He, Z.
, He, H.
, Cole, K.
, Konigshofer, Y.
, Garlick, R.
, Ramprakash, J.
, Young, M.
, Hall, E.
, Corner, A.
, Clarissa, M.
, Wright, J.
, Cannon, V.
, Yu, M.
, Grady, W.
, Yeung, C.
, Heimer, Z.
, Wang, Z.
, Zou, S.
, Jia, S.
, Liu, F.
, Bonora, G.
, Bomsztyk, K.
and Mar, D.
(2026),
Development, characterization, and inter-laboratory validation of methylated human cell free DNA candidate reference materials, Clinical Epigenetics, [online], https://doi.org/10.1186/s13148-026-02156-3, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=959573 (Accessed June 15, 2026)
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
Secure .gov websites use HTTPS
A lock (
) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.
Development, characterization, and inter-laboratory validation of methylated human cell free DNA candidate reference materials
Published
Author(s)
, , , Yves Konigshofer, Russell Garlick, Jayanthi Ramprakash, Matthew Young, Eric Hall, Adam Corner, Michelle Clarissa, Jocelyn Wright, Victoria Cannon, Ming Yu, William Grady, Cecilia Yeung, Zachary Heimer, Zhili Wang, ShiPing Zou, Shidong Jia, Fang Liu, Giancarlo Bonora, Karol Bomsztyk, Daniel Mar
Abstract
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). 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 based on whole genome amplification and methylated cfDNA produced by in vitro methylation. The candidate RMs were characterized, and the methylation status of three targets was confirmed by droplet digital PCR (dPCR) 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 qPCR, chamber dPCR, droplet dPCR, matrix methylated DNA immunoprecipitation-based assays, and whole-genome bisulfite sequencing. 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 statuses associated with assay-specific biases were observed. This study clearly demonstrates the value of candidate RMs as calibrators for evaluating assay performance, as well as for increasing confidence in reporting cfDNA methylation status for clinical applications.Citation
Clinical Epigenetics
Pub Type
Journals
Download Paper
Keywords
Cell free DNA, DNA methylation, reference material, characterization, interlaboratory study, cancer detection, epigenetics
Citation
Issues
If you have any questions about this publication or are having problems accessing it, please contact [email protected].
Created May 18, 2026, Updated June 11, 2026