Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.

细胞外囊泡（EVs）是在生理和病理过程中具有关键功能的细胞间通讯体，由于其诊断和治疗潜力，最近引起了人们的关注。过去的十年带来了从血清中分离EV的多种方法的开发和商业化。捕获电动汽车的哪些亚群在很大程度上取决于隔离方法，而隔离方法又决定了所得样本对于各种下游应用的适合程度。为了帮助临床医生和科学家为他们的实验选择最合适的方法，需要对分离方法进行比较表征。很少有人尝试全面分析患者生物流体中的水泡微RNA（miRNA）进行生物标记研究。为了解决这种差异，我们着手对健康个体和重症患者血清EV的几种隔离原则的性能进行基准测试。在这里，我们比较了五种不同的EV分离方法与两种RNA提取方法相结合的方式，以探讨它们对以生物标记物为中心的miRNA测序的适用性以及所捕获囊泡的生物学特性。我们的发现揭示了分离的囊泡的性质以及相关miRNA在生物标记研究中发挥作用的能力方面的显着方法特异性差异。尽管通过沉淀和膜亲和力进行分离非常适合基于miRNA的生物标记物发现，但基于尺寸排阻色谱的方法未能将患者与健康志愿者区分开。分离的囊泡的大小，数量，纯度和组成各不相同，表明每种方法都可以捕获独特的电动汽车种群以及其他污染物。即使这项工作的重点是EV-miRNA的转录组分析，我们的见识也适用于其他研究领域。我们为当今可用的多种EV隔离方法提供指导，并帮助研究人员和临床医生明智地选择针对重症患者的实验应采用哪种策略。