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WO2024229114A1 - Dosage au point d'intervention pour thrombose plaquettaire occlusive - Google Patents

Dosage au point d'intervention pour thrombose plaquettaire occlusive Download PDF

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Publication number
WO2024229114A1
WO2024229114A1 PCT/US2024/027245 US2024027245W WO2024229114A1 WO 2024229114 A1 WO2024229114 A1 WO 2024229114A1 US 2024027245 W US2024027245 W US 2024027245W WO 2024229114 A1 WO2024229114 A1 WO 2024229114A1
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Prior art keywords
blood
platelet
vwf
subject
sample
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English (en)
Inventor
David N. Ku
Christopher BRESETTE
Viviana CLAVERIA
Gian Rivera CRESPO
Saagar BAKSHI
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Georgia Tech Research Institute
Georgia Tech Research Corp
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Georgia Tech Research Institute
Georgia Tech Research Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G01N11/02—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material
    • G01N11/04—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material through a restricted passage, e.g. tube, aperture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/4905—Determining clotting time of blood

Definitions

  • Ischemic events are the leading cause of death in the United States, despite major advances in prevention, detection and treatment in the past decades. Ischemic events are a subtype of Major Adverse Cardiovascular Events (MACE) and affect millions of Americans annually, with an estimated 720,000 individuals having a primary myocardial infarction (MI), 335,000 having a secondary /recurring MI, 610,000 cases of new ischemic strokes and 185,000 recurrent ischemic strokes.
  • MI myocardial infarction
  • MI myocardial infarction
  • Ischemic stroke is the number one cause of disability in adults and damage to heart tissue following an MI can cause heart failure and require significant lifestyle changes.
  • financial burden of ischemic events is significant, over $195.6 billion in direct costs. This cost reflects the severity of the problem and includes preventative measures (the prescription of anti-thrombotic drugs >$3 billion in 2020) and the cost of treating existing clots (-$20,000 cost of treating one patient, with catheterization lab costs accounting for 45%).
  • Arterial thrombosis is the process of forming a blood clot, or thrombus, in the high-pressure vessels supplying blood to tissues.
  • the thrombus forms in the coronary arteries. These arteries wrap around the heart and supply the muscle tissue with oxygen and nutrients.
  • thrombi form in the carotid or cerebral arteries, which supply the brain with blood. In both cases thrombi can form in a matter of minutes and require treatment as soon as possible.
  • Thrombi can form through two main pathways: coagulation and shear-induced platelet aggregation (SIPA). Thrombi formed through the process of arterial thrombosis are different from clots formed by the coagulation cascade and have a unique composition and structure, which has been previously documented.
  • a coagulation clot can form from a cascade of coagulation proteins under static conditions and exposure to a pro -thrombotic surface such as glass or collagen. These surfaces differ from the endothelial cells that blood typically contacts in that protein absorption can occur and the contact activation pathway of coagulation is initiated. Flowing blood inhibits coagulation from occurring by washing away reagents before they can react. Coagulation can also be inhibited chemically using heparin or citrate.
  • a platelet-rich clot can occlude an artery under fast-flowing blood conditions.
  • These blood clots are different from the red, gelatinous clots formed through the coagulation cascade. Instead, these clots are platelet-rich and contain few red blood cells, giving them a white appearance. They also have unique mechanical properties, being significantly stiffer and stronger than coagulation clots. Most importantly, they form through a completely different mechanism known as SIPA.
  • SIPA involves the blood protein von Willebrand Factor (vWF), which is not part of the coagulation cascade. During SIPA, vWF elongates under high shear stress and elongational flow conditions.
  • vWF von Willebrand Factor
  • vWF adheres to fibrillar collagen at a surface, forming a pro-thrombotic surface. Then, vWF strands form platelet aggregates by binding platelets away from the wall and capture these platelet aggregates at the surface to occlude the lumen of a blood vessel. Captured platelets can activate and release additional vWF, rapidly forming a clot that can span a stenotic artery in less than an hour. Research has shown that three criteria are critical for the formation of a SIPA clot: high shear rate of greater than approximately 3000 s' 1 at the wall; presence of platelets and vWF to form the clot; and a disrupted endothelial surface to which the clot can attach and grow.
  • SIPA serotonin deprivation
  • This SIPA process is not strongly inhibited by anticoagulants such as heparin and citrate.
  • This SIPA process does not need to be initiated by pharmacologic stimulants such as exogenous norepinephrine or ADP.
  • SIPA does not require platelets to be activated prior to being captured. [0007] This process plays an important role in various areas of clinical medicine in relation to hemostasis. Traumatic, post-partum, and intra-operative hemorrhage, along with occlusive thrombosis can be affected by dysfunctional SIPA-based blood clot formation.
  • Platelet function assays such as PFA-100, VerifyNowTM, TEG/ROTEM, GTT, and T-TAS have been developed to address this problem, but each test either fails to incorporate all three of the criteria for SIPA listed above or causes SIPA artificially, obscuring the measure of a patient’s natural ability to form SIPA clots.
  • T-TAS does incorporate all criteria, but the dimensions of the test section do not permit the formation of a clot representative of one found in an artery.
  • the T- TAS method of driving flow in the assay prevents the individual channels that make up the device from being independent measurements, causing significant variability in the measured outcome.
  • thrombosis tests measure coagulation function with no platelets, e.g., PT/PTT/INR/D-Dimers/C-reactive protein levels/VWF antigen.
  • existing thrombosis tests apply low shear to blood and biochemically activate clotting.
  • Some tests e.g., TEG/ROTEM
  • Platelet function tests e.g., Verify Now, Light Transmission Aggregometry
  • clots can form too slowly for the mechanism to be SIPA, the time scale matches that of coagulation and GTT is strongly influenced by anti-coagulants.
  • PFA-100 can create clots that seal a single membrane hole at higher shear stress from agonists such as NE or ADP but is strongly dependent on platelet activation.
  • T-TAS drives a constant flow through multiple channels coated with collagen, which causes some channels to occlude, and others never occlude. Having a single constant flow inlet for multiple channels also means the shear rates in the channels will vary.
  • the TTAS channel dimensions of 40 microns assure that occlusion is from surface adhesion, not a bulk accumulation of a platelet thrombus.
  • the present invention relates to a system for measuring occlusive blood clots and the use of the system in a method of determining that a subject is at risk of developing a major adverse cardiac event (MACE) and/or a method of determining whether a subject is a candidate for anti-platelet therapy.
  • MACE major adverse cardiac event
  • the present invention provides a system for measuring occlusive blood clots, the system comprising a testing device having at least one channel through which a sample of blood flows; a pump for generating a pressure in the testing device to facilitate the flow of blood; and a controller configured to measure a total volume or mass of blood that flows through the plurality of channels prior to occlusion, wherein the total volume or mass of blood can be indicative of occlusion time.
  • each of the at least one channel(s) can have a cross-sectional area of at least 50 pm 2 and/or a channel size greater than 100 microns in length with a minimum dimension of at least 70 microns.
  • the testing device comprises a cartridge.
  • the pump generates constant pressure.
  • occlusive platelet thrombosis can be evaluated.
  • the sample can be non-citrated or non-heparinized whole blood.
  • the system is configured as a point-of-care device that creates the formation of platelet-rich thrombi present at greater than 10% platelets by volume.
  • the point-of-care device can be configured to create high initial shear rates to induce von Willebrand Factor (VWF) elongation. In some embodiments, the high initial shear rates are above 3000/s.
  • VWF von Willebrand Factor
  • the point-of-care device includes a constant pressure-driven system, wherein the constant pressure-driven system allows for the occlusion of flow in multiple channels.
