Urine tests for Down's syndrome screening

Down’s syndrome

Down’s syndrome affects approximately one in 800 live-born babies (Cuckle 1987a). It results from a person having three, rather than two, copies of chromosome 21—or the specific area of chromosome 21 implicated in causing Down's syndrome, as a result of trisomy or translocation. If not all cells are affected, the pattern is described as 'mosaic'. Down’s syndrome can cause a wide range of physical and mental problems. It is the commonest cause of mental disability, and is also associated with a number of congenital malformations, notably affecting the heart. There is also an increased risk of cancers such as leukaemia, and numerous metabolic problems including diabetes and thyroid disease. Some of these problems may be life-threatening, or lead to considerable ill health, while some individuals with Down’s syndrome have only mild problems and can lead a relatively normal life.

There is no cure for Down’s syndrome, and antenatal diagnosis allows for preparation for the birth and subsequent care of a baby with Down’s syndrome, or for the offer of a termination of pregnancy. Having a baby with Down’s syndrome is likely to have a significant impact on family and social life, relationships and parents’ work. Special provisions may need to be made for education and care of the child, as well as accommodating the possibility of periods of hospitalisation.

Definitive invasive tests (amniocentesis and chorionic villus sampling (CVS)) exist that allow the diagnosis of Down's syndrome before birth, but carry a risk of miscarriage. No test can predict the severity of problems a person with Down’s syndrome will have. Noninvasive screening tests based on biochemical analysis of maternal serum or urine, or fetal ultrasound measurements, allow an estimate of the risk of a pregnancy being affected and provide parents with information to enable them to make choices about definitive testing. Such screening tests are used during the first and second trimester of pregnancy.

Screening tests for Down's syndrome

Initially, screening was determined solely by using maternal age to classify a pregnancy as high or low risk for trisomy 21, as it was known that older women had a higher chance of carrying a baby with Down’s syndrome (Penrose 1933).

Further advances in screening were made in the early 1980s, when Merkatz et al investigated the possibility that low maternal serum alpha-fetoprotein (AFP), obtained from maternal blood in the second trimester of pregnancy could be associated with chromosomal abnormalities in the fetus. Their retrospective case-control study showed a statistically significant relationship between fetal trisomy, such as Down’s syndrome, and lowered maternal serum AFP (Merkatz 1984). This was further explored by Cuckle et al in a larger retrospective trial using data collected as part of a neural tube defect (NTD) screening project (Cuckle 1984). This work was followed by calculation of risk estimates using maternal serum AFP values and maternal age, which ultimately led to the introduction of the two screening parameters in combination (Alfirevic 2004).

In 1987, in a small case-control study of women carrying fetuses with known chromosomal abnormalities, Bogart and colleagues investigated maternal serum levels of human chorionic gonadotrophin (hCG) as a possible screening tool for chromosomal abnormalities in the second trimester (Bogart 1987). This followed the observations that low hCG levels were associated with miscarriages, which are commonly associated with fetal chromosomal abnormalities. They concluded that high hCG levels were associated with Down’s syndrome and because hCG levels plateau at 18 to 24 weeks, that this would be the most appropriate time for screening. Later work suggested that the ß sub-unit of hCG was a more effective marker than total hCG (Macri 1990; Macri 1993).

Second trimester unconjugated oestriol (uE3), produced by the fetal adrenals and the placenta, was also evaluated as a potential screening marker. In another retrospective case-control study, uE3 was shown to be lower in Down’s syndrome pregnancies compared with unaffected pregnancies. When used in combination with AFP and maternal age, it appeared to identify more pregnancies affected by Down’s syndrome than AFP and age alone (Canick 1988). Further work suggested that all three serum markers (AFP, hCG and uE3) showed even higher detection rates when combined with maternal age (Wald 1988a; Wald 1988b) and appeared to be a cost-effective screening strategy (Wald 1992a).

