用于慢性免疫缺陷特征分析的T细胞、B细胞和NK细胞亚群的八色免疫表型分析

Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies.

作者信息

Boldt Andreas, Borte Stephan, Fricke Stephan, Kentouche Karim, Emmrich Frank, Borte Michael, Kahlenberg Franka, Sack Ulrich

机构信息

Institute of Clinical Immunology, Universität Leipzig, Medical Faculty, Leipzig, Germany; Translational Centre for Regenerative Medicine (TRM), Universität Leipzig, Leipzig, Germany.

出版信息

Cytometry B Clin Cytom. 2014 May;86(3):191-206. doi: 10.1002/cyto.b.21162. Epub 2014 Jan 31.

DOI:10.1002/cyto.b.21162

PMID:24488780

Abstract

BACKGROUND

The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations.

METHODS

Peripheral blood samples from healthy adult volunteers (n = 25) were collected and split into eight panel fractions (100 µl each). Subsequently, premixed eight-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants (RTE), (vii) NK-cell subpopulations, and (viii) NK-cell activation markers. All samples were lysed, washed, and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, and percentile ranges).

RESULTS

Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, nonswitched/switched, memory, (activated) CD21(low) , transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like, and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) α/β- and γ/δ-T-cells, RTE in CD4/CD8 cells; (vii) immature/mature CD56(bright) , CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46, and CD57+NK-cells. Clinical examples and quadrant statistics are provided.

CONCLUSION

The present study represents a practical approach to standardize the immunophenotyping of most T-, B-, and NK-cell subpopulations. That allows differentiating whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance.

摘要

背景

原发性和继发性免疫缺陷的异质性要求开发一种全面的流式细胞术筛查系统，该系统基于支持大多数淋巴细胞亚群标准化免疫表型特征的参考值。

方法

收集健康成年志愿者（n = 25）的外周血样本，并分成八个样本组（每组100微升）。随后，针对每个特定的全血样本组，孵育预混的八色抗体鸡尾酒，以检测和区分以下细胞亚群：（i）一般淋巴细胞概况；（ii）B细胞亚群；（iii）CD4 +亚群；（iv）CD8 +亚群；（v）调节性T细胞；（vi）近期胸腺迁出细胞（RTE）；（vii）NK细胞亚群；以及（viii）NK细胞活化标志物。所有样本均进行裂解、洗涤，并通过流式细胞术进行检测。使用FACS DIVA软件进行数据分析和象限统计（平均值、平均标准误差和百分位数范围）。

结果

淋巴细胞的全血染色可分析：（i）CD3 +、4 +、8 +、19 +、16/56 +和活化的CD4/8细胞；（ii）未成熟、幼稚、未转换/已转换、记忆、（活化的）CD21（低）、过渡性B细胞、浆母细胞/浆细胞；（iii和iv）幼稚、中枢记忆、效应、效应记忆、TH1/TH2/TH17样和CCR5 + CD8细胞；（v）CD25 +调节性T细胞（幼稚/记忆，HLA-DR +）；（vi）α/β和γ/δT细胞，CD4/CD8细胞中的RTE；（vii）未成熟/成熟的CD56（明亮）、CD94/NKG2D + NK细胞；以及（viii）Nkp30、44、46和CD57 + NK细胞。提供了临床实例和象限统计数据。