically significant. The prevalence of such 'atypical abnormal karyotypes' was increased in women over 45 years of age, in pregnancies with increased nuchal translucency (NT) thickness (> 3.5 mm), with abnormal levels of free chorionic gonadotropin (<0.2 or > 5.0 multiples of the median [MoM]) or pregnancy-associated plasma MoM. One or more of these factors was present in 3% of women, and the prevalence of atypical abnormal karyotypes in this high-risk cohort was 1.6%. Study assumed that cfDNA testing would have 100% sensitivity for trisomies 21, 18 and 13 and sex chromosome aneuploidy.
() Cohort study III-2 143,051 Aim: To compare the number and types of chromosome anomalies prenatally diagnosed and the number of invasive procedures between current prenatal testing pathways (a combination of first or second trimester screening plus ultrasound and/or diagnostic testing) and a pathway where cfDNA testing replaces Down syndrome screening tests. Setting: Victoria, Australia Population: women participating in prenatal diagnostic testing Methods: Numbers and of chromosome anomalies for each referral category were extracted from prenatal diagnostic testing reports routinely collected in Victoria, Australia, in 2006 and 2007. These data were then applied to the proposed implementation strategy. If cfDNA testing for Down syndrome had replaced Down syndrome screening tests in 2006 and 2007, in Victoria, there would have been 25 (7%) additional Down syndrome diagnosed, a 28% reduction in detection of chromosomal anomalies. Specifically, 56% non-Down syndrome chromosome anomalies would no longer detected (including trisomy 13, trisomy 18, sex chromosome anomalies, balanced and unbalanced rearrangements, polyploidy, and mosaic results). With the inclusion of trisomy 13, trisomy 18, and sex chromosome aneuploidy in the cfDNA panel, the number of missed anomalies would decrease to 11%. There would have been 6,896 (84%) fewer invasive procedures with cfDNA testing for Down syndrome; 83% fewer with an aneuploidy panel.
() Modelling — 100,000 Aim: to conduct a quantitative analysis of different cfDNA testing strategies. Setting: The Netherlands Population: Theoretical cohort of pregnant women Methods: Decision were created to illustrate all plausible alternatives in five screening programmes: classical screening by the first-trimester combined test (FCT), pre-selection of high-risk women prior to cfDNA by the FCT, cfDNA as the first screening test at 10 weeks and at 13 weeks, and the simultaneous conductance of cfDNA and the FCT. Outcomes: Detection of trisomy 21, rates of amniocentesis and procedure-related fetal loss. cfDNA as the first screening test detects almost all fetal Down syndrome cases (87.5% at 10 weeks, 82.5% at 13 weeks compared to 78.5% with classical first trimester screening). Rates of fetal loss due to amniocentesis complications are lower (n=2 at 10 weeks or 13 weeks versus 25 with classical first trimester screening) as fewer women undergo amniocentesis (n=234 vs 4,056). Model assumed a sensitivity of 99.6% and specificity of 99.9% for cfDNA testing for trisomy 21 in high- and pregnancies and a sensitivity of 89% and specificity of 95.4% for cFTS.
