Apparatus and method for clot brake pressure measurement

Abstract

Devices and methods for clot bursting pressure measurement are disclosed herein. Particular embodiments include a pump, a pressure measurement device, and a container having an inlet, an outlet, and an orifice. In particular embodiments, a pump is coupled to an inlet of a container, a pressure measurement device is coupled to an outlet of the container, and an orifice is configured to hold fluid extending across the orifice.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 483,590, filed February 7, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] Embodiments of this disclosure relate to apparatus and methods for measuring clot burst pressure. Background Technology

[0004] Hemorrhagic shock accounts for 40% of all trauma deaths and leads to the fatal trauma triad. Due to the rapid turnover of clotting factors, newly formed clots occur in a state of hypocoagulability and hyperfibrinolysis (excessive fibrinolysis). A better understanding of clot integrity can help optimize resuscitation strategies, such as the use of permissive hypotension.

[0005] Trauma is the leading cause of death in people under 46 years of age in the United States. While mortality rates from cancer and heart disease have declined, trauma mortality increased from 140,951 cases in 2000 to 173,140 cases in 2010 (Rhee, 2014). Trauma mortality is largely influenced by poor hemorrhage control in both pre-hospital and hospital settings (Wade, 2006). In trauma, hemorrhagic shock accounts for 40% of all deaths resulting from the fatal triad of complications.

[0006] Bleeding can be caused by blunt or penetrating injury to blood vessels. At the site of damage to the vessel wall, exposed subendothelial collagen interacts with platelets, erythrocytes, and various coagulation factors to form a clot. Due to the rapid turnover of coagulation factors and the significant volume loss associated with bleeding, clots in early traumatic conditions form under hypocoagulable, hyperfibrinolytic, and hemodiluted conditions (Rossaint, 2010). However, as hemorrhagic shock progresses, increased production of prothrombotic (prethrombotic) factors and decreased plasmin activity induce a hypercoagulable state (Sumislawski, 2018). Therefore, the intensity profile of clots formed in trauma differs from that of clots formed under hemostatic conditions. A better understanding of the integrity of clots in trauma can help guide acute care and reduce mortality and related morbidity.

[0007] Current strategies for managing hemorrhagic shock include permissive hypotensive resuscitation, which involves a more conservative fluid resuscitation approach that uses less fluid and fewer components to maintain a lower mean arterial pressure (MAP). Lower MAP is thought to prevent new clots from dislodging by reducing hydrostatic pressure (Rezende-Neto, 2010), by maintaining peripheral vasoconstriction with less preload to the heart (Smith, 2014), and by avoiding dilutional coagulopathy in the setting of already impaired coagulation (Bolliger, 2010). Previous multicenter studies have shown that the use of permissive hypotensive resuscitation in trauma patients can reduce overall mortality and morbidity, such as major organ failure (MOF) and acute respiratory distress syndrome (ARDS) (Owatatapanich, 2018). These studies were outcome-based and did not explore the effect of individual components on the resuscitation strategy in terms of MAP.

[0008] Thromboelastography (TEG) (Haemonetics, MA) is a quantitative test that measures coagulation kinetics of whole blood. Whole blood is allowed to clot around a pin, and torsional deflections are recorded, with increasing deflections indicating better coagulation kinetics. TEG-based resuscitation has been shown to actively guide resuscitation strategies to avoid coagulopathy (Suliburk, 2012), but these strategies have certain limitations. Clinical studies have shown that patients treated with heparin can have false negative TEG graphs that fail to detect heparin coagulopathy (Dunham, 2014). SUMMARY

[0009] Accordingly, embodiments of the present disclosure provide apparatuses and methods for measuring clot burst pressure. Particular embodiments include examples of pumping a fluid (e.g., water) through a clot and into a measurement column above the clot, where the fluid continues to exert increasing pressure on the clot until it bursts. It is believed that exemplary embodiments of the present disclosure can distinguish between thrombi in a hypocoagulable state and thrombi in a hypercoagulable state by observing differences in clot burst pressure (CBP) in a regulated group versus a control CBP.

[0010] Particular embodiments of the device include a sensor cartridge or container having a polymeric test surface. In particular embodiments, the interior surface of the container has a shallow depression with a small hole or aperture in the center. During operation of the device, a drop of fluid (e.g., blood or plasma) is placed on the depression so that it covers the aperture, and the blood or is induced to clot by a drug. An adhesive film can be applied to the exterior surface of the container to cover the aperture to help hold the drop of fluid in place so that the blood or plasma does not fall through the aperture before clotting. The adhesive film will be removed prior to the pressurization process. The aperture can also include a grid to retain the blood or plasma.

[0011] After the blood sample clots, the inner surface of the cartridge is subjected to progressively higher pressure until the clot fails and breaks through the orifice. This manifests as a sudden drop in pressure as measured by a pressure gauge in fluid communication with the interior volume of the vessel. In certain embodiments, the fluid is water and the pressure gauge is a water column, although other embodiments can use air as the measurement fluid.

