Measurement and Quality Control Issues in Multiplex Protein Assays: A Case Study
Measurement and Quality Control Issues in Multiplex Protein Assays: A Case Study
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DOI：
10.1373/clinchem.2008.120717
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发表时间：
2009-06-01
影响因子：
9.3
通讯作者：
Klee, George G.
中科院分区：
文献类型：
--
作者：
Ellington, Allison A.;Kullo, Iftikhar J.;Klee, George G.
背景：多重阵列越来越多地用于测量蛋白质生物标志物。这种方法的优点包括标本保存，有限的样品处理，并减少时间和成本，但优化每种蛋白质的测定格式，选择共同的稀释因子，并建立强大的质量控制算法的挑战是巨大的。在这里，我们使用来自一项大型研究的15种蛋白质生物标志物的测量值来说明多重免疫测定的处理、分析和质量控制问题。我们与ThermoScientific签订合同，对来自社区队列的2322名参与者的15种蛋白质进行重复测量，血浆对照，和重组蛋白对照，使用2个定制平面微阵列，每个孔中印刷有6个（图A）或9个（图B）捕获抗体。我们根据内源性浓度和检测试剂盒可用性选择每组中的组分分析物。对样品处理、储存和冻融暴露的方案进行了标准化。我们分析了数据的处理方案，精密度和偏差的偏差的影响。结果：测量值在可报告的范围内为每个分析，但是，浓度为7的1.5蛋白质不集中的剂量-反应曲线。一个样本子集的额外冻融循环和错误的样本稀释产生了显著不同的结果。观察到重复测定之间存在较大差异的测量值按分析物、平板和参与者聚类。传统的单变量质量控制算法拒绝了许多板。血小板特异性中位数的队列和血浆对照数据显着covariated，观察替代质量控制algorithm.CONCLUSIONS：多重测量提出了困难的挑战，需要进一步的分析和统计的发展。(C)2009年美国临床化学协会
BACKGROUND: Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays.METHODS: We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias.RESULTS: Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 1.5 proteins were not centered on the dose-response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms.CONCLUSIONS: Multiplex measurements present difficult challenges that require further analytical and statistical developments. (C) 2009 American Association for Clinical Chemistry