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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 抽象的 现在，流式细胞仪是一个标准分析平台，用于诊断人类血液样本，主要是通过使用荧光标记抗体标记的细胞表面标记标记来使用免疫表型。这些应用的相关性涵盖了各种各样的领域，包括白血病/淋巴瘤诊断和特定于患者的管理，传染病，移植药物（干细胞枚举/表征），阵发性夜间夜间血红蛋白尿（PNH）诊断和胎儿血红蛋白检测。尽管这种分析是常规的，但仍有一些问题可以改善流量仪器。 在与查尔斯·古尔斯比（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中开发的独特仪器直接解决这两个问题：在多色流量分析中重叠的荧光团分离，这些流程在多色流动分析中通常是六十八次的六级彩色和在红色蜂窝溶解过程中白细胞子的损失。 与光学过滤器的标准使用和复杂的补偿矩阵相比，项目3中开发的改进的光谱分辨率仪器将用于确定从多染色的细胞对完全发射光谱的反卷积是否可以改善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色和白色细胞的声场分离是否可以消除对红细胞裂解步骤的需求，其所致的某些白细胞（尤其是脆弱的异常细胞）的结果丢失。 通过这种合作的几种类型的临床样本的可用性将直接测试这两种工具在现实情况下解决常规流式细胞仪的局限性。 背景 查尔斯·古尔斯比（Charles Goolsby）博士是弗洛伊德·埃尔罗伊·帕特森（Floyd Elroy Patterson）病理学教授，也是西北大学流式细胞仪临床和核心设施主任，专门研究B慢性淋巴细胞性白血病（CLL）的基本细胞生物学，使用流动细胞仪（1-3）。 Goolsby博士还领导着基于诊断目的和患者特定治疗的决策/监测目的的复杂，多参数流式细胞仪的发展，包括在激酶抑制剂疗法中评估信号转导途径的评估（4）。临床实验室环境中采用了六到八种颜色分析的常规利用。尽管流式细胞仪是对各种疾病的人类血液样本进行免疫表型分析的“黄金标准”，但至少有两个领域可以改善该技术对诊断和治疗监测的实用性。 第一个涉及使用多个与抗体相关的重叠的荧光色素，以在单个样品中标记许多细胞类型。 流式细胞仪软件上的复杂补偿算法估计了重叠的荧光色素的相对贡献，并且无法直接测试各个样品补偿矩阵的成功。 在项目3中开发的升级的全光谱分辨率细胞仪将用于解决常规流式细胞仪的这种局限性。 第二个问题是在流动分析之前需要在血细胞样品中裂解红细胞。尽管红细胞裂解方案的选择性远不及梯度分离技术，但在某些患者中，仍然可以看到特定细胞子集的选择性丢失，这是一种并发症，在临床环境中，很难跟踪单个样本。 在分析某些急性白血病，大细胞淋巴瘤以及具有较高凋亡指数的标本中，白细胞子集的选择性丧失是一个特殊的问题，在该标本中，异常，癌细胞在红细胞裂解过程中可能特别脆弱，损失。这种不一致且难以在患者特定的样本基础上进行预测，这使使用流式细胞仪用于诊断和治疗监测复杂。 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子集是否可以在分析之前通过基于现场的操作分离，从而消除了对红细胞裂解步骤的需求。 方法 改善多重染色血细胞的光谱分离 升级的全光谱分辨率细胞仪将在此协作中使用，以确定通过测量细胞的完整发射光谱来改善重叠荧光素的分离。 与Goolsby博士实验室的固定临床样品的初步工作表明，当前的光谱仪器可以在通常用于多色临床分析的两个激发波长下收集单个细胞的完整发射光谱。 我们还证明，我们可以从单个单元格收集的大量完整发射光谱中生成标准列表模式数据文件。 我们将同时使用傅立叶变换和小波算法将发射光谱从单个荧光染色体中反应到其成分发射光谱，并使用与每个单个细胞表面标记物染色的细胞以及未染色的细胞，作为一组对照光谱。 将光谱的每个组件的信号强度与从Goolsby实验室中分析的相同临床样本中获得的标准补偿矩阵分析进行比较。 作为进一步的控制，我们将使用与使用实际带通滤波器和长期滤波器使用的相同波长范围在Los Alamos获得的发射光谱上设置“虚拟过滤器”，以获取临床数据的标准流式细胞仪。 然后，这些数据将通过与标准数据相同的补偿矩阵运行，以确定光谱仪器是否忠实地复制了在标准细胞仪上测量的发射数据。 我们还将使用与Goolsby实验室相同的设置来测量内部商业仪器上的样品，以确保样品提供可再现的结果。 光谱仪器改善光谱分辨率的能力将通过使用光谱反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。 我们将构建由不同比例不同细胞类型的已知混合物组成的测试样品，并使用这些样品比较标准和光谱反卷积方法。 改善血细胞分离 项目1主要基于声学焦点技术在流动样品流中操纵颗粒的能力。 我们已经证明，混合物中的不同尺寸的颗粒可以通过调节线路驱动器中的声频和振幅来分离流动流（请参阅项目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造成细胞膜物理损害的阈值，这意味着我们应该能够在保持其生存能力的同时操纵血细胞。 项目1中开发的声学仪器将用于确定我们是否可以基于这两个粒子之间的细胞大小的较大差异在混合流中分离白细胞。 如项目1的特定目标3（见图13）所述，我们的第一种方法是将较大的白色细胞集中到样品流的中心，在那里将使用中央管去除它们，而红色细胞在外部围绕外部流动。 最初的研究将使用本地获得的常规血液样本以及从血库进行。 重要的是要注意，只需要高度的纯化：对于随后的流式细胞仪分析，白细胞样品对白细胞样品的小污染不会成为问题。 如果我们无法单次获得足够的纯度，我们可以使用多个通过声学室。 一旦我们验证了分离红色和白色细胞的能力，我们将获得临床正常和造血恶性肿瘤样本，如上述从Goolsby博士的实验室所述，以确定我们是否可以对这些样品进行分离。 将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，以验证改善的细胞回收率。 一旦证明和优化了该过程，就可以实施一个简单的声学室进行血细胞分离，该室可以用作独立的准备仪器，或在标准流量分析仪的前端进行改造。