Sage Journals: Discover world-class research

Abstract

Objective

Maternal plasma DNA analysis has a high but imperfect antenatal Down’s syndrome screening performance. We aimed to determine the effect of combining DNA testing with current tests.

Methods

In our modelled screening protocol, women provide two samples, one serum sample for a Combined test, and a plasma sample for a possible DNA test. Women with a Combined test risk above a specified level have a DNA test using the plasma sample without the need to recall them for another sample and counselling (ie. in a reflex manner). Women with a failed DNA test after a second attempt using a fresh plasma sample have an Integrated test. Screening performance was estimated according to the proportion of women reflexed to DNA testing and compared with universal DNA testing.

Results

Reflexing 10% of women to a DNA test yields a 91% detection rate (DR) for a 0.025% false-positive rate (FPR) and no failed tests, compared with a 98% DR, 0.2% FPR and a 2.5% test failure rate with universal DNA testing (94% for 0.046% if 20% reflexed). DNA test failure rate has little influence on screening performance

Conclusion

Reflex DNA testing substantially reduces the FPR with a relatively small loss in detection compared with universal DNA testing, and reduces patient anxiety by avoiding the recall of women for DNA testing.

Keywords

Down’s syndrome antenatal screening DNA analysis reflex testing maternal plasma

Introduction

Maternal plasma DNA analysis has the highest screening performance of any single test used in the antenatal detection of Down’s syndrome.^1–11 These DNA testing methods achieve a Down’s syndrome detection rate (proportion of Down’s syndrome pregnancies with positive results) (DR) of 98–99% for a false-positive rate (proportion of unaffected pregnancies with positive results) (FPR) of about 0.2%, but, in a few percent of samples received, the test fails. The precise screening performance and failure rate is likely to depend on the particular DNA analysis used. The FPR, though low, is not low enough for the test to be used diagnostically; chorionic villus sampling (CVS) or amniocentesis is still needed to make the diagnosis.

Incorporating a DNA test into current screening methods, so that only a proportion of women have a DNA test, could have medical advantages as well as saving costs compared with universal DNA testing, by substantially reducing the FPR, with a relatively small reduction in DR. We conducted a modelling study to investigate this, by estimating the DRs and FPRs according to the percentages of women that have a DNA test on the basis of the screening result from a first trimester Combined test (free β-human chorionic gonatrophin [hCG], pregnancy associated plasma protein-A [PAPP-A], and nuchal translucency [NT] with maternal age). Collecting a plasma sample at the same time as the serum sample and using a “reflexing” approach^12,13 in which, if necessary, the second blood sample is automatically used for DNA testing avoids the need to recall women for counselling and the collection of another blood sample. We here estimate the screening performance of this reflexing protocol (see Figure 1) and consider the implications. We compare its screening performance with screening using the Combined test or the Integrated test (first trimester PAPP-A and NT with second trimester free β-hCG, alphafetoprotein [AFP], unconjugated oestriol [uE₃], and inhibin-A). We also compare it with screening based on universal DNA testing.

Figure 1.

Flow chart of reflex DNA testing with the Combined test.

Methods

The screening performance of reflex DNA testing was estimated using the multivariate Gaussian distribution parameters (means, standard deviations and correlation coefficients) of the screening markers from a large cohort study (the Serum Urine and Ultrasound Screening Study [SURUSS]),¹⁴ revised to incorporate subsequent improvements.¹⁵ Two million Down’s syndrome pregnancies and two million unaffected pregnancies were simulated, each with a set of marker values (first trimester marker values measured at 11, 12, or 13 completed weeks’ gestation). Each simulated pregnancy was assigned a maternal age based on the distribution of maternities in England and Wales from 2006 to 2008¹⁶ inclusive and the maternal age-specific odds of an affected livebirth.^17–19

