IMPROVED FLOW ANALYSIS OF CLINICAL BLOOD SAMPLES

改进临床血样的流量分析

• 批准号: 8169401
• 负责人: Charles L. Goolsby
• 金额: $ 3.34万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169401
• 关键词: Abnormal Cell; Acoustics; Acute leukemia; Address; Algorithms; Antibodies; Apoptotic; Area; Blood Banks; Blood Cells; Blood specimen; Cell Separation; Cell Size; Cell surface; Cells; Cellular Membrane; Cellular biology; Chronic Lymphocytic Leukemia; Clinical; Clinical Data; Collaborations; Color; Communicable Diseases; Complex; Complication; Computer Retrieval of Information on Scientific Projects Database; Computer software; Core Facility; Cytolysis; Data; Data Files; Detection; Development; Devices; Diagnosis; Diagnostic; Disease; Ensure; Erythrocytes; Fetal Hemoglobin; Financial compensation; Flow Cytometry; Fluorochrome; Fourier Transform; Frequencies; Funding; Gold; Grant; Hematopoietic Neoplasms; Housing; Human; Immunophenotyping; Individual; Institution; Investigation; Label; Laboratories; Large-Cell Lymphomas; Lead; Leukemic Cell; Leukocytes; Link; Mammalian Cell; Measures; Methods; Monitor; Optics; Particle Size; Pathology; Patients; Preparation; Procedures; Protocols documentation; Recovery; Relative (related person); Research; Research Personnel; Resolution; Resources; Running; Sampling; Signal Transduction; Signal Transduction Pathway; Source; Specimen; Staining method; Stains; Stem cells; Stream; Techniques; Technology; Testing; Therapeutic; Tube; United States National Institutes of Health; Universities; Work; abstracting; base; cancer cell; cell type; fluorophore; improved; indexing; instrument; instrumentation; kinase inhibitor; leukemia/lymphoma; particle; peripheral blood; professor; rat Piga protein; success; transplantation medicine; virtual

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. Background Dr. Charles Goolsby is the Floyd Elroy Patterson Professor of Pathology and Director of the Flow Cytometry Clinical and Core Facilities at Northwestern University, specializing in the investigation of the basic cell biology of B chronic lymphocytic leukemia (CLL) using flow cytometry (1-3). Dr. Goolsby has lead also the development of complex, multiparametric flow cytometry based clinical analyses both for diagnostic purposes and for patient specific therapeutic decision/monitoring purposes including assessment of signal transduction pathways in the setting of kinase inhibitor therapies (4). Routine utilization of six to eight color analyses are employed in the clinical laboratory setting. Although flow cytometry is the 'gold standard' for the immunophenotypic analysis of human blood samples for a wide variety of diseases, there are at least two areas in which technical advances would improve the utility of this technology for diagnostics and therapy monitoring. The first concerns the use of multiple, overlapping fluorochromes linked to antibodies to label numerous cell types in a single sample. A complex compensation algorithm on the flow cytometer software estimates the relative contribution of the overlapping fluorochromes, and there is no way to directly test the success of the compensation matrix for individual samples. The upgraded full spectral resolution cytometer to be developed in Project 3 will be used to address this limitation of conventional flow cytometers. A second problem is the requirement to lyse red cells in blood cell samples prior to flow analysis. Although red blood cell lysis protocols are far less selective than gradient separation techniques, in some patients selective loss of specific cell subsets can still be seen, a complication that it is difficult to track for individual samples in a clinical setting. The selective loss of subsets of white cells is a particular problem in the analysis of some acute leukemias, large cell lymphomas, and