Two other serum markers, produced by the placenta, have been linked with Down’s syndrome, namely pregnancy-associated plasma protein A or PAPP-A, and Inhibin A. PAPP-A has been shown to be reduced in the first trimester of Down’s syndrome pregnancies, with its most marked reduction in the early first trimester (Bersinger 1995). Inhibin A is high in the second trimester in pregnancies affected by Down’s syndrome (Cuckle 1995a; Wallace 1995). There are some issues concerning the biological stability and hence reliability of this marker, and the effect this will have on individual risk.

In addition to serum and ultrasound markers for Down’s syndrome, work has been carried out looking at urinary markers. These markers include invasive trophoblast antigen, ß-core fragment, free ßhCG and total hCG (Cole 1999a). There is controversy about their value (Wald 2003a).

Screening and parental choice

Antenatal screening is used for several reasons (Alfirevic 2004), but the most important is to enable parental choice regarding pregnancy management and outcome. Before a woman and her partner opt to have a screening test, they need to be fully informed about the risks, benefits and possible consequences of such a test. This includes the choices they may have to face should the result show that the woman has a high risk of carrying a baby with Down’s syndrome and the implications of both false positive and false negative screening tests. They need to be informed of the risk of a miscarriage due to invasive diagnostic testing, and the possibility that a miscarried fetus may be chromosomally normal. If, following invasive diagnostic testing, the fetus is shown to have Down’s syndrome, further decisions need to be made about continuation or termination of the pregnancy, the possibility of adoption and finally, preparation for parenthood. Equally, if a woman has a test that shows she is at a low risk of carrying a fetus with Down’s syndrome, it does not necessarily mean that the baby will be born with a normal chromosomal make up. This possibility can only be excluded by an invasive diagnostic test (Alfirevic 2003).The decisions that may be faced by expectant parents inevitably engender a high level of anxiety at all stages of the screening process, and the outcomes of screening can be associated with considerable physical and psychological morbidity. No screening test can predict the severity of problems a person with Down's syndrome will have.

Types of studies

We included studies in which all women from a given population had one or more index test(s) compared to a reference standard. Both consecutive series and diagnostic case-control study designs were included. Randomised trials where individuals were randomised to different screening strategies and all verified using a reference standard were also eligible for inclusion. Studies in which test strategies were compared head-to-head, either in the same women, or between randomised groups were identified for inclusion in separate comparisons of test strategies. Studies were excluded if they included less than five Down's syndrome cases, or more than 20% of participants were not followed up.

Participants

Pregnant women at less than 24 weeks' gestation confirmed by ultrasound, who had not undergone previous testing for Down’s syndrome in their pregnancy were eligible. Studies were included if the pregnant women were unselected, or if they represented groups with increased risk of Down’s syndrome, or difficulty with conventional screening tests including maternal age greater than 35 years old, multiple pregnancy, diabetes mellitus and family history of Down’s syndrome.

Index tests

The following index tests were examined; AFP; ITA; ß-core fragment; free ßhCG; total hCG; oestriol (also termed as uE3); gonadotropin peptide and various marker ratios and combinations of these markers combined with maternal age. Combinations without maternal age were not included in the test comparisons (Table 1; Table 2), however, information on such test combinations is provided.

We looked at comparisons of tests in isolation and in various combinations. These included single (one marker), double (two markers), triple (three markers), test strategies, all maternal age-adjusted.

Where tests were used in comparison, we looked at the performance of test comparisons according to predicted probabilities computed using risk equations and dichotomised into high risk and low risk.

Target conditions

Down's syndrome in the fetus due to trisomy, translocation or mosaicism.

Reference standards

We considered several reference standards, involving chromosomal verification and postnatal macroscopic inspection.

Amniocentesis and CVS are invasive chromosomal verification tests undertaken during pregnancy. They are highly accurate, but the process carries a 1% miscarriage rate, and therefore they are only used in pregnancies considered to be at high risk of Down's syndrome, or on the mother's request. All other types of testing (postnatal examination, postnatal karyotyping, birth registers and Down’s syndrome registers) are based on information available at the end of pregnancy. The greatest concern is not their accuracy, but the loss of the pregnancy to miscarriage between the urine test and the reference standard. Miscarriage with cytogenetic testing of the fetus is included in the reference standard where available. We anticipated that older studies, and studies undertaken in older women were more likely to have used invasive chromosomal verification tests in all women.

