Miniaturized Automated Whole Blood Cellular Analysis System

 描述（由申请人提供）：细胞免疫测定是理解病毒和其他传染病的发病机理和控制机制的关键。但是，这种检测很难作为临床研究的一部分进行，因为它们是：不稳定的：它们必须在新鲜血液上进行，或者在血液采集后相对较短的时间内冷冻保存的PBMC上进行。费力：它们需要大量的手工劳动，以及在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上进行时。这些挑战限制了细胞免疫功能测定的实施，特别是在儿童（抽血量最有限）和偏远地区。然而，偏远地区的儿童往往受重大传染病的影响最大，也是疫苗和其他控制方法进步的最大受益者。因此，我们建议开发一种样品保留和全自动系统，用于刺激和稳定全血功能细胞测定。该系统将基于Smart Tube公司开创的概念。在他们现有的全血刺激系统中，使用的血液将减少80%，并提供完全自动化，从而消除了所有移液步骤。它将直接从收集管中抽取血液，使用少至2 cc，将血液分配到多个孵育室中，在那里将用冻干的预配置试剂（抗原，有丝分裂原等）进行刺激。到 测定细胞功能，然后用蛋白质组稳定剂稳定以供以后分析。在Specifi Aim 1中，我们将开发此原型系统（通过与Smart Tube，Inc.的合作）。并在斯坦福大学人类免疫监测中心的对照样本上进行测试。两个beta单元将用于进一步测试。在具体目标2中，我们将通过与蒙巴萨技术大学的合作，在肯尼亚的两个临床地点测试这些β单位。这些临床中心将从感染基孔肯雅和/或登革热病毒的儿童中采集血液样本。他们将使用β系统来刺激和稳定这些血液样本，并将其运送到斯坦福大学，通过最先进的CyTOF质谱仪进行分析。这项研究的结果不仅将验证血液采集系统的性能，而且将提供有关儿童对这些病毒的细胞反应的宝贵生物学数据。血液收集系统，反过来，将B用于许多不同种类的细胞免疫研究，特别是在远程设置的情况下，样品是有限的。最终的结果应该是我们对疾病发病机理的理解和重要传染因子的疫苗开发的更快进步。

 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.