Review
Non-invasive fetal RHD genotyping tests: A systematic review of the quality of reporting of diagnostic accuracy in published studies

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Abstract

Articles reporting the diagnostic accuracy of non-invasive prenatal diagnostic (NIPD) tests for RHD genotyping using fetal material extracted from maternal blood have been published steadily for over a decade. Health care providers in Europe have started to use this technology for management of the small number of sensitised pregnancies (ca. 220–600 per annum in the Netherlands, Germany, France and the UK). Scientists and clinicians are also advocating widespread implementation for the far larger number of non-sensitised RhD-negative pregnancies (ca. 34,000–125,000 per annum in the same countries). Large-scale, prospective trials are only now underway. Estimates of the technical performance of these tests are currently based on results from small-scale studies, together with formal meta-analysis. The issue of early assessment of test performance is one faced by many new genetic tests. As part of a wider study we have investigated the quality of reporting of diagnostic accuracy in publications and produced guidelines for future studies.

A systematic search of the literature identified 27 papers which met predefined inclusion criteria. All 27 papers were, first, assessed against an international quality (STARD) checklist for reporting of diagnostic accuracy and, second, against our own in-house NIPD proforma to assess the implications of the quality of reporting specifically for the RhD NIPD test. Authors were found to generally present an optimistic view of NIPD, bearing in mind weaknesses identified in reporting and conduct of their studies and the analysis of results, as evidenced by the low STARD scores. The NIPD proforma identified that specific biases were potentially introduced through selective population sampling and/or failure to report the make-up of the population tested, omission of inconclusive results, inconsistencies in the handling of repeat results on a sample, and lack of adequate controls. These factors would inevitably affect the validity of diagnostic accuracy as reported in individual publications, as well as any subsequent meta-analyses. Together, published reports to date may provide a biased picture of the actual potential of NIPD testing for fetal RHD genotyping. Generalisation of the available evidence on diagnostic accuracy, especially to large-scale implementation of NIPD testing of non-sensitised women, will also require that decision makers consider further aspects such as test reliability and cost of routine testing in clinical practice.

It is recommended that all studies of diagnostic accuracy of NIPD tests adhere to the STARD quality checklist in order to improve reporting, thereby, minimising bias and increasing the comparability of studies. Researchers should also consider specific shortcomings for NIPD and avoid selective participant sampling; report population characteristics; report handling of replicate sampling as well as their failure rates; and include controls for genotypes tested in the study. Furthermore, meta-analyses should consider the quality, as well as the sample size, of NIPD studies in their analysis. Larger trials, required to produce results that are valid and meaningful for clinical practice, must also adhere to these reporting standards.

Introduction

Fetal RHD genotyping from maternal whole blood [1], fetal cells [2] or cell free fetal DNA [3] in maternal blood has been feasible for over a decade. Following the first papers, research interest in this form of non-invasive prenatal diagnosis (NIPD) has become global. Publications in Europe, America and Australia aim to improve the management of pregnancies of women who are rhesus negative [2], [4], [5], [6], [7], [8], [9]. In white Caucasian populations about 10% of all pregnancies involve an RhD-negative mother and an RhD-positive fetus, potentially placing the mother at risk of sensitisation and future babies at risk of haemolytic disease of the fetus and newborn (HDFN).

Anti-D immunoglobulin can be given to prevent a woman producing antibodies against fetal RhD-positive blood cells. Introduction of postnatal anti-D prophylaxis (within 72 h of delivery) in the late 1960s reduced sensitisation and HDFN rates considerably internationally. Combined with antenatal prophylaxis for high-risk events, such as amniocentesis, this form of targeted prophylaxis has proved highly effective and cost-effective [10]. Consequently, the number of sensitised RhD-negative women who give birth every year is lower than 1% of total births.

For non-sensitised women, routine antenatal anti-D prophylaxis (RAADP) at 28–30 weeks gestation was introduced in the mid-1990s, and is now available in most European countries. However, 40% of RhD-negative women receive this blood product unnecessarily because the fetus is not RhD-positive. Fetal RHD genotyping has the potential to enable targeted antenatal prophylaxis only for women whose pregnancy is at risk. This might address any issues of limited anti-D supplies and perceived risk associated with unnecessary administration of a blood product.

The NIPD RHD genotyping test has already started to have an impact on the management of sensitised women, in countries where this technology is available, by replacing invasive procedures like amniocentesis for the determination of fetal RhD status. Replacement of amniocentesis is desirable since the procedure can promote feto–maternal blood exchange as well as being associated with a small increased risk of miscarriage [11], [12].

The current state of development of the RhD NIPD test is promising. Several studies have reported high accuracy rates [13], [14], [15], [16] and these have prompted the initiation of larger scale trials in the Netherlands, Germany and the UK [17], [18]. Reports in the literature have already led to initial use of the test for management of sensitised women. An increasing focus of debate is now on widespread implementation of the test in non-sensitised pregnancies [19]. Discussion of markers (exons and/or introns) to be used for RHD genotyping, and the applicability of NIPD tests for routine clinical use in diverse populations form part of this debate [13], [17].

A recent meta-analysis undertaken by Geifman-Holtzman et al. has reported sensitivity and specificity values of 0.986 and 0.954 respectively, and an overall positive predictive value of 0.990 and negative predictive value of 0.921 [20]. Some studies were excluded from this meta-analysis on the basis of sample numbers, but aspects such as study quality were not considered. Because assessment of the quality of reporting of diagnostic accuracy can be problematic, a world-wide Delphi panel has identified agreed international quality criteria for assessing the reporting of such studies [21]. The resulting checklist (STARD -standards for reporting studies of diagnostic accuracy) is now required from all authors of health technology assessment reports of diagnostic technologies in the UK, and increasingly requested by editors of international journals. We have undertaken an independent systematic literature search and assessed the quality of reporting of all identified articles using the STARD checklist. We further investigated the implications of any shortcomings on the generalisability of NIPD study results, in order to identify key aspects influencing test reliability and any underlying reporting biases. An NIPD assessment proforma has been produced as well as recommendations that are specific and relevant for reporting diagnostic accuracy of RhD NIPD tests.

Section snippets

Literature search strategy

Published articles were identified by systematic searches of electronic databases from 1966 until January 2007; these included PubMed, Ovid Medline, Ovid Embase, the Cochrane Library, the National Library for Health (UK), Online Computer Library Center (OCLC) and the Conference Papers Index. Text words and MeSH headings used separately and in combination included: prenatal diagnosis, Rh, fetal cells, fetal DNA, maternal blood, serum, plasma, Rh alloimmunis(z)ation. Bibliographies of all papers

Results

The literature search resulted in the identification of 51 publications. The selection procedure described above excluded 24 papers (Fig. 1) leaving 27 studies for final quality assessment (Table 2). Of these, 25 were in English, 1 in French and 1 in Polish. Publication dates ranged from 1996 to the end of 2006. Findings were published in a wide range of journals and originated from 15 countries.

Discussion

Articles which report the diagnostic accuracy of new non-invasive prenatal diagnostic tests for RHD genotyping are difficult to identify through systematic searches of the literature. Indexing of the articles identified was found to be poor, as has been reported by other diagnostic test reviews [40]. Of those finally identified, less than one-third were found to have included the correct MeSH headings for diagnostic accuracy.

A detailed appraisal of all retrieved articles has identified a

Acknowledgement

This work is supported by the European Commission funds allocated to the SAFE Network of Excellence under the 6th Framework. Project Number: LSHB-CT-2004-503243.

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