Standardization of cytokine flow cytometry assays
Standardization of cytokine flow cytometry assays
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DOI：
10.1186/1471-2172-6-13
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发表时间：
2005-06-24
期刊：
影响因子：
3
通讯作者：
Sekaly, RP
中科院分区：
文献类型：
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作者：
Maecker, HT;Rinfret, A;Sekaly, RP
背景：细胞因子流式细胞术（CFC）或细胞内细胞因子染色（ICS）可在诸如实验性疫苗接种等情况下对特异性抗原T细胞反应进行定量。在开展疫苗研究的实验室之间对ICS进行标准化，将提供一个通用平台，借此可在多个开展临床试验的国际组织之间比较不同候选疫苗的免疫原性。因此，在几个参与艾滋病临床试验的实验室之间开展了一项研究，以确定使用各种样本类型以及针对每个实验采用通用方案时ICS的实验室间精密度（见在线补充文件）。
结果：三种样本类型（活化、固定和冷冻全血；新鲜全血；以及冻存的外周血单个核细胞）被运送到各个地点，在这些地点使用巨细胞病毒（CMV）pp65肽混合物或对照抗原在96孔板中平行进行ICS检测。在一项实验中，抗原和抗体混合液被冻干到96孔板中，以简化和标准化检测设置。每个地点测定结果（CD4⁺细胞因子⁺细胞和CD8⁺细胞因子⁺细胞）。原始数据也被发送到一个中心地点，使用动态设门模板进行批量分析。
实验室间平均变异系数（C.V.）根据样本类型和分析方法的不同，介于17 - 44%之间。冻存的外周血单个核细胞（PBMC）产生的实验室间C.V.比全血低。集中分析（使用动态设门模板）根据实验的不同，可使实验室间C.V.降低5 - 20%。对于平均干扰素γ⁺T细胞>0.5%的样本，实验室间C.V.最低（18 - 24%），而对于平均干扰素γ⁺细胞<0.1%的样本，实验室间C.V.最高（57 - 82%）。
结论：多个实验室可使用通用方案进行ICS检测，且具有良好的实验室间精密度，随着反应细胞频率的增加，精密度会提高。冻存的PBMC可能比运送的全血产生更一致的结果。分析，尤其是设门，是变异的一个重要来源，可通过集中分析和/或使用标准化动态设门模板来减少变异。使用预先分装的冻干试剂进行刺激和染色可为这些检测提供进一步的标准化。
Background: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment ( see additional files online).Results: Three sample types ( activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4(+) cytokine(+) cells and CD8(+) cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template.Mean inter-laboratory coefficient of variation ( C. V.) ranged from 17 - 44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells ( PBMC) yielded lower inter-lab C. V.' s than whole blood. Centralized analysis ( using a dynamic gating template) reduced the inter-lab C. V. by 5 - 20%, depending upon the experiment. The inter-lab C. V. was lowest ( 18 - 24%) for samples with a mean of > 0.5% IFN gamma + T cells, and highest ( 57 - 82%) for samples with a mean of < 0.1% IFN gamma+ cells.Conclusion: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.