() Cohort study III-2 99,619 Aim: To evaluate and benefits of different scenarios for second-line cfDNA screening. Setting: Belgium Population: Pregnancies screened by combined first trimester screening (78.5%) or second trimester screening (21.5%) in 2011 Methods: Data from the Belgian National Institute for Health and Disability Insurance and the Study Centre for Perinatal Epidemiology were used in modeled calculations of medical and economic impact of cfDNA testing after prior conventional screening (1) at thresholds 1:300 and 1:600, and (2) at current and improved screening sensitivity. Outcomes: Detection of trisomy 21, rate of procedure-related miscarriage, costs Second-line cfDNA screening under current screening conditions would maintain today's 7.90/000 live birth prevalence of Down syndrome (LBPD) at an 11% reduction of overall short-term costs. Lowering the screening threshold to 1:600 or increasing sensitivity by 10% would reduce LBPD to 70/000 at a maximum 3% increase of overall short-term costs. Rates of procedure-related miscarriage were lower with cfDNA than with current screening (0.03 vs 0.06%) with a risk threshold of 1:300 or 1:600. The study assumed that cfDNA testing has a sensitivity of 99.3% and a specificity of 99.84% for trisomy 21 ()
Trisomy 21 and other chromosomal anomalies Study ref Design LoE N Aim, setting, population, methods Results Comments () Modelling study — — Aim: To use a decision-analytic model to assess a comprehensive set of outcomes of prenatal genetic testing strategies among women of varying ages. Setting: United States Population: theoretical cohort of women desiring screening Methods: We assessed outcomes of six testing strategies incorporating diagnostic testing with chromosomal microarray, multiple marker screening, cell-free DNA screening, and nuchal translucency screening alone, in combination, or in sequence. Outcomes: Clinical outcomes included prenatal detection or birth of a neonate with a significant chromosomal anomaly and diagnostic procedures performed. Other outcomes included maternal life-years and costs. Sensitivity analyses were conducted to examine the robustness of the findings. At all ages assessed, screening strategies starting with multiple marker screening offered the highest detection rate when all chromosomal anomalies were considered Incorporating cfDNA as an optional secondary screen decreased the number of diagnostic procedures, but also decreased the number of anomalies diagnosed prenatally, resulting in a similar number of procedures per case diagnosed at age 30 years; the option of secondary cfDNA screening becomes more favorable at older ages. Potential conflict of interest Impact of cell-free fetal DNA testing on screening practices and invasive procedures Study ref Design LoE N Aim, setting, population, methods Results Comments adjusted
() Historical control III-3 15,418 tests Aim: To describe the changes over a 9-year period in the number and rate of diagnostic testing after the introduction of the combined first-trimester screen and subsequent cfDNA testing. Setting: United States Population: women participating in prenatal screening at a maternal-fetal medicine group practice. Methods: The number of prenatal screening and diagnostic tests was recorded over a period from billing records. Three time intervals were considered: 20 months before a combined first-trimester screen was offered; 72 months after a combined screen was offered; and 16 months after cfDNA testing introduction. Prenatal testing was compared per year, per time interval, and per 100 morphologic ultrasonograms to account for fluctuations in patient number. Outcomes: rates of combined first trimester screening, CVS and amniocentesis Combined screen peaked at 1,836 in 2009-2010 but declined by 48.1% after cfDNA testing was introduced. Combined first-trimester screen per 100 morphologic ultrasonograms also significantly decreased (P<0.05) after cfDNA testing introduction. CVS peaked after combined first-trimester screen introduction in 2007-2008 with 100 procedures, representing an 81.8% increase from prefirst-trimester screen. After the introduction of cfDNA, CVS declined by 68.6% during 2012-2013. CVS per 100 morphologic ultrasonograms followed the same trend. Amniocentesis declined every year of the study period (78.8% overall), including 60.3% after combined first-trimester screen and a further 46.7% after noninvasive prenatal testing. Monthly amniocentesis procedures per 100 morphologic ultrasonograms significantly decreased (P<.05) after introduction of a combined first-trimester screen and cfDNA. year
() Historical control III-3 Aim: to assess changes in rates of invasive diagnostic prenatal procedures associated with combined first trimester screening and cfDNA Setting: Australia Population: invasive procedures claimed through Medicare from 1994 to 2014 Method: analysis of data from the Medicare Australia Medical Benefits Scheme (MBS) database on procedural item numbers 16600 (diagnostic amniocentesis) and 16603 (chorionic villus sampling [CVS] by any route) for the period 1994 to 2014 inclusive, by calendar year Outcomes: changes in of invasive diagnostic tests Following the introduction of the number of amniocenteses performed in Australia fell by 51% between the first quarter of 2013 and the final quarter of 2014 (1,560 vs 758. OR 0.48; 95% CI: 0.44–0.53; 2 = 282, P<0.005). The total number of CVS fell by 37% over the same period (767 vs 481. OR 0.63; 95% CI: 0.56–0.70; 2 = 66, P<0.005). This represents the largest annual decrease in invasive procedures during the 20-year study period. This change has important implications for training in, and maintenance of, the procedural skills of amniocentesis and CVS. () Historical control III-3 500 Aim: To assess the impact of cfDNA testing on choice of invasive procedures. Setting: United States Population: Women with a positive first trimester screening result. Method: A retrospective chart review was performed. Data were collected prior to (2011) and after (2012) the introduction of cfDNA. Outcomes: gestational trimester at positive screen, whether cfDNA screening was requested or offered, and any additional tests pursued The percentage of participants who chose not to pursue further testing after a positive screen decreased significantly between 2011 and 2012, from 44% (110/250) to 32% (79/250) (p=0.006). Of those participants who chose to pursue additional testing in 2011, 47% (117/250) chose invasive testing (either CVS or amniocentesis) and only 29% chose invasive testing in 2012 (p<0.001).
