Impact of Preanalytical Variables on Urinary Cell-Free DNA Quality: Centrifugation, EDTA Concentration, Storage and Thawing Strategies

Background Urinary cell-free DNA (ucfDNA) holds promise for liquid biopsies, yet standardized preprocessing protocols are lacking. This study evaluated the impact of several preanalytical variables on ucfDNA quality: ethylenediaminetetraacetic acid (EDTA) concentration, centrifugation, storage conditions, and thawing methods. Results Untreated urine exhibited complete ucfDNA degradation within 1 day at room temperature (RT). No significant differences in ucfDNA yield were detected among any of the EDTA-treated whole urine samples after 1 day and 3 days of RT storage, whereas the 40 mM group presented significantly lower ucfDNA yields than did the 10 mM group after 7 days of RT storage. In particular, the %cfDNA scores of all EDTA-treated whole urine samples decreased significantly (61.51 ± 1.54 to 32.38 ± 7.04) because of high-molecular-weight DNA contamination from cell lysis. The urine supernatant with 10 mM EDTA optimally preserved ucfDNA over 7 days, but 40 mM EDTA impaired yields and %cfDNA scores. In the thawing experiment with/without EDTA, the frozen urine supernatant showed thawing-method-independent stability, but frozen whole urine thawed at 4°C lost > 60% yield. In the absence of EDTA, nearly all frozen urine samples presented higher %cfDNA scores than did the baseline samples after thawing, and postthawing secondary centrifugation significantly reduced the yields. With 10 mM EDTA, the frozen urine supernatant maintained the baseline-equivalent ucfDNA quality, whereas the whole urine samples presented significantly lower %cfDNA scores, and the frozen urine supernatant subjected to postthawing secondary centrifugation significantly reduced the ucfDNA yield when thawed at 4°C. The 10 mM EDTA-treated urine supernatant stored at -80°C maintained baseline-equivalent ucfDNA for 3 months, whereas samples stored at -20°C degraded ucfDNA after 2 months. Conclusions This work offers practical guidelines for optimizing ucfDNA quality in clinical settings. Without centrifugation, preservatives other than EDTA should be further explored for whole-urine biobanking. The addition of 10 mM EDTA to the urine supernatant is recommended, followed by temporary storage at RT for up to 7 days or freezing at -20°C for less than one month. For long-term storage, ultralow temperatures are necessary. Rapid thawing at RT/37°C can yield good ucfDNA quality.