Assessment of pre-analytical sample handling conditions for comprehensive liquid biopsy analyses.

Liquid biopsies as minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet while protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multi-center clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. Our study aimed at testing the impact of commonly used (EDTA, heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA (cfDNA) for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leucocytes. For genomic analyses EDTA-tubes showed good results if stored for maximum 4 hours at RT or for up to 24 hours when stored at +4°C. Spike-in experiments revealed that EDTA-tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leucocytes and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA samples for LB analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.