Schumacher Sara, Malchau Lauesgaard Jacob, Carlsson Therese, Linder Anna, Sundfeldt Karin
Sahlgrenska Center for Cancer Research, Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Sahlgrenska Center for Cancer Research, Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; Sahlgrenska University Hospital, Region Västra Götaland, Gothenburg, Sweden.
J Mol Diagn. 2025 Mar;27(3):199-208. doi: 10.1016/j.jmoldx.2024.12.008. Epub 2025 Jan 17.
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°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 room temperature was evaluated in comparison to storage at 4°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°C. Consistent with the in vitro findings, prospectively collected samples showed reduced fragmentation when stored at 4°C compared with room temperature (P = 0.007). Long-term storage at 4°C had a significant negative effect on DNA stability (P = 0.013), whereas freezing slowed down fragmentation. Immediate storage at 4°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.
在生殖道样本中可检测到源自卵巢癌的游离DNA（cfDNA）。本研究旨在评估分析前处理如何影响巴氏（Pap）试验中的DNA谱和完整性，以优化其检测卵巢癌（OC）的潜力。对来自OC患者的存档Pap试验进行分析，这些样本在室温下保存48小时后储存在-80°C，同时辅以体外实验。评估了在4°C、-20°C或-80°C保存的样本中温度相关的DNA片段化效应。与在4°C保存相比，评估了室温下随时间的DNA降解情况。结果在前瞻性收集的Pap试验中得到验证。通过片段分析评估DNA完整性。在来自OC患者的存档Pap试验中观察到短DNA片段的积累。在体外，室温下100至350 bp的片段在48小时内增加了11.5倍，而在4°C保存时增加了1.7倍。与体外研究结果一致，前瞻性收集的样本在4°C保存时与室温相比片段化减少（P = 0.007）。在4°C长期保存对DNA稳定性有显著负面影响（P = 0.013），而冷冻减缓了片段化。采样后立即在4°C保存可显著减少DNA降解，这表明了一种优化分析前处理并减少Pap试验中cfDNA分析不必要片段化的简单方法。
