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: 7740289
• 负责人: Michael John Levene
• 金额: $ 15.4万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740289
• 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）：我们建议发展荧光波动光谱（FFS）来评估血管性血友病因子（vWF）多聚体，作为血管性血友病（vWD）诊断、分类和监测中更准确和可重复的技术，以阐明血栓性血小板减少性紫癜（TTP）的发病机制，并作为评估各种常见全身疾病中凝血或出血风险的新工具。所开发的方法将广泛适用于寡聚化的测量，包括淀粉样蛋白和朊病毒疾病的研究。VWF是一种血液蛋白，对正常凝血至关重要，存在于由2到80个单体组成的多聚体中。vWF多聚体的功能和分布缺陷导致vWD，这是最常见的遗传性凝血疾病。了解vWF多聚体的浓度和大小分布将有助于vWD的临床分型和管理，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时间测量方法存在实验室间差异和低重复性的问题。多重过程也很费力，技术上具有挑战性，并且依赖于辐射，因此通常只能从参考实验室获得。FFS包括几种单分子灵敏度技术，包括荧光相关光谱（FCS）和光子计数直方图（PCH）。FFS监测小共聚焦观测体积内自由扩散粒子数量的波动，比最大的vWF多路计时器还要小。我们将优化仪器的观察体积，以便使用荧光珠在大范围的粒径范围内使用。最大熵正则化被用于多参数FCS （MEMFCS）拟合；我们将开发我们自己的MEMFCS算法，以提高准确性和可重复性，并将采用类似的方法开发第一个广泛分布的PCH拟合。将在正常健康对照、I型vWD、IIA型vWD和TTP患者的样本中证明FFS对vWF测量的适用性。vWF分布振幅、均值、宽度、峰度和偏度的聚类将用于建立诊断相关性。目前预测vWF活动紊乱患者临床行为的方法是不够的。我们在不同环境下检查vWF生理的能力受到传统方法多聚体测试所带来的挑战的限制。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，努力生产一种实用的工具，用于常规测量许多凝血异常疾病中的vWF多聚体。公共卫生相关性：开发一种简单易用、更准确和可重复的方法来测量血液蛋白血管性血友病因子的大小分布，将增加我们对血管性血友病（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状况）的理解和改善诊断和治疗。以及广泛的与凝血异常相关的常见全身性疾病。