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. In particular, genomic DNA (gDNA) released from lysed white blood cells (WBCs) can contaminate plasma and compromise downstream cfDNA analyses. This 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 (Cell-Free DNA BCT, Streck; S-Monovette cfDNA Exact, Sarstedt) and stored at room temperature for 1, 5, or 10 days before plasma isolation. CfDNA was extracted using either a magnetic bead-based method or a silica column-based approach. DNA quantity and quality were assessed by fluorometric quantification, automated fragment analysis, and gene-specific quantitative PCR. Streck-based workflows maintained stable cfDNA yields and characteristic mononucleosomal fragmentation profiles across all storage times. In contrast, Sarstedt tubes showed reduced cfDNA concentrations after 5 days and a pronounced increase at 10 Days, accompanied by high-molecular weight DNA patterns consistent with WBC lysis. These trends were largely independent of the extraction method. Overall, the results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing. Streck tubes, particularly when combined with QIAamp, provided the most robust and reproducible workflow for routine molecular diagnostics, whereas Sarstedt tubes produced physiologically implausible results after extended storage.