Gerber Teresa, Taschner-Mandl Sabine, Saloberger-Sindhöringer Lisa, Popitsch Niko, Heitzer Ellen, Witt Volker, Geyeregger René, Hutter Caroline, Schwentner Raphaela, Ambros Inge M, Ambros Peter F
Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz and Christian Doppler Laboratory for Liquid Biopsies for Early Detection of Cancer, Graz, Austria.
J Mol Diagn. 2020 Aug;22(8):1070-1086. doi: 10.1016/j.jmoldx.2020.05.006. Epub 2020 Jun 1.
Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.
液体活检作为一种微创方法，有潜力彻底改变分子诊断。然而，尽管样本处理和循环肿瘤DNA（ctDNA）分离的方案众多，但目前尚无考虑现实生活中多中心临床研究所有方面的诊断和研究综合指南。这些方面包括样本量、运输和采血管的限制。我们测试了常用的（乙二胺四乙酸和肝素）和专用采血管以及储存条件对无细胞DNA产量和纯度的影响，以用于下游分析。此外，我们评估了一种用于ctDNA和肿瘤细胞基因组检测以及白细胞平行流式细胞术分析的联合工作流程的可行性。对于基因组分析，如果在室温下最多储存4小时或在4°C下储存长达24小时，乙二胺四乙酸管显示出良好的结果。加标实验表明，乙二胺四乙酸管与密度梯度离心相结合，能够并行分离ctDNA、白细胞和少量肿瘤细胞（0.1%），并通过流式细胞术对其进行免疫表型分析，以及通过全基因组测序进行下游基因组分析。总之，遵守时间和温度限制允许使用常规乙二胺四乙酸血样进行液体活检分析。我们还提供了一种工作流程，能够对无细胞和细胞特征进行并行分析，用于疾病监测和克隆进化研究。