[0012] The depressions on the inner surface can be coated with one or more chemicals or proteins. These can enhance or selectively bind the drop of blood or plasma in a particular way. Certain embodiments can include multiple such depressions and orifices, enhanced with different chemicals to provide more specific information to guide the transfusion of platelets, plasma, or clotting factors. In certain embodiments, multiple depressions and orifices can be pressurized together, such that one orifice breaks first, or they can be pressurized independently, such that each orifice breaks at its representative pressure. In particular embodiments, multiple depressions and orifices can be filled individually, or they can use microfluidic techniques to help partition a drop of blood or plasma into multiple depressions / orifices.

[0013] Exemplary embodiments of the present disclosure provide advantages over existing functional assays by performing the clot breakage pressure test more quickly. The test can be completed in less than a minute (e.g., tens of seconds) once the clot reaches the desired state. Exemplary embodiments of the present disclosure also provide another advantage by physically measuring a quantity that reflects the ability of a blood clot to resist breakage pressure, which is a characteristic of interest for patients with bleeding.

[0014] Exemplary embodiments of the present disclosure provide a direct, low-cost clot breakage pressure (CBP) test and require much less volume of blood than other tests such as thromboelastography (TEG). Assays performed with exemplary embodiments of the present disclosure can test the effect of various combinations and permutations of resuscitation products and clotting factors on patients in clinically relevant parameters: mmHg (pressure).

[0015] Exemplary embodiments include a device for measuring clot breakage pressure, wherein the device comprises: a pump, a pressure measurement device, and a vessel comprising an inlet, an outlet, and an orifice. In certain embodiments, the pump is coupled to the inlet of the vessel, the pressure measurement device is coupled to the outlet of the vessel, and the orifice is configured to retain a fluid extending across the orifice. In particular embodiments, the fluid comprises blood or plasma, fibrinogen, heparin, platelets, tranexamic acid, and / or thrombin. In some embodiments, the fluid is in a clotted state. In some embodiments, the orifice has a diameter greater than or equal to 0.05 inches and less than or equal to 0.20 inches. In specific embodiments, the orifice is a first orifice of a plurality of orifices in the vessel, each orifice of the plurality of orifices is configured to retain the fluid. In certain embodiments, each orifice of the plurality of orifices has a diameter greater than or equal to 0.05 inches and less than or equal to 0.20 inches.

[0016] In particular embodiments, the pump comprises a syringe. In some embodiments, the pressure measurement device comprises a column comprising a measurement fluid. In specific embodiments, the measurement fluid is water, saline, or air. In certain embodiments, the pressure measurement device comprises a liquidless pressure gauge. In particular embodiments, the pump is configured to pump the measurement fluid from the pump to the container and into the column. In some embodiments, the pump is configured to pump the measurement fluid at a rate greater than or equal to 0.2 milliliters per second (mL / s) and less than or equal to 2.5 mL / s.

[0017] Specific embodiments further comprise an indicator for a level of the measurement fluid in the column. In certain embodiments, the indicator is configured to detect a maximum level of the measurement fluid in the column. Particular embodiments further comprise a first conduit and a second conduit, wherein the first conduit is coupled to the pump and an inlet of the container, and wherein the second conduit is coupled to an outlet of the container and the pressure measurement device.

[0018] In some embodiments, the surface around the orifice is chemically treated. In specific embodiments, the surface around the orifice is coated with collagen, fibronectin, thrombin, fibrinogen, lyophilized platelets, and / or plasma proteins. In certain embodiments, the surface around the orifice is coated with specific factors from the coagulation cascade to detect deficiencies in these factors. In particular embodiments, the surface around the orifice comprises a recess.

[0019] Exemplary embodiments include a method of measuring clot lysis pressure, wherein the method comprises: applying a fluid across an orifice of a container; forming a clot from the fluid, wherein the clot extends across the orifice of the container; increasing a pressure inside the container; and measuring the pressure inside the container when the clot detaches from the orifice. In certain embodiments, forming the clot comprises: providing a support surface across the orifice prior to applying the fluid across the orifice; applying the fluid to the support surface; and removing the support surface after a period of time. In particular embodiments, forming the clot further comprises: providing a temperature inside the container greater than 25 degrees Celsius prior to removing the support surface. In some embodiments, forming the clot further comprises providing a temperature inside the container greater than or equal to 35 degrees Celsius and less than or equal to 40 degrees Celsius prior to removing the support surface. In specific embodiments, forming the clot further comprises: providing a temperature inside the container of approximately 37 degrees Celsius prior to removing the support surface. In certain embodiments, the period of time is greater than or equal to 5 minutes and less than or equal to 50 minutes. In particular embodiments, the period of time is approximately 30 minutes.

[0020] In certain embodiments, forming the clot further comprises: adding heparin to the fluid. In particular embodiments, forming the clot further comprises: adding fibrinogen to the fluid. In some embodiments, increasing the pressure inside the container comprises: pumping the measurement fluid into the container. In specific embodiments, measuring the pressure inside the container as the clot detaches from the orifice comprises: measuring a column of the measurement fluid.

