Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Department of Pathology, Amsterdam University Medical Centres, University of Amsterdam, Amsterdam, The Netherlands.
Clin Chem. 2022 Jul 3;68(7):963-972. doi: 10.1093/clinchem/hvac069.
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.
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.
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.
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.
循环肿瘤 DNA（ctDNA）的高效回收依赖于循环无细胞 DNA（ccfDNA）的数量和质量。在此，我们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，使用了相同的肺癌患者衍生血浆样本。
将 4 份高容量诊断白细胞分离血浆样本和 1 份人工参考血浆样本的等分试样分配给 14 家荷兰实验室。根据当地的常规标准操作程序进行 ccfDNA 的提取，并在中央参考实验室进行分析，以检测突变体并评估 ccfDNA 的数量和完整性。
从提取的 ccfDNA 样本中突变分子水平在实验室之间差异很大，但没有一致的高于或低于平均水平的表现。与基于硅胶膜的方法相比，基于磁性珠的试剂盒提取的样本总体上总 ccfDNA 产量较低（-29%；P<0.0001），并且回收的突变分子较少（-41%；P<0.01）。变异等位基因频率和样本完整性相似。在总 ccfDNA 产量高于平均水平的样本中，观察到突变分子的回收增加。
在荷兰，我们发现了前分析工作流程的多样性，这可能对临床实践中突变 ctDNA 的检测产生影响。基于硅胶膜的方法导致了最高的总 ccfDNA 产量，因此更适合检测相关突变的低拷贝数。需要对提取工作流程进行协调，以实现准确的定量和敏感的检测，以防止在预分析阶段引入技术差异，并减少实验室间的差异。
