外周血人单核细胞亚群分析中样品采集和样品制备的标准化。

Standardization of sampling and sample preparation for analysis of human monocyte subsets in peripheral blood.

机构信息

Department of Biomedical Laboratory Science and Chemical Engineering, Western Norway University of Applied Sciences, Bergen, Norway.

Department of Clinical Science, University of Bergen, Bergen, Norway; Haukeland University Hospital, Bergen, Norway.

出版信息

J Immunol Methods. 2018 Oct;461:53-62. doi: 10.1016/j.jim.2018.06.003. Epub 2018 Jun 12.

DOI:10.1016/j.jim.2018.06.003

PMID:29906454

Abstract

INTRODUCTION

Monocytes are important for innate immunity and include the classical (CD14CD16), intermediate (CD14CD16) and non-classical (CD14CD16) monocyte subsets. The quantification of these functionally different subsets in peripheral blood may become useful for diagnosis and follow-up in human diseases. The aim of the present study was to investigate how different pre-analytical parameters influence analysis of monocyte subsets in peripheral blood samples.

METHODS

We determined relative levels of monocytes and monocyte subsets by flow cytometry of peripheral blood samples derived from healthy individuals. A gating strategy exclusively extracting viable CD14 monocytes and focusing on the three monocyte subsets was applied. We investigated the effects of (i) various anticoagulants (i.e. Li-Heparin, ACD-A, KEDTA), (ii) insufficient filling of blood sampling tubes, (iii) cryopreservation. In addition, we analysed expression of the CCR2 chemokine receptor.

RESULTS

The relative numbers of CD14 monocytes depended on the anticoagulant used, whereas the fraction of the three monocyte subsets did not. Insufficient filling of blood sampling tubes altered the relative levels of monocytes out of leukocytes, but not the relative levels of the monocyte subsets. Finally, the fraction of CD14 monocytes out of isolated peripheral blood mononuclear cells was not significantly altered by cryopreservation, but the relative percentages of monocyte subsets was altered (similar effects for ACD-A and KEDTA samples) and this was observed in correlation to a decreased CD16 expression.

CONCLUDING REMARKS

Analysis of the monocyte subsets (i.e. classical, intermediate, non-classical) in peripheral blood samples requires a careful standardization of peripheral blood sampling and pre-analytic handling of the samples with respect to the anticoagulant used, filling of sample tubes, and cryopreservation of cells prior to analysis.

摘要

简介

单核细胞在先天免疫中起着重要作用，包括经典（CD14CD16）、中间（CD14CD16）和非经典（CD14CD16）单核细胞亚群。在外周血中定量这些功能不同的亚群可能对人类疾病的诊断和随访有用。本研究旨在探讨不同的预分析参数如何影响外周血样本中单核细胞亚群的分析。

方法

我们通过对来自健康个体的外周血样本进行流式细胞术来确定单核细胞和单核细胞亚群的相对水平。应用一种专门提取活 CD14 单核细胞并专注于三个单核细胞亚群的门控策略。我们研究了以下因素的影响：（i）不同的抗凝剂（即 Li-Heparin、ACD-A、KEDTA），（ii）采血管填充不足，（iii）冷冻保存。此外，我们还分析了 CCR2 趋化因子受体的表达。

结果

CD14 单核细胞的相对数量取决于使用的抗凝剂，而三个单核细胞亚群的比例则不受影响。采血管填充不足会改变白细胞中外周血单核细胞的相对水平，但不会改变单核细胞亚群的相对水平。最后，冷冻保存不会显著改变分离的外周血单个核细胞中外周血单核细胞的比例，但会改变单核细胞亚群的相对百分比（ACD-A 和 KEDTA 样本也有类似的影响），并且这种变化与 CD16 表达的降低有关。