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Implementing a prenatal-specific reference markedly improves call quality and reduces repeat testing for clinical CMA

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction:
In 2016, ACOG and SMFM issued joint guidelines recommending chromosomal microarray (CMA) as a first-tier genetic test for prenatal cases undergoing invasive procedures with major structural abnormalities identified by fetal ultrasound and instances of fetal death or stillbirth. Since then, the UCLA cytogenetics laboratory has routinely performed prenatal testing on chorionic villus sampling (CVS), amniotic fluid (AF), and products of conception (POC) specimens, with an annual volume of approximately 400 tests per annum. With sufficient starting material, testing on a direct prenatal specimen is preferred for several reasons, including faster turnaround time and reduced risk of cultural artifacts. However, it has been noted that direct prenatal specimens, particularly POCs, typically generate noisier data, with as much as 15-20% of cases resulting in data unsuitable for analysis. While repeat testing on cultured POC specimens often yields acceptable results, this incurs substantial time and cost. Here, we sought to evaluate how incorporating a custom prenatal reference file in CMA analysis could improve data quality and optimize downstream analysis.

Methods:
A representative set of 285 direct prenatal CMA cases (142 AF, 132 POC, and 11 CVS) previously performed at UCLA from 2021 to 2023 were retrospectively selected for reference file generation using Affymetrix ChAS software. Inclusion criteria were limited to cases with no reportable variants and passing all established CMA QC metrics, with an effort to balance both sex and ethnicity. A mutually exclusive set of prenatal cases was assembled to assess the performance of this prenatal reference file on downstream analysis, by comparing CMA assay QC metrics and number of calls to those generated using our existing reference based on blood specimens from 200 normal individuals. To assess for clinical sensitivity, we reprocessed and reanalyzed 94 cases with previously reported variants.

Results:
Data analysis using prenatal-specific reference resulted in a dramatic reduction of noise signal and lower the number of artifact calls significantly. The average number of calls/sample was reduced by 21% (24.5 to 19.4), and this improvement was greatest for POCs, showing a 30% reduction (24.7 to 17.2). Even more strikingly, approximately 65% of samples deemed unsuitable for analysis, which is likely due to overwhelming artifact calls, resulted in acceptable or even clean data simply through reprocessing with the prenatal reference file. Among direct prenatal specimens already suitable for analysis, the prenatal reference outperformed blood-based reference in every established CMA QC metric. These improvements came without a loss of sensitivity, as we achieved 100% clinical concordance for prenatal specimens with previously reported variants, both direct and cultured.

Conclusion:
For prenatal CMA testing on the Affymetrix platform, incorporation of a custom reference file based on a representative set of direct specimens is an effective and cost-efficient way to improve CMA call quality, reduce repeat testing, improve turnaround time, and lower the overall burden of interpretation.

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