For each simulated pregnancy, a risk of being affected with Down’s syndrome based on the Combined test was calculated by multiplying the maternal age specific odds of having an affected live birth by the likelihood ratio of being affected (for the simulated set of marker values) which were calculated from the multivariate Gaussian distributions of NT, free β-hCG and PAPP-A levels in affected and unaffected pregnancies. The risk cut-offs for proportions reflexed to DNA testing from 1% to 99% were determined. A DNA result for those with a risk greater than the calculated risk cut-off levels was generated. The DNA test failure rate was taken as 2.5%, assuming a higher initial failure rate of 5%, with half of these successfully re-tested in a fresh blood sample collected later in pregnancy. Of those tests in which a DNA result was assumed to have been successfully obtained, the screening performance was taken from Palomaki et al,² so 98.6% of the simulated Down’s syndrome pregnancies and 0.2% of the simulated unaffected pregnancies were randomly classified as being screen positive based on DNA sequencing. If the DNA test failed, an Integrated test was performed, re-using the first trimester Combined test markers with the second trimester markers AFP, uE₃ and inhibin-A in the multivariate Gaussian model to obtain an Integrated risk estimate of Down’s syndrome. Pregnancies with an Integrated test risk greater than or equal to 1 in 25 were classified as being screen positive. Overall DRs and FPRs were estimated, and compared with the corresponding estimates for women having either a Combined test or an Integrated test only (no DNA testing) and with the estimates for universal DNA testing. The latter involves either offering women with failed tests (i) a diagnostic amniocentesis (or CVS), or (ii) a second trimester Quadruple screening test (AFP, uE₃, hCG, inhibin-A, and maternal age). The results are given for women having first trimester markers measured at 11, 12, or 13 completed weeks of pregnancy. All analyses were performed in Stata version 12.

Results

Figure 2 is a flow diagram of the reflex screening protocol, in which 10% of women have a DNA test. The DR is 91% and the FPR is 0.025%. Figure 3 shows the DR and FPR according to the percentage of women reflexed to a DNA test. As this increases, the DR increases (upper part of figure 3), as well as the FPR (lower part of figure 3). Figure 4 shows the DRs and FPRs according to three policies: (i) no DNA testing (Combined or Integrated test only), (ii) reflex DNA testing with 10% or 20% of women reflexed to a DNA test, and (iii) universal DNA testing, with women who have DNA test failures offered an amniocentesis (or CVS) or have the second trimester Quadruple screening test. The figure shows that the reflexing policy has a relatively small loss in detection but a substantial reduction in the FPR compared with universal DNA testing. For example, a universal DNA testing policy with test failures having an amniocentesis (or CVS) has a DR of 98.7% and a FPR of 2.70%, compared with a DR of 94.2% and a FPR of 0.046% with a 20% reflexing policy.

Figure 2.

Reflex DNA screening with the Combined test: 10% of women have DNA test. First trimester markers measured at 11 completed weeks of gestation (DR = detection rate; FPR = false-positive rate).

Figure 3.

Detection rate and false-positive rate according to proportion of women reflexed to DNA testing; DNA test linked to Combined test. First trimester markers measured at 11 completed weeks of gestation.

Figure 4.

Detection rate and false-positive rates of selected screening policies. First trimester markers measured at 11 completed weeks of gestation.

Table 1 shows DRs and FPRs according to the proportion of women reflexed, together with the first trimester risk cut-off required to achieve the specified reflexing proportions according to the week of pregnancy (11, 12, or 13 completed weeks). Table 2 shows, for measurements at 11, 12, and 13 weeks of pregnancy, the reflexing proportion according to the Combined test risk cut-off. The reflexing proportion increases as the risk cut-off is decreased and is greater with increasing gestation.

Table 1.

Reflex DNA screening linked to the Combined test: detection rate (DR) and false-positive rate (FPR) according to proportion of women reflexed to DNA testing (with risk cut-off specified) and completed week of gestation for the Combined test (DNA test failures have Integrated test using a risk cut-off 1 in 25).