[0021] Any embodiment discussed in relation to a single aspect of the application is equally applicable to other aspects of the application.

[0022] The embodiments in the example section are to be understood as applying to embodiments of all aspects of the application.

[0023] Although this disclosure supports referring only to the alternative and the definition of "and / or", the use of the term "or" in the claims is used to mean "and / or" unless specifically indicated otherwise, or the alternative is mutually exclusive.

[0024] In this application, the terms "about" and "approximately" are used to indicate that a value includes the standard deviation of error for a device or method being used to determine the value. If the standard deviation of error for a device or method used to determine the value is unknown, these terms are interpreted that the value is plus or minus five percent.

[0025] According to long-standing patent law, the words "a" and "an" when used in conjunction with the term "comprising" in the claims of the present disclosure are used to mean one or more unless specifically indicated otherwise.

[0026] Other objects, features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to one of ordinary skill in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0027] These and other objects, features and advantages will become apparent from the following detailed description and accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] Figure 1 An overview photograph showing an exemplary embodiment according to the present disclosure.

[0029] Figure 2 is Figure 1 An exploded schematic view of a container of an embodiment.

[0030] Figure 3 is Figure 2 A cross-sectional view of a container.

[0031] Figure 4 Graph of normalized burst pressure for different examples of conditioned donor blood.

[0032] Figure 5 Assembly and use photographs are shown in panels A-D according to embodiments of the disclosure.

[0033] Figure 6 Assembly and use photographs are shown in panels A-L according to embodiments of the disclosure.

[0034] Figures 7-10 Graph of burst pressure versus constriction according to exemplary embodiments of the disclosure. DETAILED DESCRIPTION

[0035] Reference is now made to Figures 1-3 Exemplary embodiments of the disclosure include a device 100 for measuring clot burst pressure. For clarity, not all elements are labeled with reference numerals in every figure. In the illustrated embodiment, the device 100 includes a pump 110; a pressure measurement apparatus 120; and a container 130 including an inlet 131, an outlet 132, and an orifice 135. As shown in the schematic embodiment, the pump 110 is coupled to the inlet 131 of the container 130 via a conduit 111, while the pressure measurement apparatus 120 is coupled to the outlet 132 of the container 130 via a conduit 121.

[0036] As shown in Figure 2 and Figure 3 the orifice 135 is configured to retain fluid 140 extending across the orifice 135. As explained further below, in certain embodiments, the fluid 140 can initially be in a liquid state, and then solidify to form a clot. In particular embodiments, the fluid 140 can include blood or plasma, and can also include agents that promote or inhibit clotting, including, for example, fibrinogen or thrombin or heparin. In particular embodiments, the recess 136 (as Figure 2 and Figure 3The recess 136 can be chemically implemented to assist in the analysis of the blood sample in certain embodiments. In particular embodiments, the recess 136 can be a dimple that is used to hold a blood clot in place over the orifice 135. The surface around the orifice 135 (including the recess if present) can be coated with collagen or fibronectin to assist clot adhesion and mimic in vivo surfaces. In certain embodiments, the surface around the orifice 135 can be coated with thrombin to generate a more rapidly formed clot, resulting in faster results. In particular embodiments, the surface around the orifice 135 can be coated with fibrinogen for comparison to a control clot to detect low fibrinogen states. Similarly, the surface around the orifice 135 can be coated with lyophilized platelets or plasma proteins to detect deficiencies in platelets or plasma in the test sample. In certain embodiments, the surface around the orifice 135 can be coated with specific factors in the clotting cascade to detect deficiencies in these factors.

[0037] The container 130 includes a body portion 138 and a lid 139. In certain embodiments, the body portion 138 can be transparent so that the user can see the fluid 140 during testing. In particular embodiments, a support surface 150 can initially be provided across the orifice 135 to support the fluid 140 during clot formation. After a period of time sufficient for the fluid 140 to form a clot extending across the orifice 135, the support surface 150 can be removed to allow a pressure test to determine the clot break pressure. In particular embodiments, the period of time for the fluid 140 to form a clot can be greater than or equal to 5 minutes and less than or equal to 50 minutes. In specific embodiments, the period of time for the fluid 140 to form a clot can be approximately 30 minutes.

[0038] In specific embodiments, the temperature inside the container 130 can also be raised above ambient temperature (e.g., 25 degrees Celsius) to assist the fluid 140 in forming a clot. For example, the temperature inside the container 130 can be raised to greater than or equal to 35 degrees Celsius and less than or equal to 40 degrees Celsius before the support surface 150 is removed. In specific embodiments, the temperature inside the container 130 can be raised to approximately 37 degrees Celsius before the support surface 150 is removed.

