A flow cytometric protocol for enumeration of endothelial progenitor cells and monocyte subsets in human blood
A flow cytometric protocol for enumeration of endothelial progenitor cells and monocyte subsets in human blood
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DOI：
10.1016/j.jim.2012.04.003
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发表时间：
2012-07-31
影响因子：
2.2
通讯作者：
Weber, Christian
中科院分区：
文献类型：
--
作者：
Hristov, Mihail;Schmitz, Susanne;Weber, Christian
背景：越来越多的证据强烈建议循环内皮祖细胞和单核细胞亚群作为心血管和癌症疾病的替代细胞生物标志物。然而，它们的定量仍然缺乏一个通用的标准，从而排除了对临床研究的常规监测和比较解释。目的：我们打算开发一种先进的、快速的流式细胞术方法，用于适当地体外定量人类血液中的单核细胞亚群和内皮祖细胞。我们在适当的补偿下使用了三色抗体面板。EPC实验中罕见事件和低抗原表达的分析通过排除死亡细胞的序贯门控、高强度荧光与低密度标记物的匹配以及实施荧光减一对照来加强。结果：对10名健康献血者外周血的分析显示，中位数(IQR)为1.88(1.35-2.85)活CD45(DIM)CD34(+)VEGFR2(+)内皮祖细胞/微升。对单核细胞的分析显示，经典CD14(++(高))CD16(-)、中等CD14(++)CD16(+(中))和非经典CD14(+(低))CD16(++)单核细胞每微升分别有329.5(264.5-374.8)、16.0(8.0-22.2)和26.5(19.8-36.3)个细胞。因此，这种分析方法高度适用于常规使用。(C)2012爱思唯尔B.V.保留所有权利。
Background: Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) and monocyte subsets as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative interpretation of clinical studies.Objective: We intend to develop an advanced and express flow cytometric protocol for proper ex vivo quantification of monocyte subsets and EPCs in human blood.Methods: We employ now lyse/no-wash procedure and bead-based determination of absolute cell counts. We use three-color antibody panels at appropriate compensation. Analysis of rare events and low antigen expression in the EPC experiment is strengthening by sequential gating with exclusion of dead cells, as well as by matching high-intensity fluorochromes to low-density markers and by implementing the fluorescence-minus-one control.Results: Analysis of peripheral blood of ten healthy donors revealed median (IQR) value of 1.88 (1.35-2.85) viable CD45(dim)CD34(+)VEGFR2(+) EPCs per microliter. Analysis of monocytes revealed 329.5 (264.5-374.8), 16.0 (8.0-22.2) and 26.5 (19.8-36.3) cells per microliter for classical CD14(++(high))CD16(-), intermediate CD14(++)CD16(+(mid)) and non-classical CD14(+(low)) CD16(++) monocytes.Conclusion: Our current protocol provides quantitative information under a simple gating logic while using commonly accepted fluorochromes. This assay is therefore highly adapted for routine use. (C) 2012 Elsevier B.V. All rights reserved.