Enabling pediatric leukapheresis with high-throughput microfluidic technology

项目摘要 白细胞分离术是一种专门的医疗程序，在此过程中，患者的全血（WB）通过 单采机的体外回路，其从WB中提取白色血细胞（WBC），并返回 红细胞（RBC）和血小板（PLT）返回给患者。从患者血液中分离白细胞 通过白细胞分离术是越来越多的高效细胞治疗的关键初始步骤， 影响数百万成人和儿童的一些最具破坏性的血液和免疫系统疾病 国际吧目前，使用基于离心的单采机进行白细胞单采，其具有 大量的体外容量（ECV）。虽然大多数成年人和年龄较大的儿童都能很好地耐受， 体重小于约10 kg（或22 lbs）的幼儿的白细胞去除术在技术上具有挑战性， 冒险了由于ECV占其总血容量（TBV）的特别大的部分，这些易受伤害的人 患者发生低血压、症状性低钙血症、过敏性 反应、导管相关血栓形成、感染、严重贫血甚至死亡。目前没有实际的 替代成人尺寸的血液分离机，用于对新生儿和低体重婴儿进行白细胞分离。 为了解决这一重大限制，我们将开发和验证新的高通量微流体装置， 空隙体积非常低，能够以足够的体积通量和效率从WB中分离WBC 最终能够实现无离心、低ECV白细胞分离。这些设备将利用新的细胞 分离技术（“受控增量过滤”，或CIF），我们以前应用于分离 来自浓缩血细胞悬液的WBC，具有高效率，最小的RBC和PLT损失， 与常规白细胞分离术的比率相当。在这里，我们将应用这种方法来分离白细胞直接从 WB通过完成三个互补的目标，范围从迭代设计优化， 确认工作，在动物模型中测试基于CIF的白细胞去除装置的性能。第一、 我们将优化CIF设计参数，以最大限度地分离WBC，同时最大限度地减少RBC/PLT损失， 处理WB时的器械流体阻力。其次，我们将把单个CIF设备模块复用成完整的- 缩放设备原型，优化其在再循环制度中的操作，并验证其处理能力 体外大体积WB。第三，我们将全面评估设备性能和CIF的效果- 基于小鼠模型中的血细胞特性的处理。详细的血细胞计数和细胞标志物 将在整个项目中测量激活和损坏，以帮助迭代设计过程并验证 使用CIF技术进行白细胞分离。通过完成这项研究，我们将开发出功能化的设备， 原型并生成关键数据，以支持在最终确定前在临床前模型（猪）中进行进一步测试 该器械设计用于由热塑性塑料制造并在人类受试者中进行临床试验。

PROJECT SUMMARY Leukapheresis is a specialized medical procedure during which patient's whole blood (WB) is passed through the extracorporeal circuit of an apheresis machine, which extracts white blood cells (WBCs) from WB, and returns red blood cells (RBCs) and platelets (PLTs) back to the patient. The separation of WBCs from patient's blood via leukapheresis is the key initial step for an increasing number of highly effective cell-based treatments for some of the most devastating hematologic and immune system disorders affecting millions of adults and children worldwide. Currently, leukapheresis is performed using centrifugation-based apheresis machines, which have a substantial extracorporeal volume (ECV). Although well-tolerated by most adults and older children, leukapheresis in young children weighing less than about 10 kg (or 22 lbs) is technically challenging and clinically risky. Because ECV represents a particularly large fraction of their total blood volume (TBV), these vulnerable patients experience a significantly higher incidence of hypotension, symptomatic hypocalcemia, allergic reactions, catheter-related thrombosis, infections, severe anemia and even death. There is currently no practical alternative to adult-size apheresis machines for performing leukapheresis in neonates and low-weight infants. To address this significant limitation, we will develop and validate novel high-throughput microfluidic devices with very low void volume, capable of separating WBCs from WB with volumetric throughput and efficiency sufficiently high to ultimately enable centrifugation-free, low-ECV leukapheresis. These devices will utilize new cell separation technology (`controlled incremental filtration', or CIF) which we have previously applied to separating WBCs from concentrated blood cell suspensions with high efficiency, minimal RBC and PLT loss, and at flow rates on par with conventional leukapheresis. Here we will apply this approach to separating WBCs directly from WB by completing three complementary aims with scope ranging from iterative design optimization and validation work, to testing the performance of the CIF-based leukapheresis devices in an animal model. First, we will optimize the CIF design parameters to maximize WBC separation, while minimizing RBC/PLT losses and device fluidic resistance when processing WB. Second, we will multiplex individual CIF device modules into full- scale device prototypes, optimize their operation in the recirculation regime, and validate their ability to process large volumes of WB in vitro. Third, we will comprehensively evaluate device performance and the effect of CIF- based processing on blood cell properties in a mouse model. Detailed blood cell counts and markers of cell activation and damage will be measured throughout the project to aid the iterative design process and to validate the use of CIF technology for leukapheresis. By completing this research, we will develop functional device prototypes and generate pivotal data to support further testing in a pre-clinical model (porcine), before finalizing the device design for manufacturing from thermoplastic and clinical testing in human subjects.