Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies
Eight-color immunophenotyping of T-, B-, and NK-cell subpopulations for characterization of chronic immunodeficiencies
复制标题
DOI：
10.1002/cyto.b.21162
复制
发表时间：
2014-05-01
影响因子：
3.4
通讯作者：
Sack, Ulrich
中科院分区：
文献类型：
--
作者：
Boldt, Andreas;Borte, Stephan;Sack, Ulrich
原发和继发性免疫缺陷的异质性要求建立一套完整的流式细胞仪筛查系统，该系统以支持大多数淋巴细胞亚群标准化免疫表型特征的参考值为基础。方法采集健康成年志愿者(n=25)的外周血，分成8组(每组100亩L)。随后，每个特定的全血面板孵育预混合的八色抗体鸡尾酒，以检测和区分以下细胞亚群：(I)一般淋巴细胞概述，(Ii)B细胞亚群，(Iii)CD4+亚群，(Iv)CD8+亚群，(V)调节性T细胞，(Vi)最近的胸腺移民(RTE)，(Vii)NK细胞亚群，和(Viii)NK细胞激活标志物。所有样本均经裂解、洗涤和流式细胞仪检测。结果淋巴细胞全血染色可分析：(1)CD3+、4+、8+、19+、16/56+和活化的CD4/8细胞；(2)未成熟、幼稚、未转换/转换、记忆、(激活的)CD21(低)、过渡性B细胞、浆母细胞/浆细胞；(3)幼稚、中央记忆、效应器、效应记忆、TH1/TH2/TH17样细胞和CCR5+CD8-细胞；(V)CD25+调节性T细胞(naive/Memory，HLA-DR+)；(Vi)CD4/CD8细胞中的α/β和γ/β-T细胞；(Vii)未成熟/成熟的CD56(Bright)、CD94/NKG2D+NK细胞；以及(Viii)Nkp30、44、46和CD57+NK细胞。结论本研究为大多数T、B和NK细胞亚群的免疫表型标准化提供了一种实用的方法。这使得区分淋巴细胞亚群中观察到的异常或发育变化是源于初级还是继发性免疫紊乱。(C)2014国际临床细胞计数学会
BackgroundThe 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.MethodsPeripheral blood samples from healthy adult volunteers (n = 25) were collected and split into eight panel fractions (100 mu l 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).ResultsWhole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naive, nonswitched/switched, memory, (activated) CD21(low), transitional B-cells, plasmablasts/plasmacells; (iii and iv) naive, central memory, effector, effector memory, TH1/TH2/TH17-like, and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naive/memory, HLA-DR+); (vi) alpha/beta- and gamma/delta-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.ConclusionThe 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. (c) 2014 International Clinical Cytometry Society