Systems and methods for drug-related data collection and analysis

Abstract

An exemplary system for detecting and quantifying drug and / or chemical interactions with a biological sample is provided. The system includes a detection instrument having computing capabilities. The system includes an assay device capable of receiving the biological sample. Introduction of the biological sample into the assay device results in a biological process that may result in the accumulation of fibrin and platelets in a reaction zone of the assay device. The assay device is capable of receiving one or more chemical reagents and one or more drug reagents. The fibrin and platelets accumulated in the reaction zone of the assay device and their associated signals can be used to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration in the biological sample.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of co-pending, commonly assigned U.S. Provisional Patent Application No. 63 / 442,847, filed February 2, 2023. The entire contents of the aforementioned provisional application are incorporated herein by reference.

[0002] Government support statement This invention was made with government support under Grant No. R44HL149480-03 awarded by the National Institutes of Health (NIH) of the U.S. Department of Health and Human Services. The government has certain rights in this invention. [Background technology]

[0003] Evaluating bleeding and thrombus risk in critical care settings can be challenging using conventional systems. Conventional systems generally fail to create a testing environment that mimics the blood coagulation physiology found in the human body, do not provide specific data regarding key components of hemostasis (clotting via platelets and fibrin formation), and fail to deliver results in the timeframe required for critical care decision-making in emergency situations. For example, Figures 1A-1C illustrate the physiology of hemostasis in the human body. Hemostasis involves a physiological response to vascular injury to stop blood leakage, including both platelet aggregation and coagulation, while coagulation involves an enzymatic reaction system that generates thrombin and fibrin, which stabilize the clot. Figure 1A illustrates vasoconstriction due to collagen and tissue factor upon exposure to vascular injury. Figure 1B illustrates platelet plug formation accompanied by platelet aggregation. Figure 1C illustrates clot formation in the form of coagulation. Summary of the Invention

[0004] definition To clarify particular elements of this disclosure, specific terminology is defined here: The term "system" refers to the entire technology in question, including the microfluidic device, reagents, imaging equipment, software, analytical methods, and reporting.

[0005] The term "device" refers to a microfluidic apparatus for generating fibrin and platelet signals.

[0006] The term "reagents" refers to chemicals and drugs necessary to perform an assay.

[0007] The term "instrument" or "analyzer" refers to the imaging equipment and associated computing hardware used to collect and process data from a device.

[0008] The term "analysis" refers to the methodology for converting raw imaging data into clinical results.

[0009] The term "reporting" refers to the documentation of the results of an analysis.

[0010] As used herein, the term "factor IIa" is the scientific term for thrombin. Conventional nomenclature for coagulation factors uses Roman numerals, and inhibitors for those factors are designated by a lowercase I; thus, inhibitors for factor Xa are designated Xai, and inhibitors for factor IIa are designated IIai. As used herein, the term "DT" is a generic term for factor IIa and can refer to the function of DOAC drugs (direct thrombin inhibitors compared to Xai, an indirect thrombin inhibitor) on its target. Factor II (prothrombin) is activated to factor IIa (thrombin) through a pathway dependent on factor Xa. The terms "factor IIa" or "IIa" are used interchangeably herein to refer to DT (and vice versa), and the terms "factor IIai" or "IIai" are used interchangeably herein to refer to DT (and vice versa). 46-47 are diagrams of the coagulation pathways relevant to the use of the terms DTi and IIai herein.

[0011] As used herein, the term "fluidically" or "fluidic" refers to communication having static or active fluid communication along a fluid path between ports. Because the devices are fluidly coupled, flow within the device is possible at any time. As used herein, the term "active" flow or "fluid communication" refers to flow caused by pressure or vacuum applied to the fluidics of the device.

[0012] Unmodified vs. modified sample Numerous processes, actions, chemicals, and drugs are used in the systems described herein. An important distinction is made by modified or unmodified samples. A whole blood sample (whether containing a drug or not) obtained directly from a patient, and that same blood sample mixed with detection chemicals, are considered unmodified (as the term is used herein) because the behavior of the sample is not substantially altered from that of in vivo behavior. For example, the addition of a platelet label or a fibrinogen label does not essentially alter the behavior of platelets and coagulation (e.g., fibrin accumulation). A modified sample (as the term is used herein) is one whose essential behavior is altered from in vivo conditions. For example, anticoagulants or antiplatelet drugs, and / or reversals of those same drugs, applied to a sample will certainly alter the behavior of platelets and / or fibrin. This is distinct from the fact that a sample may already contain some or all of those drugs originally from a patient taking those prescribed drugs.