  • the point-of-care device includes a test section length designed to keep shear stress from going above about 1,000,000/s.
  • a method of determining that a subject is at risk of developing a major adverse cardiac event comprising: obtaining a sample of blood from the subject, exposing the sample to the system described above, and determining whether the subject has, or is at risk of developing blood clots, therefore determining if the subject is at risk of a MACE.
  • the subject has previously experienced a MACE.
  • the subject can be a trauma patient.
  • the subject can be treated to prevent a MACE.
  • a method of determining whether a subject is a candidate for anti-platelet therapy comprising: obtaining a sample from the subject, exposing the sample to the system described above and analyzing occlusion time to determine whether the subject is a candidate for anti-platelet therapy.
  • the subject can be exposed to anti-platelet therapy before the sample is obtained and in some embodiments, the sample can be taken and analyzed before and after exposure to anti-platelet therapy to determine whether the anti-platelet therapy is effective.
  • FIG. 1 (A-B) shows components of the novel thrombosis assay. Further, FIG. 1 (A-B) shows diagrams of an example configuration of a point-of-care system for measuring the formation of occlusive blood clots, according to some implementations.
  • FIG. 1A shows the entire assay with a cartridge inserted and an extra 5 mF vacutainer for scale.
  • FIG. IB shows two POC cartridges next to a glass test section for scale and a 0.7 cm long glass capillary tube with a 200-um channel used as test section for thrombus formation.
  • FIG. 1C shows a block diagram of a point-of-care system including cartridge, according to some implementations.
  • FIG. ID shows an example cross sectional schematic of a cartridge, according to some implementations .
  • FIG. IE shows an example test section that can be used with a cartridge, according to some implementations.
  • FIG. 2 is a diagram of the channels of the point-of-care system of FIGS. 1A and IB, according to some implementations.
  • FIG. 3 is a diagram of two different configurations of the channels of the point-of-care system of FIGS. 1A and IB, according to some implementations.
  • FIG. 4 is a block diagram of an example computing system, according to some implementations .
  • FIG. 5 shows a comparison of coagulation and arterial thrombosis.
  • FIG. 5 A shows arterial thrombi also termed as white clot, (top) typically have a lighter appearance than coagulation clots (bottom) also termed as red clots.
  • FIG. 5B shows that these differences in composition are apparent after H&E staining of clots.
  • White clots (left) are platelet rich (grey dots), whereas red clots (right) are dominated by RBC-rich areas (dark grey).
  • FIG. 5C shows Virchow’s triad for coagulation differs from the alternative triad for arterial thrombosis in both flow and cellular composition.
  • FIG. 6 shows the structure of Von Willebrand Factor (vWF).
  • vWF is a large protein made of repeating monomers which dimerize and are then linked to form vWF concatemers. Under low shear stress conditions these concatemers have a globular configuration.
  • FIG. 6B shows western blots that illustrate the difference in molecular weight distributions of plasma vWF between Normal Plasma (NP) and two diseases: VWD type 2A (2A), which is characterized by a loss of HMW vWF and Thrombocytopenic Purpura (TTP), which is characterized by an increase in HMW vWF.
  • NP Normal Plasma
  • TTP Thrombocytopenic Purpura
  • FIG. 7 (A-C) shows the process of arterial thrombosis.
  • FIG. 7 A shows that high shear stress caused by vessel narrowing elongates vWF and allows it to stick to thrombogenic surfaces like collagen, if present.
  • FIG. 7B shows that platelets are captured onto the thrombogenic surfaces through adhesion to vWF via GPIb.
  • FIG. 7C shows that adhered platelets activate, releasing vWF and form strong GPIIb/IIIa bonds. Additional platelets are captured by the newly released vWF.
  • FIG. 8 shows the correlation between the thrombosis timeline and the clinical timeline.
  • Untreated, thrombosis is a relatively linear pathway from plaque formation to irreversible tissue damage.
  • the clinical timeline involves preventative measures and treatment of acute events. Symptoms occur sometime between the formation of a plaque and when irreversible tissue damage from ischemia sets in but cannot correlate the onset of symptoms to a specific thrombotic state.
  • FIG. 10 shows the Shear Induced Platelet Aggregation (SIPA) mechanism and novel POC Assay.
  • FIG. 10A illustrates the overview of SIPA.
  • a damage to an atherosclerotic plaque creates a region of high shear with exposed collagen.
  • vWF can bind to platelets and adhere to the exposed collagen, forming occlusive thrombi.
  • FIG. 10B shows an alternative triad for forming arterial thrombi including platelets and vWF, high shear to elongate and activate the vWF, and a thrombogenic surface for the thrombus to grow on.
  • FIG. 11 shows techniques of generating a consistent end point.
  • FIG. 11 A shows that initial assessment of 10% variability within individual test sections including 4 test sections significantly reduces inter-assay variability.
  • FIG. 12 (A-B) are images of tubing for testing individual test section independently.
  • FIG. 12A shows a single tube connected to a vacuum source to provide an identical pressure gradient to each test section. Groups of 4 tubes were bound together to run 4 test sections using the same blood sample. End volume was calculated by measuring the distance the blood front travelled.
  • FIG. 12B shows individual glass test sections with a tubing O-ring used to ensure a good seal with the vacuum tubing.
  • FIG. 13 shows geometric constraints for cylindrical occluding test sections.
  • FIG. 13A displays the effect of test section radius on the fraction of platelet-platelet interactions (black curve) and total amount of blood used to make an occlusive thrombus (grey curve). Creation of bulk thrombi (>90% platelet-platelet interactions) with ⁇ 1 mL of blood constrains the potential radii of the test section to a narrow window, 39-114 pm.
  • FIG. 13B shows that manufactured glass capillary tubes have a tight range of inner diameters which fits within the defined window of acceptable diameters.
  • FIG. 14 (A-B) describes methods of ensuring proper shear rates.
  • FIG. 14A shows that while the chosen peristaltic pump has some natural variability, it is demonstrated that the experimental pressures which can drive flow are sufficient to achieve the desired flow rates.
  • Region I represents the time necessary for a -70hPa vacuum to be achieved.
  • Region II is the pressure measurements while the test section is in air and region III contains the pressure measurements when the test section is filled with liquid.
  • the light grey trace is from measurements taken with a 40% glycerol solution and the dark grey trace is obtained using blood.
  • FIG. 14B shows shear rate evolution during clotting as a function of test section length. By shortening test sections, the peak in shear rates can be reduced (and associated shear stresses) on the forming clot.
  • FIG. 15 is a graph showing control End Volume (EV) distribution. Distribution of the control endpoints is bimodal, with the bulk of individuals having an average EV of 0.48 mL. A subpopulation of individuals presented with higher end volumes, roughly twice as high, with an average of 0.92 mL.
  • EV Control End Volume
  • FIG. 16 shows Col/ADP and TC EV measurements in comparison with PFA- 100.
  • FIG. 17 shows the effect of antiplatelet agents on assay endpoints.
  • FIG. 17A shows end volume (EV) measurements for all testing conditions. ASA and 2MeSAMP show 36% and 52% increases in EV respectively. With Eptifibatide, a majority of assays did not occlude and ran out of blood around 1.75-2 mL.
  • FIG. 17B shows the effect of ASA and 2MeSAMP on EV, highlighted by normalizing antiplatelet EVs to each individual’s average control EV. (* p ⁇ 0.05).
  • FIG. 18 shows the effect of decreasing test section radius on blood use. Small reductions in the test section radius (from 100 pm to 80 pm) can cut the expected blood required to occlude 4 tests sections by more than half.
  • a test section of the system includes a collagen surface with no additional agonists.