Women reflexed (%)	11 completed weeks			12 completed weeks			13 completed weeks
Women reflexed (%)	Risk cut-off 1 in:	DR (%)	FPR (%)	Risk cut-off 1 in:	DR (%)	FPR (%)	Risk cut-off 1 in:	DR (%)	FPR (%)
1	65	75.0	0.006	65	73.3	0.006	58	68.6	0.006
2	130	80.3	0.008	129	78.4	0.009	113	74.5	0.010
3	200	83.2	0.011	196	81.3	0.011	170	78.0	0.012
4	275	85.2	0.013	265	83.4	0.013	229	80.4	0.015
5	353	86.6	0.016	338	84.9	0.015	290	82.3	0.016
6	437	87.8	0.017	414	86.2	0.018	355	83.8	0.018
7	524	88.7	0.020	494	87.2	0.020	423	85.0	0.020
8	617	89.5	0.022	578	88.1	0.021	494	86.1	0.023
9	715	90.2	0.023	665	88.8	0.024	567	87.0	0.024
10	815	90.8	0.025	756	89.5	0.026	644	87.7	0.027
15	1384	92.9	0.035	1265	91.9	0.036	1076	90.6	0.036
20	2075	94.2	0.046	1874	93.4	0.045	1596	92.5	0.047
25	2905	95.1	0.056	2594	94.5	0.056	2221	93.7	0.056
30	3885	95.8	0.065	3455	95.3	0.066	2969	94.7	0.067
35	5045	96.3	0.076	4464	95.9	0.075	3860	95.4	0.076
40	6418	96.7	0.086	5649	96.3	0.085	4926	96.0	0.085
45	8050	97.0	0.095	7058	96.7	0.094	6193	96.5	0.097
50	9984	97.2	0.104	8739	97.0	0.103	7739	96.8	0.106
60	15118	97.6	0.124	13190	97.5	0.125	11983	97.4	0.125
70	22839	97.9	0.144	20010	97.8	0.144	18859	97.7	0.144
80	35623	98.0	0.163	31699	98.0	0.163	31615	97.9	0.165
90	61403	98.1	0.182	57453	98.1	0.181	63371	98.0	0.183
99	168893	98.2	0.200	189035	98.1	0.200	258482	98.1	0.201

Table 2.

Reflexing proportion according to first trimester Combined test risk cut-off and week of pregnancy.

Risk cut-off 1 in:	Women reflexed (%)
Risk cut-off 1 in:	11 weeks	12 weeks	13weeks
100	1.5	1.6	1.8
200	3.0	3.1	3.5
300	4.3	4.5	5.2
400	5.6	5.8	6.7
500	6.7	7.1	8.1
600	7.8	8.3	9.4
700	8.9	9.4	10.7
800	9.9	10.5	11.9
900	10.8	11.5	13.1
1000	11.7	12.5	14.2
1100	12.6	13.5	15.2
1200	13.5	14.4	16.3
1300	14.3	15.3	17.3
1400	15.1	16.2	18.2
1500	15.9	17.0	19.1
1600	16.7	17.9	20.0
1700	17.4	18.7	20.9
1800	18.1	19.4	21.7
1900	18.8	20.2	22.5
2000	19.5	20.9	23.3

Discussion

Our results show that a high screening performance can be achieved through reflex DNA testing, with a FPR about one-eighth that of universal DNA testing, with a relatively small loss in detection.

The DNA test failure rate in the context of a reflexing policy has little impact on overall screening performance, for example if the rate were 1.25% instead of 2.5%, the DR would be 90.9% instead of 90.8% and the FPR 0.023% instead of 0.025%, and if the failure rate were 5%, the corresponding DR would be 90.5% and FPR 0.030%. With a universal DNA testing policy, test failures is a problem (see figure 4). If DNA testing had no false-positives, no failed tests, and was inexpensive, universal DNA testing could replace current screening methods. At present this is not the case, so a combination of current routine methods of screening and DNA testing offers the most effective approach.

