Proof-of-Concept for Card-based CD4 Cell Counting

描述（由申请人提供）：这项拟议研究的长期目标是开发一种简单、廉价的即时护理设备，该设备将从全血中进行CD4+ T细胞的绝对和百分比计数。这将通过适应和测试一种新的“混合”方法来完成微流控样品制备和分析。一次性使用的薄膜层压微流控盒将收集、计数和储存血液样本，而所需的鸡尾酒试剂将被储存并从台式装置注射到盒中。集成系统将执行总白细胞计数，绝对CD4+ T细胞计数和百分比CD4计数使用一个新的容量组合电阻抗/荧光传感器包含在一个一次性微流控盒。为了实现这一目标，第二阶段将扩展第一阶段的工作，并将重点放在系统优化和验证上。目标一将优化微流控盒设计和CD4血液标记方案，用于自动卡上CD4+ T细胞计数。目标二将集中于集成台式系统的制造和测试。目标三将测试和验证完整的CD4计数系统，使用改良的市售正常人类全血和HIV+患者的临床样本，并开始获得FDA批准的II类医疗设备所需的台式研究。当在拟议的使用场景中进行充分验证和评估时，所得到的设备将是简单、可靠和独特的，因为它可以提供完全定量的CD4+ T细胞分析，仪器成本低于1000美元。这比现有流式细胞仪的成本低很多倍。试剂和一次性成本也将低于任何目前可用的设备，每次测试总成本低于1.50美元。该工具将立即应用于各级公共卫生保健系统，特别是在资源贫乏地区和发展中国家，用于监测人类免疫缺陷病毒（HIV）感染者，制定鉴别诊断，并就抗逆转录病毒药物治疗方案作出治疗决定，包括高效抗逆转录病毒疗法（HAART）方法。廉价的便携式CD4计数器的成功完成将构成艾滋病毒监测和治疗方面的重大突破，为卫生保健提供者提供一个较便宜的移动平台，进行目前世界上最常见的CD4诊断测试。这一突破将极大地提高其一次性测试盒的测试效率，并使在床边、偏远地区或医疗资源有限的地区快速测试患者成为可能。最后，这种新的护理点系统可以显著减少当前的CD4设备和每次测试成本，从而为美国医疗保健和保险行业节省数百万美元，并对发展中国家的全球医疗保健产生更大的影响。

DESCRIPTION (provided by applicant): The long-term objective of this proposed research is to develop a simple, inexpensive point-of-care device that will perform absolute and percent CD4+ T cell counts from whole blood. This will be accomplished by adapting and testing a novel "hybrid" approach to microfluidic sample preparation and analysis. One-time use thin-film laminated microfluidic cassettes will collect, count, and store the blood sample while the required cocktail reagents will be stored and injected into the cassettes from a bench-top unit. The integrated system will perform a total white blood cell count, absolute CD4+ T cell count, and percent CD4 count using a new volumetric combination electric impedance/fluorescence sensor contained on a disposable microfluidic cassette. Toward this objective, Phase II will expand on our Phase I effort and focus on system optimization and validation. Aim one will optimize the microfluidic cassette design and CD4 blood labeling protocol for automated, on-card CD4+ T cell counting. Aim two will focus on fabrication and testing of the integrated bench-top system. Aim three will test and validate the completed CD4 enumeration system, using both modified commercially available normal human whole blood and clinical samples from HIV+ patients, and begin the bench-top studies required to obtain FDA clearance for a Class II medical device. The resulting device, when fully validated and evaluated in the proposed scenarios of use, will be simple, robust, and unique in that it provides a fully quantitative CD4+ T cell analysis with an instrument costing less than $1000. This is many times below the cost of established flow cytometry devices. Reagent and disposable costs will also be lower than that of any currently available device with per-test total costs of less than $1.50. The tool will find immediate application in all levels of the public health care system, particularly in resource-poor areas and developing countries, for monitoring persons with human immunodeficiency virus (HIV), formulating differential diagnoses, and making therapeutic decisions regarding anti-retroviral drug treatment regimes, including the highly active anti-retroviral therapy (HAART) approach. PUBLIC HEALTH RELEVANCE The successful completion of an inexpensive, portable CD4 counter would constitute a significant breakthrough in HIV monitoring and treatment by offering healthcare providers a less expensive, mobile platform to conduct the most common CD4 diagnostic test currently performed in the world. Such a breakthrough will dramatically improve testing efficiencies with its single-use, disposable test cartridges and will make it possible to rapidly test patients at bedside, in remote locations, or in areas with limited healthcare resources. Finally, this new point-of-care system could significantly reduce current CD4 equipment and per-test costs, thereby resulting in millions of dollars saved in the US healthcare and insurance industries, with even greater worldwide healthcare impacts in developing countries.