A multiplexed micro scale assay for real time analysis of pediatric immune cell function

摘要 可从儿科患者身上采集的生物样本数量受到限制 对于免疫功能的研究，通常不适用于常规检测方法。 开发新的、多参数的样品保留分析方法是必要的 促进对免疫系统的基本了解。在这项提案中，我们 概述三个目标，详细说明将新的生物读数添加和整合到 一种现有的微fluiic技术以及使用该扩展平台来调查 儿童过敏症和哮喘的发病机制。盖子上的工具包- 检测(考拉)是一个简单易用的自含式Microfluic平台，它能够 从全血中分离中性粒细胞，并评估其趋化能力。在……里面 目的1我们建议调整和验证测定中性粒细胞ROS的方法。 生产、网织、吞噬、细胞因子生产和基因表达(RT- QPCR)转化为考拉。我们还计划将考拉市的细胞捕获规格修改为fi 允许我们从全血中分离T细胞并评估其细胞因子的产生和 基因表达(RT-qPCR)。目标1的最后一个组件是测试和比较 一种与传统抽血相反的新的、侵入性较小的抽血方法--血链™ 方法根据免疫细胞表型进行分析。在目标2中，我们建议使用单细胞 一种称为光学代谢成像的实时无创无标记显微方法 (OMI)确定T细胞和中性粒细胞内活化的单细胞异质性 人口。OMI与其他端点兼容，可以同时运行。 在Aim 3中，我们将把我们的多路考拉分析应用于来自一群 WISC队列中患有过敏性疾病的儿童和匹配的健康对照组(AN 威斯康星大学正在进行的研究包括农场和非农场儿童)。我们将比较我们的 这些儿童的中性粒细胞和T细胞功能和表型结果 作为WISC队列研究的一部分，正在进行广泛的测量。在《目标3》中，我们将 还将这些分析扩展到来自Crew研究的一组婴儿(一项新的研究，位于 旨在确定早期生活、遗传和环境过敏风险因素的华盛顿大学 疾病和哮喘)使用HemoLink™设备以最低限度的速度抽血 不适和支持fiLE 2型T细胞功能(T细胞分化为分泌IL-4，IL-5， IL-13和其他2型细胞因子)使用考拉。如果我们的目标成功了，我们将拥有 创建了一个小型化、高度多元化、自给自足的Microfluiic平台， 能够测量与免疫状态和功能有关的生物标志物。这 该系统随后将在儿科受试者中得到验证，并准备过渡到 临床研究。

Abstract Restricted amounts of biological samples that can be collected from pediatric patients for the study of immune function are often insufficient for conventional assays. Development of novel, multi-parameter sample sparing assays is necessary for advancing the fundamental understanding of the immune system. In this proposal, we outline three aims which detail the addition and integration of new biological readouts to an existing microfluidic technology and the use of this expanded platform to investigate the mechanisms for how allergies and asthma develop in children. The kit-on-a-lid- assay (KOALA) is an easy to use self-contained microfluidic platform which is capable of isolating neutrophils from whole blood, and assessing their chemotactic abilities. In Aim 1 we propose to adapt and validate assays for measuring neutrophil ROS production, NETosis, phagocytosis, cytokine production, and gene expression (RT- qPCR) into KOALA. We also plan to modify the cell capture specificity of KOALA to allow us to isolate T cells from whole blood and assess their cytokine production and gene expression (RT-qPCR). The last component of Aim 1 is to test and compare a new, less invasive blood draw method called Hemolink™ against traditional blood draw methods in terms of immune cell phenotype. In Aim 2 we propose to use a single-cell real-time non-invasive label-free microscopy method called optical metabolic imaging (OMI) to identify single-cell heterogeneity in activation within T cell and neutrophil populations. OMI is compatible with the other endpoints and can be run simultaneously. In Aim 3 will apply our multiplexed KOALA assays to blood samples from a cohort of children with allergic disease and matched healthy controls from the WISC cohort (an ongoing study at UW that includes farm and non-farm children). We will compare our neutrophil and T cell function and phenotype results from these children with the extensive measurements being done as part of the WISC cohort study. In Aim 3 we will also extend these analyses to a group of infants from the CREW study (a new study at UW that aims to identify early life genetic and environmental risk factors for allergic diseases and asthma) using the HemoLink™ device to draw blood with minimal discomfort and profile type 2 T cell function (T cells differentiated to secrete IL-4, IL-5, IL-13 and other type 2 cytokines) using KOALA. If successful in our aims, we will have created a miniaturized, highly multiplexed, self-contained microfluidic platform that is capable of measuring biological markers related to immune status and function. This system will have then been validated in pediatric subjects and ready for transition into clinical research.