Pre-analytical pitfalls: How blood collection tubes influence exercise-induced cell-free DNA concentrations

Circulating cell-free DNA (cfDNA) is a promising biomarker for physiological stress, including exercise-induced responses. However, the lack of standardization in blood collection tubes (BCTs) for quantification of cfDNA hampers inter-study comparisons. In this study, we assessed the impact of different BCTs on exercise-induced cfDNA dynamics. Eleven participants [25 (SD 2.3) years of age] performed three different treadmill exercise protocols, including an all-out test and combinations of constant and interval load. Blood samples were collected before, 5 min and 30 min post-exercise using EDTA, lithium-heparin (LH) and serum BCTs. Concentrations of cfDNA were quantified using quantitative PCR. The cfDNA increased significantly across all protocols and BCTs. A significant effect of BCT on cfDNA concentrations (P = 0.034) was found, with serum showing higher concentrations than EDTA and LH. Although absolute differences from pre- to post-exercise were comparable across BCTs (P = 0.476), fold changes differed significantly (P = 0.012), with the highest observed in EDTA and the lowest in serum. Bland-Altman analyses demonstrated better agreement between EDTA and LH compared with serum. Significant correlations of cfDNA with energy expenditure and peak oxygen uptake were found. These correlations were stronger in EDTA and LH than in serum. Our findings highlight the crucial influence of BCT choice on cfDNA measurements in exercise settings. Given that EDTA and LH reflected exercise load better, they could be preferred for exercise physiology research. This work underscores the need to account for the choice of BCT to improve data comparability across studies. Additionally, these findings might have broader implications for clinical settings where cfDNA is used as a biomarker.