Enabling pediatric leukapheresis with high-throughput microfluidic technology

项目总结 白细胞分离是一种专门的医疗程序，在此过程中患者的全血(WB)被 分离机的体外电路，从白细胞中提取白细胞(WBC)，然后返回 将红细胞(RBC)和血小板(PLT)送回患者体内。从患者血液中分离白细胞 经白细胞分离是越来越多的基于细胞的高效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 全世界。目前，白细胞分离是使用基于离心机的分离机进行的，这种机器有 相当大的体外体积(ECV)。尽管大多数成年人和年龄较大的儿童都能很好地忍受， 对体重不足10公斤(或22磅)的幼儿进行白细胞分离在技术上和临床上都是具有挑战性的 太冒险了。由于ECV在他们的总血量(TBV)中所占的比例特别大，这些脆弱的 患者发生低血压、症状性低钙血症、过敏的发生率显著升高 反应、导管相关血栓形成、感染、严重贫血甚至死亡。目前还没有实际的 替代成人大小的分离机，用于新生儿和低体重婴儿的白细胞分离。 为了解决这一重大限制，我们将开发和验证新型高通量微流控设备 极低的空隙体积，能够以足够的体积吞吐量和效率将WBC与WB分离 高以最终实现无离心法、低ECV的白细胞分离。这些设备将利用新的电池 我们以前应用于分离的分离技术(‘控制增量过滤’，或CIF) 从浓缩的血细胞悬液中获得高效率、最小的RBC和PLT损失以及流动时的WBC 其比率与传统的白细胞分离法相当。在这里，我们将应用此方法将WBC直接从 WB通过完成三个互补的目标，范围从迭代设计优化和 验证工作，以在动物模型中测试基于CIF的白细胞分离设备的性能。第一, 我们将优化CIF设计参数，以最大限度地分离WBC，同时将RBC/PLT损失降至最低 处理WB时的设备流体阻力。第二，我们将把单个CIF设备模块多路传输到完整的 调整设备原型，优化其在再循环状态下的运行，并验证其处理能力 大量的WB在体外。第三，我们将综合评估设备性能和CIF的效果- 根据小鼠模型中的血细胞特性进行处理。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，我们将开发出功能丰富的设备 在最终确定之前，生成关键数据以支持在临床前模型(猪)中进行进一步测试 用于制造热塑性塑料的设备设计和人体临床测试。

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.