Miniaturized Automated Whole Blood Cellular Analysis System

 描述(申请人提供)：细胞免疫分析是了解病毒和其他传染病的发病机制和控制机制的关键。但这种分析很难作为临床研究的一部分进行，因为它们不稳定：它们要么必须在新鲜血液上进行，要么必须在采血后相对较短的时间内冷冻保存的PBMC上进行。：费力：他们需要大量的手工劳动，以及临床现场不常见的技能和设备。：样本密集型：它们往往需要大量的血液，特别是在冷冻保存的PBMC上进行的时候。这些挑战限制了细胞免疫功能分析的实施，特别是在儿童(抽血量最有限的地方)和偏远环境中。然而，偏远地区的儿童往往是受重要传染病影响最大的群体，也是从疫苗和其他控制方法的进步中获益最多的群体。因此，我们建议开发一种节省样本的全自动全血刺激和稳定系统，用于功能性细胞分析。该系统将基于Smart Tube，Inc.在其现有的全血刺激系统中开创的概念，但将减少80%的血液使用量，并提供完全自动化，从而消除所有移液步骤。它将直接从采集管中抽取血液，只需2毫升，将血液分配到多个孵化室，在那里它将被冷冻干燥的预先配置的试剂(抗原、有丝分裂原等)刺激。至 检测细胞功能，然后用蛋白质组稳定剂稳定下来，供以后分析。在特定的目标1中，我们将开发这个原型系统(通过与Smart Tube，Inc.的分包合同)并在斯坦福人类免疫监测中心的对照样本上进行测试。两个测试版将被建造出来进行进一步的测试。在具体目标2中，我们将通过与蒙巴萨技术大学的分包合同，在肯尼亚的两个临床站点测试这些Beta单位。这些诊所将收集感染基孔肯雅和/或登革热病毒的儿童的血液样本。他们将使用Beta系统来刺激和稳定这些血液样本，并将它们运往斯坦福大学进行最先进的细胞飞行时间质谱仪分析。这项研究的结果不仅将验证血液采集系统的性能，还将提供关于儿童对这些病毒的细胞反应的有价值的生物学数据。反过来，血液采集系统将对许多不同类型的细胞免疫研究有用，特别是在样本有限的偏远环境中。最终的结果应该是我们对疾病发病机制和重要感染性病原体疫苗开发的理解得到更快的进步。

 DESCRIPTION (provided by applicant): Assays of cellular immunity are key to understanding the pathogenesis and mechanisms of control of viral and other infectious diseases. But such assays are difficult to perform as part of clinical studies because they are: º Labile: They must either be performed on fresh blood, or on PBMC that are cryopreserved within a relatively short time after blood collection. º Laborious: They require a lot of manual effort, as well as skills ad equipment not commonly found at clinical sites. º Sample-intensive: They tend to require large volumes of blood, particularly if performed on cryopreserved PBMC. These challenges have limited the implementation of cellular immune function assays, particularly in children (where blood draw volumes are most limited) and in remote settings. Yet, children in remote settings are often the most affected by important infectious diseases, and stand the most to gain from advances in vaccines and other methods of control. Therefore, we propose to develop a sample-sparing and fully automated system for stimulation and stabilization of whole blood for functional cellular assays. This system will be based on the concepts pioneered by Smart Tube, Inc. in their existing whole blood stimulation system, but will use 80% less blood, and provide full automation, so that all pipetting steps are eliminated. It will draw blood directly from a collection tube, using as little as 2 cc, distribute the blood into multiple incubation chambers where it will be stimulated with lyophilized, pre-configured reagents (antigens, mitogens, etc.) to assay cellular function, then stabilized with a proteomic stabilizer for later analysis. In Specifi Aim 1, we will develop this prototype system (through a subcontract with Smart Tube, Inc.) and test it on control samples in the Stanford Human Immune Monitoring Center. Two beta units will be built out for further testing. In Specific Aim 2, we will test these beta units at two clinical ites in Kenya through a subcontract with the Technical University of Mombasa. These clinical sites will collect blood samples from children infected with chikungunya and/or dengue virus. They will use the beta systems to stimulate and stabilize these blood samples, and ship them to Stanford for analysis by state-of-the art CyTOF mass cytometry. The results of this study will not only validate the blood collection system's performance, but will provide valuable biological data on the cellular response to these viruses in children. The blood collection system, in turn, will b useful for many different kinds of studies of cellular immunity, particularly in remote settings an situations where samples are limiting. The end result should be much more rapid advancement of our understanding of disease pathogenesis and of vaccine development for important infectious agents.