BACKGROUND Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

中文翻译：

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荷兰国家循环试验血浆衍生的循环无细胞 DNA 提取方法通常用于分子肿瘤分析的临床病理学。

背景技术循环肿瘤DNA（ctDNA）的有效回收取决于循环游离DNA（ccfDNA）的数量和质量。在这里，我们使用相同的肺癌患者血浆样本评估了荷兰实验室常规应用的各种 ccfDNA 提取方法是否会影响 ccfDNA 产量、ccfDNA 完整性和突变 ctDNA 检测。方法 4 份大容量诊断性白细胞分离术血浆样本和一份具有预定肿瘤衍生突变的人工参考血浆样本的等分试样分布在 14 个荷兰实验室中。ccfDNA 的提取是根据当地常规标准操作程序进行的，并在中央参考实验室进行分析，用于突变检测和 ccfDNA 数量和完整性的评估。结果 提取的 ccfDNA 样本中的突变分子水平在实验室之间差异很大，但没有迹象表明其性能始终高于或低于平均水平。与基于硅胶膜的方法相比，使用基于磁珠的试剂盒提取的样品显示总 ccfDNA 产量总体较低（-29%；P < 0.0001），并且回收的突变分子更少（-41%；P < 0.01）。变异等位基因频率和样品完整性相似。在总 ccfDNA 产量高于平均水平的样品中，观察到突变分子的回收率增加。结论 在荷兰，我们遇到了分析前工作流程的多样性，这些工作流程对临床实践中的突变 ctDNA 检测具有潜在影响。基于硅胶膜的方法产生最高的 ccfDNA 总产量，因此更适合检测相关突变的低拷贝数。为了防止在分析前阶段引入技术差异并减少实验室间差异，需要协调提取工作流程以进行准确定量和灵敏检测。