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. 2022 Jan;53(1):127-132.
doi: 10.12182/20220160106.

[Establishment of 14-Color Panel for Determining Leukocyte Subsets in Human Peripheral Blood with Flow Cytometry]

[Article in Chinese]
Qiao-Rong Huang  1 , Yan-Ni Jiang  1 , Hui-Fang Li  2 , Xue Li  1 , Wen-Tong Meng  1
Affiliations

[Establishment of 14-Color Panel for Determining Leukocyte Subsets in Human Peripheral Blood with Flow Cytometry]

[Article in Chinese]
Qiao-Rong Huang et al. Sichuan Da Xue Xue Bao Yi Xue Ban. 2022 Jan.

Abstract
in English, Chinese

Objective: To establish a 14-color flow cytometry protocol for the examination of leukocyte subsets in human peripheral blood.

Methods: We used cell membrane surface antibodies CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and nucleus staining dye DAPI to establish a 14-color flow cytometry assay to determine the major cell subsets in human peripheral blood. We collected peripheral blood specimens from healthy volunteers to test for antibody titers and optimal photomultiplier tube (PMT) voltage, and to conduct single-color staining and fluorescence minus one control staining. After determining the test method and test conditions, the peripheral blood samples of 18 healthy volunteers were analyzed.

Results: According to the cell classification and staining index, optimal antibody mass concentrations selected were as follows: CD25 and CD127 at 8.0 μg/mL, CD45, CD3, CD14 and CD123 at 4.0 μg/mL, CD8, CD19, CD56, CD16, HLA-DR and CD11c at 2.0 μg/mL, CD4 at 1.0 μg/mL and DAPI at 0.1 μg/mL. The detection voltages for CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and DAPI were 450 V, 410 V, 400 V, 550 V, 405 V, 500 V, 520 V, 550 V, 550 V, 400 V, 450 V, 400 V, 580 V, and 300 V, respectively. The appropriate fluorescence compensation was determined by single-color staining and fluorescence minus one controls. The 14-color flow cytometry panel was established to analyze the main subsets of leukocytes in human peripheral blood, and peripheral blood samples from 18 healthy adults were examined, obtaining the percentages of each subset of peripheral blood leukocytes and the immunophenotypes of the main subsets.

Conclusion: We established a 14-color panel for determining leukocyte subsets in human peripheral blood by flow cytometry, which produced stable and reliable results and was easy to operate.

目的: 建立14色流式检测人外周血中白细胞亚群方案。

方法: 用细胞膜表面抗体CD45、CD3、CD4、CD8、CD19、CD56、CD16、CD14、CD25、CD127、HLA-DR、CD123、 CD11c和核酸染料DAPI建立14色流式染色方案，检测人外周血白细胞主要细胞亚群。取健康成年志愿者外周血标本分别对各抗体进行抗体滴度实验、光电倍增管（photomultiplier tube, PMT）电压优化、单色染色和减一色染色，确定检测方法和检测条件后，对18例健康成年志愿者外周血标本进行检测分析。

结果: 根据细胞分群情况和染色指数选择抗体最适质量浓度：CD25和CD127为8.0 μg/mL；CD45、CD3、CD14和CD123为4.0 μg/mL；CD8、CD19、CD56、CD16、HLA-DR和CD11c为2.0 μg/mL；CD4为1.0 μg/mL；DAPI使用质量浓度为0.1 μg/mL。CD45、CD3、CD4、CD8、CD19、CD56、CD16、CD14、CD25、CD127、HLA-DR、CD123、CD11c和DAPI检测电压分别为：450 V、410 V、400 V、550 V、405V、500 V、520 V、550 V、550 V、400 V、450 V、400 V、580 V和300 V。单色染色和减一色试验确定合适的荧光补偿。采用建立的14色流式检测人外周血白细胞主要亚群的方案检测了18例健康成年人外周血样本，可分析出外周血中各细胞亚群在白细胞中的百分比，以及主要细胞群的免疫表型。

结论: 成功建立14色流式检测人外周血白细胞亚群方案，结果稳定可靠，操作简便。

Keywords: Cell subset; Human peripheral blood; Immunophenotype; Multicolor flow cytometry; Panel optimization.

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Conflict of interest statement

利益冲突 所有作者均声明不存在利益冲突

Figures

图 1
图 1
Optimal mass concentration (A) and optimal voltage (B) of CD56 antibody CD56抗体最适质量浓度的选择（A）和最适电压选择（B）
图 2
图 2
Gating steps for peripheral blood leukocytes 外周血白细胞设门步骤
图 3
图 3
Analysis of major cell subsets of peripheral blood leukocytes 外周血白细胞主要细胞亚群分析方法
图 4
图 4
Analysis strategy of DC DC分析方法
图 5
图 5
Analysis of lymphocytes by t-SNE 淋巴细胞t-SNE分析
See this image and copyright information in PMC

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