An Optimized Flow Cytometry Protocol for Analysis of Angiogenic Monocytes and Endothelial Progenitor Cells in Peripheral Blood
An Optimized Flow Cytometry Protocol for Analysis of Angiogenic Monocytes and Endothelial Progenitor Cells in Peripheral Blood
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DOI：
10.1002/cyto.a.20772
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发表时间：
2009-10-01
期刊：
影响因子：
3.7
通讯作者：
Weber, Christian
中科院分区：
文献类型：
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作者：
Hristov, Mihail;Schmitz, Susanne;Weber, Christian
循环中的成人CD 34（+）VEGFR 2（+）内皮祖细胞（EPCs）已被证明可分化为内皮细胞，从而有助于血管稳态。此外，循环中的CD 14（+）单核细胞亚群共表达CD 16和血管生成素受体Tie 2，并在肿瘤血管生成中发挥作用。然而，外周血中EPCs和Tie 2（+）单核细胞的流式细胞术定量的临床适用方案和参考值的共识仍然难以捉摸。使用与PerCP、PE、PE-Cy 7、APC和APC-Cy 7偶联的特异性单克隆抗体，通过三色流式细胞术评估稳定型冠状动脉疾病患者外周静脉血中Tie 2（+）CD 14（+）CD 16（中）血管生成单核细胞和CD 34（+）VEGFR 2（+）CD 45（低/-）EPCs的数量。进行排除死细胞的散点多门控以解剖复杂的单核细胞群体。通过将明亮的荧光染料（PE-Cy 7、PE、APC）与微弱表达的标志物（CD 34、VEGFR 2、Tie 2）匹配，通过自动补偿以使荧光溢出最小化，并通过使用荧光减一（FMO）对照来确定阳性/阴性边界，进一步细化该分析。假设高斯分布，我们得到平均值Tie 2（+）CD 14（+）CD 16（中）单核细胞为1.45 +/- 1.29%（平均值+/- SD）（n = 11，范围：0.12-3.64%）和0.019 +/- 0.013%的CD 34（+）VEGFR 2（+）CD 45（低/-）EPC（n = 17，范围：0.003-0.042%）。试验内和试验间变异性分别为1.6%和4.5%。我们已经优化了一种快速，灵敏的流式细胞术定量检测循环血管生成单核细胞和内皮祖细胞在心血管药物。该方案可代表这些细胞亚群的标准化分析和监测的基础，以确定其在临床应用中的正常范围和预后/诊断价值。(C)2009年国际细胞计数促进学会
Circulating adult CD34(+)VEGFR2(+) endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14(+) monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2(+) monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2(+)CD14(+)CD16(mid) angiogenic monocytes and CD34(+)VEGFR2(+)CD45(low/-) EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, and APC-Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE-Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence-minus-one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean +/- SD) of 1.45 +/- 1.29% for Tie2(+)CD14(+)CD16(mid) monocytes (n = 11, range: 0.12-3.64%) and 0.019 +/- 0.013% for CD34(+)VEGFR2(+)CD45(low/-) EPCs (n = 17, range: 0.003-0.042%). The intra- and inter-assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use. (C) 2009 International Society for Advancement of Cytometry