Interlaboratory evaluation of quality control methods for circulating cell-free DNA extraction

Area of Science:

• Biochemistry
• Molecular Biology
• Clinical Diagnostics

Background:

• Circulating cell-free DNA (ccfDNA) analysis from liquid biopsies is increasingly used in clinical settings.
• Diagnostic accuracy is hindered by inconsistent sample quality and lack of standardized quality control (QC) methods for cfDNA.
• Standardized QC is crucial for reliable in vitro diagnostic assays.

Purpose of the Study:

• To evaluate nucleic acid spike-in control materials for quality control and standardization of cfDNA isolation.
• To develop and characterize an in-process QC material for cfDNA extraction.
• To assess the suitability of spike-in controls for inter-laboratory standardization of cfDNA analysis.

Main Methods:

• Designed and characterized a spike-in material with exogenous DNA and cfDNA-like fragments.
• Incorporated the spike-in material into plasma cfDNA extraction protocols.
• Quantified spike-in recovery using digital PCR (dPCR) in an inter-laboratory study across five labs.
• Assessed various blood collection devices and cfDNA extraction methods.

Main Results:

• Spiking exogenous DNA did not negatively impact endogenous cfDNA recovery.
• The spike-in approach demonstrated consistent performance across diverse extraction protocols.
• The method effectively highlighted variations in efficiency and reproducibility among different extraction techniques.
• Digital PCR quantification showed high repeatability, with coefficients of variation generally below 5%.

Conclusions:

• The developed spike-in control approach is fit for purpose in standardizing cfDNA isolation.
• Spike-in recovery, combined with other QC metrics, can monitor process performance over time.
• This method supports external quality assessment for cfDNA-based diagnostics.
• Standardized QC is essential for improving the reliability of liquid biopsy diagnostics.