Malentacchi Francesca, Pizzamiglio Sara, Verderio Paolo, Pazzagli Mario, Orlando Claudio, Ciniselli Chiara Maura, Günther Kalle, Gelmini Stefania
Clin Chem Lab Med. 2015 Nov;53(12):1935-42. doi: 10.1515/cclm-2014-1161.
Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies.
Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR).
The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile.
The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).
循环游离DNA（ccfDNA）已被确认为癌症和产前临床实践中的一种有用生物标志物。使用ccfDNA的主要关键点之一是样本处理方法、储存条件、提取程序和定量缺乏标准化，这些都会影响ccfDNA的质量和数量。我们报告了从SPIDIA-DNAplas获得的结果，SPIDIA-DNAplas是欧盟SPIDIA（体外诊断通用分析前工具和程序的标准化与改进）子项目之一，基于实施一项外部质量评估计划，以评估分析前阶段对ccfDNA的影响。这是首个针对分析前阶段研究的ccfDNA质量控制方案。
招募了欧洲各地的56个实验室。参与实验室收到相同的血浆样本，并在规定的血浆储存条件下使用各自的程序提取ccfDNA，然后将分离出的ccfDNA送至SPIDIA机构进行分析。通过使用特定的质量参数（如ccfDNA完整性（通过多重PCR）和产量（通过qPCR））评估实验室性能。
对实验室提取的ccfDNA进行分析表明，大多数实验室（56个中的53个）能够回收ccfDNA，但只有12.5%回收的是未片段化的ccfDNA。专门为ccfDNA设计的提取方法保留了完整性特征。
SPIDIA-DNAplas EQA基于证据的结果已被提议作为欧洲标准化委员会（CEN）制定技术规范的基础。
