The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray
The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray
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DOI：
10.1016/j.jim.2016.08.007
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发表时间：
2016-11-01
影响因子：
2.2
通讯作者：
Jorgensen, Malene M.
中科院分区：
文献类型：
--
作者：
Baek, Rikke;Sondergaard, Evo K. L.;Jorgensen, Malene M.
细胞外囊泡的研究领域不断扩大，其潜在的应用前景也是无限的。它们大量存在于各种体液中，血液样本可以作为液体活检。然而，这些细胞来源的小膜衍生实体在分离和表征方面并不简单。测试了广泛的相关预分析问题，重点是较小EV的表型影响。检查i）所用采血管，ii）初始离心前的孵育时间，iii）运输/物理应力，iv）储存温度和时间（短期和长期），v）离心方案的选择，vi）冻融循环，和vii）外来体分离程序（ExoQuick，TM）的影响。为了确定分析前处理的影响，使用基于多重抗体的微阵列技术（称为EV阵列）分析了小EV的相对量（检测到的CD 9阳性、CD 63阳性和/或CD 81阳性的信号强度）和表型。该分析包括15个表面或表面相关标志物，包括CD 9、CD 63、CD 81、CD 142和膜联蛋白V。该研究显示，在不同采血管中采集的样本在不同程度上受到此处测试的分析前处理的影响。对所提出的问题没有明确的答案。然而，一般而言，观察到初始离心前的时间段和预期运输、离心方案的选择和储存温度对样品有重大影响。相反，长期储存和冻融似乎没有关键的影响。因此，关于样品收集和制备的任何选择都有利弊，并且很可能取决于分析。然而，为了比较样品和结果，重要的是要确保所有样品都是相同类型的，并且经过类似的处理。(C)2016年6月，作者。由爱思唯尔公司出版。这是一篇开放获取的文章，使用CC BY许可证（http：//creativecommons.org/licenses/by/4.0/）。
The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization.A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick,TM) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V.This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).