Mladenović Danilo, Khamari Delaram, Kittel Ágnes, Koort Kairi, Buzás Edit I, Zarovni Nataša
HansaBioMed Life Sciences Ltd., Tallinn, Estonia; School of Natural Sciences and Health, Tallinn University, Tallinn, Estonia. Electronic address: https://twitter.com/DanMladenovic.
Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary; Eötvös Loránd Research Network, Translational Extracellular Vesicle Research Group, Semmelweis University, Budapest, Hungary.
J Thromb Haemost. 2023 Apr;21(4):1032-1042. doi: 10.1016/j.jtha.2023.01.007. Epub 2023 Jan 14.
Blood plasma is available with minimal invasive sampling, it has significant diagnostic utility, and it is a valuable source of extracellular vesicles (EVs). Nevertheless, rich protein content, the presence of lipoproteins (LPs) that share similar biophysical properties, and relatively low abundance of EVs, especially those of rare subpopulations, make any downstream application a very challenging task. The growing evidence of the intricate surface interactome of EVs, and the association of EVs with LPs, impose further challenges during EV purification, detection, and biomarker analyses.
In this study, we tackled the fundamental issues of plasma EV yield and LP co-isolation and their implications in the subsequent marker analyses.
Moderate acidification of plasma was combined with size exclusion chromatography (SEC) and/or differential centrifugation (DC) to disrupt LPs and improve recovery of EVs and their subsequent detection by immunoassays and single-particle analysis methods.
Our results demonstrate a surprisingly efficient enrichment of EVs (up to 3.3-fold higher than at pH 7) and partial depletion of LPs (up to 61.2%). Acidification of blood plasma samples enabled a quick single-step isoelectric precipitation of up to 20.4% of EVs directly from plasma, upon short low-speed centrifugation.
Thus, acidification holds potential as a simple and inexpensive methodological step, which improves the efficacy of plasma EV enrichment and may have implications in future biomarker discoveries.
血浆可通过微创采样获得，具有重要的诊断价值，是细胞外囊泡（EVs）的宝贵来源。然而，血浆中丰富的蛋白质含量、具有相似生物物理特性的脂蛋白（LPs）的存在以及EVs相对较低的丰度，尤其是稀有亚群的EVs，使得任何下游应用都成为一项极具挑战性的任务。越来越多的证据表明EVs存在复杂的表面相互作用组，以及EVs与LPs的关联，这在EVs的纯化、检测和生物标志物分析过程中带来了进一步的挑战。
在本研究中，我们解决了血浆EVs产量和LP共分离的基本问题及其在后续标志物分析中的影响。
将血浆适度酸化与尺寸排阻色谱（SEC）和/或差速离心（DC）相结合，以破坏LPs并提高EVs的回收率，随后通过免疫测定和单颗粒分析方法进行检测。
我们的结果表明，EVs的富集效率惊人（比pH 7时高3.3倍），LPs部分减少（高达61.2%）。血浆样品酸化后，通过短时间低速离心，可直接从血浆中快速进行单步等电沉淀，沉淀的EVs高达20.4%。
因此，酸化作为一个简单且廉价的方法步骤具有潜力，它可提高血浆EVs富集的效率，并可能对未来生物标志物的发现产生影响。
