The reflex DNA testing protocol proposed here, in which the first part of an Integrated test is used to determine who should receive a DNA test, has a number of advantages. First, it has a high screening performance; while the detection rate may be about five percentage points lower than routine DNA testing, the false-positive rate is about 30 times lower if DNA test failures are considered positive and these women have a diagnostic amniocentesis, or about 3 times lower if these women have a second trimester Quadruple test. Second, all women receive a screening result; there are no failed tests and there is no need to tell some women that they had a failed DNA test and needlessly cause them anxiety. Third, the cost of DNA sequencing for the programme is reduced by 80 or 90%, depending on which of the options is adopted. The cost of continuing the Integrated test would remain, which in a public service context is available at the Wolfson Institute of Preventive Medicine, London, for £35 (about $50), but at present this is substantially less than all women having a DNA test. Fourth, the very low false positive rate means that only about 3 per 1000 women screened would need a diagnostic amniocentesis and in about 2 in 3 a Down's syndrome pregnancy would be diagnosed. Fifth, such a protocol allows for the screening of other pregnancy complications such as pre-eclampsia, identified using immunoassays or heart defects using ultrasound markers.
In our analysis we used the results of Palomaki and colleagues (DNA test detection rate of 98.6% and false-positive rate of 0.2% 
) and a DNA test failure rate of 3%. A sensitivity analysis showed that our estimated overall detection rate of 94.8% and overall false-positive rate of 0.08% is robust to reported variations in the DNA test detection rates, false-positive rates, and test failure rates. For DNA detection rates between 97.5% and 99.5%, and false-positive rates between 0.1% and 0.3%, and DNA test failure rates between 1% and 5%, the overall detection rates were between 93.6% and 95.8% and the overall false-positive rate between 0.05% and 0.12%.
In a typical Integrated test free β-hCG is not measured in the first trimester, but either total or free β-hCG is measured in the second trimester. In the proposed protocol, the overall screening performance is marginally better by measuring hCG earlier; if total hCG were measured in the second trimester and only an NT and PAPP-A measured in the first trimester, the overall detection rate would be 93.8% (instead of 94.8%) and the overall false-positive rate 0.12% (instead of 0.08%) if 20% of women were selected for a reflex DNA test. There is no advantage in measuring hCG in both the first and second trimesters. The measurement of additional markers such as serum placental growth factor, or ultrasound ductus venosus blood flow or nasal bone would improve the screening performance of the proposed protocol, but these are not routinely used, and therefore are not considered here.
Most of the published studies on DNA sequencing as a screening test for Down's syndrome were performed on women who were, for one reason or another, at higher than average risk, but this is not a source of bias as the detection rate and false-positive rate of screening tests using markers that are the consequence of the disorder are independent of the prevalence of the disorder.
As experience is gained with the screening protocol proposed in this paper, and if the cost of DNA testing falls, with a lower rate of failed tests, DNA testing could be offered to a larger proportion of pregnancies. At the same time the need for immunoassays and ultrasound measurements currently used to screen for Down's syndrome can be reviewed to assess their value in screening for other disorders such as pre-eclampsia.
provides an indication of the costs of the proposed protocol with varying proportions having a DNA test. Expressing cost in multiples of the cost of the Integrated test makes the table generally applicable, so that, if a 4 to 5 fold greater cost per woman screened were acceptable, a 20% DNA testing percentage could be done if the DNA test were 20 times more costly than the Integrated test, or 40% if 10 times more costly. As can be seen from , the marginal cost of detecting the extra 4% (from 95% to 99%) of Down's syndrome pregnancies that would be detected if all women had DNA testing rather than adopting the proposed protocol would be extremely high, requiring about 80,000 extra DNA tests (100,000 – 20,000) for each extra Down's syndrome pregnancy detected (if 20% have reflex DNA testing). This would remain the case unless the cost of a DNA test were substantially reduced. Collecting plasma samples for DNA testing that may not be used is, of course, an expense, but a small one compared with routine DNA testing.
Reflex DNA testing with the Combined test rather than the Integrated test yields a similar screening performance (see Table S1
) with a similar overall cost (see Table S2
). The principal disadvantage in using reflex DNA testing with the first trimester Combined test instead of the first stage of the Integrated test is that it would generate a burden of anxiety in a significant number of women. This is because a negative Combined test result would be informed immediately if testing were done at the time of the ultrasound NT measurement or within a day or two if done in a central laboratory. However, because many would regard it to be wrong to artificially delay the reporting of a negative screening result, a “positive” would trigger a DNA test that would take 1–2 weeks, so if women did not receive a prompt result they would realize that they were in a higher risk category. If 20% were selected for a DNA test, as shown in Figure S2
, this would apply to 20% of women in the population. If, however, as we suggest DNA testing were conducted together with the Integrated test the corresponding proportion would be substantially less than 1%.
A feature of the proposed screening protocol is that it applies equitably to all pregnant women with the cost averaged over all women screened, regardless of who has a DNA test. This is a practical approach in the context of a screening programme, which is the appropriate way of delivering the service to a population.
Our proposed protocol could usefully be linked to interpretive software that would automatically modify the proportion of women selected for reflex DNA testing and provide the appropriate risk cut-off to achieve this. Also, all women who have a DNA test could receive an estimate of the risk of having a Down's syndrome pregnancy, based on a combination of the DNA test result and the first stage of the Integrated test, so the results would be quantitative and not simply be based on a qualitative DNA result. In deriving this risk, the DNA result could be adjusted for relevant factors such as the fetal DNA fraction and maternal weight.
In summary, the proposed protocol combines current screening methods with the newer DNA sequencing methods to provide a cost effective strategy for all pregnant women with a very high level of efficacy and safety.