Impact of preanalytical variables on urinary cell-free DNA quality: Centrifugation, EDTA concentration, storage and thawing strategies.

Urinary cell-free DNA (ucfDNA) holds promise for liquid biopsies, yet standardized preprocessing protocols remain to be fully established. Using pooled urine from healthy volunteers, we compared the effects of EDTA concentration (0-40 mM), centrifugation (0-2 spins), storage temperature and duration (RT, -20°C, -80°C), and thawing conditions (4°C, RT, 37°C) on ucfDNA yield and purity (%cfDNA score). The optimal protocol was subsequently validated using patient urine. Untreated whole urine (WU) and urine supernatant (US) both exhibited substantial ucfDNA degradation within 1 day at RT. For EDTA-treated WU, %cfDNA scores declined markedly owing to high-molecular-weight DNA contamination, and 20-40 mM EDTA compromised yields. In contrast, 10 mM EDTA-treated US optimally preserved both ucfDNA yields and %cfDNA scores for 7 days at RT, whereas 40 mM EDTA impaired yields. Frozen US showed thawing-method-independent stability, whereas frozen WU thawed at 4°C lost >60% of ucfDNA yields. Post-thawing secondary centrifugation further reduced ucfDNA recovery. For long-term storage, 10 mM EDTA-treated US maintained baseline-equivalent ucfDNA quality at -80°C for 3 months, but ucfDNA degraded after 2 months at -20°C. Patient urine validation confirmed that 10 mM EDTA-treated US frozen at -20°C and thawed at 37°C preserved ucfDNA quality comparable to baseline. This study offers practical guidelines and standardized preprocessing protocols for optimizing ucfDNA preservation. For temporary storage of urine intended for ucfDNA preservation, adding EDTA to WU is not advised; instead, we recommend supplementing 10 mM EDTA to US and storing it at RT ≤ 7 days or at -20°C ≤ one month. Long-term storage requires temperatures below -80°C. Rapid thawing can yield good ucfDNA quality.