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The research aims to develop and validate reference materials for measuring cfDNA methylation, addressing inconsistencies in current methods.

May 20, 2026Open Access

Development, characterization, and inter-laboratory validation of methylated human cell free DNA candidate reference materials

Key Points

The research aims to develop and validate reference materials for measuring cfDNA methylation, addressing inconsistencies in current methods.
Developed two candidate reference materials (RMs) comprising various methylation percentages of cfDNA simulator.
Conducted an interlaboratory study with six labs using different assays, including ddPCR and qPCR, to evaluate RM utility.
Characterized methylation status confirmation using droplet digital PCR assays.
The observed methylation levels correlated well across all laboratories, indicating effective validation.
Each assay demonstrated good reproducibility, with minor assay-specific biases in methylation proportions.

Abstract

Abstract Background Aberrant DNA methylation biomarkers have demonstrated potential for early cancer detection, multicancer detection, and determining the tissue of origin. Due to their stability, frequency, and accessibility in bodily fluids, circulating cell-free DNA (cfDNA) methylation is a promising biomarker in liquid biopsy. A reliable and quantifiable analysis of cfDNA methylation status is critical to its application. However, there are current challenges and a lack of consensus on measurement methods. To address this, we developed two candidate methylated cfDNA reference materials (RMs). Methods The National Institute of Standards and Technology (NIST) RM consists of five components, formulated by mixing in vitro methylated cfDNA simulant at fractions of 0%, 5%, 25%, 50%, and 100% with native-state cfDNA simulant derived from the GM24385 cell line. The LGC Clinical Diagnostics (LGC) RM consists of two components: non-methylated cfDNA simulant derived from GM24385 genomic DNA and whole genome amplification and methylated cfDNA produced by in vitro methylation of amplified material. The candidate RMs were characterized, and the methylation status of three targets was confirmed by droplet digital PCR (ddPCR) assays. To test the utility of these RMs, six laboratories participated in an interlaboratory study, each using their own lab-developed assays and methods, which included methylation-specific qPCR, nanoplate digital PCR (dPCR), ddPCR, matrix methylated DNA immunoprecipitation-based assays, and whole-genome bisulfite sequencing. Results The interlaboratory study results showed that the designed percentage of methylation was well correlated with the observed values across all participating labs, and good reproducibility was found for each individual method. However, slightly different methylation proportions associated with assay-specific biases were observed. Conclusions This study clearly demonstrates the value of candidate RMs as standards for evaluating assay performance, as well as for increasing confidence in reporting cfDNA methylation status for clinical applications.

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