Microfluidic Processing of Leukocytes for Molecular Diagnostic Testing

描述（由申请人提供）：用于诊断测试的白细胞的微流体处理概述/摘要：用于量化来自血液样品的关键白细胞类型的数量和功能状态的新测试提供了临床诊断、预后和治疗反应的增强的确定和个性化。例如，标记>30种细胞表面和细胞内靶分子可以通过多参数流式细胞术或将来的原子质谱法同时评估多种类型的正常白细胞与白血病细胞的信号传导途径状态。然而，目前收获血液白细胞的方法昂贵、耗时、细胞产量低、引入细胞毒性试剂、选择性地损失白细胞亚群，并且需要相当多的人类专业知识。此外，需要多个连续步骤，包括红细胞裂解、用单克隆抗体（Mab）进行表面标记、固定/透化和细胞内标记。每个步骤需要1个或多个离心洗涤步骤，导致细胞损失和研究生产率降低。开发一种自动化的、无试剂的过程，确保靶细胞的高产率，并大大简化预分析工作流程，将对研究和临床实验室产生重大益处。商业目标：销售一种系统，该系统结合了血液中白细胞的收集、洗涤和浓缩，优于标准离心方法。我们将应用微流体确定性横向位移（DLD），其中细胞通过微流体系统的路径是基于尺寸的并且是确定性的，即绝对确定的，不受随机过程的影响。该STTR项目的关键创新是使用DLD从红细胞中去除白细胞/白血病细胞，并在连续流动过程中从标记或其他试剂中去除白细胞/白血病细胞，最终浓缩目标白细胞/白血病细胞。这是一种新的用途，使这种应用在商业上新颖，在临床上有益。该研究战略利用了GPB Scientific LLC的微机械系统专家之间的合作;马里兰州大学（Civin实验室）的造血细胞生物学和细胞处理专家;以及普林斯顿大学（Sturm-Austin实验室）的微流体专家。在具体目标1中，该团队将创建微流体设备，以在几分钟内自动化过程中从含有标记单克隆抗体的流中洗涤和浓缩白细胞。在特定目标2中，该系统将用于收获和浓缩全血中标记的白细胞，去除红细胞和未结合的Mab。该项目的重点是流式细胞术的白细胞处理，但同样的技术可用于许多现有的和新的细胞和其他（如DNA，RNA）测试癌症和其他疾病。价值主张很明确：通过取代红细胞裂解和离心洗涤，GPB技术应通过自动化、无试剂的工作流程提高研究和临床生产力，提供高质量的细胞产品，实现具有可重现结果的高价值细胞分析。 公共卫生相关性：用于分子诊断检测的白细胞微流体处理该项目专注于在通过流式细胞术进行多参数分析之前实现白细胞样品制备的突破。目前涉及离心的方法是繁琐的手动过程，需要专业操作员，并导致细胞丢失和损坏。我们的微流体方法将大大提高细胞质量;简化工作流程并降低成本。应用包括癌症、传染病和炎性疾病等疾病领域的研究和临床诊断。

DESCRIPTION (provided by applicant): Microfluidic Processing of Leukocytes for Diagnostics Testing Summary/Abstract: New tests to quantify the numbers and functional states of key leukocyte types from blood samples offer enhanced determination and personalization of clinical diagnosis, prognosis, and treatment response. For example, labeling of >30 cell surface and intracellular target molecules can assess signaling pathway status of multiple types of normal leukocytes vs leukemia cells simultaneously, by multi- parameter flow cytometry or in the future, atomic mass spectrometry. However, current procedures to harvest blood leukocytes are expensive, time-consuming, have low cell yields, introduce cytotoxic reagents, selectively lose leukocyte subsets, and require considerable human expertise. Furthermore, multiple sequential steps are required including erythrocyte lysis, surface labeling with monoclonal antibodies (Mabs), fixation/permeablization, and intracellular labeling. Each step requires 1 or more centrifugal wash steps, resulting in cell loss and reduced research productivity. Development of an automated, reagent-free process that ensures high yield of target cells and greatly streamlines the pre-analytical workflow would be a significant benefit to research and clinical labs. Commercial goal: Market a system that combines the harvest, wash and concentration of leukocytes from blood, outperforming standard centrifugal methods. We will apply microfluidic deterministic lateral displacement (DLD), in which the paths cells take through the microfluidic system is based on size and is deterministic, i.e. absolutely determined, not subject to random processes. The key innovation of this STTR project is the use of DLD to remove leukocytes/leukemia cells from erythrocytes and also from labeling or other reagents in a continuous flow process, finally concentrating the target leukocytes/leukemia cells. This is a new use, making this application commercially novel as well as clinically beneficial. The Research Strategy leverages a collaboration among micromechanical systems experts at GPB Scientific LLC; hematopoietic cell biology and cell processing experts at University of Maryland (Civin lab); and microfluidics experts at Princeton University (Sturm-Austin lab). In Specific Aim 1, the team will create microfluidic devices to wash and concentrate leukocytes from a stream containing labeling Mabs in an automated process in a few minutes. In Specific Aim 2, the system will be applied to harvest and concentrate leukocytes labeled in whole blood, removing erythrocytes and unbound Mabs. This project focuses on leukocyte processing for flow cytometry, but the same technology could be used for many existing and new cellular and other (e.g. DNA, RNA) tests for cancer and other diseases. The value proposition is clear: by replacing erythrocyte lysis and centrifugal washing, GPB technology should increase research and clinical productivity through automated, reagent-free workflows that provide high quality cell products, enabling high value cell analyses with reproducible results. PUBLIC HEALTH RELEVANCE: Microfluidic Processing of Leukocytes for Molecular Diagnostic Testing This project is focused on achieving a breakthrough in the sample preparation of leukocytes prior to multi- parameter analysis via flow cytometry. Current methods involving centrifugation are tedious, manual processes that require expert operators and result in lost and damaged cells. Our microfluidic approach will greatly improve cell quality; streamline workflows, and lower costs. Applications include research and clinical diagnostics in cancer, infectious disease, and inflammatory disease, among other disease areas.