Reflex DNA screening results in a lower FPR than universal DNA testing because the DNA test is limited to a subset of the women screened, namely those with a Combined test risk high enough to lead to a DNA test. In the example shown in Figure 4, with 10% reflexed to DNA testing, and with a DNA test FPR of 0.2%, the use of both in sequence yields an expected testing of 0.2% of 10%. With an Integrated test performed when the DNA test fails, this increases the FPR from 0.02% to 0.025%

With a DNA reflexing approach,^12,13 plasma samples are collected from all women screened at the time of blood collection for the measurement of first trimester markers hCG and PAPP-A at 11, 12, or 13 weeks of pregnancy. Then women selected for DNA testing do not need to be approached; their stored plasma samples are retrieved for DNA testing. The screening strategy is explained and consent obtained when screening is initially offered. Others have reported on the advantages of incorporating DNA testing into current antenatal screening practice^20–22 but have not suggested a reflexing approach. Without such an approach, women would need to be recalled for a DNA test, causing them anxiety through being singled out for further testing. The rationale for the reflexing approach is that it minimizes the FPR, and therefore the anxiety arising from screening, without loss of screening performance. It comes with an increased cost of having to collect and store plasma samples from all women, but only testing some. However, it avoids the cost of having to recall women for another blood sample.

The estimates in Table 2, linking the Combined test risk cut-off to the reflexing proportion can help to plan a reflex DNA screening programme, influenced also by the extent to which the DNA test failure rate is influenced by gestation.

Our statistical analysis is based upon the DNA result being categorical (positive or negative) and does not take account of the risk based on the result of the Combined test. An analysis that takes account of the Combined test risk produces almost identical screening results. For example with a reflexing proportion of 20% yielding an overall DR of 94.2%, the FPR is 0.045% instead of 0.046%. While combining information from two informative tests will yield a better screening performance than from either test alone, the discrimination of the DNA test is so much greater than that of the Combined test, adding quantitative information from the Combined test to a categorical DNA result has little effect. In expectation, the performance of such sequential screening would be improved if the DNA results were expressed quantitatively, rather than categorically. To this end, it would be valuable if, in future, DNA results were expressed in the underlying units of measurement (for example, percentage DNA fragments mapped to chromosome 21), and the distributions of this measurement determined in affected and unaffected pregnancies, as has been done in the past with conventional serum markers, such as hCG.

If women with failed DNA tests were automatically offered an amniocentesis (or CVS), the FPR would be approximately equal to the test failure rate, but the increase in the DR would be negligible (eg. from 98.00 to 98.01%), If women with failed tests were not tested further, the DR would decrease by an amount equal to the test failure rate (eg. from 98 to 96.5%) and the FPR would be unchanged.

To be fully successful, reporting a DNA test result should not take longer than it takes to report a Combined test result. If it takes much longer, women will suspect that their risk was sufficiently high to warrant a DNA test and as a result may become worried. If the DNA test does take significantly longer to report, reflex DNA testing can be linked to the Integrated test. Since women are scheduled to provide a second blood sample 3–4 weeks after the first, there is no anxious waiting period for a test result. DNA results can be reported before the second stage of the Integrated test and obviate the need for a second sample. Screening performance of reflex DNA testing linked to the Integrated test is similar to that linked to the Combined test and may be an option for centres that already have established routine Integrated screening programmes. The appendix provides quantitative estimates of screening performance of reflex DNA testing linked to the Integrated test.

The reflexing DNA policy could also be adopted in relation to the second trimester Quadruple test if first trimester screening markers are not available, in which case the requirements for a rapid DNA test become even greater. This achieves a 94% DR for a 0.10% FPR with 20% of women having a reflex DNA test, a DNA test failure rate of 2.5% and in women with a failed DNA test, a Quadruple test risk cut-off of 1 in 100 to determine which women are offered an amniocentesis.

In summary, reflex DNA screening reduces the FPR substantially, and hence the anxiety arising from screening, with a relatively small loss of detection compared with universal DNA testing, and avoids the problem of failed tests. Demonstration projects in which reflex DNA testing is introduced in routine antenatal screening programmes, coupled with appropriate clinical audit, would be useful.