() Historical control III-3 1,069 Aim: To investigate how the introduction of cfDNA testing influenced women's testing choices following a positive Down syndrome screening. Setting: Hong Kong Population: (largely Chinese) with a singleton pregnancy and a positive trisomy 21 screening (first trimester combined screen or second trimester screening depending on gestational age at presentation). Methods: With the use of descriptive statistics and a χ2 test, rates of women choosing the invasive test and those who declined further testing were compared before and after the introduction of cfDNA. We also compared demographic factors with rates of different options including an invasive test, cfDNA or ‘declined further testing’. Predictors of accepting cfDNA and an invasive test were analyzed with multiple logistic regression. Conventional screening was funded publicly, but cfDNA was not. Outcomes: differences in the uptake of invasive prenatal diagnosis (IPD) or no testing before and after the introduction of cfDNA and factors affecting choices. In multivariable analysis, first trimester screening, nulliparity and working women were significant predictors of accepting cfDNA, while only nulliparity was a predictor of declining IPD (OR = 0.61).
() analysis — 300,000 Aim: To evaluate the cost-effectiveness of different strategies of for trisomy 21 screening in comparison with current practice. Setting: Australia Population: A theoretical cohort of singleton pregnancies Methods: The strategies compared were the following: current practice, cfDNA as a second-tier investigation, cfDNA only in women >35 years, cfDNA only in women >40 years and cfDNA for all women. The direct costs (low and high estimates) were derived using both health system costs and patient out-of-pocket expenses. The number of trisomy 21 cases detected and losses (PRL) were compared between strategies. The incremental cost per case detected was the primary measure of cost-effectiveness. Universal cfDNA costs an additional $134,636,832 compared with current practice, but detects 123 more trisomy 21 cases (at an incremental cost of $1,094,608 per case) and avoids 90 PRL. for women >40 years was the most strategy, costing an incremental $81,199 per additional trisomy 21 case detected and avoiding 95 PRL. The cost of needs to decrease significantly if it is to replace current practice on a purely cost-effectiveness basis. However, it may be beneficial to use cfDNA as first-line screening in selected high-risk patients. Further evaluation is needed to consider the longer-term costs and benefits of screening. Study assumed a sensitivity of 98% and specificity of 97% for cfDNA testing. No failure rate assigned. Estimates based on costs of $102.95 for cFTS, $575 for cfDNA testing and $528.95 for invasive testing.
() analysis — Aim: To examine the cost and performance implications of introducing cell-free DNA (cfDNA) testing within modeled scenarios in a publicly funded Canadian provincial Down syndrome (DS) prenatal screening program. Setting: Canada Method: Two clinical algorithms were created: the to represent the current screening program and the second to represent one that incorporates cfDNA testing. From these algorithms, eight distinct scenarios were modeled to examine: (1) the current program (no cfDNA), (2) the current program with first trimester screening (FTS) as the nuchal translucency-based primary screen (no cfDNA), (3) a program substituting current screening with primary cfDNA, (4) contingent cfDNA with current FTS performance, (5) contingent cfDNA at a fixed price to result in overall cost neutrality,(6) contingent cfDNA with an improved detection rate (DR) of FTS, (7) contingent cfDNA with higher uptake of FTS, and (8) contingent cfDNA with optimized FTS (higher uptake and improved DR). This modeling study demonstrates that introducing contingent cfDNA testing improves performance by increasing the number of cases of trisomy 21 detected prenatally, and reducing the number of amniocenteses performed and concomitant iatrogenic pregnancy loss of pregnancies not affected by trisomy 21. Contingent models of cfDNA testing can improve overall screening performance while maintaining the provision of an 11- to 13-week scan. Costs are modestly increased, but cost per prenatally detected case of DS is decreased