Assessment of fibrinolytic status in whole blood using a dielectric coagulometry microsensor.

Rapid assessment of the fibrinolytic status in whole blood at the point-of-care/point-of-injury (POC/POI) is clinically important to guide timely management of uncontrolled bleeding in patients suffering from hyperfibrinolysis after a traumatic injury. In this work, we present a three-dimensional, parallel-plate, capacitive sensor - termed ClotChip - that measures the temporal variation in the real part of blood dielectric permittivity at 1 MHz as the sample undergoes coagulation within a microfluidic channel with <10 µL of total volume. The ClotChip sensor features two distinct readout parameters, namely, lysis time (LT) and maximum lysis rate (MLR) that are shown to be sensitive to the fibrinolytic status in whole blood. Specifically, LT identifies the time that it takes from the onset of coagulation for the fibrin clot to mostly dissolve in the blood sample during fibrinolysis, whereas MLR captures the rate of fibrin clot lysis. Our findings are validated through correlative measurements with a rotational thromboelastometry (ROTEM) assay of clot viscoelasticity, qualitative/quantitative assessments of clot stability, and scanning electron microscope imaging of clot ultrastructural changes, all in a tissue plasminogen activator (tPA)-induced fibrinolytic environment. Moreover, we demonstrate the ClotChip sensor ability to detect the hemostatic rescue that occurs when the tPA-induced upregulated fibrinolysis is inhibited by addition of tranexamic acid (TXA) - a potent antifibrinolytic drug. This work demonstrates the potential of ClotChip as a diagnostic platform for rapid POC/POI assessment of fibrinolysis-related hemostatic abnormalities in whole blood to guide therapy.

A novel, point-of-care, whole-blood assay utilizing dielectric spectroscopy is sensitive to coagulation factor replacement therapy in haemophilia A patients.

BACKGROUND: Reliable monitoring of coagulation factor replacement therapy in patients with severe haemophilia, especially those with inhibitors, is an unmet clinical need. While useful, global assays, eg thromboelastography (TEG), rotational thromboelastometry (ROTEM) and thrombin generation assay (TGA), are cumbersome to use and not widely available. OBJECTIVE: To assess the utility of a novel, point-of-care, dielectric microsensor - ClotChip - to monitor coagulation factor replacement therapy in patients with haemophilia A, with and without inhibitors. METHODS: The ClotChip Tpeak parameter was assessed using whole-blood samples from children with severe haemophilia A, with (n = 6) and without (n = 12) inhibitors, collected pre- and postcoagulation factor replacement therapy. ROTEM, TGA and chromogenic FVIII assays were also performed. Healthy children (n = 50) served as controls. RESULTS: ClotChip Tpeak values exhibited a significant decrease for samples collected postcoagulation factor replacement therapy as compared to baseline (pretherapy) samples in patients with and without inhibitors. A difference in Tpeak values was also noted at baseline among severe haemophilia A patients with inhibitors as compared to those without inhibitors. ClotChip Tpeak parameter exhibited a very strong correlation with clotting time (CT) of ROTEM, endogenous thrombin potential (ETP) and peak thrombin of TGA, and FVIII clotting activity. CONCLUSIONS: ClotChip is sensitive to coagulation factor replacement therapy in patients with severe haemophilia A, with and without inhibitors. ClotChip Tpeak values correlate very well with ROTEM, TGA and FVIII assays, opening up possibilities for its use in personalized coagulation factor replacement therapy in haemophilia.

Trauma-targeted delivery of tranexamic acid improves hemostasis and survival in rat liver hemorrhage model.

BACKGROUND: Trauma-associated hemorrhage and coagulopathy remain leading causes of mortality. Such coagulopathy often leads to a hyperfibrinolytic phenotype where hemostatic clots become unstable because of upregulated tissue plasminogen activator (tPA) activity. Tranexamic acid (TXA), a synthetic inhibitor of tPA, has emerged as a promising drug to mitigate fibrinolysis. TXA is US Food and Drug Administration-approved for treating heavy menstrual and postpartum bleeding, and has shown promise in trauma treatment. However, emerging reports also implicate TXA for off-target systemic coagulopathy, thromboembolic complications, and neuropathy. OBJECTIVE: We hypothesized that targeted delivery of TXA to traumatic injury site can enable its clot-stabilizing action site-selectively, to improve hemostasis and survival while avoiding off-target effects. To test this, we used liposomes as a model delivery vehicle, decorated their surface with a fibrinogen-mimetic peptide for anchorage to active platelets within trauma-associated clots, and encapsulated TXA within them. METHODS: The TXA-loaded trauma-targeted nanovesicles (T-tNVs) were evaluated in vitro in rat blood, and then in vivo in a liver trauma model in rats. TXA-loaded control (untargeted) nanovesicles (TNVs), free TXA, or saline were studied as comparison groups. RESULTS: Our studies show that in vitro, the T-tNVs could resist lysis in tPA-spiked rat blood. In vivo, T-tNVs maintained systemic safety, significantly reduced blood loss and improved survival in the rat liver hemorrhage model. Postmortem evaluation of excised tissue from euthanized rats confirmed systemic safety and trauma-targeted activity of the T-tNVs. CONCLUSION: Overall, the studies establish the potential of targeted TXA delivery for safe injury site-selective enhancement and stabilization of hemostatic clots to improve survival in trauma.

