Pre-analytical storage effects on ALU- and LINE1-derived cell-free DNA biomarkers in whole blood and plasma

Cell-free DNA (cfDNA) biomarkers derived from Arthrobacter luteus (ALU) repeats and long interspersed nuclear elements 1 (LINE1) - including ALU-115, ALU-247, LINE1-97, and LINE1-266 concentrations, as well as the integrity ratios ALU-247/115 and LINE1-266/97 - are commonly utilized to assess cfDNA quantity and integrity. This study examined the impact of delayed blood processing and prolonged plasma storage on these biomarkers using quantitative polymerase chain reaction. Blood samples were collected from twelve healthy individuals (6 males; mean age, 65.8 ± 4.69 years) into dipotassium ethylenediaminetetraacetic acid tubes. Plasma cfDNA was extracted after various storage durations and temperatures, with aliquots from immediately processed blood subsequently stored at -80°C for different time intervals. Except for LINE1-97, most biomarkers showed significantly higher levels in plasma isolated from whole blood stored at room temperature compared to plasma processed immediately. Storage at 4°C resulted in fragment-specific effects: ALU-247/115 levels remained stable at 3 hours but decreased at 6 hours, while LINE1-266/97 levels increased at both time points. For plasma stored at -80°C, ALU-derived biomarkers remained stable for up to 12 months; however, LINE1-97 levels significantly declined, accompanied by a corresponding increase in LINE1-266/97 as early as one month after freezing. These findings indicate that both storage duration and temperature significantly impact the measured levels of ALU- and LINE1-derived cfDNA biomarkers. Consequently, standardization of pre-analytical handling of blood and plasma is crucial for studies evaluating cfDNA quantity and integrity.