Biological and preanalytical variations of cfDNA concentration in individuals

Introduction – The last years alterations in cell-free DNA concentration (cfDNA) over the time have either been studied in diseased or injured patients or analyzed in athletes after exhaustive exercise. However, no fluctuations have been examined within a short interval in healthy humans at rest so far, wherefore the aim of this study was to analyze individual variations within 75 minutes and to examine the influence of the blood drawing protocol.

Methods – Within 75 minutes, serial blood drawing was performed with 22 healthy volunteers at rest via an IV-catheter. To evaluate the influence of the sampling protocol, in 14 participants the first milliliter was discarded prior to every sampling step (“with discarding” versus “no discarding”). CfDNA was quantified by real-time qPCR and absolute levels were analyzed over the time together with fold changes in relation to the initial value.

Results – Overall cfDNA levels ranged from 0.4 to 3.8 ng/ml, whereby the median value was 1.3 ng/ml (IQR 0.9 – 2.0 ng/ml). While the minimum concentration was observed at 15min, the maximum was measured at 75min. No significant differences between concentration at the time-points and the initial value were noted (Wilcoxon-test; p > 0.05) and no trends were noted comparing absolute concentration courses. Significant lower fold changes were remarked for volunteers “with discarding” (Mann-Whitney-U-test, p=0.0001), whereby a maximum of 1.6 (10min) was calculated. Regarding the group “no discarding”, fold changes higher than 3.0 were identified after 30min, whereby the maximum was 5.6 (75min).

Conclusion – We evidenced the variability of cfDNA in healthy humans within 75minutes. However, the evaluation of natural fluctuations over time requires a standardized blood drawing, whereby the first milliliter should be discarded prior to every sample collection to avoid false alterations. The determined variations should serve in future studies to distinguish small concentration increases after minor trauma from natural fluctuations.