Clinical and Health Sciences, University of South Australia, Adelaide, Australia.
Freemasons Foundation Centre for Men's Health, Adelaide, Australia.
Biopreserv Biobank. 2020 Oct;18(5):462-470. doi: 10.1089/bio.2020.0060. Epub 2020 Aug 26.
Liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. However, these are highly affected by preprocessing variables with many protocols designed for downstream analysis of a single molecular biomarker. Here we investigate whether specialized blood tubes could be repurposed for the analysis of an increasingly valuable biomarker, extracellular vesicles (EVs). Blood was collected from three donors into K3-EDTA, Roche, or Streck cell-free DNA (cfDNA) collection tubes and processed using sequential centrifugation either immediately or after storage for 3 days. MicroEV were collected from platelet-poor plasma by 10,000 centrifugation and NanoEVs isolated using size exclusion chromatography. Particle size and counts were assessed by Nanoparticle Tracking Analysis, protein quantitation by bicinchoninic acid assay (BCA) assay, and dot blotting for blood cell surface proteins. MicroEVs and NanoEVs could be isolated from plasma collected using all three tube types. Major variations were seen with delayed time to processing. Both MicroEV particle number and protein content increased with the processing delay. The NanoEV number did not change with the time-delay but their protein quantity increased. EV-associated proteins predominantly arose from platelets (CD61) and erythrocytes (CD235a). However, leukocyte marker CD45 was only increased in NanoEVs from ethylenediaminetetraacetic acid (EDTA) tubes, suggestive of stabilization of nucleated cells by the specialized blood tubes. Epithelial cell surface marker EpCAM, often used as a marker of cancer, remained the same across conditions in both MicroEV and NanoEV preparations indicating that these EVs were stable with time. Specialized cfDNA collection tubes can be repurposed for MicroEV and NanoEV analysis; however, simple counting or using protein quantity as a surrogate of EV number may be confounded by preanalytical processing. The EVs would be suitable for disease selective EV subtype analysis if the molecular target of interest is not present in blood cells.
液体活检为患者疾病诊断和监测提供了一种微创方法。然而，这些方法受到预处理变量的极大影响，许多方案都是专为单一分子生物标志物的下游分析而设计的。在这里，我们研究了专门的采血管是否可以重新用于分析越来越有价值的生物标志物——细胞外囊泡 (EVs)。从三名供体中采集血液到 K3-EDTA、罗氏或 Streck 无细胞 DNA (cfDNA) 收集管中，并立即或在储存 3 天后使用连续离心进行处理。从血小板贫乏的血浆中通过 10000 离心收集微 EV，并使用排阻色谱法分离纳米 EV。通过纳米颗粒跟踪分析评估颗粒大小和计数，通过二辛可宁酸测定法 (BCA) 测定蛋白定量，并进行血细胞表面蛋白的斑点印迹分析。可以从使用所有三种管类型采集的血浆中分离出 MicroEV 和 NanoEV。处理时间延迟较大时会出现较大的差异。随着处理时间的延迟，MicroEV 的颗粒数和蛋白含量都增加了。NanoEV 的数量不会随时间延迟而变化，但它们的蛋白含量增加。EV 相关蛋白主要来源于血小板 (CD61) 和红细胞 (CD235a)。然而，白细胞标志物 CD45 仅在 EDTA 管中的 NanoEV 中增加，提示专用采血管稳定了有核细胞。上皮细胞表面标志物 EpCAM，常用于癌症标志物，在 MicroEV 和 NanoEV 制剂中，在所有条件下保持不变，表明这些 EV 随时间稳定。专门的 cfDNA 收集管可重新用于 MicroEV 和 NanoEV 分析；然而，简单计数或使用蛋白量作为 EV 数量的替代物可能会受到预处理的影响。如果感兴趣的分子靶标不存在于血液细胞中，则 EV 适合用于疾病选择性 EV 亚型分析。