in specimens with a high apoptotic index where the abnormal, cancer cells can be especially fragile and lost during the red cell lysis procedure. This inconsistent, and difficult to predict on a patient specific sample basis complicates the use of flow cytometry for diagnosis and therapy monitoring. The acoustic instrument developed in Project 1 will be used to determine if specific cell subsets in peripheral blood samples can be separated by field-based manipulation prior to analysis, negating the need for a red blood cell lysis step. Approach Improved Spectral Separation of Multiply Stained Blood Cells The upgraded full spectral resolution cytometer will be used in this collaboration to determine if the separation of overlapping fluorochromes can be improved by measuring the complete emission spectrum of the cell. Preliminary work with fixed clinical samples from Dr. Goolsby's laboratory has shown that the current spectral instrument can collect complete emission spectra of individual cells at the two excitation wavelengths commonly used for multi-color clinical analysis. We have also demonstrated that we can generate standard list-mode data files from large numbers of complete emission spectra collected on a single cell basis. We will use both Fourier transform and wavelet algorithms for deconvoluting the emission spectra into its component emission spectra from the individual fluorochromes, using cells stained with each of the individual cell surface markers, along with unstained cells, as a basis set of control spectra. The intensity of signal from each component of the deconvolved spectra will be compared to those obtained from standard compensation matrix analysis of the same clinical samples as analyzed in the Goolsby laboratory. As a further control, we will set 'virtual filters' on the emission spectra acquired at Los Alamos, using the same wavelength ranges as employed using actual bandpass and longpass filters on the standard flow cytometer used to acquire the clinical data. These data will then be run through the same compensation matrix as used on the standard data in order to determine if the spectral instrument faithfully replicates the emission data measured on the standard cytometer. We will also measure the samples on our in-house commercial instrument using the same settings as were employed in the Goolsby laboratory to ensure that the samples provide reproducible results. The ability of the spectral instrument to improve spectral resolution will be tested by comparing the separation of multiple cell types in mixed samples using the spectral deconvolution and standard compensation matrix analysis methods. We will construct test samples composed of known mixtures of different cell types in different ratios and use these compare the standard and spectral deconvolution methods. Improved Separation of Blood Cells Project 1 is largely based on the ability of acoustic focusing techniques to manipulate particles in a flowing sample stream. We have already demonstrated that different sized particles in a mixture can be separated in the flow stream by adjusting the acoustic frequency and amplitude in the line drive (see Preliminary Data section in Project 1). We have also already demonstrated that the acoustic energy needed to separate mammalian cells is well below the threshold for causing physical damage to cellular membranes, meaning that we should be able to manipulate blood cells while maintaining their viability. The acoustic focusing instrument developed in Project 1 will be used to determine if we can separate red blood cells from white cells in a mixed flowing stream, based on the large difference in cell size between these two particles. As described in Specific Aim 3 of Project 1 (see Figure 13), our first approach will be to concentrate the