Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents
Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents
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DOI：
10.1128/cvi.00326-08
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发表时间：
2009-02-01
影响因子：
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通讯作者：
Gilmour, Jill
中科院分区：
文献类型：
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作者：
Boaz, Mark J.;Hayes, Peter;Gilmour, Jill
伽马干扰素(干扰素-伽马)酶联免疫斑点(ELISPOT)试验通常用于评估人类免疫缺陷病毒(HIV)候选疫苗和其他候选疫苗的效力。为了比较候选人和汇集来自多个试验实验室的数据，必须在多个实验室之间应用经过验证的标准化方法。熟练程度小组是全面质量保证计划的关键部分，用于监控实验室间和实验室内的性能，以及随着时间的推移的分析性能。七个由国际艾滋病疫苗倡议赞助的试验站点参加了本研究中描述的熟练程度小组。在每个实验室，两名操作员通过使用四个盲刺激，独立处理由来自不同捐赠者的冰冻外周血单个核细胞(PBMC)样本组成的相同样本集。检测PBMC在过夜静息后的回收率和活性，以及干扰素-γELISPOT检测性能。所有站点在PBMC解冻和静息方面表现良好，中位数回收率为78%，中位数存活率为95%。这些实验室能够检测到类似的抗原特异性T细胞反应，每百万个PBMC中有50到3000个斑点形成细胞。在抗原特异性反应的比较中，操作员在站点内或跨站点的结果的大约一半的对数范围被看到。所有实验室的背景反应都很低。这些熟练程度小组的结果表明，分布在三大洲的七个实验室有能力处理PBMC样本，并对不同程度的干扰素-伽马ELISPOT反应的志愿者进行排名。这些发现还表明，当采用常见的培训方法、试剂(如胎牛血清)和标准操作程序时，能够跨多个实验室标准化干扰素-伽马ELISPOT检测。这些结果对于正在使用基于细胞的免疫学分析来测试艾滋病毒疫苗和其他疫苗的实验室来说是令人鼓舞的。
The gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter-and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-gamma ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-gamma ELISPOT responses. These findings also illustrate the ability to standardize the IFN-gamma ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.