Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
Methodology for isolation, identification and characterization of microvesicles in peripheral blood.
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DOI：
10.1016/j.jim.2011.10.012
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发表时间：
2012-01-31
影响因子：
2.2
通讯作者：
Owen, Whyte G.
中科院分区：
文献类型：
--
作者：
Jayachandran, Muthuvel;Miller, Virginia M.;Heit, John A.;Owen, Whyte G.
关键词：
循环细胞膜衍生的微泡（MV）的分析已经作为疾病的潜在诊断和预后生物标志物受到审查。然而，分离、标记和定量MV的方法既没有系统化，也没有经过验证。确定分析前、分析和分析后因素如何影响血浆MV计数、细胞来源标志物和促凝血剂表面磷脂酰丝氨酸的表达。从健康志愿者和患有心血管疾病和/或糖尿病的患者采集外周静脉血样品。使用来自这些人群的重复样本，评价了血液样本采集、抗凝剂和样本处理对无血小板血浆（PFP）的影响，以及MV分离、染色和储存（冻融）和细胞计数器设计。关键发现是使用柠檬酸盐或EDTA抗凝剂通过诱导微泡粘附于血小板或其他有形成分来减少或消除血浆中的微泡。蛋白酶抑制剂抗凝剂（包括肝素）可保存MV计数。开发了离心方案，其中分离的MV的回收率高，分辨率低至0.2微米乳胶珠的等效光散射。系统评价了每种手术对MV计数和特征的影响。这项研究提供了一个系统的方法MV的分离，鉴定和定量，必不可少的MV的发展作为诊断和预后的生物标志物的疾病。
Analyses of circulating cell membrane-derived microvesicles (MV) have come under scrutiny as potential diagnostic and prognostic biomarkers of disease. However, methods to isolate, label and quantify MV have been neither systematized nor validated. To determine how pre-analytical, analytical and post-analytical factors affect plasma MV counts, markers for cell of origin and expression of procoagulant surface phosphatidylserine. Peripheral venous blood samples were collected from healthy volunteers and patients with cardiovascular disease and/or diabetes. Effects of blood sample collection, anticoagulant and sample processing to platelet free plasma (PFP), and MV isolation, staining and storage (freeze-thaw) and cytometer design were evaluated with replicate samples from these populations. The key finding is that use of citrate or EDTA anticoagulants decreases or eliminates microvesicles from plasma by inducing adhesion of the microvesicles to platelets or other formed elements. Protease inhibitor anticoagulants, including heparin, preserve MV counts. A centrifugation protocol was developed in which recovery of isolated MV was high with resolution down to the equivalent light scatter of 0.2 micron latex beads. Each procedure was systematically evaluated for its impact on the MV counts and characteristics. This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease.
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DOI：
10.1152/ajpheart.00193.2008
发表时间：
2008-09-01
影响因子：
4.8
作者：
Jayachandran, Muthuvel;Litwiller, Robert D.;Miller, Virginia M.
通讯作者：
Miller, Virginia M.
影响因子：
20.3
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
12.8
作者：
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通讯作者：
Ziglio, Giuliano
影响因子：
10.4
作者：
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通讯作者：
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