A Flow Through Cell Counting Assay

描述（由申请方提供）：拟定研究的长期目标是开发一种用于人血液中CD 4 + T淋巴细胞计数的新型定量流通细胞计数方法。需要CD 4细胞计数来分期和监测HIV感染患者。在成人中，每微升血液中CD 4 + T细胞的绝对数量具有重要的预后和治疗意义，并用于HIV分期和治疗决策。目前用于定量CD 4细胞的金标准技术是流式细胞术。然而，流式细胞仪，包括最近开发的较小的单一用途CD 4细胞计数装置，价格昂贵，在资源有限的国家难以获得。此外，它们还需要训练有素的实验室技术人员来处理和加工样品，分析和报告数据。由于基于流式细胞术的CD 4细胞计数超出了如此多的艾滋病毒感染者的能力范围，世界卫生组织（WHO）敦促开发用于资源有限环境的简单的即时CD 4细胞计数测定。正是在这种情况下，更便宜、快速和易于执行的方法， 不受常规测定法的限制，建议开发用于测定CD 4 + T细胞的方法。 拟定方法，即流通细胞计数试验（FTCA），是基于使用特殊膜和专有流通试剂卡来测量细胞计数状态。该测试可能在不到15分钟的时间内完成，并且可以由受过低水平培训的实验室技术人员进行。第II阶段的具体目标是：1）设计和组装FTCA检测卡的预生产原型，并调整血糖仪用于FTCA信号测量; 2）探索影响FTCA CD 4 + T细胞计数方法性能的所有变量，并优化检测性能; 3）制备用于进行FTCA的冻干免疫试剂，并检查所有FTCA试剂的稳定性; 4）通过检查具有统计学显著性的临床样本数量来验证FTCA。 所提出的技术的优点，如分析的速度，方便，和低成本证实了我们的信念，FTCA将在临床实践中广泛使用。

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 potentially be completed in less than 15 minutes and can be performed by laboratory technicians with low levels of training. The specific aims of Phase II are: 1) To design and assemble a pre-production ¿-prototype of the FTCA cassette and adapt a blood glucose meter for the FTCA signal measurements; 2) To explore all variables affecting the performance of the FTCA CD4+ T-cell counting method and optimize the performance of the assay; 3) To prepare lyophilized immuno-reagents for performing FTCA and examine the stability of all FTCA reagents; 4) To validate the FTCA by examining a statistically significant number of clinical samples. 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.