Cell-free DNA-based prenatal screening via rolling circle amplification: Identifying and resolving analytic issues.

Objective: A rolling circle amplification (RCA) based commercial methodology using cell-free (cf)DNA to screen for common trisomies became available in 2018. Relevant publications documented high detection but with a higher than expected 1% false positive rate. Preliminary evidence suggested assay variability was an issue. A multi-center collaboration was created to explore this further and examine whether subsequent manufacturer changes were effective.

Methods: Three academic (four devices) and two commercial (two devices) laboratories provided run date, chromosome 21, 18, and 13 run-specific standard deviations, number of samples run, and reagent lot identifications. Temporal trends and between-site/device consistency were explored. Proportions of run standard deviations exceeding pre-specified caps of 0.4%, 0.4% and 0.6% were computed.

Results: Overall, 661 RCA runs between April 2019 and July 30, 2022 tested 39,756 samples. In the first 24, subsequent 9, and final 7 months, proportions of capped chromosome 21 runs dropped from 39% to 22% to 6.0%; for chromosome 18, rates were 76%, 36%, and 4.0%. Few chromosome 13 runs were capped using the original 0.60%, but capping at 0.50%, rates were 28%, 16%, and 7.6%. Final rates occurred after reformulated reagents and imaging software modifications were fully implemented across all devices. Revised detection and false positive rates are estimated at 98.4% and 0.3%, respectively. After repeat testing, failure rates may be as low as 0.3%.

Conclusion: Current RCA-based screening performance estimates are equivalent to those reported for other methods, but with a lower test failure rate after repeat testing.