Studies undertaken in younger women and more recent studies were likely to use differential verification as they often only used prenatal karyotypic testing on fetuses considered screen positive/high risk according to the screening test; the reference standard for most unaffected infants being observing a phenotypically normal baby. Although the accuracy of this combined reference standard is considered high, it is methodologically a weaker approach as pregnancies that miscarry between the index test and birth are likely to be lost from the analysis, and miscarriage is more likely to occur in Down's than normal pregnancies. We investigated the impact of the likely missing false negative results in sensitivity analyses.

Electronic searches

We applied a sensitive search strategy to search the following databases. We used one broad generic search strategy to identify studies for all reviews in this series.

Databases searched included;

• MEDLINE via OVID (1980 to 25 August 2011)

• EMBASE via Dialog Datastar (1980 to 25 August 2011)

• BIOSIS via EDINA (1985 to 25 August 2011)

• CINAHL via OVID (1982 to 25 August 2011)

• The Database of Abstracts of Reviews of Effectiveness (The Cochrane Library 2011, Issue 7)

• MEDION (25 August 2011)

• The Database of Systematic Reviews and Meta-Analyses in Laboratory Medicine (www.ifcc.org/) (25 August 2011)

• The National Research Register (archived 2007)

• Health Services Research Projects in Progress database (HSRPROJ) (25 August 2011)

The search strategy combined three sets of search terms (see Appendix 1). The first set was made up of named tests, general terms used for screening/diagnostic tests and statistical terms. Note that the statistical terms were used to increase sensitivity and were not used as a methodological filter to increase specificity. The second set was made up of terms that encompass Down syndrome and the third set made up of terms to limit the testing to pregnant women. All terms within each set were combined with the Boolean operator OR and then the three sets were combined using AND. The terms used were a combination of subject headings and free text terms. The search strategy was adapted to suit each database searched.

We attempted to identify cumulative papers that reported data from the same data set, and we contacted authors to obtain clarification of the overlap between data presented in these papers, in order to prevent data from the same women being analysed more than once.

Searching other resources

In addition, we examined references cited in studies identified as being potentially relevant, and those cited by previous reviews. We contacted authors of studies where further information was required. We did not apply a diagnostic test filter, and we did not apply language restrictions to the search.

We carried out forward citation searching of relevant items, using the search strategy in ISI citation indices, Google scholar and Pubmed ‘related articles’.

Selection of studies

Two review authors screened the titles and abstracts (where available) of all studies identified by the search strategy. We obtained full-text versions of studies identified as being potentially relevant and two review authors independently assessed these for inclusion, using a study eligibility screening pro forma according to the pre-specified inclusion criteria. Any disagreement between the two review authors was settled by consensus, or where necessary, by a third party.

Data extraction and management

We developed a data extraction form and piloted the form using a subset of 20 identified studies (from all identified studies in this suite of reviews). Two review authors independently extracted data, and where disagreement or uncertainty existed, a third review author validated the information extracted.

Data on each marker were extracted as binary test positive/test negative results for Down's and non-Down's pregnancies, with a high risk-result, as defined by each individual study, being regarded as test positive (suggestive or diagnostic of Down's syndrome), and a low-risk result being regarded as test negative (suggestive of absence of Down's syndrome). Where results were reported at several thresholds, we extracted data at each threshold.

We noted those in special groups that posed either increased risk of Down’s syndrome or difficulty with conventional screening tests, including maternal age greater than 35 years old, multiple pregnancy, diabetes mellitus and family history of Down’s syndrome.