[0013] Indirect vs. Direct Drug Action An indirect drug effect is one in which the physiological response to a drug can be measured at a process that is distinct and / or distal to the molecular target the drug is designed to target. A direct drug effect is one in which the physiological response to a drug can be measured at the target for which the drug is intended. For example, direct thrombin inhibitors (DTi, or IIai) such as dabigatran directly target the activity of thrombin, while factor Xa inhibitors (Xai) such as apixaban target factor Xa, an important upstream activator of thrombin. With respect to thrombin, dabigatran has a direct effect on thrombin activity, while apixaban has an indirect effect on thrombin activity, even though both reduce thrombin activity.

[0014] inhibition vs attenuation In the above example, thrombin inhibition by either class of DOAC reduces fibrin formation. Neither class of drug inhibits fibrin directly; therefore, the reduction is an attenuation of fibrin development. Dabigatran inhibits thrombin directly, while Xa inhibitors such as apixaban inhibit factor Xa; both attenuate fibrin by reducing the activity of thrombin (and acting indirectly).

[0015] Drugs vs. Chemicals The usefulness of the system described herein relies on the distinction between drugs and chemicals. Drugs are specific compounds formulated to induce targeted biological effects. For example, the DOAC drug dabigatran specifically targets (inhibits) thrombin. Chemicals in this context are substances that exert a specific, targeted function within an assay, but are not designed or intended to direct or affect a biological pathway or function. For example, platelet-labeling chemicals are used to fluorescently tag platelets. However, these chemicals do not affect the way platelets interact biologically in the formation of a blood clot.

[0016] Levels, Concentrations and Thresholds Generally, the usefulness of evaluating patient samples for the presence of drugs can be divided into two paths: semi-analytical and analytical. A more common approach to understanding the effect of drugs on hemostasis is to determine the level of the drug in a patient, typically compared to a threshold value. As used herein, the term "threshold value" refers to a specific target value or range of values ​​that is clinically meaningful when considering whether the level or concentration of a drug (or platelet or fibrin activity) exceeds, falls below, or is equal to this specific value, or is within a range of values.

[0017] The level is a semi-quantitative assessment because it may not be possible to definitively determine the absolute amount present in the blood. For example, a clinician may want to know whether a patient's drug level is above or below a given threshold (e.g., > or < 30 ng / mL) to determine whether a DOAC reversal agent should be administered. In this case, the value of the result is not what the exact amount is, but whether the patient's drug level is above or below a given threshold (or within a given range of possible concentrations (e.g., > 30 ng / mL but < 100 ng / mL)). In contrast, a concentration determination is quantitative in the sense that the actual amount of drug in the blood sample, plus or minus some level of imprecision, is determined.

[0018] Molar excess vs supratherapeutic For purposes of this document, a supratherapeutic dose of a drug refers to any drug concentration that exceeds the typical range of concentrations achieved in plasma with normal compliance with the drug's dosing regimen. A "molar excess" of a drug refers to any dose at which the corresponding assay signal (platelet fluorescence or fibrin fluorescence or other) altered by the presence of the drug would not be further attenuated by a higher dose of the drug. As such, a "molar excess" can be considered a "signal-saturating" dose of the drug.

[0019] Embodiments of the present disclosure provide general and specific systems capable of determining a patient's hemostatic status by simultaneously measuring platelet and fibrin accumulation, with platelet and fibrin accumulation as reporting signals. Because hemostasis involves both platelet function and coagulation, the system assesses both together to obtain a clear and accurate sense of an individual's hemostatic status. Although platelets (the subcellular component of hemostasis) are distinct from coagulation (the enzymatic reaction cascade), the two are closely related. Platelet binding to collagen and subsequent activation are early steps in the initiation of a stabilized coagulation cascade, and degranulating platelets release numerous compounds that promote and stimulate coagulation and additional platelet accumulation. This process is influenced by numerous cofactors, including exposed membrane phospholipids, which aid in the assembly of the tenase and prothrombinase complexes. Thrombin, a terminal coagulation protease, cleaves fibrinogen to form fibrin monomers, initiating the formation of fibrin polymers. Fibrin formation generates a polymeric mesh around the aggregating platelets to form a stable clot. In addition to catalyzing fibrin, thrombin is also a potent activator of platelets. Therefore, modulators of platelet function, specifically (such as targeted drugs), can also have a differential level of influence on fibrin formation, and vice versa. The two are closely related.

