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流经细胞的定量计数方法来计数人血中的CD4T淋巴细胞。对HIV感染者进行分期和监测需要进行CD4细胞计数。在成人中，每微升血液中CD4T细胞的绝对数量具有重要的预后和治疗意义，并可用于艾滋病毒的分期和治疗决策。目前用于CD4细胞定量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的CD4细胞计数装置，价格昂贵，在资源有限的国家无法获得。此外，它们还需要训练有素的实验室技术人员来处理和处理样品以及分析和报告数据。由于基于流式细胞仪的CD4细胞计数超出了这么多艾滋病毒感染者的能力范围，世界卫生组织(WHO)敦促开发适用于资源有限环境的简单护理点CD4细胞计数分析方法。正是在这种情况下，成本更低、快速和易于执行的方法，即 不受常规检测方法的限制，CD4T细胞的检测方法有待发展。提出的流式细胞计数分析方法(FTCA)是基于使用特殊的膜和专有的流动试剂盒来测量细胞计数状态。测试可能在不到15分钟内完成，并且可以由受过较低培训的实验室技术人员进行。第二阶段的具体目标是：1)设计和组装FTCA试剂盒的试生产原型，并采用血糖仪进行FTCA信号测量；2)探索影响FTCA CD4 T细胞计数方法性能的所有变量，并优化分析性能；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)通过检查大量统计显著的临床样本来验证FTCA。该技术具有快速、方便、成本低等优点，使我们相信FTCA技术将在临床上得到广泛应用。

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.