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 14 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 18 index tests were examined; ADAM12, AFP, inhibin, PAPP‐A, ITA, free βhCG, PlGF, SP1, total hCG, progesterone, uE3, GHBP, PGH, hyperglycosylated hCG, ProMBP, hPL, free αhCG, and free ßhCG to AFP ratio, and combinations of these markers combined with maternal age.

We looked at comparisons of tests used in isolation and in 78 various combinations. These included single (one marker), double (two markers), triple (three markers), quadruple (four markers) and quintuple (five markers) tests, some of which were adjusted for maternal age.

Where tests were used in combination, we looked at the performance of test combinations 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. Chromosomal verification is considered preferential but because of the risks involved, often not feasible. Where macroscopic inspection or examination raises a question about the possibility of an individual being affected by Down's syndrome, in clinical practice this is usually confirmed or refuted by formal karyotyping.

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 high risk of Down's, 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. For the purposes of meta‐analysis they are considered equivalent. The greatest concern is not their accuracy, but the loss of the pregnancy to miscarriage between the timing of serum testing 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 or high risk according to the screening test; the reference standard for most unaffected infants is likely to be observation of a phenotypically normal baby. Although the accuracy of this combined reference standard is considered high, it is methodologically a weaker approach because 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.

Electronic searches

We applied a sensitive search strategy to search the following databases using the text words and MeSH terms detailed in Appendix 1, adapting the search strategy for each different database.

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 (25 August 2011)

• 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's 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 which reported data from the same data set, and 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 carried out forward citation searching of relevant items, using the search strategy in ISI citation indices, Google scholar and Pubmed ‘related articles’.

We did not apply language restrictions to the search.

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

A data extraction form was developed and piloted using a subset of 20 identified studies. 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 made a note of 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 (Quality Assessment of Diagnostic Accuracy Studies) 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 if 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 be marked as ‘yes’, ‘no’ or ‘unclear’, and scores are presented graphically and in tables. We did not use a summary quality score. See Appendix 3 for QUADAS questionnaire.

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 two or three studies, but the individual study results indicated performance likely to be superior to a sensitivity of 70% and specificity of 90%.

Investigations of heterogeneity

We planned to undertake investigations of heterogeneity if there were 10 or more studies available for a test. We planned to investigate the effect of a covariate by adding covariate terms to the HSROC model to assess differences in accuracy and threshold.

Sensitivity analyses

Mothers with pregnancies identified as high risk for Down's syndrome by serum testing are often offered immediate definitive testing by amniocentesis, whereas those considered low risk are 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 serum 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 or 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.