larger white cells to the center of the sample stream, where they will be removed using a central tube while the red cells flow around the outside. Initial studies will be performed using routine blood samples obtained locally as well as from blood banks. It is important to note that only a high degree of purification is required: a small contamination of the white cell sample with red cells will not be a problem for subsequent flow cytometric analysis. If we cannot obtain sufficient purity with a single pass, we can use multiple passes through the acoustic focusing chamber. Once we have verified the ability to separate red and white cells, we will obtain clinical normal and hematopoietic malignancy samples as noted above from Dr. Goolsby's laboratory to determine if we can perform the separation on these samples. The fraction, and quality, of leukemic cells obtained via the acoustic separating procedure will be compared to samples prepared using standard red cell lysis techniques in order to verify improved cell recovery. Once the procedure has been demonstrated and optimized, it will be possible to implement a simple acoustic chamber for blood cell separation that could be used as a stand-alone preparative instrument or retrofit onto the front end of a standard flow analyzer.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 抽象的 流式细胞术现已成为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抗体的细胞表面标记进行免疫表型分析。这些应用的相关性涵盖广泛的领域，包括白血病/淋巴瘤诊断和患者具体管理、传染病、移植医学（干细胞计数/表征）、阵发性睡眠性血红蛋白尿（PNH）诊断和胎儿血红蛋白检测。尽管这种分析是常规的，但仍然存在需要改进流量仪表的问题。 在与 Charles Goolsby 博士的临床免疫表型实验室的合作中，我们将使用项目 1 和 3 中开发的独特仪器来直接解决其中两个问题：多色流分析中重叠荧光团的分离，现在通常为六八色以及红细胞裂解过程中白细胞亚群的损失。 项目 3 中开发的改进的光谱分辨率仪器将用于确定与光学滤光片和复杂补偿矩阵的标准使用相比，多重染色细胞的完整发射光谱的解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装置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，该步骤会导致某些白细胞亚群的损失，特别是脆弱的异常细胞。 通过此次合作获得的多种类型的临床样本将直接测试这两种仪器在现实情况下解决传统流式细胞术局限性的实用性。 背景 Charles Goolsby 博士是西北大学 Floyd Elroy Patterson 病理学教授兼流式细胞术临床和核心设施主任，专门从事使用流式细胞术研究 B 型慢性淋巴细胞白血病 (CLL) 的基础细胞生物学 (1-3 ）。 Goolsby 博士还领导开发了基于复杂、多参数流式细胞术的临床分析，用于诊断目的和患者特定治疗决策/监测目的，包括评估激酶抑制剂治疗中的信号转导途径 (4)。临床实验室环境中常规使用六到八种颜色分析。尽管流式细胞术是对多种疾病的人体血液样本进行免疫表型分析的“金标准”，但至少在两个领域，技术进步将提高该技术在诊断和治疗监测方面的实用性。 第一个涉及使用与抗体连接的多种重叠荧光染料来标记单个样品中的多种细胞类型。 流式细胞仪软件上的复杂补偿算法可估计重叠荧光染料的相对贡献，并且无法直接测试单个样品的补偿矩阵是否成功。 项目3中将开发的升级版全光谱分辨率细胞仪将用于解决传统流式细胞仪的这一局限性。 第二个问题是在流式分析之前需要裂解血细胞样品中的红细胞。尽管红细胞裂解方案的选择性远不如梯度分离技术，但在一些患者中仍然可以看到特定细胞亚群的选择性丢失，这是一种在临床环境中很难跟踪单个样本的并发症。 白细胞亚群的选择性丢失是某些急性白血病、大细胞淋巴瘤以及细胞凋亡指数高的样本分析中的一个特殊问题，其中异常癌细胞可能特别脆弱并在红细胞裂解过程中丢失。这种不一致且难以根据患者特定样本进行预测使得使用流式细胞术进行诊断和治疗监测变得复杂。 项目 1 中开发的声学仪器将用于确定外周血样本中的特定细胞亚群是否可以在分析之前通过现场操作进行分离，从而无需红细胞裂解步骤。 方法 改进多重染色血细胞的光谱分离 此次合作将使用升级后的全光谱分辨率细胞仪，以确定是否可以通过测量细胞的完整发射光谱来改善重叠荧光染料的分离。 对古尔斯比博士实验室固定临床样本的初步研究表明，当前的光谱仪器可以在多色临床分析常用的两种激发波长下收集单个细胞的完整发射光谱。 我们还证明，我们可以从单细胞收集的大量完整发射光谱中生成标准列表模式数据文件。 我们将使用傅立叶变换和小波算法将发射光谱解卷积为其来自各个荧光染料的分量发射光谱，使用用每个单独的细胞表面标记染色的细胞以及未染色的细胞作为控制光谱的基础集。 来自解卷积光谱的每个分量的信号强度将与由古尔斯比实验室分析的相同临床样品的标准补偿矩阵分析获得的信号强度进行比较。 作为进一步的控制，我们将在洛斯阿拉莫斯采集的发射光谱上设置“虚拟滤光片”，使用与用于采集临床数据的标准流式细胞仪上的实际带通和长通滤光片相同的波长范围。 然后，这些数据将通过与标准数据上使用的相同的补偿矩阵运行，以确定光谱仪器是否忠实地复制在标准细胞仪上测量的发射数据。 我们还将使用与古尔斯比实验室相同的设置在我们的内部商业仪器上测量样品，以确保样品提供可重复的结果。 将通过使用光谱解卷积和标准补偿矩阵分析方法比较混合样品中多种细胞类型的分离来测试光谱仪器提高光谱分辨率的能力。 我们将构建由不同比例的不同细胞类型的已知混合物组成的测试样本，并使用这些样本来比较标准和光谱反卷积方法。 改善血细胞分离 项目 1 主要基于声学聚焦技术操纵流动样品流中的粒子的能力。 我们已经证明，通过调整线路驱动器中的声频和振幅，可以在液流中分离混合物中不同尺寸的颗粒（参见项目 1 中的初步数据部分）。 我们还已经证明，分离哺乳动物细胞所需的声能远低于对细胞膜造成物理损伤的阈值，这意味着我们应该能够操纵血细胞，同时保持其活力。 项目 1 中开发的声学聚焦仪器将用于确定我们是否可以根据混合流动流中的红细胞和白细胞分离这两种颗粒之间的细胞大小差异。 如项目 1 的具体目标 3 中所述（参见图 13），我们的第一个方法是将较大的白细胞集中到样品流的中心，在那里使用中心管将其去除，而红细胞则在样品流周围流动。外部。 初步研究将使用当地和血库获得的常规血液样本进行。 值得注意的是，只需要高度纯化：白细胞样品被红细胞少量污染不会对后续流式细胞术分析造成问题。 如果单次无法获得足够的纯度，我们可以多次通过声聚焦室。 一旦我们验证了分离红细胞和白细胞的能力，我们将从 Goolsby 博士的实验室获取如上所述的临床正常和造血系统恶性肿瘤样本，以确定我们是否可以对这些样本进行分离。 通过声学分离程序获得的白血病细胞的分数和质量将与使用标准红细胞裂解技术制备的样品进行比较，以验证细胞回收率的提高。 一旦该程序得到论证和优化，就可以实现一个用于血细胞分离的简单声室，该声室可以用作独立的制备仪器或改装到标准流动分析仪的前端。