Flow cytometric analysis of circulating microparticles in plasma.
Flow cytometric analysis of circulating microparticles in plasma.
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DOI：
10.1002/cyto.a.20886
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发表时间：
2010-06
期刊：
影响因子：
3.7
通讯作者：
Lewis, Dorothy E.
中科院分区：
文献类型：
--
作者：
Orozco, Aaron F.;Lewis, Dorothy E.
关键词：
微颗粒包括外泌体、微囊泡、凋亡小体和凋亡微颗粒，它们是小的（直径0.05 ~ 3 μm）膜状囊泡，可以包含DNA、RNA、miRNA、细胞内蛋白，并表达来自亲本细胞的细胞外表面标记物。它们可以从细胞内多泡体分泌或从起泡膜表面释放。正常人血浆中含有大量循环微粒，可来源于循环血细胞，如血小板、红细胞和白细胞，也可来源于组织，如内皮组织和胎盘组织。微颗粒水平升高与多种疾病有关，如血栓形成（血小板微颗粒）、充血性心力衰竭（内皮细胞微颗粒）、乳腺癌患者（白细胞微颗粒）和子痫前期妇女（合体滋养细胞微颗粒）。虽然微颗粒可以通过显微镜、酶联免疫测定和功能测定来检测，但流式细胞术是首选的方法，因为它具有定量的能力（荧光珠或基于流量的方法），而且具有多色能力。然而，分离、计数和荧光标记微粒的分析前和分析操作方法的标准化仍然是一个挑战。本文的主要重点是回顾优化研究体内循环微颗粒所需的初步步骤，包括：1)使用的离心速度，2)抗体标记前的微颗粒定量，3)抗体标记微颗粒的荧光强度水平，4)微颗粒亚群的多色流式细胞分析和5)用于流式细胞术的Western blotting设计的多克隆抗体的使用。这些研究确定了开发微颗粒作为各种条件下的生物标志物的路线图。©2010国际细胞术进步学会
Microparticles, which include exosomes, micro‐vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 ‐ 3 μm in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzyme‐linked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent bead‐ or flow rate‐based method) and because of polychromatic capabilities. However, standardization of pre‐analytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulatingin vivomicroparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibody‐labeled microparticles, 4) polychromatic flow cytometric analysis of microparticle sub‐populations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions. © 2010 International Society for Advancement of Cytometry
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