Fluorescence Fluctuation Spectroscopy for von Willebrand Factor Multimer Analysis

用于冯维勒布兰德因子多聚体分析的荧光波动光谱

• 批准号: 7888313
• 负责人: Michael John Levene
• 金额: $ 24.83万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-10 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888313
• 关键词: Acute; Address; Adhesions; Adult Respiratory Distress Syndrome; Affect; Algorithms; Alzheimer' s Disease; Amyloid; Behavior; Biological; Biological Models; Biophysics; Blinded; Blood Coagulation Disorders; Blood Platelets; Blood Proteins; Cardiomyopathies; Carrier Proteins; Classification; Cleaved cell; Clinical; Cluster Analysis; Coagulation Process; Collection; Data; Development; Diagnosis; Diagnostic; Diagnostics Research; Diffuse; Diffusion; Disease; Electrophoresis; Entropy; Evaluation; Factor VIII; Factor VIII-Related Antigen; Fluorescence; Fluorescence Spectroscopy; Functional disorder; Gel; Goals; Gold; Hemorrhage; Heterogeneity; Inherited Blood Coagulation Disorders; Knowledge; Laboratories; Lasers; Lead; Liver diseases; Malignant Neoplasms; Measurement; Measures; Medical; Medicine; Methods; Molecular Analysis; Monitor; Particle Size; Patients; Peptide Hydrolases; Photons; Physiology; Pilot Projects; Preparation; Prion Diseases; Process; Property; Purpura; Radiation; Reporting; Reproducibility; Research; Risk; Role; Sampling; Sepsis; Signal Transduction; Specimen; Spectrum Analysis; Spottings; Systemic disease; Techniques; Testing; Thrombocytopenia; Time; Validation; Width; base; clinical care; cofactor; disease diagnosis; enzyme activity; improved; instrument; interest; monomer; particle; peripheral blood; public health relevance; research clinical testing; single molecule; tool; von Willebrand Disease; von Willebrand Factor

DESCRIPTION (provided by applicant): PROJECT SUMMARY We propose the development of fluorescence fluctuation spectroscopy (FFS) for the evaluation of von Willebrand Factor (vWF) multimers as a more accurate and reproducible technique in von Willebrand Disease (vWD) diagnosis, classification, and monitoring, for elucidating specifics about the pathogenetic mechanism of thrombotic thrombocytopenia purpura (TTP), and as a new tool for assessment of coagulation or bleeding risk in a variety of common systemic conditions. The developed approach will have broad applicability to the measurement of oligomerization, including the study of amyloid and prion diseases. VWF is a blood protein that is critical for proper clotting and exists in multimers composed of between 2 and 80 monomers. Deficiencies in the function and distribution of vWF multimers lead to vWD, the most prevalent group of inherited coagulation disorders. Knowledge of the concentration and size distribution of vWF multimers would aid in the clinical subtyping and management of vWD and would be valuable as a diagnostic and research tool in TTP. Traditional testing parameters for vWF testing and current gel-based methods for multimer measurement suffer from significant problems of inter-laboratory variability and low reproducibility. The multimer process is also laborious, technically challenging, and radiation dependent, so it is typically only available from reference laboratories. FFS includes several techniques with single-molecule sensitivity, including fluorescence correlation spectroscopy (FCS) and photon counting histograms (PCH). FFS monitors fluctuations in the number of freely- diffusing particles within small confocal observation volumes - smaller than the largest vWF multimers. We will optimize the observation volume in our instrument for use with a large range of particle sizes using fluorescence beads. Maximum entropy regularization has been used for multi-parameter FCS (MEMFCS) fits; we will develop our own MEMFCS algorithm to improve accuracy and reproducibility, and will apply a similar approach to develop the first PCH fits to broad distributions. The FFS applicability to vWF measurement will be demonstrated on samples from normal healthy controls, Type I vWD, Type IIA vWD, and TTP patients. Clustering of vWF distribution amplitude, mean, width, kurtosis and skew will be used to establish diagnostic relevance. Present day methods for predicting clinical behavior from patients with disordered vWF activity are inadequate. Our ability to examine the physiology of vWF in different settings is limited by the challenges that multimeric testing by traditional methods present. We are adapting the techniques of single molecule analysis from the field of biophysics to address these shortcomings in an effort to produce a practical tool for the routine measurement of vWF multimers in the many diseases with abnormal coagulation. PUBLIC HEALTH RELEVANCE: Relevance Statement The development of a simple-to-use, more accurate, and reproducible method for measuring the distribution of sizes of the blood protein von Willebrand Factor will increase our understanding and improve diagnosis and treatment of von Willebrand Disease (the most common inheritable bleeding disorder), thrombotic thrombocytopenia purpura (a serious condition of abnormal clotting), and the broad range of common systemic diseases that are associated with abnormalities in coagulation.

描述(由申请人提供)：项目摘要我们建议开发荧光波动光谱(FFS)来评估von Willebrand因子(VWF)多聚体，作为von Willebrand病(VWD)诊断、分类和监测的更准确和可重复性的技术，用于阐明血栓性血小板减少性紫癜(TTP)的发病机制，并作为一种新的工具来评估各种常见全身情况下的凝血或出血风险。所开发的方法将在寡聚化的测量中具有广泛的适用性，包括对淀粉样蛋白和普里恩疾病的研究。VWF是一种对正常凝血至关重要的血液蛋白质，存在于由2到80个单体组成的多聚体中。VWF多聚体功能和分布的缺陷导致vWD，这是最常见的遗传性凝血障碍组。了解vWF多聚体的浓度和大小分布将有助于vWD的临床分型和治疗，并将作为TTP的诊断和研究工具。传统的vWF测试参数和当前基于凝胶的多聚体测量方法存在实验室间变异性和重复性低的严重问题。多聚体过程也很费力，技术上具有挑战性，而且依赖于辐射，所以它通常只在参考实验室才能获得。FFS包括几种具有单分子灵敏度的技术，包括荧光相关光谱(FCS)和光子计数直方图(PCH)。FFS监测小共焦观测体积内自由扩散粒子数量的波动--小于最大的vWF多聚体。我们将优化仪器中的观察量，以便使用荧光珠处理大范围的颗粒尺寸。最大熵正则化已被用于多参数FCS(MEMFCS)拟合；我们将开发自己的MEMFCS算法以提高精度和重复性，并将使用类似的方法将第一个PCH拟合开发到广泛的分布。FFS对vWF测量的适用性将在正常健康对照组、I型vWD、IIA型vWD和TTP患者的样本上进行验证。VWF分布幅度、平均值、宽度、峰度和偏斜度的聚集性将用于建立诊断相关性。目前预测vWF活性紊乱患者临床行为的方法是不够的。我们在不同环境中检查vWF生理学的能力受到传统方法多聚体测试带来的挑战的限制。我们正在采用生物物理学领域的单分子分析技术来解决这些缺点，以努力生产一种实用的工具，用于许多凝血异常疾病的vWF多聚体的常规测量。公共卫生相关性：相关声明开发一种简单易用、更准确和可重复性的测量血液蛋白von Willebrand因子大小分布的方法将增加我们对von Willebrand病(最常见的遗传性出血性疾病)、血栓性血小板减少性紫癜(一种严重的凝血异常情况)以及与凝血功能异常有关的各种常见系统性疾病的诊断和治疗。