Intravenous synthetic platelet (SynthoPlate) nanoconstructs reduce bleeding and improve 'golden hour' survival in a porcine model of traumatic arterial hemorrhage.

Traumatic non-compressible hemorrhage is a leading cause of civilian and military mortality and its treatment requires massive transfusion of blood components, especially platelets. However, in austere civilian and battlefield locations, access to platelets is highly challenging due to limited supply and portability, high risk of bacterial contamination and short shelf-life. To resolve this, we have developed an I.V.-administrable 'synthetic platelet' nanoconstruct (SynthoPlate), that can mimic and amplify body's natural hemostatic mechanisms specifically at the bleeding site while maintaining systemic safety. Previously we have reported the detailed biochemical and hemostatic characterization of SynthoPlate in a non-trauma tail-bleeding model in mice. Building on this, here we sought to evaluate the hemostatic ability of SynthoPlate in emergency administration within the 'golden hour' following traumatic hemorrhagic injury in the femoral artery, in a pig model. We first characterized the storage stability and post-sterilization biofunctionality of SynthoPlate in vitro. The nanoconstructs were then I.V.-administered to pigs and their systemic safety and biodistribution were characterized. Subsequently we demonstrated that, following femoral artery injury, bolus administration of SynthoPlate could reduce blood loss, stabilize blood pressure and significantly improve survival. Our results indicate substantial promise of SynthoPlate as a viable platelet surrogate for emergency management of traumatic bleeding.

Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding.

Bleeding complications arising from trauma, surgery, and as congenital, disease-associated, or drug-induced blood disorders can cause significant morbidities and mortalities in civilian and military populations. Therefore, stoppage of bleeding (hemostasis) is of paramount clinical significance in prophylactic, surgical, and emergency scenarios. For externally accessible injuries, a variety of natural and synthetic biomaterials have undergone robust research, leading to hemostatic technologies including glues, bandages, tamponades, tourniquets, dressings, and procoagulant powders. In contrast, treatment of internal noncompressible hemorrhage still heavily depends on transfusion of whole blood or blood's hemostatic components (platelets, fibrinogen, and coagulation factors). Transfusion of platelets poses significant challenges of limited availability, high cost, contamination risks, short shelf-life, low portability, performance variability, and immunological side effects, while use of fibrinogen or coagulation factors provides only partial mechanisms for hemostasis. With such considerations, significant interdisciplinary research endeavors have been focused on developing materials and technologies that can be manufactured conveniently, sterilized to minimize contamination and enhance shelf-life, and administered intravenously to mimic, leverage, and amplify physiological hemostatic mechanisms. Here, a comprehensive review regarding the various topical, intracavitary, and intravenous hemostatic technologies in terms of materials, mechanisms, and state-of-art is provided, and challenges and opportunities to help advancement of the field are discussed.

ClotChip: A Microfluidic Dielectric Sensor for Point-of-Care Assessment of Hemostasis.

This paper describes the design, fabrication, and testing of a microfluidic sensor for dielectric spectroscopy of human whole blood during coagulation. The sensor, termed ClotChip, employs a three-dimensional, parallel-plate, capacitive sensing structure with a floating electrode integrated into a microfluidic channel. Interfaced with an impedance analyzer, the ClotChip measures the complex relative dielectric permittivity, ϵr , of human whole blood in the frequency range of 40 Hz to 100 MHz. The temporal variation in the real part of the blood dielectric permittivity at 1 MHz features a time to reach a permittivity peak, , as well as a maximum change in permittivity after the peak, , as two distinct parameters of ClotChip readout. The ClotChip performance was benchmarked against rotational thromboelastometry (ROTEM) to evaluate the clinical utility of its readout parameters in capturing the clotting dynamics arising from coagulation factors and platelet activity. exhibited a very strong positive correlation ( r = 0.99, p < 0.0001) with the ROTEM clotting time parameter, whereas exhibited a strong positive correlation (r = 0.85,  p < 0.001) with the ROTEM maximum clot firmness parameter. This paper demonstrates the ClotChip potential as a point-of-care platform to assess the complete hemostatic process using <10 µL of human whole blood.