This implementation project showed that routine reflex DNA screening for trisomies 21, 18, and 13 achieved a detection rate of 95% with a false-positive rate of 0.02% and an odds of being affected given a positive result of 25:1. Only 2 in 10,000 women with unaffected pregnancies had an invasive diagnostic test. No other method of prenatal screening for these disorders has such a high detection rate for such a low false-positive rate. While the detection rate is a few percentage points lower than with universal DNA screening,9,10,12 the greater proportional reduction in the false-positive rate results in a greater discrimination between affected and unaffected pregnancies.

Chitty et al.3 described a similar two-step screening protocol but, instead of performing a reflex DNA test on a previously collected plasma sample, women with a combined test risk ≥1 in 1,000 were recalled for counseling with the offer of a DNA screening test or, if the risk was ≥1 in 150, the choice of a DNA screening test or an invasive diagnostic test. Twelve percent of women were recalled in this way and informed that they were in this higher risk group, and 18% chose to proceed directly to an invasive diagnostic test. This increases the false-positive rate and consequently also increases the number of invasive diagnostic tests in women with unaffected pregnancies; this is avoided with the reflex method. We can be confident that the reflex DNA screening strategy benefits women by reducing the chance that they will be made acutely anxious. Measuring anxiety levels directly in such circumstances is, in our view, neither appropriate nor necessary; imparting potentially distressing information when this can be completely avoided is self-evidently of benefit.

The technical DNA test failure rate is a problem with DNA screening. The reflex DNA approach with 10% of women having a DNA test means that, among all women screened, about 2 per 1,000 (10% × 1.8%) needed to have an extra blood collection, a much lower recall rate than with contingent DNA screening without reflexing.3 The 68% reduction in the failure rate between tests using the first and second aliquot from the initial blood sample indicates that the failure is mainly technical, and not due to factors associated with the woman and her pregnancy. As the technical aspects of the test improve, the initial failure rate is likely to fall significantly. With the reflex DNA screening approach, all pregnancies have a screening result and in this implementation project only 3 pregnancies out of 2,480 reflexed to a DNA test (0.12%) had an integrated test after a DNA test failure using the second aliquot of the extra blood collection.

Compared with established screening methods, reflex DNA screening reduces the clinical workload involved in counseling women with screen-positive results; in the implementation audit 105 (101 + 4 from Table 2) women required counseling following a screen-positive result whereas 635 (549 + 86) would have required counseling if the combined test alone or the two-step (recall) method 3 had been used. The two-step method 3 will further increase the clinical workload because in addition to counseling after a positive combined test result, some women would need counseling again after a positive DNA test result.

Women identified as being screen positive in the implementation audit had a high odds of having an affected pregnancy (25:1, see Table 2), which is likely to reduce uncertainty over the decision to have an invasive diagnostic test (1:6 with the combined test alone, see Table 2). Reflex DNA screening can potentially achieve cost savings because of the reduction in the number of invasive diagnostic tests needed and the reduced need for patient counseling associated with the two-step approach. These savings could be used to pay for the reflex DNA screening tests to secure the clinical benefits. Depending on local costs, a combined test cutoff level could be selected so that the costs of the screening program are affordable and cost-effective. As the cost of reflex DNA screening declines, the combined test cutoff can be lowered, resulting in an increase in the proportion of women having a reflex DNA test and hence an increase in the detection rate.

In this implementation project Streck tubes were used to reduce white blood cell lysis and cellular DNA leakage into the plasma of whole blood before plasma separation. The effect of such leakage would be to diminish the fetal fraction and possibly increase the proportion of failed tests. The use of inexpensive ethylenediaminetetraacetic acid (EDTA) tubes rather than the more expensive Streck tubes would lead to cost savings. Such a switch should be acceptable as there is evidence that the separation of plasma from cells up to at least 48 hours after blood collection does not significantly degrade the sample needed for DNA analysis.13,14

Integrating information from combined test markers with sequencing information from DNA analysis enhances screening performance. While the improvement in screening performance is small, with suitable interpretive software this can readily be implemented without additional cost. A source of false positives associated with the DNA test arises from maternal mosaicism,15 confined placental mosaicism,16 and maternal copy-number variation.17 Though they are rare occurrences, this problem is mitigated in reflex DNA screening. For example, if 10% of women have a reflex DNA test the problem is reduced 10-fold. A practical point affecting any reflex DNA screening program is that the invasive diagnostic test should be amniocentesis, not chorionic villus sampling, which will replicate the confined placental mosaicism observed in the maternal plasma.

With universal DNA screening the detection rate would have been 99%, but an extra blood sample would be required for a repeat DNA test in 1.8% of pregnancies based on the failure rate in pregnancies tested at the Wolfson Institute (see Figure 3). With the reflex approach, in which 10% have a DNA test, the detection rate is 95% with a 10-fold lower rate of requesting an extra blood collection (0.18%) than universal DNA screening. There is a trade-off between small incremental increases in detection for increasing proportions of women required to provide an extra blood sample. In every 100,000 pregnancies undergoing reflex DNA screening, based on a 10% reflexing proportion, 180 would be recalled for an extra blood collection compared with 1,800 such return visits with universal DNA screening.

The reflex DNA policy makes prenatal screening for trisomies 21, 18, and 13 safer than other policies because of the reduced false-positive rate. Taking the risk of fetal loss due to an invasive diagnostic test as 1 in 100,18 among 1 million unaffected pregnancies that undergo reflex DNA screening, 200 would have a diagnostic amniocentesis and about 2 of these would result in a fetal loss due to the diagnostic procedure. If all pregnant women were screened using the reflex DNA approach, this would amount to eight procedure-related unaffected fetal losses in the United States and about two in the United Kingdom, each year. If the fetal loss rate from an invasive diagnostic test is less than 1 in 100, these estimates of the number of procedure-related fetal losses would be even lower. As well as improved safety, 19 out of 20 pregnancies with trisomy 21, 18, or 13 are detected by reflex DNA screening.

The benefits of reflex DNA screening arise mainly from the substantially lower false-positive rate compared with other methods of screening, the avoidance of recall-induced anxiety associated with non-reflex contingent screening, and a detection rate similar to universal DNA testing. These clinical benefits, together with the reduced cost compared with universal DNA testing, make the reflex approach a preferred method of screening. The results of this implementation project show that the benefits of reflex DNA screening were achieved in routine screening practice.