Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)
Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)
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DOI：
10.1007/s00262-007-0380-6
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发表时间：
2008-03-01
影响因子：
5.8
通讯作者：
Hoos, A.
中科院分区：
文献类型：
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作者：
Janetzki, S.;Janetzki, S.;Hoos, A.
萨宾疫苗研究所癌症疫苗联盟（CVC/SVI）正在通过其成员和附属协会进行大规模的免疫监测协调计划。2005年，通过与36个实验室进行国际Elispot熟练度小组，这项工作成为一项外部验证计划，随后在2006年与29个参与实验室进行了第二次小组，允许应用第一次小组的学习。通过详细的调查，对关键的协议选择以及实验室之间的标准化和验证做法进行了评估。虽然小组成员必须遵守一般准则，以便比较结果，但每个实验室都能够使用自己的方案、材料和试剂。第二组记录了整体显著改善的性能，如通过正确检测所有预定义响应的能力所测量的。确定了可能对总体测定结果产生显著影响的方案选择和实验室实践，并提出了以下建议：（A）建立Elispot检测程序的实验室SOP，包括（A1）凋亡细胞的计数方法，用于确定接种的适当细胞稀释度，以及（A2）在接种和孵育前过夜静置细胞，（B）仅使用针对低背景：高信号比优化的预检测血清，（C）建立平板阅读的实验室SOP，包括（C1）阅读过程中的人工审核和（C2）技术伪影的适当调整，以及（D）仅允许经过培训的人员（根据实验室SOP进行认证）进行测定。发现（A）中描述的建议在测定性能方面具有统计学显著性差异，而其余建议基于经小组结果证实的实际经验，无法进行统计学检验。这些结果提供了初步的协调准则，以优化免疫治疗界的Elispot测定性能。正在与正在进行的小组进行进一步优化。
The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.