[0039] In certain embodiments, after the fluid 140 is applied across the orifice 135 and a clot is formed (and the support surface 150 is removed), the pressure inside the container 130 can be increased until the clot dislodges from the orifice 135. The pressure at which the clot formed by the fluid 140 dislodges from the orifice 135 can be measured and recorded as the clot break pressure. In the illustrated embodiment, the pump 140 can be operated to pump the measurement fluid 112 into the container 130 via the conduit 111 and the inlet 131. In particular embodiments, the measurement fluid 112 can be water or other suitable fluid. As the pump 110 continues to pump the measurement fluid 112 into the container 130, the measurement fluid 112 fills the container 130 and flows from the outlet 132 into the pressure measurement device 120 via the conduit 121. In the illustrated embodiment, the pressure measurement device 120 is a liquid column with graduated markings indicating the level of liquid in the column.

[0040] As the level of the measurement fluid 112 in the pressure measurement device 120 rises, the pressure exerted on the clot formed by the fluid 140 increases until the clot eventually dislodges from the orifice 135.

[0041] While Figures 1-3 The illustrated and described embodiments include a syringe pump and a measurement column using water as the measurement fluid, but other embodiments can include different components. For example, other embodiments can use air as the measurement fluid and can include an air pump (e.g., a compressor) to pressurize a container containing an orifice with a clot formed by a drop of blood or plasma. Further, the pressure measurement device can be a pressure gauge or other suitable device for measuring pressure. In particular embodiments, the pressure measurement device 120 can be a liquidless pressure gauge, including embodiments that use air as the measurement fluid 112, for example.

[0042] Test results and description of one particular embodiment

[0043] The following test data was obtained using an exemplary embodiment of the present disclosure. Blood from donors (n=7) was collected into 3.2% sodium citrate tubes. The blood was aliquoted and treated to achieve the following conditions: for low coagulation states, 0.10, 0.15, and 0.20 USP heparin per milliliter of blood; for high coagulation states, the blood was treated with 1.0 or 2.0 grams per liter of fibrinogen added. A 2 millimeter hole was drilled in a 6-well polystyrene plate and the bottom was covered with packaging tape. The blood was recalcified with CaCl2and pipetted into the well. The plate was incubated at 37 degrees Celsius for 30 minutes to allow a thrombus to form. The tape was removed after incubation. A pressure cap was designed with an inlet for fluid from a syringe pump and an outlet for effluent and was secured in the well with a waterproof seal. The effluent was directed to a vertical graduated measurement column, where the pressure was converted from centimeters of water to millimeters of mercury for data analysis.

[0044] CBP was normalized to the baseline value for the individual donor to account for inherent differences in clot strength. The 1.0 g / L and 2.0 g / L fibrinogen were 1.78 ± 0.11 (p < 0.0005) and 2.66 ± 0.36 (p < 0.005) times stronger, respectively, compared to the baseline value for CBP. The 0.10, 0.15, 0.20 USP heparin / mL were 0.54 ± 0.19 (p < 0.005), 0.41 ± 0.17 (p < 0.0005), 0.25 ± 0.02 (p < 0.0005) times stronger, respectively, compared to the baseline value. Data were analyzed using one-way ANOVA with multiple comparisons to Tukey's test.

[0045] Researchers were able to create a model that allowed for testing clot strength in a parameter not seen in existing devices such as TEG, namely burst pressure. The CBP model was able to show significant differences between a control group, a hypercoagulation group, and a hypocoagulation group of multiple donors. Future exploration of this device can involve using blood from trauma patients to better understand the pathophysiology of trauma.

[0046] As shown in FIG. 1, Figure 1 The device includes four main components: a syringe pump, a pressure cap, a 6-well plate assembly, and a water column. A programmable syringe pump was programmed to pump a 100 cc syringe at a continuous rate of 1 mL / sec. The syringe pump was coupled to the pressure cap assembly.

[0047] Figure 2 As shown in FIG. 2, Figure 3 The pressure cap assembly directs fluid directly through the clot and prevents pressure loss to the environment. The cap has two female ports to allow for inlet connection from the syringe pump and outlet connection to the water column. The cap has three feet to avoid direct contact with the clot. An O-ring (Dash 242) fits in a groove around the pressure cap, ensuring a tight fit with a standard 6-well plate. As fluid enters the pressure cap, it is directed into a compartment where the formed clot is suspended. The backflow fluid raises a 150 cm water column, which is obtained by a clear PVC tube marked at 5 cm intervals. All connections use luer locks and barb connections to prevent fluid loss.

[0048] A 20 inch x 20 inch sheet of high-density polyethylene was used as the base for the device containing the 6-well plate assembly and water column. The 6-well plate assembly was elevated using 4 2-inch screws to allow for easy visual inspection after a run of trials. A single 5 / 64 inch hole was drilled in the center of each well of the 6-well plate. 220 grit sandpaper was used in a circular fashion on the interior surface of the well plate to provide a sheared surface for platelet adhesion. The wells on the bottom of the plate were covered with clear packing tape before the start of the assay.

