Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is essential to the accurate analysis of EV cargo. Unfortunately, generally used isolation techniques do not offer good separation of EVs from non-EV contaminants. Hence, it is important to have a standardized method to characterise the properties of EV preparations, including size distribution, particle concentration, purity and phenotype. Employing a laboratory-built nano-flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm, here we report a new benchmark to the quality and efficiency assessment of EVs isolated from plasma, one of the most difficult body fluids to work with. The performance of five widely used commercial isolation kits was examined and compared with the traditional differential ultracentrifugation (UC). Two to four orders of magnitude higher particle concentrations were observed for EV preparations from platelet-free plasma (PFP) by kits when compared with the EV preparation by UC, yet the purity was much lower. Meanwhile, the particle size distribution profiles of EV preparations by kits closely resembled those of PFP whereas the EV preparation by UC showed a broader size distribution at relatively large particle size. When these kits were used to isolate EVs from vesicle-depleted PFP (VD-PFP), comparable particle counts were obtained with their corresponding EV preparations from PFP, which confirmed again the isolation of a large quantity of non-vesicular contaminants. As CD9, CD63 and CD81 also exist in the plasma matrix, single-particle phenotyping of EVs offers distinct advantage in the validation of EVs compared with ensemble-averaged approaches, such as Western blot analysis. nFCM allows us to compare different isolation techniques without prejudice.

中文翻译：

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通过纳米流式细胞术评估六种细胞外囊泡分离方法的质量和效率。

细胞外囊泡（EVs）由于其在诊断和治疗中的巨大潜力而​​引起了极大的兴趣。从高纯度的复杂生物流体中分离出电动汽车对于准确分析电动汽车货物至关重要。不幸的是，通常使用的隔离技术不能很好地将电动汽车与非电动汽车污染物隔离开。因此，重要的是要有一种标准化的方法来表征电动车制剂的特性，包括尺寸分布，颗粒浓度，纯度和表型。利用实验室建造的纳米流式细胞仪（nFCM），可以对小至40 nm的单个EV进行多参数分析，在这里，我们报告了从血浆（最难的体液之一）中分离出的EV的质量和效率评估的新基准跟...共事。检查了五种广泛使用的商业分离试剂盒的性能，并将其与传统的差异超速离心（UC）进行了比较。与通过UC制备的EV制剂相比，通过试剂盒从无血小板血浆（PFP）制备的EV制剂观察到的颗粒浓度高出2到4个数量级，而纯度却低得多。同时，通过试剂盒制备的EV制剂的粒度分布曲线与PFP的粒度分布图非常相似，而通过UC制备的EV制剂在相对较大的粒度下显示出较宽的粒度分布。当使用这些试剂盒从泡囊贫化的PFP（VD-PFP）中分离出EV时，从PFP中获得相应的EV制剂可获得相当的颗粒计数，这再次证实了大量非泡囊污染物的分离。作为CD9，CD63和CD81也存在于血浆基质中，与整体平均方法（例如Western印迹分析）相比，电动汽车的单颗粒表型在电动汽车的验证中提供了明显的优势。nFCM允许我们比较不同的隔离技术而不会产生偏见。