Assessment of methodological quality

We used a modified version of the QUADAS tool (Whiting 2003), a quality assessment tool for use in systematic reviews of diagnostic accuracy studies, to assess the methodological quality of included studies. We anticipated that a key methodological issue would be the potential for bias arising from the differential use of invasive testing and follow-up for the reference standard according to index test results, bias arising due to higher loss to miscarriage in false negatives than true negatives. We chose to code this issue as originating from differential verification in the QUADAS tool: we are aware that it could also be coded under delay in obtaining the reference standard, and reporting of withdrawals. We omitted the QUADAS item assessing quality according to length of time between index and reference tests, as Down's syndrome is either present or absent rather than a condition that evolves and resolves, and disregarding the differential reference standard issue thus any length of delay is acceptable. Two review authors assessed each included study separately. Any disagreement between the two authors was settled by consensus, or where necessary, by a third party. Each item in the QUADAS tool was marked as ‘yes’, ‘no’ or ‘unclear’, and scores were summarised graphically. We did not use a summary quality score.

QUADAS criteria included the following 10 questions.

1. Was the spectrum of women representative of the women who will receive the test in practice? (Criteria met if the sample was selected from a wide range of childbearing ages, or selected from a specified ‘high-risk’ group such as over 35s, family history of Down’s syndrome, multiple pregnancy or diabetes mellitus, provided all affected and unaffected fetuses included that could be tested at the time point when the screening test would be applied; criteria not met if the sample taken from a select or unrepresentative group of women (i.e. private practice), was an atypical screening population or recruited at a later time point when selection could be affected by selective fetal loss).

2. Is the reference standard likely to correctly classify the target condition? (Amniocentesis, CVS, postnatal karyotyping, miscarriage with cytogenetic testing of the fetus, a phenotypically normal baby or birth registers are all regarded as meeting this criteria).

3. Did the whole sample or a random selection of the sample receive verification using a reference standard of diagnosis?

4. Did women receive the same reference standard regardless of the index test result?

5. Was the reference standard independent of the index test result (i.e. the index test did not form part of the reference standard)?

6. Were the index test results interpreted without knowledge of the results of the reference standard?

7. Were the reference standard results interpreted without knowledge of the results of the index test?

8. Were the same clinical data (i.e. maternal age and weight, ethnic origin, gestational age) available when test results were interpreted as would be available when the test is used in practice?

9. Were uninterpretable/intermediate test results reported?

10. Were withdrawals from the study explained?

Statistical analysis and data synthesis

We initially examined each test or test strategy at each of the common risk thresholds used to define test positivity by plotting estimates of sensitivity and specificity from each study on forest plots and in receiver operating characteristic (ROC) space. Test strategies were selected for further investigation if they were evaluated in four or more studies or, if there were three or fewer studies, but the individual study results indicated performance likely to be superior to a sensitivity of 70% and specificity of 90%.

Investigations of heterogeneity

Had there been 10 or more studies available for a test, we planned to investigate heterogeneity by adding covariate terms to the HSROC model to assess the effect of a covariate on accuracy and threshold.

Sensitivity analyses

In many of the included studies, mothers with pregnancies identified as high risk for Down's syndrome by the urine testing were offered immediate definitive testing by amniocentesis, whereas the remainder were assessed for Down's syndrome by inspection at birth. Such delayed and differential verification will introduce bias most likely through there being greater loss to miscarriage in the Down's syndrome pregnancies that were not detected by the urine testing (the false negative diagnoses). Testing and detection of miscarriages is impractical in many situations, and no clear data are available on the magnitude of these miscarriage rates.

To account for the possible bias introduced by such a mechanism, we planned to perform sensitivity analyses by increasing the percentage of false negatives in studies where delayed verification in test negatives occurred (Mol 1999). We planned to incrementally increase the percentage from 10% to 50%, the final value representing a scenario where a third of more Down's pregnancies than normal pregnancies were likely to miscarry, thought to be higher than the likely value. We intended to conduct the sensitivity analyses on the analysis investigating the effect of maternal age on test sensitivity.

1) Second trimester ß-core fragment

Results for this single test were derived from six studies (Cole 1999b; Cuckle 1995b; Cuckle 1999a; Isozaki 1997; Spencer 1996; Wald 2003), and included 9615 women in whom 193 pregnancies were known to be affected by Down's syndrome. Two studies (Cole 1999b; Cuckle 1999a) contributed over 7000 pregnancies to the data. Six studies (Cole 1999b; Cuckle 1995b; Cuckle 1999a; Isozaki 1997; Spencer 1996; Wald 2003) presented data for a cut-point of 5% FPR and the estimated sensitivity was 41% (95% confidence interval (CI) 20 to 66).

