Background Maintaining DNA integrity in blood samples, from transport to storage to processing, is crucial to the success of downstream applications, especially regarding diagnostic applications. Additionally, due to the invasive nature of collecting blood samples, minimizing resampling to avoid excess patient pain and cost of collection and transportation are important to consider. Having the option to store and maintain portions of blood samples in the freezer enables future retesting without recollection. Since -20°C freezers generally are more affordable, require less energy, and have smaller footprints than -80°C freezers, demonstrating frozen blood stability at -20°C temperatures could decrease sampling burdens for labs with facility constraints. Methods Whole blood was collected into 9mL vacuum tubes (HemaSure-OMXP) from healthy donors. Aliquots of whole blood and buffy coat samples were stored at -20°C and thawed for genomic DNA extractions (QIAamp DNA Blood Mini Kit, Qiagen) at a range of different timepoints; frozen whole blood samples were processed at 0, 49, 141, and 148 days and frozen buffy coat samples were processed at 7, 27, 36, 185, 246, 373, 394, 584, and 604 days. Purified gDNA was quantified with Qubit (Broad Range dsDNA Assay, ThermoFisher) and qualified with Nanodrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher) and TapeStation (Genomic DNA ScreenTape, Agilent Technologies). Results No significant decrease was observed for DNA yield and quality between fresh and frozen whole blood samples. Average DNA yields from 200 μL whole blood were 4.5 ± 2.2 μg and 5.3 ± 1.2 μg for fresh and frozen blood respectively. Average DNA integrity (DIN) was 6.9 ± 0.4 and 7.1 ± 0.3 for fresh and frozen blood respectively. Donor differences in blood samples can help explain the variance in DNA yields reported. No significant decrease was observed for DNA yield and quality in buffy coat samples frozen for longer at -20°C. In total, 84 whole blood and 25 buffy coat samples were processed in this study. Conclusions Although buffy coat samples tended to have higher yields than whole blood, due to the higher amount of white blood cells present, both sample types provided sufficient amounts of gDNA for downstream applications. Freezing blood samples for future testing can be effective at reducing the need for resampling without sacrificing the nucleic acid yield or quality of fresh samples. Additionally, as technological advancements are made for equipment and assays, frozen blood samples can be retested and compared to previous results for improved diagnostic decisions, done without requiring recollection from labs and patients.

中文翻译：

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B-184 从 -20°C 保存长达 1.5 年的冷冻血液样本中纯化高质量基因组 DNA


背景 保持血液样本中 DNA 的完整性，从运输到储存再到处理，对于下游应用（尤其是诊断应用）的成功至关重要。此外，由于采集血液样本的侵入性，因此必须考虑尽量减少重新采样以避免患者过度痛苦以及采集和运输的成本。可以选择在冰箱中存储和保存部分血液样本，以便将来重新测试而无需重新收集。由于 -20°C 冷冻机通常比 -80°C 冷冻机更便宜、需要更少的能源并且占地面积更小，因此证明冷冻血液在 -20°C 温度下的稳定性可以减轻设施有限的实验室的采样负担。方法将健康献血者的全血收集到9mL真空管(HemaSure-OMXP)中。将全血和血沉棕黄层样品的等分试样储存在 -20°C 下，并在一系列不同时间点解冻用于基因组 DNA 提取（QIAamp DNA Blood Mini Kit，Qiagen）；在第 0、49、141 和 148 天处理冷冻全血样本，在第 7、27、36、185、246、373、394、584 和 604 天处理冷冻血沉棕黄层样本。使用 Qubit（宽范围 dsDNA 检测，ThermoFisher）对纯化的 gDNA 进行定量，并使用 Nanodrop One 微量紫外-可见分光光度计 (ThermoFisher) 和 TapeStation（基因组 DNA ScreenTape，Agilent Technologies）进行鉴定。结果 新鲜全血样本和冷冻全血样本之间的 DNA 产量和质量未观察到显着下降。 200 μL 全血的平均 DNA 产量分别为 4.5 ± 2.2 μg 和 5.3 ± 1.2 μg（新鲜血液和冷冻血液）。新鲜血液和冷冻血液的平均 DNA 完整性 (DIN) 分别为 6.9 ± 0.4 和 7.1 ± 0.3。 血液样本中供体的差异有助于解释所报告的 DNA 产量的差异。在 -20°C 下冷冻较长时间的血沉棕黄层样品中，未观察到 DNA 产量和质量显着下降。本研究总共处理了 84 个全血和 25 个血沉棕黄层样本。结论 虽然血沉棕黄层样品的产量往往高于全血，但由于白细胞数量较多，两种样品类型都为下游应用提供了足够量的 gDNA。冷冻血液样本以供将来测试可以有效减少重新采样的需要，而不会牺牲新鲜样本的核酸产量或质量。此外，随着设备和检测技术的进步，可以重新测试冷冻血液样本并与之前的结果进行比较，以改进诊断决策，而无需从实验室和患者重新收集。