Towards a Robust cell-free DNA Isolation Protocol for NGS Applications in a Clinical Molecular Diagnostics Setting

Cell-free DNA (cfDNA) released from apoptotic and necrotic cells into body fluids represents a non-invasive source of genetic information for disease prediction, diagnosis, and monitoring. However, its low physiological abundance makes cfDNA highly susceptible to pre-analytical influences, particularly contamination by genomic DNA from lysed white blood cells. The study evaluated the impact of different blood collection tubes and isolation methods on cfDNA stability and yield. Blood samples from 13 healthy donors were collected using cfDNA-stabilizing tubes (Streck and Sarstedt) and stored at room temperature for 1, 5, or 10 days. CfDNA was extracted using magnetic bead-based or silica column-based methods. Streck tubes maintained stable cfDNA yields and characteristic fragmentation profiles across all storage times, while Sarstedt tubes showed reduced concentrations after 5 days and pronounced increases after 10 days with high-molecular-weight DNA patterns. The results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing.