Optimization of pre-analytical handling to maintain DNA integrity in diagnostic Papanicolaou tests.

Abstract

Cell-free DNA of ovarian tumor origin can be detected in samples from the gynecologic tract. This study aims to evaluate how pre-analytical handling, and storage conditions affect DNA profile and integrity in Pap tests, to optimize its potential for detection of ovarian cancers (OC). Analysis of archived Pap tests from OC patients, kept at RT for 48h and stored at -80°C was complemented by in vitro experiments. Temperature-associated effects on DNA fragmentation were evaluated in samples stored at 4°C, -20°C or -80°C. Time-dependent DNA degradation at RT was evaluated in comparison to storage at 4°C (0-96h). 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 OC patients. In vitro, fragments of 100-350bp increased 11.5-fold within 48h at RT compared to 1.7-fold when stored at 4°C. Consistent with the in vitro findings, prospectively collected samples showed reduced fragmentation when stored at 4°C compared to RT (p=0.007). Long-term storage at 4°C had a significant negative effect on DNA stability (p=0.013), while freezing slowed down fragmentation. This study highlights the need for optimization of pre-analytical handling for cfDNA analysis. Immediate storage at 4°C after sampling markedly reduces DNA degradation suggesting a simple way to decrease unwanted fragmentation for cfDNA analysis in Pap tests.