[0020] Generally, blood products administered to patients can have an immediate effect on platelet and / or fibrin function. Packed red blood cells affect hematocrit, which directly affects platelet margination and therefore concentration in the acellular layer. The direct addition of purified platelets immediately increases platelet function. Fresh frozen plasma contains all of the cofactors involved in clotting, and 4-factor prothrombin concentrate (4-FPCC) contains important cofactors involved in clotting that can be used both for bleeding events and to reverse the effects of anticoagulants such as warfarin (see, e.g., Horstman, EE et al., Plasma Products for Transfusion: An Overview, Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA, Vol. 7 (March 2022)). Therefore, blood product use and management directly dictate the need to monitor both platelet and coagulation function.

[0021] Hemostatic equilibrium is an important aspect of normal hemostatic function, in which the body regulates both platelet function and coagulation so as not to produce too weak or too strong a physiological response to injury or disease states. While there are generally "normal" measurable amounts of platelets, fibrinogen, and other coagulation factors circulating in the blood, there are numerous additional factors that can influence any one individual's hemostatic response to injury or disease, thereby creating a wide range of "normal" function. This represents a major challenge for diagnostic assessment of hemostatic function, which evaluates only one aspect of the hemostatic cascade.

[0022] In addition to tissue injury, numerous factors can interfere with the normal functioning of platelets and the coagulation cascade. These include, for example, drugs, illness, disease, genetics, certain foods, physical injury, or a combination thereof. Published research indicates that physical trauma to the body, for example, can result in dysfunction of platelet activity. COVID-19 infection has been shown to result in hypercoagulation, which can lead to inappropriate clot formation and death. Inhibition or promotion of platelet function and / or coagulation can result in either severe bleeding events or hyperthrombotic events, respectively, which can complicate medical care, lead to iatrogenic injury, and even death. Some medical conditions requiring medications or procedures that alter platelet function and / or coagulation (such as anticoagulants or antiplatelet drugs, procoagulants, transfusions, packed red blood cells, platelet therapy, etc.) can also result in abnormal thrombosis and / or bleeding events, which, with rapid onset, can cause iatrogenic injury or even death.

[0023] From a medical perspective, general thrombus formation (hemostasis) is distinct from the medical process of preventing abnormal thrombus formation (thrombosis) not associated with injury. For example, direct-acting oral anticoagulants (DOACs), which directly inhibit coagulation function, are taken by patients to prevent venous thromboembolism (VTE) complicated by long-term medical conditions such as cardiac abnormalities and arrhythmias like aFib. In some cases, preventing thrombus formation may be paramount, for example, to avoid stroke or heart attack. This differs from preventing thrombus formation due to direct physical injury (a short-term process), as many patients are on DOAC therapy chronically (for months or years) and therefore have perturbed thrombogenic behavior over the long term. Consequently, in critical care settings, detailed and accurate determination of whether a patient is taking a DOAC and their anticoagulation status may be essential for properly managing the patient's increased bleeding risk caused by DOAC medications. In a traditional critical care setting, the only available information may be whether a patient has been prescribed a DOAC and perhaps when the last dose was taken (if this information is even available). In such situations, accuracy related to the frequency of DOAC use and the actual state of anticoagulation in the patient's body may be difficult, if not impossible, to obtain. The exemplary systems discussed herein provide accurate and detailed determinations of whether a patient is taking a DOAC and their anticoagulation status. In some embodiments, the systems discussed herein can be used for similar determinations regarding novel oral anticoagulants (NOACs), target-specific oral anticoagulants (TSOACs), and / or novel platelet-targeting drugs, and / or drugs that target other upstream aspects of the hemostatic cascade.

[0024] In its simplest form, a patient's blood is passed through a reaction zone where platelets and coagulation activators, such as collagen and lipidated tissue factor (LTF), are located. As whole blood flows through the reaction zone, platelets can bind to collagen via their collagen receptors, activating them. LTF, combined with factor VIIa and other cofactors, activates the coagulation cascade. This activation then recruits more platelets to the thrombus formation zone, where activated thrombin catalyzes the formation of fibrin from soluble fibrinogen. This reaction is self-sustaining, with additional platelets and fibrin accumulation increasing over time until blood flow is occluded. The reaction zone could contain only one reactant (collagen) or more than collagen and LTF.