  • a bulk thrombus that is greater than thousands of platelets on a surface is created.
  • the disclosed thrombus has dimensions greater than 50 microns at its largest extent.
  • the pressure-driven system allows for independent flow cessation in all channels. In some implementations, pressure does not exceed 200 mmHg in any channel.
  • the disclosed system is different from the other methodologies and systems described above in forming large occlusive thrombi without inducing pre-activation of platelets and not causing blow-outs from pressures exceeding 200 mmHg. It should be appreciated that the disclosed system activates through shear stress, not artificial agonists.
  • the disclosed system is realized through a point-of-care device that creates the formation of platelet-rich thrombi (e.g., >10% platelets by volume) using collagen adhered to a wall, high shear rates above 3000/s, and stable occlusion by thrombus formation.
  • the disclosed point-of-care device creates high initial shear rates above 3000/s to induce VWF elongation.
  • the disclosed point-of-care device has a channel size greater than 100 microns in length and a minimum dimension of at least 70 microns to create occlusion by bulk volume accumulation away from the wall, not surface occlusion.
  • the disclosed point-of- care device includes a constant pressure-driven system, e.g., which allows for the occlusion of flow in multiple channels.
  • the disclosed point-of-care device includes a test section length designed to keep shear stress from going above 1,000,000/s.
  • Additional features of the disclosed system include, but are not limited to: endpoint averaging between more than one independent channel to improve accuracy and reduce variability; a point-of-care device that relates to the SIPA thrombotic potential in patient’s arteries; a point-of-care device that inversely relates to bleeding; a disposable cartridge that utilizes a metal-free non-sharp interface; a direct interface to most widely used blood collection tubes (e.g., vacutainer), obviating the need for manual transfer or removal of blood from collection tubes; more than one independent channel to reduce intrasample variability to less than 5% (e.g., increased accuracy of about 60% to 95%); a single-button interface for test for platelet-rich thrombus formation; a point-of-care device which can test for platelet function using a time-independent endpoint; a point-of-care device which accurately tests for post-stenosis blood volume accumulation;
  • the disclosed system and device can be used for assessing the bleeding risk from the lack of occlusive platelet thrombosis in trauma patients. In some implementations, the disclosed system and device can be used for assessing bleeding risk in postpartum patients. In some implementations, the disclosed system and device can be used for measuring thrombotic risk for myocardial infarction and cerebral vascular events. In some implementations, the disclosed system and device can be used for quantifying the effect of anti-platelet pharmaceutical agents on an individual’s blood.
  • FIGS. 1A and IB diagrams of an example configuration of a point- of-care system for measuring the formation of occlusive blood clots, according to some implementations.
  • FIG. 1A shows an example configuration of the system - sometimes referred to herein as “Thrombocheck” - described herein and
  • FIG. IB shows an example test cartridge that is adapted to worth with the system shown in FIG. 1A.
  • the system in some implementations, is designed to address various limitations of current platelet function assays, as discussed above, and creates a functional SIPA assay.
  • the system includes a plurality of channels configured to facilitate blood flow for obtaining measurements.
  • each channel has a tolerance of less than 3% (e.g., with respect to height/radius) to ensure consistent test results.
  • the system may include more than one channel due to the stochasticity in the process of SIPA. Multiple channels allow for several independent measurements to be made and averaged together to give a reliable endpoint. Additionally, the dimensions of each channel may be small enough so that all channels can occlude using blood from a single venous blood draw, e.g., approximately 6mL. In some implementations, blood flow is driven through the channels using a pressure-controlled system.
  • flow-controlled systems can easily achieve higher shear rates, they also can cause the SIPA clot to break since the pump is stronger than the clot.
  • Using a pressure- controlled system can create clots that occlude flow and do not break.
  • clotting in one channel diverts flow to other channels and changes the fluid dynamics of the non-occluded channels. This means the channels are not independent measurements.
  • the occlusion of one channel has no effect on the flow through other channels in parallel.
  • the shear force that vWF experiences is dependent not only on the shear rate but also on the blood viscosity. Therefore, the disclosed point-of-care system may utilize various calibration methods to remain valid for a wide range of hematocrits and blood viscosities.
  • a test section of the disclosed system is connected to a cartridge that interfaces with the test cartridge (e.g., vacutainer) or other container containing the sample.
  • the system stores the blood that has passed through the test section and connects to an external device that provides the vacuum pressure and times the test.
  • the cartridge also serves to provide the assay endpoint of the total blood volume, which is a good measure of the amount of blood flow required to form an occlusive SIPA clot. This endpoint of total blood volume can be made more precise by correcting for blood viscosity.
  • the dimensions of the test unit have a small footprint, the system does not require connections to any external computers, and the system is straightforward to operate.
  • the disclosed point-of-care system is unique from other platelet function tests in that it has high shear flow, the appropriate geometry described above, multiple independent measurements providing a robust endpoint, and no addition of platelet agonists.
  • the disclosed system creates a platelet-rich thrombus in a miniature test section outside of the body using non-citrated whole blood.
  • Other devices use citrated blood, artificial agonists, nonbiologic surfaces, and flow-controlled pumps that do not allow occlusion.
  • the test section mentioned above includes a microfabricated, biologically active surface without biochemical agonists such as epinephrine or ADP agonists.
  • the test channel has a passage for flow that is greater than 70 microns in diameter or lumen width.
  • the flow of blood through the system is driven by a pressure head instead of a constant volume pump to allow for flow cessation or occlusion.
  • the pressure does not exceed 300 mmHg in the system.
  • the system creates a bulk thrombus that accumulates more than ten thousand platelets with a volume greater than 1 nL. Table 1, below, illustrates various characteristics of the disclosed system compared to other techniques and devices.
  • a diagnostic device e.g., the point-of-care system described above
  • the system described herein perfused in a way that the results from each channel are independent of parallel channels
  • the system described herein with a method for using pressure measurements to calibrate the final measured outcomes based on blood viscosity; using continuous pressure measurements along with a built-in pump, pressure can be maintained for reliable results
  • the system described herein that uses a polymer-based, non- metal, non-sharp interface with a blood container
  • the system described herein using direct interface with widely-used blood collection tubes e.g., vacutainer
  • the system described herein uses an interface that prevents contamination of the blood by utilizing direct attachment of a blood collection tube.
  • FIG. 1C is a block diagram of an example system 200.
  • the system 200 can be configured to perform the methods described according to various implementations of the present disclosure, for example performing point-of-care tests (as a point-of-care device) to determine if a subject is at risk of developing a major adverse cardiac event.
  • point-of-care tests as a point-of-care device
  • the system 200 includes a testing device 210, a controller 220, and a pump 230.
  • the controller 220 can optionally include any or all of the components of the computing system 100 shown in FIG. 4.
  • the controller 220 can be operably coupled (e.g., by wired or wireless communications) to the testing device 210 and the pump 230, so that the controller 220 can control the operation of the testing device 210 and/or the pump 230.
  • the testing device 210 can include one or more channels 232a-232n, as shown in Fig. 1C.
  • the channels 232a-232n can be configured for blood flow into the testing device 210 by the channels 232a-232n.
  • the channels can have a cross sectional area of 50 square pm.
  • any or all of the channels 232a-232n can be longer than 100 microns in length.
  • the channels can optionally be configured as a cartridge 250 that can be attachably and releasably connected from the testing device 210.
  • the cartridge 250 can optionally include the channels 232a-232n, as shown in FIG. 1C. Additional descriptions of cartridges that can be used according to implementations of the present disclosure are described with reference to the examples herein.
