Probing basophil function in microfluidic systems for allergic disease diagnosis

项目摘要 嗜碱性粒细胞在过敏性疾病中发挥重要作用：它们的激活越来越多地用于食物 过敏诊断，它们迁移到组织中与过敏症的发生有关。 过敏反应然而，由于嗜碱性粒细胞是罕见的，他们很少研究和利用诊断 特别是在离体微流体系统中。本项目的总体目标 是通过开发微流控技术来更好地量化嗜碱性粒细胞， 功能，并通过使用食物过敏作为模型疾病。 食物过敏已达到流行病的比例，并已成为一个重要的来源， 医疗负担。常见的体外过敏试验缺乏足够的准确性。口服食物挑战， 食物过敏评估的黄金标准，往往不执行，因为它把病人在 过敏反应的风险。因此，食物过敏通常仅在发生不良反应后才被发现， 可能会危及生命此外，发病与死亡之间没有可靠的相关性。 过敏反应和最小过敏原剂量。预测过敏反应风险 病人基本上不存在。 我们的长期目标是通过开发过敏诊断测试来应对这些挑战 准确、安全、快速和可访问的指标，以及2）更好地对高风险患者进行分层的指标 过敏反应这项试验拨款的总体目标是测试以下假设：1）阻抗 嗜碱性粒细胞活化后的变化，暴露带负电荷的外置颗粒 蛋白聚糖，是嗜碱性粒细胞活化的准确和简单的无标记指示剂，和2） 嗜碱性粒细胞迁移和分泌特征与反应严重程度相关。我们的方法包括 使用来自经临床证实和充分表征的花生过敏受试者的全血样品 和健康控制，并利用我们的跨学科团队的专业知识， 微流控技术和人类免疫学。我们将：（目标1）设计和验证一个无标签的 在食物过敏原存在下使用全血的基于阻抗的嗜碱性粒细胞活化试验 体内，和（目的2）设计和验证耦合嗜碱性粒细胞迁移和多重分泌 离体测定以确定与患者过敏史的相关性。

PROJECT SUMMARY Basophils play important roles in allergic diseases: their activation is increasingly used for food allergy diagnosis, and their migration into tissues has been correlated with the onset of anaphylaxis. However, since basophils are rare, they are less studied and utilized for diagnostics than other leukocytes, especially in ex vivo microfluidic systems. The overall goal of this project is to fill this gap by developing enabling microfluidic technologies to better quantify basophil function, and by using food allergy as a model disease. Food allergy has reached epidemic proportions and has become a significant source of healthcare burden. Common in vitro allergy tests lack sufficient accuracy. Oral food challenge, the gold standard for food allergy assessment, is often not performed as it places the patient at risk of anaphylaxis. As such, food allergy is often identified only after an adverse reaction that could be life-threatening. In addition, there is no reliable correlation between the onset of anaphylaxis and a minimum offending dose of allergen. Metrics to predict anaphylaxis risk in a patient are essentially non-existent. Our long-term goal is to address these challenges by developing 1) allergy diagnostic tests that are accurate, safe, rapid, and accessible, and 2) metrics to better stratify patients at high risk of anaphylaxis. The overall objective of this pilot grant is to test the hypotheses that: 1) impedance changes in basophils upon activation, which exposes negatively-charged exteriorized granule proteoglycans, is an accurate and simple label-free indicator for basophil activation, and 2) basophil migration and secretion profile correlates with reaction severity. Our approach includes using whole blood samples from clinically-proven and well-characterized peanut allergic subjects and healthy controls, and leveraging our interdisciplinary team's expertise in innovative microfluidic technology and human immunology. We will: (Aim 1) design and validate a label-free impedance-based basophil activation test using whole blood in the presence of food allergen ex vivo, and (Aim 2) design and validate a coupled basophil migration and multiplexed secretion assay ex vivo to determine the correlation with patient history of anaphylaxis.