IMPROVED FLOW ANALYSIS OF CLINICAL BLOOD SAMPLES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。对该分项目和该分项目首席调查员的主要支持可能是由其他来源提供的，包括国家卫生研究院的其他来源。列出的子项目总成本可能代表子项目使用的中心基础设施的估计数量，而不是NCRR拨款提供给子项目或子项目工作人员的直接资金。 摘要 流式细胞术现在是人类血液样本诊断分析的标准分析平台，主要通过使用荧光标记的抗体标记细胞表面标记的免疫表型来进行。这些应用的相关性涉及广泛的领域，包括白血病/淋巴瘤诊断和患者特定处理、传染病、移植医学(干细胞计数/鉴定)、阵发性睡眠性血红蛋白尿(PNH)诊断和胎儿血红蛋白检测。虽然这样的分析是常规的，但仍然存在一些问题，可以改进流动仪表。在这次与Charles Goolsby博士的临床免疫表型实验室的合作中，我们将使用在项目1和3中开发的独特仪器来直接解决其中的两个问题：多色流动分析中重叠荧光团的分离(目前通常为6-8种颜色)和红细胞溶解过程中白细胞亚群的丢失。项目3中开发的改进的光谱分辨率仪器将用于确定，与标准使用滤光片和复杂的补偿矩阵相比，多重染色细胞的完整发射光谱的去卷积是否可以提高不同细胞类型的分辨率和定量。项目1中开发的在线样品制备设备将用于确定流动样品流中红细胞和白细胞的声场分离是否可以消除红细胞裂解步骤的需要，从而导致某些白细胞亚组的损失，特别是脆弱的异常细胞。通过这种合作获得几种类型的临床样本将直接测试这两种仪器在现实世界中解决传统流式细胞术局限性的效用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract Flow cytometry is now a standard analysis platform for diagnostic analysis of human blood samples, primarily through the use of immunophenotyping via cell surface marker labeling with fluorescently-tagged antibodies. Relevance of these applications covers a wide range of areas including leukemia/lymphoma diagnosis and patient specific management, infectious disease, transplant medicine (stem cell enumeration/ characterization), paroxysmal nocturnal hemoglobinuria (PNH) diagnosis and fetal hemoglobin detection. Although such analysis is routine, there are still problems that are amenable to improve flow instrumentation. In this collaboration with Dr. Charles Goolsby's clinical immunophenotyping laboratory, we will use the unique instrumentation developed in Projects 1 and 3 to directly address two of these problems: separation of overlapping fluorophores in multi-color flow analysis which are now routinely six-eight color and loss of subsets of white cells during red cell lysis procedures. The improved spectral resolution instrument developed in Project 3 will be used to determine if deconvolution of complete emission spectra from multiply-stained cells can improve the resolution and quantitation of different cell types as compared to the standard use of optical filters and a complex compensation matrix. The in-line sample preparation device developed in Project 1 will be used to determine if acoustic field separation of red and white cells in a flowing sample stream can eliminate the need for a red cell lysis step with its resultant loss of certain subsets of white cells, particularly fragile abnormal cells. The availability of several types of clinical samples through this collaboration will directly test the utility of these two instruments to address limitations of conventional flow cytometry in a real-world situation.