Footnotes

Conflict of interest disclosure

Nicholas Wald holds patents for the Integrated test, and is Director of Logical Medical Systems Ltd, which produces software for the interpretation of Down’s syndrome screening tests.

References

Chiu

RWK

Akolekar

Zheng

YWL

. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 2011; 342: c7401–c7401.

Palomaki

Kloza

Lambert-Messerlian

. DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study. Genet Med 2011; 13: 913–920.

Bianchi

Platt

Goldberg

. Maternal Blood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) Study Group. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol 2012; 119: 890–901.

Sparks

Stuble

Wang

. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012; 206: 319.e1–9–319.e1–9.

Nicolaides

Syngelaki

Ashoor

. Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol 2012; 207: 374.e1–6–374.e1–6.

Dan

Wang

Ren

. Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11105 pregnancies with mixed risk factors. Prenat Diag 2012; 32: 1225–1232.

Zimmerman

Hill

Gemelos

. Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X and Y, using targeted sequencing of polymorphic loci. Prenat Diagn 2012; 32: 1233–1241.

Bianchi

Parker

Wentworth

. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med 2014; 370: 799–808.

Norton

Brar

Weiss

. Non-invasive chromosomal evaluation (NICE) study: results of a multicentre prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012; 207: 137.e1–8–137.e1–8.

10.

Chiu

RWK

. Noninvasive prenatal testing by maternal plasma DNA analysis: Current practice and future applications. J Clin Lab Invest 2014; 74(Supp 244): 48–53.

11.

Pergament

Cuckle

Zimmermann

. Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort. Obstet Gynecol 2014; 124: 210–8.

12.

Wald

. Community Corner: Opening the Pandora’s box of prenatal genetic testing. Nature Medicine 2011; 17: 250–251.

13.

Wald NJ, Bestwick JP. Incorporating DNA Sequencing into Current Prenatal Screening Practice for Down’s Syndrome. PLoS ONE 8:e58732. doi:10.1371/journal.pone.0058732.

14.

Wald

Rodeck

Hackshaw

. First and Second trimester Antenatal screening for Down's syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen 2003; 10: 56–104.

15.

Wald

Bestwick

Huttly

. Improvements in antenatal screening for Down’s syndrome. J Med Screen 2013; 20: 7–14.

16.

Office for National Statistics Birth Statistics. England and Wales; 2006, 2007, 2008 (FM1, Nos 35–37). London: The Stationary Office, 2008.

17.

Morris

Mutton

Alberman

. Revised estimates of the maternal age specific live birth prevalence of Down’s syndrome. J Med Screen 2002; 9: 2–6.

18.

Morris

Wald

Mutton

. Comparisons of models of maternal age-specific risk for Down syndrome live births. Prenat Diagn 2003; 23: 252–258.

19.

Morris

Mutton

Alberman

. Corrections to maternal age-specific live birth prevalence of Down’s syndrome. J Med Screen 2005; 12: 202–202.

20.

Okun

Teitelbaum

Huang

. The price of performance: a cost and performance analysis of the implementation of cell-free fetal DNA testing for Down syndrome in Ontario, Canada. Prenat Diagn 2013; 34: 350–6.

21.

Nicolaides

Wright

Poon

. First-trimester contingent screening for trisomy 21 by biomarkers and maternal blood cell-free DNA testing. Ultrasound Obstet Gynecol 2013; 42: 41–50.

22.

Morris

Karlsen

Chung

. Model-based analysis of costs and outcomes of non-invasive prenatal testing for Down’s syndrome using cell free fetal DNA in the UK National Health Service. PLoS One 2014; 9(4): e93559. doi: 10.1371/journal.pone.0093559.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.38 MB

Performance of antenatal reflex DNA screening for Down’s syndrome

Abstract

Objective

Methods

Results

Conclusion

Keywords

Introduction

Methods

Results

Discussion

Footnotes

Conflict of interest disclosure

References

Supplementary Material