Pre-analytical optimization of cell-free DNA and extracellular vesicle-derived DNA for mutation detection in liquid...

Liquid biopsies enable noninvasive tumor profiling and longitudinal disease monitoring. Their analytical performance is strongly influenced by pre-analytical factors, yet direct comparisons between cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (evDNA) remain scarce. We prospectively evaluated four pre-analytical variables: processing delay, storage temperature, tube type, and plasma input volume, on cfDNA and evDNA from cancer patient plasma (n = 244) using ddPCR, Qubit, and TapeStation. Key findings were validated in archived plasma samples (n = 723). In the prospective cohort, cfDNA concentrations increased after 24 h and evDNA after 48 h at room temperature, while retrospective analysis revealed earlier changes (cfDNA: 6 h; evDNA: 24 h). Storage conditions influenced both analytes, as short-term refrigeration (4 °C) better preserved DNA quality than -80 °C freezing, while extracted DNA remained stable at -80 °C. Acid citrate dextrose (ACD) and K₂EDTA tubes performed similarly under prompt processing. Higher plasma volumes improved evDNA, but not cfDNA, for mutation detection. evDNA demonstrates greater resilience than cfDNA under suboptimal conditions. Standardized workflows and prompt processing are essential to ensure reliable mutation detection in clinical liquid biopsy applications.