[0025] Blood from healthy, non-injured individuals will produce a specific profile of platelet and fibrin accumulation over time. Individual variation among healthy, non-injured individuals will produce a distribution of "normal" hemostatic function around the mean overall population function (see, e.g., Figures 43 and 44). This will provide a boundary for states where signals outside of this "normal" distribution constitute "abnormal" platelet or coagulation function. In some embodiments, the assay will be able to distinguish normal from abnormal platelet and coagulation function by percentage or percentile (for example) from the "normal" mean (however, this will not specify the origin of the abnormal behavior (e.g., drug, injury, disease, etc.)).

[0026] Determining the Presence of Drugs: If a drug targeting platelet or fibrin function is present, then the presence of the drug can be determined by the system or assay through a functional test using a reversing agent specific for that drug. The reversing agent could be a specific drug formulated to reverse the activity of the target drug. If a drug reversing agent is added to a patient blood sample containing the drug, then an increase in platelet and / or fibrin signal would be expected. In the absence of the drug, the reversing agent would have no effect (positive or negative) on platelet function or coagulation.

[0027] Determining Drug Class: When there are two or more classes of drugs targeting the same function (platelets or coagulation), the drug class can be determined through functional testing of platelet function and / or coagulation. For example, if there are two classes of drugs (A and B) that reduce fibrin formation (clotting), and a reversing agent for Class A is added to a patient blood sample containing the Class A drug, an increase in platelet and / or fibrin signal would be expected for that reaction. More specifically, if a second reaction is performed in which a Class B reversing agent is added to a patient blood sample containing the Class A drug, an increase in platelet and / or fibrin signal would not be expected for that reaction (and vice versa). In combination, using two separate reactions using reversing agents A and B, simultaneous data will accurately identify which class of drug a patient is using. In the absence of the drug, neither reversing agent will show any effect (positive or negative) on platelet function or coagulation. The systems or assays discussed herein will rely on a similar concept to identify one or more classes of drugs present in a patient blood sample.

[0028] IC50 Curves for Determining Drug Concentrations: In some embodiments, the systems or assays discussed herein can determine unknown drug concentrations by comparing the amount of inhibition found in a patient sample being evaluated against a known standard curve generated from a population of healthy donors analyzed by the device / system. In such embodiments, a series of healthy blood samples are treated with different concentrations of a drug that reduces platelet or fibrin function. A series of drug dilutions is generated across the expected range of clinically functional drug activity. This standard curve of drug activity can then be used by the system or assay to compare against the patient sample being evaluated. The amount of inhibition seen in the patient being evaluated is compared to the inhibition found in the standard curve. This percent inhibition is then related to the drug concentration that demonstrated the same level of inhibition in a healthy population. Figure 41 provides an example of an IC50 curve that could be used in the described manner to determine drug concentrations.

[0029] Methods for Determining Clinical Cutoffs for Specific Drugs: In some embodiments, the system can determine useful clinical values ​​by discriminating whether a drug concentration is above or below a specific threshold. This is important because there are clinical guidance documents that promote the use of reversal medications in the event that a patient exceeds a specific threshold for clot inhibitors. Statistical methods such as binary logistic regression or classification and regression tree (CART) discriminant analysis can be used by the system. In such embodiments, a set of donor samples (spiked samples or patient samples) can be evaluated using a true analytical approach, such as liquid chromatography / mass spectrometry (LCMS) analysis, to determine the actual concentration of the drug as an analyte. Drug concentrations are then classified against a binary criterion (below threshold and above threshold) compared to a threshold concentration. Binary logistic regression of the device's fibrin and platelet signals under various conditions is used to derive a regression equation that is used to classify samples containing unknown amounts of drug compared to a threshold concentration. In a similar approach, CART classification utilizes various fibrin and platelet signals from the device to determine or create an algorithm used to classify unknown amounts of drug compared to a threshold concentration. This approach allows for the use of simultaneously analyzed fibrin and platelet (and potentially other) signals of interest to improve agreement between the LCMS and the test device and therefore the ability to determine unknowns.

[0030] Statistical Methods for Determining Drug Concentration: In some embodiments, the system is capable of more precisely determining drug concentration. Statistical methods such as regression (e.g., linear regression) or classification and regression tree (CART) regression analysis can be used. In such embodiments, a set of donor samples (spiked samples or patient samples) can be evaluated using a true analytical approach, such as liquid chromatography / mass spectrometry (LCMS) analysis, to determine the actual concentration of the drug as an analyte. Using regression with device fibrin and platelet signals under various conditions, the system derives a regression equation that provides an estimate of the actual drug concentration. In a similar approach, CART regression utilizes various fibrin and platelet signals from the device to determine or create an algorithm used to calculate the estimated concentration of the drug in the sample. This approach allows for the use of simultaneously analyzed fibrin and platelet (and potentially other) signals of interest to improve agreement between the LCMS and the test device and, therefore, the ability to determine unknowns.

