Cross-laboratory evaluation of multiplex bead assays including independent common reference standards for immunological monitoring of observational and interventional human studies

BackgroundMultiplex assays are increasingly applied to analyze multicomponent signatures of human immune responses, including the dynamics of cytokine and chemokine production, in observational as well as interventional studies following treatment or vaccination. However, relatively limited information is available on the performance of the different available multiplex kits, and comparative evaluations addressing this important issue are lacking.Study designTo fill this knowledge gap we performed a technical comparison of multiplex bead assays from 4 manufacturers, each represented by 3 different lots, and with the assays performed by 3 different laboratories. To cross compare kits directly, spiked samples, biological samples and a newly made reference standard were included in all assays. Analyses were performed on 324 standard curves to allow for evaluation of the quality of the standard curves and the subsequent interpretation of biological specimens.ResultsManufacturer was the factor which contributed most to the observed variation whereas variation in lots, laboratory or type of detection reagent contributed minimally. Inclusion of a common reference standard allowed us to overcome observed differences in cytokine and chemokine levels between manufacturers.ConclusionsWe strongly recommend using multiplex assays from the same manufacturer within a single study and across studies that are likely to compare results in a quantitative manner. Incorporation of common reference standards, and application of the same analysis method in assays can overcome many analytical biases and thus could bridge comparison of independent immune profiling (e.g. vaccine immunogenicity) studies. With these recommendations taken into account, the multiplex bead assays performed as described here are useful tools in capturing complex human immune-signatures in observational and interventional studies.

背景：背景多重检测技术日益被应用于分析人类免疫反应的多组分特征，包括细胞因子和趋化因子生产的动态变化，这些分析既包括观察性研究，也包括治疗或疫苗接种后的干预性研究。然而，关于不同多重检测试剂盒性能的信息相对有限，针对此重要问题的比较评估尚显不足。研究设计：为了填补这一知识空白，我们进行了四家制造商的多重磁珠检测试剂的比较技术评估，每家制造商提供三个不同批次的产品，并由三个不同的实验室进行检测。为了直接进行试剂盒之间的交叉比较，所有检测中均包含了加标样品、生物样品以及新制备的参考标准。通过分析324条标准曲线，以评估标准曲线的质量及随后对生物样本的解释。结果：制造商是导致观察到的变异性最大的因素，而批次、实验室或检测试剂类型的变异性相对较小。引入共同参考标准使我们能够克服不同制造商间细胞因子和趋化因子水平观察到的差异。结论：我们强烈建议在单一研究中以及在可能进行定量结果比较的研究中，使用同一制造商的多重检测试剂。引入共同参考标准，并在检测中应用相同分析方法，可以克服许多分析偏差，从而弥合独立免疫谱分析（例如疫苗免疫原性）研究之间的比较。考虑这些建议，正如所述的多重磁珠检测试剂在观察性和干预性研究中捕捉复杂的人类免疫特征方面是有效的工具。