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

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

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。

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.