[0049] Blood sample collection

[0050] Venipuncture whole blood (WB) was collected from donors (male and female) who were at least 18 years of age and had not taken anticoagulant medications in the past 14 days. Blood was drawn from the antecubital fossa using a 19G needle into standard blue topped 3.2% sodium citrate vacuum blood collection tubes. Tubes were placed on a rocker at room temperature until used for assays, but all assay runs were initiated within 30 minutes of blood draw.

[0051] Blood conditioning

[0052] Whole blood was conditioned to mimic hypercoagulable and hypocoagulable states. RiaSTAP, a lyophilized fibrinogen concentrate, was used to generate hypercoagulable conditions. Whole blood was conditioned with a 1.0 mg / mL or 2.0 mg / mL fibrinogen bolus. Heparin sodium salt (Sigma-Aldrich, OH) was reconstituted and added to whole blood in amounts of 0.10, 0.15, 0.20 USP / mL to generate hypocoagulable states. 6 mM CaCl2 was added to each conditioned blood group and control group to recalcify and promote clotting.

[0053] CBP assay

[0054] Thirty-five microliters of conditioned whole blood or control whole blood was placed in a 5 / 64 inch hole above the packaging tape in each well of a 6-well plate. The entire plate was covered with a plate lid and incubated at 37 degrees Celsius for 30 minutes. After 30 minutes, the plate was removed from the incubator and the packaging tape on the bottom was removed. A pressure lid was placed in the individual well in a screw fashion and airtight sealed with an O-ring. One port of the pressure lid was connected to a syringe pump and the other port was connected to a vertical water column. The syringe pump was turned on and the assay was run until the clot detached or broke. The maximum height of the water column was recorded as the clot break pressure in centimeters. This process was repeated for each test and the syringe was refilled with DI H20 (deionized water) between tests. The data for centimeters of water column was converted to millimeters of mercury.

[0055] Performance testing

[0056] Tests were performed to validate the performance of the assay. The blood used in the validation tests came from 3 donors and the tests were run in duplicate for 6 runs. The preliminary tests were designed to identify the ideal syringe pump flow rate, the optimal diameter of the thrombus formation in the well plate, the incubation time for thrombus formation, the use of whole blood versus conditioned plasma, and the material to condition the donor blood. The following Tables 1A-1D detail the combinations tested and a summary of the notes.

[0057] Table 1A

[0058]

[0059] Table 1B

[0060]

[0061] Table 1C

[0062]

[0063] Table 1D

[0064]

[0065] CBP data

[0066] CBP was normalized to control CBP measurements to account for inherent differences in baseline clotting factors of each donor, as shown in Figure 4 Figure 4 Increased concentrations of heparinized blood produced weaker clots compared to the control group (p < 0.0005) and within groups (p < 0.05). Increased concentrations of short-term administered (bolus) fibrinogen produced stronger clots compared to the control group, 1.0 g / L group (p < 0.0005), 2.0 g / L group (p < 0.005), and within groups (p < 0.005). Seven donors were used for the control group, 1.0 g / L fibrinogen group, 2.0 g / L fibrinogen group, and two of the seven for heparinized conditions. All experiments were performed in triplicate. The adjusted mean whole blood was 1.78 ± 0.11 (p < 0.0005) and 2.66 ± 0.36 (p < 0.005) times stronger compared to baseline CBP for 1.0 g / L or 2.0 g / L fibrinogen bolus, respectively. The adjusted mean whole blood was 0.54 ± 0.19 (p < 0.005), 0.41 ± 0.17 (p < 0.0005), 0.25 ± 0.02 (p < 0.0005) times stronger compared to baseline CBP for 0.10, 0.15, or 0.20 USP heparin / mL, respectively. All conditions were significantly different compared to baseline, and fibrinogen bolus exhibited a dose-dependent increase in strength under hypercoagulable conditions. Heparin bolus exhibited a dose-dependent decrease in strength between the 0.10 USP heparin / mL group and the 0.20 USP heparin / mL group, with a general downward trend across the group.

[0067] The goal of this project was to create a low-cost prototype that could quickly measure clot strength in a clinically relevant manner. The development of the device was done in stages, with certain aspects of the design iteratively improved. Additionally, the optimization of the assay presented certain challenges. Five key areas were focused on: syringe pump flow rate, fluid type, well diameter, clot incubation time, and blood adjustment to generate a dose-dependent curve (Table 1A-Table 1D).

[0068] ​In TEG, the sample runs for approximately 45 minutes on average to run completion, so the device seeks to limit time similarly or shorter. The flow rate chosen should allow for timely testing while not being so strong as to cause the clot to shear. For the most accurate pressure readings, the device measurements should be affected only by the compression force normal to the clot. Ultimately, a flow rate of 1 mL / s was chosen because it satisfied both criteria (Note: 1.5 mL / s is also a viable flow rate).

