Department of Clinical Immunology, Oxford University Hospitals NHS Trust, Oxford, UK.
School of Immunity and Infection, University of Birmingham Medical School, Birmingham, UK.
J Extracell Vesicles. 2014 Sep 30;3. doi: 10.3402/jev.v3.25348. eCollection 2014.
Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results.
ONE HUNDRED SAMPLES FROM HEALTHY INDIVIDUALS AND PATIENTS WITH OBSTRUCTIVE SLEEP APNOEA WERE ANALYSED BY CONVENTIONAL FLOW CYTOMETRY (FACSCALIBUR) AND BY THREE FUNCTIONAL MV ASSAYS: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA(®) Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation.
Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001); correlated with ETP (r=0.7444, p<0.0001); negatively correlated with STA(®) Phospholipid Procoag Assay clotting time (-0.7872, p<0.0001), reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results.
Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive information on the role of MVs in health and disease.
流式细胞术是测量微泡（MVs）最常用的技术。尽管该技术存在已报道的局限性，但使用常规流式细胞术获得的 MV 水平产生了许多具有临床相关性的发现，例如与疾病严重程度的关联以及预测临床结局的能力。本研究旨在确定流式细胞术测量的 MV 计数是否与它们的功能能力相关，因为这可能解释了流式细胞术如何产生具有临床相关性的结果。
对 100 名健康个体和阻塞性睡眠呼吸暂停患者的样本进行了常规流式细胞术（FACSCALIBUR）和三种功能性 MV 分析：Zymuphen MP-活性，以磷脂酰丝氨酸当量表示数据；STA(®) Phospholipid Procoag Assay，以凝血时间表示；内源性凝血酶潜能（ETP），反映体外凝血酶生成。通过 Spearman 相关性确定相关性。
流式细胞术产生的乳酰脱氢酶+促凝 MV 的绝对计数与 Zymuphen MP-活性的结果呈弱相关（r=0.5370，p<0.0001）；与 ETP 相关（r=0.7444，p<0.0001）；与 STA(®) Phospholipid Procoag Assay 凝血时间呈负相关（-0.7872，p<0.0001），反映了凝血活性与流式细胞术之间的正相关。 Annexin V+促凝和血小板衍生 MV 的水平也与功能测定相关。源自其他细胞类型的 MV 的绝对计数与功能结果无关。
常规流式细胞术获得的促凝和血小板衍生 MV 的定量结果与 MV 的功能能力相关，这是通过三种功能性 MV 测定来定义的。流式细胞术是一种用于从不同细胞来源定量 MV 的有价值的技术；然而，几种分析技术的组合可能会提供关于 MV 在健康和疾病中的作用的最全面信息。