2) Second trimester ß-core fragment and maternal age

Results for this single test were derived from five studies (Bahado-Singh 1999; Bahado-Singh 1999a; Cole 1999b; Hsu 1999; Spencer 1996), and included 3419 women in whom 155 pregnancies were known to be affected by Down's syndrome. Cole 1999b contributed over 1000 pregnancies to the data. The studies presented data at a cut-point of 5% FPR and the summary sensitivity was 56% (95% CI 45 to 66).

3) Other test combinations

Of the 22 test combinations evaluated in three or fewer studies, nine test combinations demonstrated estimated sensitivities of more than 70% and estimated specificities of more than 90%. Six of these were evaluated in single studies (see Summary of findings ), and the following three test combinations were evaluated in two or more studies.

1. Second trimester ß-core fragment to oestriol ratio evaluated in two studies (Cole 1997b; Cole 1999b), with a summary sensitivity of 74% (95% CI 58 to 86) at a cut-point of 5% FPR.

2. Second trimester ß-core fragment to oestriol ratio and maternal age evaluated in three studies (Bahado-Singh 1999; Cole 1999b; Hsu 1999), with a summary sensitivity of 71% (95% CI 51 to 86) at a cut-point of 5% FPR.

3. Second trimester ß-core fragment, oestriol and maternal age evaluated in two studies (Cole 1999b; Hsu 1999), with a summary sensitivity of 73% (95% CI 57 to 85) at a cut-point of 5% FPR.

Comparative analyses of the five selected test strategies

For each test we obtained the detection rate (sensitivity) for a fixed FPR (1-specificity), a metric which is commonly used in Down’s syndrome screening to describe test performance. We chose to estimate detection rates at a 5% FPR in common with much of the literature. Figure 2 shows point estimates of the detection rate (and their 95% CIs) at a 5% FPR based on all available data for the five test strategies; the test strategies are ordered according to decreasing detection rates. The plot shows that all five test strategies have detection rates between 56% and 90%. The combination of second trimester AFP and ß-core fragment to oestriol ratio with maternal age showed the highest detection rate with an estimated detection rate of 90% (CI 55 to 100), based on data from one study with 10 affected cases out of a total of 356 pregnancies. The worst performing strategy was the combination of ß-core fragment to oestriol ratio and maternal age, with an estimated detection rate of 56% (CI 45 to 66), based on data from five studies with 155 affected cases out of a total of 3419 pregnancies.

Table 1 shows pair-wise direct comparisons (head-to-head) where studies were available. Such comparisons are regarded as providing the strongest evidence as they are unconfounded. The table shows the ratio of diagnostic odds ratio (RDOR) with 95% CI and P values for each test combination, the number of studies (K) for which data were available. The table shows that the diagnostic accuracy of the double marker combination of second trimester ß-core fragment and oestriol with maternal age was significantly better (RDOR 2.2 (95% CI 1.1 to 4.5); P = 0.02) than the single marker second trimester ß-core fragment and maternal age test strategy but was not significantly better (RDOR 1.5 (95% CI 0.8 to 2.8); P = 0.21) than that of the second trimester ß-core fragment to oestriol ratio and maternal age test strategy. However, the comparisons in this table were based on two or three studies and are unlikely to be powered to detect differences in detection rates.

Table 2 shows the same comparisons made using all available data (as used to create Figure 2). Results are in agreement with the direct comparisons, and in addition, showed no significant differences between any of the other pair of tests for which direct comparisons were not available. However, these comparisons are potentially confounded by differences between the studies, and the evidence is limited.

Investigation of heterogeneity and sensitivity analyses

None of the tests was evaluated by 10 or more studies and so we were unable to investigate the effect of maternal age or any other potential source of heterogeneity. The planned sensitivity analyses, looking at differential verification and any resultant bias, were also not possible.