  • the pump 230 can be configured to control the fluid flow (e.g., pressure, flow rate, shear rates, initial shear rates, shear stresses, and other properties of the fluid) through the channels 232a-232n.
  • the controller 220 can be configured to control the pump 230 to control the fluid flow, for example based on sensors configured to sense the pump 230 and/or testing device (e.g., flow sensors, pressure sensors, etc.) (not shown).
  • the system 200 is configured so that the pressure in the system is a constant pressure (e.g., by controlling the pump 230 to generate a constant pressure, and/or by configuring the pump 230 to output a constant pressure).
  • the system can be configured so that the constant pressure is maintained when one or more channels 232a-232n are occluded.
  • the system 200 can be configured to create a high initial shear rate, in order to induce von Willebrand Factor elongation.
  • An example high initial shear rate is a shear rate above 3000/s.
  • the system 200 can be configured such that the shear stress in a fluid in the test system is prevented from exceeding 1,000,000/s.
  • the geometry of the channels 232a- 232n can be configured to increase or decrease the shear stress.
  • the system 200 can be configured to operate using different fluid samples.
  • the test system is configured to evaluate occlusive platelet thrombosis.
  • the system 200 is configured to process a sample of non-citrated whole blood and/or non-heparinized whole-blood.
  • the system 200 can optionally be configured to create platelet-rich thrombi (e.g. platelet-rich thrombi present at greater than 10% platelets by volume.)
  • platelet-rich thrombi e.g. platelet-rich thrombi present at greater than 10% platelets by volume.
  • FIG. ID illustrates an example microfluidic device 500 that can be used as part of the assays described herein.
  • the microfluidic device 500 can be part of the channels 232a-232n and/or the cartridge 250 shown in FIG. 1C.
  • the microfluidic device can include an inlet 510 and an outlet 512, where a fluid (e.g., blood) flows through the device from the inlet to the outlet, as marked by flow arrow 514.
  • a fluid e.g., blood
  • the test section 520 can be configured to generate shear stresses on the fluid, as described throughout the present disclosure.
  • a chamber 516 can be disposed between the inlet 510 and outlet 512 as shown in FIG. ID.
  • FIG. ID illustrates an example microfluidic device 500 that can be used as part of the assays described herein.
  • the microfluidic device 500 can be part of the channels 232a-232n and/or the cartridge 250 shown in FIG. 1C.
  • the microfluidic device
  • ID further includes non-limiting example dimensions of the microfluidic device 500.
  • the device is approximately 132 mm long, and the test section 520 is approximately 7 mm long, as shown in FIG. ID.
  • the microfluidic device 500 can include multiple test sections 520.
  • FIG. IF illustrates an example test section 600.
  • the test section 600 shown in FIG. IF can be used as a test section 520 shown in FIG. ID, or the channels 232a-232n and/or cartridge 250 shown in FIG. 1C.
  • the test section 600 is a tube with a diameter of approximately .2 mm, and a length of approximately 7 mm.
  • the dimensions of the test section 600 are used to generate shear stresses in the fluid, for example as part of the assays described herein. Therefore, different geometries of test section can be used to generate different amounts of shear forces, or to generate the same ranges of shear forces using different combinations of length, diameter, cross sectional shape, and any other structural configuration, according to various embodiments of the present disclosure.
  • FIG. 4 is a block diagram of an example computing system 100 that can implement various methods and processes described herein, according to some implementations.
  • computing system 100 is a controller that is part of or that is communicably coupled to the point-of-treatment system described above.
  • Computing system 100 is shown to include a processing circuit 102 that includes a processor 104 and a memory 410.
  • Processor 104 can be a general-purpose processor, an ASIC, one or more FPGAs, a group of processing components, or other suitable electronic processing structures.
  • processor 104 is configured to execute program code stored on memory 106 to cause computing system 100 to perform one or more operations, as described below in greater detail.
  • computing system 100 may be part of another computing device, the components of computing system 100 may be shared with, or the same as, the host device.
  • computing system 100 may utilize the processing circuit, processor(s), and/or memory of the server to perform the functions described herein.
  • Memory 106 can include one or more devices (e.g., memory units, memory devices, storage devices, etc.) for storing data and/or computer code for completing and/or facilitating the various processes described in the present disclosure.
  • memory 106 includes tangible (e.g., non-transitory), computer-readable media that stores code or instructions executable by processor 104.
  • Tangible, computer-readable media refers to any physical media that is capable of providing data that causes computing system 100 to operate in a particular fashion.
  • Example tangible, computer-readable media may include, but is not limited to, volatile media, non-volatile media, removable media and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data.
  • memory 106 can include RAM, ROM, hard drive storage, temporary storage, non-volatile memory, flash memory, optical memory, or any other suitable memory for storing software objects and/or computer instructions.
  • Memory 106 can include database components, object code components, script components, or any other type of information structure for supporting the various activities and information structures described in the present disclosure.
  • Memory 106 can be communicably connected to processor 104, such as via processing circuit 102, and can include computer code for executing (e.g., by processor 104) one or more processes described herein.
  • processor 104 and/or memory 106 can be implemented using a variety of different types and quantities of processors and memory.
  • processor 104 may represent a single processing device or multiple processing devices.
  • memory 106 may represent a single memory device or multiple memory devices.
  • computing system 100 may be implemented within a single computing device (e.g., one server, one housing, etc.). In other embodiments, computing system 100 may be distributed across multiple servers or computers (e.g., that can exist in distributed locations).
  • computing system 100 may include multiple distributed computing devices (e.g., multiple processors and/or memory devices) in communication with each other that collaborate to perform operations.
  • an application may be partitioned in such a way as to permit concurrent and/or parallel processing of the instructions of the application.
  • the data processed by the application may be partitioned in such a way as to permit concurrent and/or parallel processing of different portions of a data set by the two or more computers.
  • virtualization software may be employed by computing system 100 to provide the functionality of a number of servers that is not directly bound to the number of computers in computing system 100.
  • Computing system 100 is also shown to include a communications interface 110 that facilitates communications between computing system 100 and any external components or devices, including one or more remote devices 112.
  • communications interface 110 can be or can include a wired or wireless communications interface (e.g., jacks, antennas, transmitters, receivers, transceivers, wire terminals, etc.) for conducting data communications, or a combination of wired and wireless communication interfaces.
  • communications via communications interface 110 are direct (e.g., local wired or wireless communications) or via a network (e.g., a WAN, the Internet, a cellular network, etc.).
  • communications interface 110 may include one or more Ethernet ports for communicably coupling computing system 100 to a network (e.g., the Internet).
  • communications interface 110 can include a Wi-Fi transceiver for communicating via a wireless communications network.
  • communications interface 110 may include cellular or mobile phone communications transceivers.
  • computing system 100 is communicably coupled to one or more remote devices 112 via communications interface 110 (e.g., via a wireless network).
  • remote device 112 is any remote computing device.
  • Remote device 112 may be a computing device including a memory (e.g., RAM, ROM, Flash memory, hard disk storage, etc.), a processor (e.g., a general-purpose processor, an application specific integrated circuit (ASIC), one or more field programmable gate arrays (FPGAs), a group of processing components, or other suitable electronic processing components).
  • remote device 112 is or includes a user interface (e.g., a touch screen), allowing a user to interact with computing system 100.
  • a user interface e.g., a touch screen
  • remote device 112 is any electronic device that allows a user to interact with computing system 100, e.g., by presenting and/or receiving user inputs through a user interface.
  • remote device 112 represents a user interface positioned on computing system 100 itself.
  • remote device 112 is configured to execute (i.e., run) a software application that presents the various user interfaces described herein.
  • the present disclosure contemplates methods, systems, and program products on any machine-readable media for accomplishing various operations.
