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Cell-free DNA-based prenatal screening via rolling circle amplification: Identifying and resolving analytic issues
Glenn E Palomaki1, Geralyn M Lambert-Messerlian1,2, Donna Fullerton3
1Department of Pathology and Laboratory Medicine, Women & Infants Hospital and Alpert Medical School at Brown University, Providence, RI, USA.
Journal of Medical Screening
|May 17, 2023
Summary
Rolling circle amplification (RCA) screening for trisomies improved significantly after manufacturer updates, reducing assay variability. This cell-free DNA (cfDNA) screening now shows performance comparable to other methods with fewer failures.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- A commercial rolling circle amplification (RCA) method for cell-free DNA (cfDNA) screening of common trisomies was introduced in 2018.
- Initial studies showed high detection rates but also a concerning 1% false positive rate, attributed to assay variability.
- A multi-center collaboration was established to investigate these variability issues and assess manufacturer improvements.
Purpose of the Study:
- To evaluate the performance and consistency of a commercial rolling circle amplification (RCA) based cell-free DNA (cfDNA) screening assay for common trisomies.
- To determine the effectiveness of manufacturer modifications in reducing assay variability and improving accuracy.
- To compare the updated RCA screening performance with established methods.
Main Methods:
- Data from three academic and two commercial laboratories were collected, including run dates, chromosome-specific standard deviations (chromosomes 21, 18, 13), sample numbers, and reagent lot information.
- Temporal trends and inter-laboratory/inter-device consistency were analyzed.
- Proportions of runs exceeding pre-defined standard deviation caps were calculated to assess variability.
Main Results:
- Across 39,756 samples tested between April 2019 and July 2022, significant reductions in variability were observed over time.
- Proportions of runs exceeding variability caps for chromosomes 21, 18, and 13 decreased substantially after the implementation of reformulated reagents and software updates.
- Revised estimates indicate a detection rate of 98.4% and a false positive rate of 0.3%, with potential failure rates as low as 0.3% after repeat testing.
Conclusions:
- Post-modification RCA-based screening demonstrates improved consistency and reduced variability.
- The current performance of RCA screening is comparable to other established methods for common trisomies.
- The updated RCA assay offers a lower test failure rate, particularly after implementing repeat testing protocols.
