Development of methods and technology for human blood hemostasis assessment

Abstract

The primary response of the human body to an injury or trauma is the formation of a thrombus (clot) to stop bleeding, which is referred to as hemostasis. It is a complex biological process involving multiple blood components including platelets, plasma coagulation factors and erythrocytes (red blood cells). Rapid, accurate, and comprehensive assessment of hemostasis is essential in many clinical settings for managing patients who undergo invasive procedures, experience hemorrhages, or receive antithrombotic therapies to treat clotting disorders. Currently available hemostasis assays can be broadly classified into platelet function assays, tests for coagulation function, and recently developed ‘global’ hemostasis assays which include thromboelastography (TEG) and thromboelastometry (ROTEM). Major challenges or drawbacks of these assays include incomplete or partial evaluation of individual faceted clotting elements, being expensive, time-consuming, and requiring highly trained personnel to conduct, analyze and interpret the results. In this dissertation, two new techniques for blood hemostasis assessments are introduced and tested for various hemostatic conditions. The first approach is a comprehensive platelet functional assay using a nano thin-film sensor. The second approach is a multiparameter whole blood hemostasis assay using a carbon nanotube paper-composite sensor. Finally, the development of an automated hemostasis analyzer for potential clinical applications is also presented in this dissertation. Platelets are highly specialized discoid-shaped blood cells that play a vital role in the hemostatic response. In the event of a vascular injury, platelets undergo a series of highly complex, regulated, and multi-stage functional changes, including adhesion to the intimal matrix, intracellular calcium influx leading to platelet conformational change of morphology and integrins, activation, granule secretion, aggregation, and cytoskeletal contraction to form and stabilize a hemostatic plug that occludes the site of damage to stop bleeding. One of the major limitations of existing platelet assays is that they focus on a single functional response and thereby provide only a partial analysis of platelet function. The first approach presented in this dissertation is to address this long-standing technological limitation in platelet function assays. The newly developed approach evaluated platelets based on multiple functional stages of hemostasis using a sensitive nano thin-film capacitance sensor. Human platelet-rich plasma-based platelet function evaluations were conducted to demonstrate the feasibility of this approach for various platelet physiological conditions including drug-induced functional disorders. Sensor responses to platelet adhesion and after activation showed excellent sensitivities towards platelet counts, platelet activation levels, and platelet activation pathways. Close similarities between the obtained signal and platelet cytoskeletal reorganization after activation were observed through a fluorescence microscopy study. In comparison to the conventional optical-based evaluation system, the developed low-cost electrical-based sensing platform offers superior performance characteristics in assaying platelet function, such as high sensitivity, continuous multiparameter evaluation, label-free and easy-to-use. The second approach presented in this thesis is focused on providing a more comprehensive analysis of the overall blood hemostatic potential. Even though the newly developed platelet assay can offer a good snapshot of the in vivo blood hemostatic status by providing a comprehensive analysis of platelet function, it did not evaluate the contributions from other cellular components and plasma coagulation factors. Additionally, the sensor system is expensive to fabricate, and the assay is technically challenging and time-consuming. Therefore, an all-encompassing hemostasis assay with a more convenient workflow using a novel carbon nanotube-paper composite (CPC) capacitance sensor was developed. The CPC capacitance response to blood clotting at 1.3 MHz provided three sensing parameters with distinctive sensitivities towards coagulation function (factors), platelets, and erythrocytes. Whole blood-based hemostasis assessments were conducted to demonstrate the potential functionality of the device for various hemostatic conditions, including pathological conditions such as hemophilia and thrombocytopenia. Results showed good agreements when compared to a conventional thromboelastography assay. Overall, the presented sensor system is a promising new biomedical device for convenient whole blood-based global assessment of hemostasis. Plans for further scientific explorations, including studying the effects of other physiological clotting parameters are also presented in this dissertation. Most clinical hemostasis assays are still restricted to specialized laboratories because they need highly trained personnel to conduct, analyze and interpret the results. To address this issue, an automated bench-top prototype using the CPC sensor was developed. The automated hemostasis analyzer was devised with an intent to meet the growing needs in the healthcare landscape for an inexpensive routine hemostasis assessment and triage of patients with bleeding or risks for bleeding. The presented first version of the device is fully automatic, starting from blood aspiration to discard after measurements. The system was designed using conventional solid modeling software and fabricated using evolving low-cost rapid prototyping techniques. Further improvements in the system and a roadmap for extensive clinical studies to establish the functional equivalence of the analyzer to an existing U.S. FDA-approved device are also presented in this dissertation.