  • the implementations of the present disclosure may be implemented using existing computer processors, or by a special purpose computer processor for an appropriate system, incorporated for this or another purpose, or by a hardwired system.
  • Implementations within the scope of the present disclosure include program products including machine-readable media for carrying or having machineexecutable instructions or data structures stored thereon.
  • Such machine-readable media can be any available media that can be accessed by a general purpose or special purpose computer or other machine with a processor.
  • machine-readable media can comprise RAM, ROM, EPROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to carry or store desired program code in the form of machine-executable instructions or data structures, and which can be accessed by a general purpose or special purpose computer or other machine with a processor.
  • Machine-executable instructions include, for example, instructions and data which cause a general-purpose computer, special purpose computer, or special purpose processing machines to perform a certain function or group of functions.
  • the Thrombocheck has the advantage that it does not require chemical agonists to induce clotting and forms large, stable thrombi. It is shown that the Thrombocheck is a functional test for high shear clot formation, is sensitive to anti-platelet use and provides a unique endpoint for arterial thrombosis.
  • N-acetylcysteine is an existing drug currently used for treating acetaminophen overdose and cystic fibrosis which also cleaves vWF.
  • NAC N-acetylcysteine
  • coagulation occurs when coagulation factors are stagnant in a region with endothelial disruption.
  • arterial thrombi form in the high-pressure, high flow arterial environment, where stagnant regions are rare.
  • vWF protein von Willebrand Factor
  • platelets are responsible for creating arterial thrombi.
  • Atherosclerosis gradually affects the shape, mechanical properties, and cellular composition of arteries. Certain locations in the body are particularly prone to atherosclerosis, such as the branch points in the common carotid arteries, femoral arteries, or coronary arteries. It has been shown that unique flow conditions at these areas leads to an increased likelihood of arterial thrombosis. Over the course of multiple decades, the arteries locally stiffen, and an atherosclerotic plaque develops in the artery, creating a narrowed section of the artery known as a stenosis. The presence of a plaque in the otherwise smooth and gradually tapered arteries creates stenosis.
  • Stenotic arteries have pathologically high shear rates which increase the shear stress applied on endothelial cells lining the plaque. Through mechanisms still being researched these plaques can erode or rupture, exposing extracellular collagen and initiating the acute process of acute arterial thrombosis.
  • Von Willebrand Factor is a protein critical to the formation arterial thrombosis and part of the arterial thrombosis triad.
  • the important role of vWF is emphasized by the fact that the concentration of vWF is one of the strongest predictors of future MACE, with levels in the upper 4 th quartile being significantly more predictive of MACE than traditional risk factors such as a family history of cardiovascular disease, previous myocardial infarctions, or diabetes mellitus type 2.
  • VWD Von Willebrand Disease
  • Von Willebrand Factor is a long, multimeric protein present in the plasma of blood.
  • Plasma vWF is released by the endothelial cells lining the vasculature as Ultra Long vWF (ULVWF).
  • ULVWF strands are the largest proteins in blood, and can be up over 20,000 kDa or 15um in length.
  • ULVWF is gradually cleaved into smaller and smaller fragments until it is eventually cleared through the liver, in a process involving CD68+ Kupffer Macrophages.
  • AD AMTS 13 A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13
  • ADAMTS13 a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13
  • vWF is stored in alpha granules of platelets. These granules are released into the extracellular space when platelet activate and can significantly increase the local concentration of vWF in a growing thrombus.
  • An atherosclerotic plaque reduces the cross-sectional area of an arterial lumen and can increase shear rates from ⁇ 1,000 s' 1 in a healthy artery to >5,000 s' 1 or even up to 100,000 s' 1 in severe stenoses.
  • vWF conformation is sensitive to this increase in shear rate and in a high shear environment globular vWF unravels into an elongated conformation. The unravelling of vWF exposes key functional domains that bind to collagen and platelets, activating the vWF. If there is a thrombogenic surface present, like the extracellular matrix collagen exposed after an atherosclerotic plaque rupture, then vWF can bind to and coat the thrombogenic surface.
  • activated vWF can bind to platelets wrapping around platelets and forming small aggregates in flow.
  • the initial interaction between platelets and vWF occurs between the Al domain in vWF and the GPIb receptor on platelets.
  • vWF tethered to a thrombogenic surface they can roll across the surface and become stably attached.
  • individual platelets can also be captured onto a vWF coated surface.
  • platelets Once attached to the surface, platelets are activated either directly by high shear stresses acting on their cell membrane, or by chemical agonists such as calcium and ADP. Activated platelets release vesicles known as alpha granules and dense granules.
  • Dense granules contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), calcium and serotonin and release of these chemicals can lead to activation of nearby platelets.
  • Alpha granules contain a number of cytokines, growth factors, clotting proteins in addition to vWF. The release of alpha granules leads to a significant increase in the local concentration of vWF. A local increase in vWF concentration causes the capture of additional platelets, which release even more vWF, and a positive feedback loop is initiated where platelets are quickly aggregated to form an occlusive thrombus. (FIG. 7) Because of the role of alpha granule vWF, alpha granule release is crucial for making occlusive thrombi.
  • a second type of bond can form, between the Cl domain on vWF and GPIIb/IIIa (also known as cq/£> ? 3 ) receptors on platelets.
  • the Cl:GPIIb/IIIa bond is stronger than the Al:GPIb bond, and serves to strengthen the clot.
  • coagulation can occur in regions of low flow, further strengthening the clot and reducing permeability through the formation of a fibrin network. This process results in a large, strong, platelet-rich clot which is both porous and occlusive and can be interspersed with and surrounded by coagulative regions.
  • ischemic events Predicting when ischemic events will occur is complicated by their infrequent nature. Despite being a leading cause of death, these are acute events which can happen only a handful of times per lifetime. For example, in patients who have just undergone a Percutaneous Coronary Intervention (PCI), a population of patients known to be at a significantly increased risk of having a secondary ischemic event, monitoring 297 patients for 12 months resulted in the observation of only 40 ischemic events. Any tools available to help proactively identify subpopulations that are most at risk means additional monitoring and preventative measures can be taken to prevent the ischemic events from occurring. ii. Risk Scores and Symptoms
  • Risk scores are one of the oldest methods for predicting MACE events. Multiple risk scores have been developed to predict a patient’s risk of forming arterial thrombi using easily measured characteristics. These include the HEART, GRACE, TIMI, and CADILLAC. Several factors reoccur throughout multiple risks scores including age, smoking, chronic kidney disease, diabetes mellitus and a history of thrombosis. The area under the curve (AUC), a measure of predictive value, for the HEART score in predicting primary MACE at 6 months is 0.83. AUC scores for predicting secondary MACE are higher, highlighting the strong predictive value of previous thrombotic events.
  • AUC area under the curve
  • Scores listed above use general patient characteristics to predict MACE.
  • Some clinical measurements used in risks scores that directly relate to the theoretical model of SIPA include pressure drop across a stenosis, Ankle Brachial Index (AB I), degree of stenosis, blood velocity and shear rate. These metrics relate to the severity of an occlusive artery and are correlated with an increased shear rate which can activate vWF.
  • AB I Ankle Brachial Index
  • MACE blood velocity
  • shear rate which can activate vWF.
  • antiplatelet drugs reduce the incidence of MACE highlights the fact that elevated shear rates alone are not sufficient to predict ischemic events.
  • Based on the alternative triad for arterial thrombosis there is a need to incorporate information on how thrombogenic a person’s blood is and the likelihood of creating a thrombogenic surface, i.e., having a plaque rupture.
