基于多色流式降维聚类方法的自发性细菌性腹膜炎患者T淋巴细胞亚群分析

doi:10.3877/cma.j.issn.1674-1358.2023.02.004

中华实验和临床感染病杂志(电子版) ›› 2023, Vol. 17 ›› Issue (02) : 92 -101. doi: 10.3877/cma.j.issn.1674-1358.2023.02.004

论著

基于多色流式降维聚类方法的自发性细菌性腹膜炎患者T淋巴细胞亚群分析

王锃涛, 王宪波, 曹钰, 郝禹, 韩俊燕, 曾辉^†(

)

100015　北京，首都医科大学附属北京地坛医院传染病研究所
100015　北京，首都医科大学附属北京地坛医院中西医结合中心
100038　北京，首都医科大学附属北京世纪坛医院生物医学创新中心
100015　北京，首都医科大学附属北京地坛医院传染病研究所；100038　北京，首都医科大学附属北京世纪坛医院生物医学创新中心

收稿日期:2022-10-24 出版日期:2023-04-15
通信作者: 曾辉
基金资助:
国家自然科学基金面上项目(No. 81971862); 北京市医院管理中心"登峰"计划(No. LDFL20191801); 北京市科学技术委员会2019年"首都临床诊疗技术研究及转化应用"项目(No. Z201100005520035)

Dimensionality reduction and clustering method of flow cytometric data for measuring T cell subpopulations in patients with spontaneous bacterial peritonitis

Zengtao Wang, Xianbo Wang, Yu Cao, Yu Hao, Junyan Han, Hui Zeng^†()

Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
Center of Integrative Medicine, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
Biomedical Innovation Center, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China
Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China; Biomedical Innovation Center, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China

Received:2022-10-24 Published:2023-04-15
Corresponding author: Hui Zeng

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引用本文：

王锃涛, 王宪波, 曹钰, 郝禹, 韩俊燕, 曾辉. 基于多色流式降维聚类方法的自发性细菌性腹膜炎患者T淋巴细胞亚群分析[J]. 中华实验和临床感染病杂志(电子版), 2023, 17(02): 92-101.

Zengtao Wang, Xianbo Wang, Yu Cao, Yu Hao, Junyan Han, Hui Zeng. Dimensionality reduction and clustering method of flow cytometric data for measuring T cell subpopulations in patients with spontaneous bacterial peritonitis[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2023, 17(02): 92-101.

目的

建立多色流式降维聚类分析方法，检测自发性细菌性腹膜炎（SBP）患者外周血和腹水中T细胞亚群，探讨SBP患者外周以及腹腔局部T淋巴细胞的表面标志物表达差异。

方法

选取2021年12月至2022年1月就诊于首都医科大学附属北京地坛医院的4例SBP患者为研究对象。根据多色流式荧光搭配原则，初步确立检测外周血及腹水T淋巴细胞亚群比例的多色流式配色方案。利用健康对照者外周血单个核细胞（PBMCs）调节最适检测电压及荧光补偿；设定条件后，检测4例SBP患者的外周血及腹水样本。建立多色流式降维聚类分析方案以检测SBP患者外周血和腹水中T淋巴细胞的差异。

结果

采用统一流形逼近和投影（UMAP）结合FlowSOM插件的多色流式降维分析法将CD4⁺ T细胞和CD8⁺ T细胞均分为12个细胞群。该降维聚类方法通过可视化UMAP图定位，并结合统计学分析初步发现，与外周血相比，SBP患者腹水中CD69⁺PD-1⁺ CD4⁺ T_EM（t = 3.427、P = 0.0416）以及CD25^－TIGIT^－CD4⁺ T_EM（t = 3.203、P = 0.0492）比例较高，差异有统计学意义。

结论

多色流式降维聚类分析法用于检测SBP患者外周血及腹水样本中T淋巴细胞分群及功能，具有快速、准确、多维度并且可视化强的优点。

关键词: 多色流式, 自发性细菌性腹膜炎, 降维聚类分析, T细胞亚群

Objective

To establish a dimensionality reduction and clustering method of flow cytometric data for measuring T cell subpopulations in peripheral blood and ascites of patients with spontaneous bacterial peritonitis (SBP), and to explore the differences in the expression of markers of T lymphocytes in the peripheral blood and ascites.

Methods

Total of 4 patients with SBP were recruited in Beijing Ditan Hospital, Capital Medical University from December 2021 to January 2022. The monoclonal antibodies targeting cell surface antigen were used to establish the multi-color flow cytometry analysis panel. The peripheral blood mononuclear cell (PBMC) sample from one healthy donor was applied to adjust optimal detection voltage and fluorescence compensation. This panel was applied to detect peripheral blood and ascites samples from 4 patients with SBP. Uniform manifold approximation and projection (UMAP) and FlowSOM were used to analyze the results from multicolor flow cytometric data.

Results

Using UMAP combined with FlowSOM, CD4⁺ T cells and CD8⁺ T cells were both divided into 12 clusters, respectively. Through the visual UMAP map positioning and statistical analysis, compared with the peripheral blood, the proportion of CD69⁺PD-1⁺CD4⁺ T_EM cells (t = 3.427, P = 0.0416) and CD25^-TIGIT^-CD4⁺T_EM cells (t = 3.203, P = 0.0492) in ascites of patients with SBP were increased, with significant differences.

Conclusions

The dimensionality reduction and clustering method of flow cytometric data can be used to detect the groups and functions of T lymphocyte in peripheral blood and ascites samples of patients with SBP, which has the advantages of rapidity, accuracy, multi-dimensionality and strong visualization.

Key words: Muti-color flow cytometry, Spontaneous bacterial peritonitis, Dimensionality reduction and cluster analysis, T cell subset

图1 多色流式降维聚类分析流程注：A：T细胞亚群圈门策略；B：FlowJo软件中T细胞流式数据UMAP降维聚类分析法流程图

图2 SBP患者CD4⁺ T细胞降维聚类分析注：A：外周血（PB）和腹水（AS）CD4⁺ T细胞总UMAP降维图；B：各个CD4⁺ T细胞群不同标志物的表达直方图；C：各个CD4⁺ T细胞群不同标志物的表达以及命名

图3 SBP患者CD8⁺ T细胞降维聚类分析注：A：外周血（PB）和腹水（AS）CD8⁺ T细胞总UMAP降维图；B：各个CD8⁺ T细胞群上不同标志物的表达直方图；C：各个CD8⁺ T细胞群上不同标志物表达以及命名

图4 SBP患者CD4⁺ T细胞统计分析注：A：外周血（PB）CD4⁺ T细胞UMAP降维图以及腹水（AS）CD4⁺ T细胞UMAP降维图；B：外周血（PB）及腹水（AS）中CD4⁺ T细胞各个细胞群的比例差异

图5 SBP患者CD8⁺ T细胞统计分析注：A：外周血（PB）CD8⁺ T细胞UMAP降维图以及腹水（AS）CD8⁺ T细胞UMAP降维图；B：外周血（PB）及腹水（AS）中CD8⁺ T细胞各个细胞群的比例差异

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Dimensionality reduction and clustering method of flow cytometric data for measuring T cell subpopulations in patients with spontaneous bacterial peritonitis

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Dimensionality reduction and clustering method of flow cytometric data for measuring T cell subpopulations in patients with spontaneous bacterial peritonitis