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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

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Summary
This summary is machine-generated.

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.

Keywords:
Down syndromePrenatal screeningcell-free DNAcommon trisomiesquality controlrolling circle amplification

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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.