  • VerifyNowTM was created in 2006 and is one of the primary platelet function tests currently in use. It is based off an older laboratory technique called light transmittance aggregometry.
  • chemical agonists are used to induce platelet aggregation onto fibrinogen coated beads. As aggregates form on the beads, they come out of solution, which increases the light transmittance through the sample. This can be detected with a combination of a light source and sensor. The rate of change in transmittance and final transmittance values are measured outputs that indicate how quickly and to what extent platelets activate and aggregate.
  • the two chemical agonists used in the VerifyNowTM are ADP for the PRU tests and Arachidonic Acid for the Aspirin Resistance test.
  • the Platelet Function Analyzer- 100 measures the time required to form an occlusive clot in a ⁇ 150um diameter hole in a membrane. This hole is collagen coated to provide a thrombogenic surface. As in the VerifyNowTM, chemical agonists are added to induce platelet activation. There are two unique test cartridges with different agonists, either Collagen and ADP (Col/ADP) or Epinephrine and ADP (Epi/ADP). Blood is pumped through the membrane hole using a constant pressure until flow ceases. The time to flow cessation is the output, labeled Closure Time (CT).
  • CT Closure Time
  • T-TAS Total-Thrombosis Analysis System
  • BAPA benzylsulfonyl-d-arg-pro- 4-amidinobenzylamide
  • Blood is perfused through the channels at a constant flow rate to achieve wall shear stresses ranging from 1000 to 2000 s’ 1 .
  • platelets aggregate in the channels the resistance to flow increases and a larger pressure drop is measured across the channels.
  • the pressure drop across the channels is graphed over time and various measurements from that graph are used as outputs, including the time to reach 10 kPa or 60 kPa and the area under the pressure curve.
  • GTT Global Thrombosis Test
  • ROTEM Rotational Thromboelastometry
  • TEG Thromboelastography
  • antiplatelet drugs Most current antithrombotic medications prescribed are antiplatelet drugs. Aspirin and clopidogrel (Plavix) have the largest market share, ⁇ 4.5 million patients each, with other drugs such as ticagrelor (Brilinta) and prasugrel (Effient) being used in roughly half a million cases. Because platelets play a large role in amplifying the coagulation cascade as well as arterial thrombosis, anti-platelet drugs are associated with increased rates of significant bleeding. While some bleeding issues are relatively harmless, such as increased incidence of bruising, other bleeding, such as cranial and GI bleeding, can be life-threatening. The rate of major bleeding events roughly doubles when taking aspirin and doubles again for patients on dual antiplatelet therapy (DAPT).
  • DAPT dual antiplatelet therapy
  • Aspirin is by far the most widely used drug for prevention of thrombosis. In 2020 it was prescribed to an estimated 4.7 million patients and was taken without a prescription by millions more. Aspirin irreversibly inactivates the enzyme cyclooxygenase (COX), which prevents the formation of thromboxane A2 (TXA2). TXA2 contributes to the activation and aggregation of platelets and reduction of TXA2 has an inhibitory effect on platelet activation and thrombosis.
  • COX cyclooxygenase
  • Clopidogrel tradename Plavix
  • Plavix is the second most prescribed drug for thrombosis. Approximately 4.4 million patients had prescriptions for clopidogrel in 2020. Enzymes in the cells lining the gut transform clopidogrel into an active metabolite which irreversibly inactivate the P2Y 12 receptor on platelets. This receptor is part of a signaling chain which links increased extracellular ADP concentration to platelet activation. As a result, platelet activation from ADP is inhibited. v. vWF Specific Therapeutics
  • Eptifibatide is a small peptide derived from a protein found in the venom of the southeastern pygmy rattlesnake. Eptifibatide reversibly binds to the GPIIb/IIIa receptor on platelets and prevents it from forming a bond with vWF. Since these bonds are essential to forming a strong bond between activated platelets and vWF, GPIIb/IIIa inhibition has a strong effect on preventing large, occlusive thrombi.
  • GPIIb/IIIa inhibitors Similar to Eptifibatide, other GPIIb/IIIa inhibitors have been developed, including the antibody abciximab and a small molecule, tirofiban. These drugs are associated with an increased risk of hemorrhage, which may be explained by the fact that GPIIb/IIIa binds not only to vWF but also to fibrinogen meaning it plays a key role in both coagulation and thrombosis. The increased risk of bleeding in these patients is so severe that use of GPIIb/IIIa inhibitors is generally limited to a hospital setting, where clinicians can monitor patients for adverse events. Additionally, GPIIb/IIIa inhibitor are much more expensive than antiplatelets, the cost of a single treatment ranges from $400-$ 1,300.
  • a novel point-of-care device that measures arterial thrombosis formation was created. Design inputs were chosen to ensure the assay reliably forms arterial thrombi by recreating the shear rate, surface and blood chemistry described in the arterial thrombosis triad. Additionally, it operates with less than 10 mL of blood, has low variability, and can be run in a clinical setting.
  • the assay was split into three components: a test section, a method for driving flow and an outcome measure. Each component was designed 1 and tested separately, then combined to form the final assay.
  • the Thrombocheck provides a measurement that is unique compared to the PFA-100.
  • Thrombotic activity in a healthy population was observed to have a bimodal distribution, with a subpopulation of ⁇ 30 having increased measurements indicating a decreased platelet aggregation rate.
  • the Thrombocheck was found to be sensitive to anti-platelet treatment with aspirin, a P2Y12 inhibitor and eptifibatide and able to distinguish the degree of individual response to those therapies.
  • MACE Adverse Cardiovascular Events
  • SIPA Shear Induced Platelet Aggregation
  • RPA rapid platelet accumulation
  • Anti-platelet drugs are currently used to reduce the risk of arterial thrombosis formation by preventing platelet activation or inhibiting platelet- vWF bonds.
  • Common anti-platelets include aspirin, clopidogrel and GPIIb/IIIa inhibitors such as eptifibatide.
  • aspirin and clopidogrel inhibit platelet activation through thromboxane A2 and P2Y12, respectively. Both of their mechanisms of action are through irreversible inactivation of proteins which play a role in platelet activation pathways. Since they effect different pathways of platelet activation, they can be taken individually or together as a therapy known as Dual Antiplatelet Therapy (DAPT). Because clopidogrel’ s active agent is a metabolite, adding clopidogrel directly to blood does not properly mimic its effect in vivo. For in vitro testing another P2Y12 inhibitor, 2MeSAMP, is used to model the effect of clopidogrel. Because aspirin and clopidogrel both have modest effects on reducing arterial thrombosis, it is expected that an assay of arterial thrombosis will be sensitive to both compounds.
  • DAPT Dual Antiplatelet Therapy
  • Eptifibatide was used as a positive control for inhibiting platelet aggregation. Eptifibatide reversibly binds to the GPIIb/IIIa receptor on platelets, preventing platelets and vWF from bonding. This has a strong effect on preventing large, occlusive thrombi and can completely prevent arterial thrombosis at micromolar doses. iii. Point-of-Care Devices
  • thrombosis formation sensitive to all changes would be useful in diagnosing the risk of ischemic events, bleeding dysfunctions in trauma patients and tailoring anti-platelet therapies for individuals.
  • SIPA SIPA there are three things that need to be incorporated into a test of arterial thrombosis: platelets/vWF, high shear stresses, and thrombogenic surface.
  • LTA Light Transmission Aggregometry
  • various chemical agonists are added to plasma, causing aggregates to form, and come out of solution.
  • Chemical activation of platelets differentiates LTA from SIPA. Additionally, LTA experiments are performed in the absences of flow, so these assays fail to the high shear stresses required for vWF elongation.