[0069] Based on prior literature regarding clot kinetics testing using microvolumes of blood, 35 μL of blood was used as the initial starting point (George, 2018). To generate clots of 8 / 64 inch and 10 / 64 inch, a square:cube relationship was observed, and volumes of 89.6 μL and 140 μL of blood were used to generate clots. No significant difference in CBP was observed between diameters, and to conserve donor blood volume, all subsequent testing used a diameter of 5 / 64 inch. Additionally, no difference in CBP was observed when using 1.0x PBS (pH = 7.4) versus DI H20 as the fluid in the syringe pump, and to reduce costs, DI H20 was used for all testing.

[0070] Specific embodiments of the disclosed device rely on the formation of a non-porous clot. While the clot is not continuously perfused during incubation, it is important to establish an ideal incubation time that generates a clot that is neither too weak nor evaporated from incubation in the incubator for too long. For a 5 / 64 inch diameter clot, 30 minutes of incubation at 37 degrees Celsius achieves these goals. In trials with longer incubation times, the clots were too weak and had dried out. During testing, these clots would either break along the edges or fall out of the well directly under minimal pressure. Conversely, at shorter time points, the clots had not had enough time to complete the cross-linking of fibrinogen to fibrin, and the fluid would permeate through the clot under minimal pressure. These immature clots were not solid and exhibited a higher viscosity. Successful clots operated between these two extremes.

[0071] Under physiological conditions, clot shape can be influenced by a variety of factors, including fluid velocity, tissue factor exposure, venous shear forces, and the relative availability of clotting factors in the microenvironment (Govindarajan, 2018). However, for comparison of clot strength between donors and conditions, the clots need to be consistently shaped. Careful placement of the blood pre-incubation ensures that the clots break at the midpoint, which indicates that the pressure is evenly distributed normal to the clot surface, and the formation of misshapen clots does not affect premature shedding.

[0072] Fibrinogen consists of three pairs of polypeptide chains. Activation of thrombin from the coagulation cascade cleaves fibrinopeptides from two of the polypeptide chains, allowing the soluble fibrinogen to cross-link and form interlaced fibrin polymers. Subsequently, Factor XIIIa, also activated by thrombin, cross-links glutamine residues on these interlaced fibrin polymers to one another, creating a multilayered clot. Increased amounts of fibrinogen increase clot strength, and studies have shown that the use of fibrinogen concentrates in trauma therapy can restore hemostatic function and increase clot strength (Machlus, 2011; Schochl, 2009). Therefore, to test the effectiveness of the CBP device in hypercoagulable or enhanced coagulation states, a large dose of fibrinogen was added to the baseline blood sample. Normal physiological levels of fibrinogen range from 2.0 g / L to 4.5 g / L (Levy, 2015). The timing of the experiment did not allow for the testing of the baseline fibrinogen levels of the donors. All values were normalized according to the individual baseline CBP to account for inherent differences in coagulation factors. The short-term administration of large doses of 1.0 g / L and 2.0 g / L represent a 50% and 100% increase, respectively, on the lower end of the physiological spectrum. As shown in FIG. 6, fibrinogen linearly increased clot strength in a dose-dependent manner. Figure 4

[0073] Conversely, the use of heparin generates weaker clots, representing a hypocoagulable state. Heparin binds to antithrombin III (ATIII) and causes a conformational change, increasing enzymatic activity. Activated ATIII inactivates thrombin and factor Xa, leading to a direct stop in clot formation and subsequent coagulation activity. For a standard patient (5 L volume), a 5000 USP / mL dose of heparin sodium corresponds to a 0.35 USP / mL dose using 35 μL of blood in the present device. However, this dose completely inhibits clot generation. The dose is lowered to the 0.20-0.10 USP / mL range, which allows for the formation of weaker but more stable clots. Heparin was chosen to weaken the clots because its mechanism of action is well known, it has been shown to be suitable for a variety of coagulopathies, and because previous literature has detailed the limitations of TEG in detecting heparin-related coagulopathies (Dunham, 2014). The device was able to record meaningful readings in a hypocoagulable state, indicating that it is effective when using heparin-modulated samples.

[0074] ​Plasma with ATIII deficiency and short-term administration of large doses of ATIII is believed to produce a dose-dependent curve. However, the clot formed by the plasma is porous and will not resist fluid pressure in the device properly. Recent studies have shown that red blood cells (RBCs) play an important role in clot formation and hemostasis (Gillespie, 2021). Red blood cells act as a plug that captures the cross-linked fibrin network that exposes the vascular system, generating a non-porous clot (Weisel, 2019). The use of plasma without red blood cells in this device further validates this concept.

[0075] Test results and description of the second specific embodiment assembly:

[0076] Use a transwell insert with no pre-loaded membrane as the support structure (Corning TM Transwell TM Multiwell plate with permeable polycarbonate membrane insert, 6 per plate, item number: 07-200-165, Corning TM 3412). (Other) The membrane is removed after use and the insert is reusable.

