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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Exploratory/Developmental Grants (R21)
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Microscopic Imaging Study Section (MI)
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Link, Rebecca P
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Yale University
Engineering (All Types)
Schools of Engineering
New Haven
United States
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Olson, Eben; Torres, Richard; Levene, Michael J (2013) Integrated fluorescence correlation spectroscopy device for point-of-care clinical applications. Biomed Opt Express 4:1074-82
Torres, Richard; Genzen, Jonathan R; Levene, Michael J (2012) Clinical measurement of von Willebrand factor by fluorescence correlation spectroscopy. Clin Chem 58:1010-8