  • VerifyNow works in a similar manner to LTA. In the VerifyNow cartridge, ADP causes platelets to aggregate around fibrinogen coated beads and the light transmittance of the solution changes as a result. VerifyNow runs into the same limitations as LTA; platelet activation is precipitated by chemical agonists and the test is performed in static conditions.
  • Platelet Function Analyzer- 100 is a platelet assay that measures the amount of time required for blood to occlude a collagen-coated hole in a cellulose membrane.
  • the PFA- 100 uses whole blood but requires samples to be collected into citrate - a known antiplatelet agent. Further, the shear rates generated during testing are not precise because the cellulose membrane has indefinite dimensions and edges. Thus, the high shear conditions may vary widely as this value depends strongly on diameter to the third power.
  • Blood is activated in the PFA- 100 cartridges by the addition of either ADP or Epinephrin. The addition of chemical agonists to the blood means the PFA- 100 is more of a biochemical platelet activation assay than a true SIPA assay.
  • T-TAS Total-Thrombosis Assessment System
  • a constant flow condition means any variation in thrombus formation between channels is amplified. This is because growth in one channel affects the shear rate of all the other channels and can lead to extremely high shear rates in some channels, while others may have close to stagnant conditions, preventing a stable, SIPA clot from forming.
  • the endpoint is not cessation of flow, which would be most analogous to the clinical pathology, but instead the area under the pressure curve. This outcome is difficult to interpret physically.
  • TEG Thromboelastography
  • ROTEM Rotational Thromboelastometry
  • GTT Global Thrombosis Test
  • the aim is to create a unique assay of arterial thrombosis that can be used in a variety of clinical situations, such as tailoring antiplatelet therapies to individual patients, stratifying patients into different risk levels for ischemic events or identifying trauma patients with platelet dysfunctions.
  • test section size The most important design consideration when developing an occluding test section is the size of the region where a clot will grow. This parameter will affect many other variables, including the composition of the clot, amount of blood required to form an occlusive clot, the pressures required to achieve sufficient flow, and the run time of the test. Choosing an appropriate test section size is fundamental to building a point-of-care device for arterial thrombosis. In this study, the focus is on circular cross-sections because of the availability of glass tubes with a wide range of radii.
  • An upper limit for the test section radius is determined by setting an upper limit on the amount of blood that the POC can require to run. 5mL is a reasonable amount of blood to draw using standard venipuncture techniques and setting an upper limit to be 1 mL per test section allows to run up to four tests in parallel with a single 5 mL vacutainer.
  • thrombus growth rates Using a computational model of thrombus growth rates, the total volume required to occlude a circular tube of diameter D can be calculated. For these simulations it is assumed that flow is driven by a constant pressure head such that the initial shear rate is 10,000 s’ 1 . Blood volume necessary for occlusion can be calculated by using the thrombus growth model to compute the change in radius base on the shear stress in the test section, then updating the geometry and iterating.
  • Platelets interactions within a thrombus are divided into two categories: platelet-surface interactions that adhere a thrombus to the thrombotic surface and platelet-platelet interactions that form the bulk of a thrombus.
  • the occlusive thrombi should be primarily composed of platelet-platelet interactions, not platelet- surface interactions.
  • the percentage of platelets involved in platelet-platelet interactions for a circular cross section is given by equation 1 assuming that platelets are spherical.
  • v is the volume of an individual platelet
  • a is the surface area occupied by a single adherent platelet
  • p is volume fraction of platelets in a thrombus
  • D is the diameter of the test section.
  • D is the diameter of the test section in pm.
  • P c is set to 0.9, to ensure a significant majority of platelet interactions are platelet-platelet.
  • shear stress is the force that causes vWF to elongate
  • shear rate is reported and assuming an average blood viscosity of 3.5 cP for all modelling, appropriate for simple geometries.
  • Shear rates around an atherosclerotic plaque can span multiple orders of magnitude from -1,000 s' 1 to -100,000 s’ 1 .
  • it is known that clot growth occurs fastest in the range of 10,000 to 30,000 s’ 1 so the device was designed to stay within this range.
  • the other main parameter which affects shear rate is the flow rate.
  • the magnitude of the increase in shear rates depends on the ratio of variable resistance to fixed resistance.
  • a computational model of arterial thrombosis previously described was modified to mimic a constant pressure system with two resistors in series, a variable resistor that increases in resistance as a clot forms, and a constant resistor.
  • the microfluidic assay that the POC device is based on has an intra-assay variability of -10%.
  • intra-assay variability outcomes from multiple test sections can be averaged.
  • variability analysis was performed. According to a Monte-Carlo simulation of 100 expected outcomes from assays with between 1 and 5 test sections, a significant decrease in intra-assay variability was observed by incorporating 4 test sections, but there are diminishing improvements in intra- assay variability when including more than 4 sections (FIG. 11A). Since blood volume scales linearly with the number of test sections, only 4 test sections were used.
  • the measured endpoint of the assay needs to be chosen.
  • mass flow rates through the stenotic sections were directly measured using mass balances. This mass measurement is a noisy endpoint when measuring small flows and is impractical for the flow rates used in the cartridge.
  • Another common endpoint used in platelet aggregation assays is occlusion time (OT in T-TAS and GTT, CT in PFA-100). Occlusion time is defined differently in each system but is meant to represent the time required to significantly reduce flow through the assay. However, determining occlusion time requires constant monitoring of either pressure or flow rates in the system. Instead, a novel endpoint was developed, called End Volume (EV).
  • EV is defined as the total amount of blood which passes through the test section prior to occlusion. Unlike mass flow rates, measuring EV does not require constant monitoring; a test can be started and then read any time after flow has ceased. Additionally, by providing markings on outlet reservoir of the transparent cartridge, EV can be simply read off by the user. Since EV is the integration of the flow rate vs. time curve, it was hypothesized that it is strongly correlated to occlusion time. To validate that EV is a good proxy for occlusion time, data from the laboratory microfluidic assay was used to look at the fit between the two. The r 2 value for the correlation is 0.883, indicated a strong correlation between EV and OT (FIG. 11B). ii. Experimental Methods
  • Vacuum strength of the point of care device was measured using a built-in pressure sensor (BMP180). After turning the POC device on, pressure measurements were recorded as the desired vacuum was created. The test cartridge was kept out of the solution for roughly 30 s to determine vacuum stability in air, then it was placed in either blood or a 40% glycerol solution for at least 90 s.
  • Blood Draw and Handling [0131] Blood was drawn from healthy adult volunteers using standard venipuncture techniques under a Georgia Tech IRB approved protocol (H17315). Using a %” 21G needle (BD), blood was first collected into a vacutainer containing EDTA (BD) for running the Complete Blood Count (CBC). Blood was then slowly drawn into a 50 mL syringe containing 3.5 lU/mL sodium heparin (Fisher Bioreagents). All experiments were performed within 4hrs of the blood draw.
  • BD vacutainer containing EDTA
  • CBC Complete Blood Count
  • test sections Up to 16 individual test sections were tested simultaneously using tubing to connect the test sections to a single vacuum pump (FIG. 12). Four test sections were placed in a single 15 mL conical vial containing 10 mL of blood. Five conditions were tested to measure the spread of endpoints in the healthy population and determine whether the assay is sensitive to anti-platelet therapies known to reduce MACE. For each of 10 individuals, 12 test sections were run on untreated whole blood.