[0077] Attach an elastic collagen membrane to the bottom of the transwell insert and secure in place with glue (Bondic - UV LED soldering starter kit with liquid plastic). Make a 1.5 mm hole in the center of the membrane and stick a center strip of 1 mm hole acrylic resin to the collagen membrane, centering the two holes (A) in Figure 5

[0078] The top (A, B, C) in Figure 6 is attached to a syringe pump (F) in Figure 6 at one end (designated as the inlet) and to a pressure column (K, L) in Figure 6 at the other end (designated as the outlet) via two luer locks (D and F) in Figure 6 . The connection between the luer locks and the plastic tubing is a luer lock to ensure that the tubing is securely attached.

[0079] Insert the top into the transwell insert (I, J) in Figure 6 and secure in place with a clamp. Attach the connector leading to the pressure column to the designated outlet (C, K) in Figure 5 , respectively. Figure 6 Start the pump and pump the medium from the syringe through the top into the transwell insert with the blood clot and out into the pressure column.

[0080] Troubleshooting mentioned in the test and limitations of materials and procedures:

[0081] ​1. The collagen membrane can stretch significantly, making the original 1 mm hole much larger, resulting in clot loss.

[0082] 2. Attaching an acrylic insert to the collagen membrane helps maintain the opening (1 mm) consistently sized and repeatable, reproducible.

[0083] 3. The collagen membrane is water resistant. If no holes are made in the collagen membrane and the intact membrane is used to flow media, the membrane will stretch but will not leak.

[0084] 4. After pipetting blood, the transport well plate must be immediately flipped upside down. If not flipped, the blood will leak out before it fully clots and the sample will be lost.

[0085] Procedure:

[0086] A blood sample (35 uL) is pre-incubated with a platelet activator or platelet activation inhibitor for 15-30 minutes. The blood is then mixed with 6 mM calcium and immediately pipetted onto the acrylic to allow it to attach and stabilize the clot. The transport well plate is immediately flipped upside down to "hang" the drop of blood. The blood is allowed to clot for 15 minutes (B in Figure 5 The top is inserted into the transport well plate and the tubing to the syringe pump and pressure column is attached.

[0087] The media is sent through the top into the transport well plate at a flow rate of 15 ml / min and the pressure at which the clot breaks is recorded (L in Figure 6

[0088] Data:

[0089] Blood from trauma samples and healthy controls is activated with 6 mM calcium (A in Figure 7 ), 10 mM calcium (B in Figure 8 ), or 250 pM U46619 with 6 mM calcium (C in Figure 9 and Figure 10 ). U46619 causes shape change but does not induce aggregation. The TxA2 receptor in platelets is only TP alpha (TPalpha), no beta (TPbeta), and the concentration of U46619 required to induce shape change is lower than that required to cause aggregation.

[0090] Exemplary embodiments of the present disclosure can include: conversion to a digital data collection system linked to a microcontroller for user input / output; changing the water column to a peristaltic pump to simulate pulsatile flow in the vasculature; and adding a channel to continuously perfuse the clot during incubation.

[0091] ​Exemplary embodiments of the present disclosure can also investigate the effect of additional coagulation factors and various resuscitation therapies on clot strength. Researchers expect to identify coagulation strength (CBP) in whole blood samples from primary trauma patients and correlate these values with TEG data as well as mortality and morbidity. Expansion of the data set will help validate the effectiveness of the device in a larger population.

[0092] Conclusions

[0093] Exemplary embodiments of the present disclosure are able to show significant differences between control, hypercoagulation, and hypocoagulation groups of multiple donors. Exemplary embodiments of the present disclosure are easy to use, low cost, and can investigate certain parameters not seen in existing devices such as TEG. Better understanding of clot strength helps guide resuscitation strategies and improve the prognosis of patients experiencing coagulopathy. Identifying the exact effect of coagulation factors / therapies or combinations of factors on clot strength can prevent the sloughing of immature clots during resuscitation and potentially save lives.

[0094] It should be noted that while the foregoing detailed description of the application has been given with respect to various embodiments of the application, the application is not limited to these details and that devices, kits, and methods according to the application can vary from those disclosed. It should be understood that the functions performed by the specific embodiments disclosed herein can be performed in different manners in alternative embodiments. It should also be noted that the above distinctions in functionality are for purposes of explanation and clarity; structural distinctions in systems or methods according to the application can not be drawn along the same lines. Accordingly, proper scope of the application should be deemed to be consistent with the following claims.

Claims

1. An apparatus for measuring clot bursting pressure, the apparatus comprising: Pump; Pressure measuring equipment; and A container, comprising an inlet, an outlet, and an orifice, wherein: The pump is connected to the inlet of the container; The pressure measuring device is connected to the outlet of the container; and The orifice is configured to retain fluid extending across the orifice.

