Certified Reference Materials for Standardization of Cell-Free DNA Isolation Recovery in Liquid Biopsy.

Reliable quantification of cell-free DNA (cfDNA) in liquid biopsy diagnostics critically depends on unbiased and reproducible isolation workflows. Fragment-length-dependent recovery during isolation represents a major source of pre-analytical bias. This variability limits method validation, interlaboratory comparability, and the reliability of cfDNA measurements in clinical and research laboratories. However, certified reference materials (CRMs) specifically designed to evaluate cfDNA isolation recovery are currently lacking. In this study, two synthetic cfDNA CRMs were developed to enable fragment-length-resolved assessment of isolation recovery. UME CRM 3022 comprises 80- and 240-bp double-stranded DNA fragments, whereas UME CRM 3024 includes fragments of 80, 120, 160, and 240 bp. The materials were prepared gravimetrically and characterized using optimized duplex and multiplex droplet digital PCR assays. Homogeneity, short-term stability, and long-term stability were evaluated. Certified copy number concentrations with stated measurement uncertainties were assigned for each fragment. Applicability in a plasma matrix was demonstrated to support practical workflow integration. These CRMs provide a traceable, fragment-resolved framework for recovery-based standardization of cfDNA isolation, supporting improved analytical reliability in liquid biopsy applications.