A Flow Through Cell Counting Assay

描述（由申请人提供）：拟议研究的长期目标是开发一种新的定量流动细胞计数方法，用于枚举人血液中CD4+ T淋巴细胞。需要CD4细胞计数来对艾滋病毒感染患者进行分期和监测。在成人中，每微升血液中CD4+ T细胞的绝对数量具有关键的预后和治疗意义，并用于HIV分期和治疗决策。目前用于定量CD4细胞的金标准技术是流式细胞术。然而，流式细胞仪，包括最近开发的小型单用途CD4细胞计数设备，在资源有限的国家价格昂贵且难以获得。此外，他们还需要训练有素的实验室技术人员来处理和处理样品，并分析和报告数据。由于基于流式细胞术的CD4细胞计数超出了许多艾滋病毒感染者的能力范围，世界卫生组织（世卫组织）敦促开发用于资源有限环境的简单的即时护理CD4细胞计数测定方法。正是在这种背景下，提出了一种更便宜、快速和易于执行的方法，该方法不受传统测定法的限制，用于测定CD4+ T细胞。所提出的方法，流式细胞计数法（FTCA）是基于使用特殊的膜和专有的流式盒来测量细胞计数状态。该测试可在不到15分钟内完成，并可由培训水平较低的实验室技术人员执行。第一阶段的具体目标包括设计和测试FTCA盒的概念验证原型，使用几种市售膜和各种抗cd4抗体结合不同来源的标签来检查FTCA的性能，以及优化试剂浓度和执行FTCA的分析条件。所提出的技术的优点，如分析的速度，方便和低成本，使我们相信FTCA将广泛应用于临床实践。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to develop a novel quantitative flow through cell counting method for enumeration of CD4+ T lymphocytes in human blood. CD4 cell counts are needed to stage and monitor HIV-infected patients. In adults, the absolute number of CD4+ T cells per microlitre of blood has critical prognostic and therapeutic implications, and is used for both HIV staging and treatment decisions. The gold standard technique currently used for quantitation of CD4 cells is flow cytometry. However, flow cytometers, including smaller single-purpose CD4 cell counting devices developed recently are expensive and inaccessible in resource-limited countries. In addition, they require trained laboratory technicians to handle and process samples and to analyze and report data. Because flow cytometry-based CD4 cell counting is beyond the reach of so many HIV-infected people, the World Health Organization (WHO) has urged the development of simple point-of-care CD4 cell count assays designed for resource-limited settings. It is in this context that the less expensive, rapid and easy-to- perform method, which is free from the limitations of the conventional assays, for determination of CD4+ T cells is proposed to be developed. The proposed method, flow-through cell counting assay (FTCA) is based on the use of the special membrane and a proprietary flow-through cassette to measure cell count status. The test can be completed in less than 15 minutes and can be performed by laboratory technicians with low levels of training. The specific aims of Phase I include designing and testing a proof-of-concept prototype of the FTCA cassette, examination of the performance of the FTCA using several commercially available membranes and various anti-CD4 antibodies conjugated with labels of different origin and optimization of concentrations of the reagents and assay conditions for performing of FTCA. Advantages of the proposed technology such as the speed, convenience, and low cost of the analysis substantiates our belief that the FTCA will be widely used in clinical practice. PUBLIC HEALTH RELEVANCE: The development of the proposed method will allow using CD4 cell counting tests for staging and monitoring HIV infection. Successful completion of these studies will be beneficial for public health, and make available all of the technology needed for a substantial business opportunity to license the technology and manufacture commercial products.