2. The apparatus as claimed in claim 1, characterized in that, The fluid includes blood or plasma.

3. The apparatus as described in claim 1 or 2, characterized in that, The fluid includes fibrinogen.

4. The apparatus according to any one of claims 1-3, characterized in that, The fluid includes heparin.

5. The apparatus according to any one of claims 1-4, characterized in that, The fluid includes platelets.

6. The apparatus according to any one of claims 1-5, characterized in that, The fluid includes tranexamic acid.

7. The apparatus according to any one of claims 1-6, characterized in that, The fluid includes thrombin.

8. The apparatus according to any one of claims 1-7, characterized in that, The fluid was frozen.

9. The apparatus as described in any one of claims 1-8, characterized in that, The diameter of the orifice is greater than or equal to 0.05 inches and less than or equal to 0.20 inches.

10. The apparatus according to any one of claims 1-9, characterized in that: The orifice is the first of a plurality of orifices in the container; and Each of the plurality of orifices is configured to retain the fluid.

11. The apparatus according to any one of claims 1-10, characterized in that, The diameter of each of the plurality of orifices is greater than or equal to 0.05 inches and less than or equal to 0.20 inches.

12. The apparatus according to any one of claims 1-11, characterized in that, The pump includes a syringe.

13. The apparatus according to any one of claims 1-12, characterized in that, The pressure measuring device includes a column containing the measuring fluid.

14. The apparatus as claimed in claim 13, characterized in that, The measuring fluid is water.

15. The apparatus as claimed in claim 13, characterized in that, The measuring fluid is salt water.

16. The apparatus as claimed in claim 13, characterized in that, The measuring fluid is air.

17. The apparatus as claimed in claim 16, characterized in that, The pressure measuring device includes a liquid-free pressure gauge.

18. The apparatus according to any one of claims 14-16, characterized in that, The pump is configured to pump the measuring fluid from the pump to the container and into the column.

19. The apparatus as claimed in claim 18, characterized in that, The pump is configured to pump the measuring fluid at a rate greater than or equal to 0.2 ml / s and less than or equal to 2.5 ml / s.

20. The apparatus as claimed in claim 19, characterized in that, It also includes an indicator for the level of the measured fluid in the column.

21. The apparatus as claimed in claim 20, characterized in that, The indicator is configured to detect the maximum level of the measuring fluid in the column.

22. The apparatus according to any one of claims 1-21, characterized in that, Also includes: First catheter; and The second catheter, wherein: The first conduit is connected to the inlet of the pump and the container; and The second conduit is connected to the outlet of the container and the pressure measuring device.

23. The apparatus according to any one of claims 1-22, characterized in that, The surface around the orifice is treated with a chemical method.

24. The apparatus according to any one of claims 1-22, characterized in that, The surface around the orifice is coated with collagen.

25. The apparatus according to any one of claims 1-22, characterized in that, The surface around the orifice is coated with fibronectin.

26. The apparatus as claimed in any one of claims 1-22, characterized in that, The surface around the orifice is coated with thrombin.

27. The apparatus according to any one of claims 1-22, characterized in that, The surface around the orifice is coated with fibrinogen.

28. The apparatus as claimed in any one of claims 1-22, characterized in that, The surface around the orifice is coated with freeze-dried platelets or plasma proteins.

29. The apparatus as claimed in any one of claims 1-22, characterized in that, The surface around the orifice is coated with specific factors from the coagulation cascade to detect defects in these factors.

30. The apparatus as claimed in any one of claims 23-29, characterized in that, The surface surrounding the orifice includes a recess.

31. A method for measuring clot bursting pressure, the method comprising: Apply fluid across the orifice of the container; A clump forms from the fluid, wherein the clump extends across the orifice of the container; Increase the pressure inside the container; and Measure the pressure inside the container when the clot detaches from the orifice.

32. The method as described in claim 31, characterized in that, The formation of the clot includes: Provide a supporting surface across the orifice before applying the fluid across the orifice; Apply the fluid to the support surface; and The support surface is removed after a period of time.

33. The method as described in claim 32, characterized in that, The formation of the clot also includes providing a temperature greater than 25 degrees Celsius inside the container before removing the support surface.

34. The method as described in claim 32, characterized in that, The formation of the clot further includes providing a temperature inside the container that is greater than or equal to 35 degrees Celsius and less than or equal to 40 degrees Celsius before removing the support surface.

35. The method as described in claim 32, characterized in that, The formation of the clot also includes providing the interior of the container with a temperature of approximately 37 degrees Celsius before removing the support surface.

36. The method according to any one of claims 32-35, characterized in that, The time period is greater than or equal to 5 minutes and less than or equal to 50 minutes.

37. The method as described in claim 36, characterized in that, The time period is approximately 30 minutes.

38. The method according to any one of claims 31-37, characterized in that, The formation of the clot also includes adding heparin to the fluid.

39. The method according to any one of claims 31-38, characterized in that, The formation of the clot also includes adding fibrinogen to the fluid.

40. The method according to any one of claims 31-39, characterized in that, Increasing the pressure inside the container includes pumping a measuring fluid into the container.

41. The method according to any one of claims 31-40, characterized in that, Measuring the pressure inside the container when the clump detaches from the orifice includes measuring a column of the measuring fluid.

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