Monitoring peripheral blood leukocyte and immune responses in health and disease

 描述(由申请人提供)：拟议的实验将检验假设：使用Dean Flow Fractionation根据白细胞的固有属性分离白细胞将能够分析微升全血中的先天和获得性免疫反应。急性和慢性炎症反应是许多疾病临床表现的组成部分，但常规的临床免疫功能评估仅基于白细胞计数。这通常是通过完整的血细胞计数和白细胞分类计数来衡量的。在目前的临床实践中，关于免疫调节的关键临床决策是基于白细胞和白细胞亚类的计数，而不是评估白细胞功能。此外，外周静脉全血细胞计数的测量使用3-5毫升。根据完成血细胞计数和其他常规实验室检测所需的大量血量，患者可能会患上医源性贫血。迫切需要开发能够评估免疫功能的分析方法，并且需要的血量要少得多。在初步结果中，我们开发了一种惯性微流控方法，基于非标记的细胞类型特定的物理特征选择性地分离不同的细胞靶点。这种新方法可以从外周静脉血中分离白细胞和红细胞、血小板、白细胞亚类，并通过串联等电分离来区分激活的中性粒细胞和未激活的细胞。这种多功能的体外分离系统是高通量的，能够在系统和细胞免疫功能的耦合分析之前对白细胞进行预备分离。为了解决我们的假设，提出了两个具体的目标：1.设计微升规模的惯性微流控分离 检测外周血中的白细胞；2.测定系统免疫功能和细胞免疫功能的微升。高通量微流控工程的最新进展使这一项目成为可能，为复杂免疫监测的新方法铺平了道路。

 DESCRIPTION (provided by applicant): The proposed experiments will test the hypothesis: The separation of leukocytes by their intrinsic properties using Dean Flow Fractionation will enable analyses of innate and adaptive immune responses in microliter quantities of whole blood. Acute and chronic inflammatory responses are integral to the clinical expression of many diseases, yet routine clinical assessment of immune function is based solely on leukocyte count. This is typically measured with a complete blood count and leukocyte differential count. In current clinical practice, critical clinical decisions regarding immune modulation are made based on the count of leukocytes and leukocyte subclasses without assessment of leukocyte function. In addition, the measurement of a peripheral venous complete blood count uses 3-5 mL. Based on the significant blood volume required for the complete blood count and other routine laboratory testing, patients can develop iatrogenic anemia. There is a critical need to develop assays that enable assessment of immune function and require significantly smaller volumes of blood. In preliminary results, we have developed an inertial microfluidic approach to selectively separate diverse cellular targets that is based on non-labeled, cell type specific physical features. From peripheral venous blood, this new approach can separate leukocytes from erythrocytes and platelets, leukocyte subclasses and distinguish activated neutrophils from unactivated cells by tandem iso-dielectric separation. This multi-functional, extracorporeal separation system is high throughput and capable of preparatory separation of leukocytes prior to coupled analyses of systemic and cellular immune function. To address our hypothesis, two specific aims are proposed: 1. Design microliter scale preparatory inertial microfluidic separation of leukocytes in peripheral blood; 2. Determine microliter scale assays of systemic and cell-based immune function. Recent advances in high-throughput microfluidic engineering enable this project, paving the way for a new approach to complex immune monitoring.