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

摘要 可从儿科患者中采集的生物样本数量有限 用于研究免疫功能的方法通常不足以进行常规测定。 有必要开发新的多参数样品保留测定法， 推进对免疫系统的基本理解。在本提案中，我们 概述了三个目标，详细说明了新的生物读数的添加和整合， 现有的微生物技术和使用这个扩展的平台来研究 儿童过敏和哮喘的发病机制。盖上的工具箱 检测（KOALA）是一种易于使用的自含式微电泳平台， 从全血中分离中性粒细胞，并评估其趋化能力。在 目的1：我们建议调整和验证用于测量中性粒细胞ROS的方法 产生、NETosis、吞噬作用、细胞因子产生和基因表达（RT- qPCR）转化为KOALA。我们还计划修改KOALA的细胞捕获特性， 使我们能够从全血中分离T细胞并评估其细胞因子的产生， 基因表达（RT-qPCR）。目标1的最后一个组成部分是测试和比较 一种新的、侵入性更小的抽血方法，称为Hemolink™，与传统抽血方法不同 方法在免疫细胞表型方面。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非侵入性无标记显微镜方法 (OMI)鉴定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，将我们的多重KOALA检测应用于来自以下队列的血液样本： 来自WISC队列的过敏性疾病儿童和匹配的健康对照组（一项研究， 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 中性粒细胞和T细胞的功能和表型结果，这些儿童与 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， 我还将这些分析扩展到CREW研究中的一组婴儿（ UW旨在确定过敏性疾病的早期遗传和环境风险因素， 疾病和哮喘）使用HemoLink™装置以最小的 不适和轮廓2型T细胞功能（T细胞分化分泌IL-4，IL-5， IL-13和其他2型细胞因子）。如果我们的目标实现了， 创建了一个小型化，高度多路复用，自包含的微处理器平台， 能够测量与免疫状态和功能相关的生物标志物。这 然后，系统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.