A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols
A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols
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DOI：
10.1186/1479-5876-9-108
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发表时间：
2011-07-11
影响因子：
7.4
通讯作者：
Britten, Cedrik M.
中科院分区：
文献类型：
--
作者：
Attig, Sebastian;Price, Leah;Britten, Cedrik M.
背景：在基于流式细胞术的检测中，引入抗体标记物来识别不需要的细胞群，即所谓的DUMP通道标记物，已经成为越来越多的实验室进行hla肽多聚体检测的实践。然而，在多次测定中引入DUMP通道的影响迄今尚未在各种协议中进行系统研究。方法：癌症研究所的癌症免疫治疗联盟（CRI-CIC）进行了一个多时间熟练小组，特别关注DUMP通道使用的影响。面板设计允许各个实验室使用自己的方案进行解冻，染色，门控和数据分析。每个实验并行执行两次，使用和不使用转储通道策略。结果：DUMP通道的引入是减少非特异性MULTIMER与T细胞结合量的有效措施。在广泛的个体实验室和所有被测试的供体-抗原组合中观察到使用DUMP通道的有益效果。在48%的实验中，我们观察到背景多聚体结合的减少。在这个亚组实验中，引入DUMP通道后观察到的中位背景降低为0.053%。结论：我们得出结论，适当使用DUMP通道可以显著减少大部分方案中的背景染色，并提高通过多试剂准确检测和量化抗原特异性T细胞频率的能力。因此，使用DUMP通道可能对检测低频抗原特异性免疫反应至关重要。还提供了有关分析性能和发表multitimer实验数据的数据呈现指南的进一步建议。
Background: The introduction of antibody markers to identify undesired cell populations in flow-cytometry based assays, so called DUMP channel markers, has become a practice in an increasing number of labs performing HLA-peptide multimer assays. However, the impact of the introduction of a DUMP channel in multimer assays has so far not been systematically investigated across a broad variety of protocols.Methods: The Cancer Research Institute's Cancer Immunotherapy Consortium (CRI-CIC) conducted a multimer proficiency panel with a specific focus on the impact of DUMP channel use. The panel design allowed individual laboratories to use their own protocol for thawing, staining, gating, and data analysis. Each experiment was performed twice and in parallel, with and without the application of a dump channel strategy.Results: The introduction of a DUMP channel is an effective measure to reduce the amount of non-specific MULTIMER binding to T cells. Beneficial effects for the use of a DUMP channel were observed across a wide range of individual laboratories and for all tested donor-antigen combinations. In 48% of experiments we observed a reduction of the background MULTIMER-binding. In this subgroup of experiments the median background reduction observed after introduction of a DUMP channel was 0.053%.Conclusions: We conclude that appropriate use of a DUMP channel can significantly reduce background staining across a large fraction of protocols and improve the ability to accurately detect and quantify the frequency of antigen-specific T cells by multimer reagents. Thus, use of a DUMP channel may become crucial for detecting low frequency antigen-specific immune responses. Further recommendations on assay performance and data presentation guidelines for publication of MULTIMER experimental data are provided.