Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions
Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions
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DOI：
10.1128/cvi.00435-06
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发表时间：
2007-05-01
影响因子：
--
通讯作者：
Caulfield, Michael J.
中科院分区：
文献类型：
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作者：
Smith, Jeffrey G.;Joseph, Heather R.;Caulfield, Michael J.
酶联免疫斑点（ELISPOT）检测是一种测量抗原特异性细胞免疫反应的强大工具。使用冷冻外周血单个核细胞（PBMC）的能力有助于在多中心临床试验中检测样品；然而，如果样品处理不当，可能会导致不可靠的ELISPOT反应。通过细胞活力和功能ELISPOT反应测量评估，将冷冻PBMC暴露于次优储存温度（-20℃）或在更优储存温度（低于-130℃和-70℃）之间反复循环会降低冷冻PBMC的质量。通过台盼蓝染色排除评估的细胞活力降低，通过番石榴连接蛋白测定的凋亡细胞百分比在这些事件发生后显着增加。功能性γ干扰素ELISPOT对植物血凝素（PHA）丝裂原、CD4 t细胞特异性抗原（水痘-带状疱疹病毒）和CD8 t细胞特异性抗原（包含已知巨细胞病毒、eb病毒和流感病毒肽的库）的反应均在次优化储存事件后显著降低。然而，对于给定的次优存储事件，减少的幅度在个体之间甚至在个体出血内的等量之间都是不同的，这表明需要特定样品的接受标准（AC）。解冻后的活细胞百分比或凋亡细胞百分比，以及对PHA的功能性ELISPOT反应，都是有效的，当限制为AC时，用于将储存期间损坏的样品与有效对照样品分离。虽然这三种AC方法都可以有效地应用，但所应用的凋亡AC限度最适合于分离对抗原刺激有反应的样品和对抗原刺激没有反应的样品。
The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for measuring antigen-specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMC) facilitates testing samples in multicenter clinical trials; however, unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMC to suboptimal storage temperature (-20 degrees C) or repeated cycling between more optimal storage temperatures (less than -130 degrees C and -70 degrees C) reduced the quality of frozen PBMC, as assessed by cell viability and functional ELISPOT response measures. Cell viability as assessed by trypan blue dye exclusion was reduced, and the percentage of apoptotic cells, as determined by the Guava Nexin assay, was significantly increased after these events. The functional gamma interferon ELISPOT responses to phytohemagglutinin (PHA) mitogen, a CD4 T-cell-specific antigen (varicella-zoster virus), and a CD8 T-cell-specific antigen (pool containing known cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) were all significantly reduced after suboptimal storage events. However, for a given suboptimal storage event, the magnitude of the reduction varied between individuals and even among aliquots within an individual bleed, indicating the need for sample-specific acceptance criteria (AC). The percent viable or percent apoptotic cells after thaw, as well as the functional ELISPOT response to PHA, were all effective when applied with limits as AC for separating samples damaged during storage from valid control samples. Although all three AC measures could be effectively applied, the apoptosis AC limit applied was best for separating samples that could respond to antigenic stimulation from samples that could not effectively respond.