  • ASA Acetyl- Salicylic Acid
  • 2MeSAMP 2-methylthioadenosine 5 ’-monophosphate triethylammonium salt hydrate
  • ASA Acetyl- Salicylic Acid
  • 2MeSAMP 2-methylthioadenosine 5 ’-monophosphate triethylammonium salt hydrate
  • Anti-platelet drugs were sourced from Fisher Scientific. Anti-platelet drugs were added through the addition of 0.1 mL of concentrated solution to 9.9 mL of whole blood to achieve a final concentration of 0.02 mg/mL ASA, 50 uM 2MeSAMP or 2 uM Eptifibatide.
  • 2MeSAMP and Eptifibatide concentrations were chosen based on previous literature and the ASA concentration was chosen to mimic a realistic clinical dose.
  • Anti-platelet treatments were allowed to incubate in the blood for 40 minutes, 2x the reaction half-life of ASA in blood. Additionally, to account for any effects of dilution or the time from the blood draw to testing, a 10 mL sample of whole blood diluted with 0.1 mL PBS was run at the same time as antiplatelet treatments. The total volume of blood drawn through the test section prior to occlusion was defined as the End Volume. Test sections that did not occlude within >1.6 mL of blood were stopped.
  • test section with a diameter of 228 pm is expected to require 1.0 mL of blood to form an occlusive clot. Based on a desired P c of 0.9, the test section diameter must be at least 78 pm. The combination of these two limits provides a relatively small window of ideal diameters for a circular test section capable of forming large, occlusive, stable thrombi: from 78-228 pm. (FIG. 12A)
  • Capillary tubes with a diameter of 200 pm were chosen to be used as the POC test section.
  • the capillary tubes were made of glass, which is known to absorb collagen and create a surface that promotes vWF attachment and arterial thrombosis.
  • the diameters of the capillary tubes were verified to be within 199.5 and 202.5 pm using optical measurements with an average of 200.90 ⁇ 0.61 pm. (FIG. 12B) If the initial wall shear rate for a 200.0 pm diameter capillary is 10,000 s' 1 the measured range in actual diameters would result in a range of initial shear rates from 9,634 s' 1 to 10,075 s’ 1 . iv. Pressure System
  • the peristaltic pump was sufficient to create and maintain the pressures necessary to create high shear flow in the microcapillaries.
  • a 50 mL vacuum reservoir was placed between the pump and the test section to dampen any variability in pressure. It was observed that the device can reach and maintain a vacuum pressure of - -70 hPa (52.5 mmHg) in each of these scenarios.
  • FIG. 13A Final pressures were reached within 20 s and varied by -10 hPa during operation. No significant difference in flow rates was observed between blood and glycerol.
  • test sections were run using untreated blood for 10 individuals to define a range of healthy values.
  • thrombosis plays a critical role in thrombosis and bleeding, multiple populations are expected to benefit from the POC test.
  • One potential use case is in determining an individual’s response to antiplatelet therapy.
  • a patient’s blood could be dosed with antiplatelets such as aspirin and 2MeSAMP to determine the appropriate dose for them. This would identify patients who respond to ASA or Plavix. Non-responders could avoid these treatments and not expose themselves to the risks of major bleeding and the cost of purchasing the drug.
  • the Thrombocheck device could be used to predict the risk of secondary MACE after a primary event.
  • the Thrombocheck could stratify patients into different risk levels and identify subpopulations that require additional anti-platelet/anti-vWF therapies to reduce the risk of MACE.
  • Thrombocheck Another population that can benefit from screening with the Thrombocheck is trauma patients. Arterial bleeding involves high shear rates and untreated hemostatic clots likely form through a hemodynamic process similar to arterial thrombosis. Trauma patients often have platelet dysfunctions leading to bleeding pathologies. In addition to coagulation assays determining whether a platelet dysfunction exists may help guide physicians on which blood products to give patients.
  • thrombi aggregate by elongated vWF under high shear conditions at a very high rate of growth that correlates with clinical thrombosis.
  • the results are independent of and do not correlate with the PFA- 100.
  • the PFA-100 test section depends on ADP or epinephrine for closure while ours does not, and the hole is made of fibrous cellulose without known dimensions such that shear rates may be variable.
  • the Light Transmission Aggregometry and ROTEM/TEG tests do not aggregate platelets by shear.
  • the T-TAS has channels that are sensitive to platelet-collagen surface interaction instead of platelet-platelet volume growth of arterial thrombi, and the constant flow arrangement makes occlusion a noisy outcome.
  • the invention disclosed herein can transform a complex research laboratory microfluidic test into a simple POC assay that can be used in a clinical setting.
  • the test section, method of driving flow, and the measured endpoint were redesigned to become a scalable POC test.
  • the Thrombocheck forms arterial thrombi without the need for exogenous chemical activation.
  • Choosing a properly sized test section ensured that the clot is representative of larger clots without using too much blood.
  • the resulting assay provides a unique measurement of SIPA thrombosis potential. This system has been used to characterize physiological ranges of a healthy population, without and with antiplatelet treatment. This assay can be used to assess arterial thrombosis risk in a variety of patient populations, from traumatic bleeding to ischemic stroke.
  • Thrombocheck is sensitive to anti-platelet medications and could be used to quantify an individual’s response to drugs such as aspirin or a P2Y12 inhibitor.
  • Use of the assay can be extended to other populations, determining MACE risk after a primary event, identifying trauma-induced platelet dysfunctions, or even predicting thrombotic complications from systemic inflammatory events, such as COVID- 19.
  • millimeter-scale platelet architecture in arterial thrombi was quantified for the first time revealing how discrete structures combine to form a single clot. These structures were recreated computationally using an agent-based model with emergent behaviors. Modelling how complex clot architecture emerges from 4 simple platelet behaviors allows to predict how changing platelet behavior affects clot structure. Using the model, how to modify clot structure can be defined to prevent thrombosis or improve treatment by thrombolysis and thrombectomy.
  • T-TAS Total Thrombus-Formation System
  • GTT Global Thrombosis Test

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Abstract

L'invention concerne un système de mesure de caillots sanguins occlusifs, le système comprenant : un dispositif de test ayant au moins un canal à travers lequel un échantillon de sang s'écoule; une pompe pour générer une pression dans le dispositif de test pour permettre l'écoulement de sang; et un dispositif de commande conçu pour mesurer un volume total ou une masse totale de sang qui s'écoule à travers la pluralité de canaux avant l'occlusion, le volume total ou la masse de sang indiquant un temps d'occlusion. L'invention concerne également des procédés d'utilisation du système.
PCT/US2024/027245 2023-05-01 2024-05-01 Dosage au point d'intervention pour thrombose plaquettaire occlusive Pending WO2024229114A1 (fr)

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US202363499380P 2023-05-01 2023-05-01
US63/499,380 2023-05-01

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WO2024229114A1 true WO2024229114A1 (fr) 2024-11-07

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160069913A1 (en) * 2014-09-09 2016-03-10 Perosphere Inc. Microfluidic chip-based, universal coagulation assay
US20160258968A1 (en) * 2013-10-16 2016-09-08 President And Fellows Of Harvard College A microfluidic device for real-time clinical monitoring and quantitative assessment of whole blood coagulation
WO2019099342A1 (fr) * 2017-11-16 2019-05-23 Georgia Tech Research Corporation Procédés et systèmes de formation de thrombus consistant et de mesure de celui-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160258968A1 (en) * 2013-10-16 2016-09-08 President And Fellows Of Harvard College A microfluidic device for real-time clinical monitoring and quantitative assessment of whole blood coagulation
US20160069913A1 (en) * 2014-09-09 2016-03-10 Perosphere Inc. Microfluidic chip-based, universal coagulation assay
WO2019099342A1 (fr) * 2017-11-16 2019-05-23 Georgia Tech Research Corporation Procédés et systèmes de formation de thrombus consistant et de mesure de celui-ci

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