Optimization of Pre-Analytical Handling to Maintain DNA Integrity in Diagnostic Papanicolaou Tests

Cell-free DNA (cfDNA) of ovarian carcinoma origin can be detected in samples from the gynecologic tract. This study aims to evaluate how pre-analytical handling affects DNA profile and integrity in Papanicolaou (Pap) tests, to optimize their potential for detection of ovarian cancers (OCs). Analysis of archived Pap tests from patients with OC, kept at room temperature for 48 hours and stored at-80 degrees C, was complemented by in vitro experiments. Temperature-associated effects on DNA fragmentation were evaluated in samples stored at 4 degrees C,-20 degrees C, or-80 degrees C. Time-dependent DNA degradation at room temperature was evaluated in comparison to storage at 4 degrees C. Results were validated in prospectively collected Pap tests. The DNA integrity was assessed by fragment analysis. Accumulation of short DNA fragments was observed in archived Pap tests from patients with OC. In vitro, fragments of 100 to 350 bp increased 11.5-fold within 48 hours at room temperature compared with 1.7-fold when stored at 4 degrees C. Consistent with the in vitro findings, prospectively collected samples showed reduced fragmentation when stored at 4 degrees C compared with room temperature (P = 0.007). Long-term storage at 4 degrees C had a significant negative effect on DNA stability (P = 0.013), whereas freezing slowed down fragmentation. Immediate storage at 4 degrees C after sampling markedly reduces DNA degradation, suggesting a simple way to optimize pre-analytical handling and decrease unwanted fragmentation for cfDNA analysis in Pap tests. (J Mol Diagn 2025, 27: 199e208; https://doi.org/10.1016/j.jmoldx.2024.12.008)