Preanalytical framework for routine clinical use of liquid biopsies: combining EVs and cfDNA

Aim: Liquid biopsies hold significant potential for the minimally invasive diagnosis of tumors and other diseases. While the clinical application of cell-free DNA (cfDNA) methodologies is emerging, the implementation of tumor-derived extracellular vesicles (EVs) as validated biomarkers is hindered by substantial preanalytical variations. In this work, we standardized the preanalytical procedures of blood collection for subsequent serial isolation of plasma cfDNA and EVs from a single blood collection tube. Methods: We compared the impact of blood preservation tubes and storage to enable proteomic profiling of resulting EVs in addition to cfDNA extraction and sequencing. Following a stringent method of large EV (lEV) and small EV (sEV) isolation, consisting of differential ultracentrifugation and size exclusion chromatography, we evaluated the protein concentration, particle number, quality and integrity of the isolated EVs. Subsequent proteomic analyses of EV isolates revealed the complexity of the respective tube-biased proteomes, allowing the interpretation of EV origins as well as contamination sources. Results: While ACD-A and Citrate tubes yield satisfactory results in the preservation of EV proteomes, only Streck RNA, Norgen, and PAX tubes can maintain high cfDNA purity for up to 7 days. When aiming for multiomics analyses, Streck RNA tubes showed the most stable performance across the tested parameters for both bioanalytes. Furthermore, we detected greater variability in protein composition in sEVs than in lEVs after 7 days of storage; thus, sEVs might be more susceptible to storage effects. Conclusion: Our clinically applicable workflow provides the basis for informed choice of liquid biopsy tubes along with a ready-to-use protocol to retrieve both genomic and EV proteomic biomarker information for multiomics biomarker-based liquid biopsy studies.