[0031] In some embodiments, it may be advantageous to utilize a regression equation for each specific drug that can be run through the system. For example, Figure 50A shows the results of a regression analysis for each drug based on its own unique clinical and LC-MS / MS data. The data uses the unaltered and fully inverted log10 fibrin signal as a continuous variable and the drug identity (apixaban (A), dabigatran (D), or rivaroxaban (R)) as a categorical variable using log10 concentration. Each equation is based on data for each of the three specific drugs. By comparison, when only drug class is used as the categorical input to predict concentration as log10, the result is only two equations (i.e., two drug classes), with poor correlation demonstrated by the Xa regression equations for the drugs apixaban and rivaroxaban (see Figure 50B). When analyzing the same data (unknown drugs but known drug classes) using CART regression, correlation is evident (similar to the individual drug equations) (see, e.g., Figure 50C).

[0032] Using the identity of the unknown specific drug and the DOAC class determined by the exemplary system / device, the average accuracy was 22.8% using the regression method to determine the concentration and 12.0% using the CART regression method. When the specific drug was known, the average accuracy was 9.9%. This ability to select for accuracy is useful for several reasons. If the clinician knew the exact drug the patient was taking, they could select for the individual drug regression model that best fits that drug, thereby resulting in greater accuracy in determining the patient's drug concentration. This ability to select the regression model can most easily be achieved through a GUI interface and associated system software. When the drug is unknown to the clinician, the CART regression method can generate quantitative or semi-quantitative drug concentration results. This shows that in CART, the CART model specifically moderates individual Xa drug behavior, thereby allowing drug type to have a much lower impact than it does in conventional regression. In all cases, the drug class is determined by the reversal agent of the DOAC test.

[0033] Methods for assessing platelet function and coagulation can be performed by systems in several ways but should utilize flowing blood. Conventional systems often utilize restrained blood samples with minimal or controlled agitation (e.g., stirring) to measure thrombus formation function. However, these methods cannot distinguish individual coagulation activity from platelet function and may utilize fractionated blood products (e.g., plasma) that are only surrogates for whole blood samples and do not mimic the effective behavior of blood in vivo. The very act of fractionating blood into its components also creates an artificial environment that differs from that found in the body. Therefore, platelet activity and coagulation pathways in this context are not representative of in vivo blood behavior or the biochemical distribution of reactants found in the body. Other conventional systems that attempt to analyze flowing blood generally do not attempt to replicate the body's actual blood flow characteristics, resulting in assays that are not representative of blood activity within the body. Conventional devices that attempt to replicate blood flow often do not attempt to simultaneously assess both platelet function and coagulation, providing an incomplete answer to overall hemostatic function. Therefore, the ability to simultaneously and accurately determine platelet and coagulation function under physiological conditions is absolutely crucial for assessing hemostatic function in real time. The systems discussed herein provide such determinations.

[0034] Several important in vivo characteristics determine blood behavior as they relate to platelet function and coagulation: (i) shear rate at the surface of the reaction zone; (ii) viscosity; (iii) temperature; (iv) blood flow / volume; (v) surface area of ​​the injury site; (vi) hematocrit; (vii) platelet number and function; (viii) fibrinogen level; and / or (ix) coagulation cofactor levels. For example, platelets have more than 10 different classes of receptors on their surface that play multiple roles in platelet function (see, e.g., Saboor, M. et al., Platelet receptors; an instrumental of platelet physiology, Pak. J. Med. Sci., 29(3): 891-896 (May-June 2013)). The number of receptors, the distribution of classes, and the overall function of the receptors all play a role in overall platelet function, which is influenced by blood flow and reactant delivery to platelets at binding sites. Coagulation is driven by numerous cofactors, many of which are delivered to (and removed from) the clotting site by the bloodstream (and subsequent diffusion into and through the clot). These clotting factors can also be influenced by genetic variation and their overall concentration in the blood (as many are produced in the liver), and are mediated by many types of drugs, foods, and hormones, to name a few. Thus, the interaction between platelets and clotting factors at the site of clot formation is dynamic, with diffusion influenced by the bloodstream and directly affected by the distribution / concentration of blood components (such as fibrinogen).

[0035] Platelet hemodynamic properties also affect hemostasis: platelets, in particular, are affected by hemodynamics. Platelets are generally 1–3 microns in size, compared to biconcave red blood cells (RBCs), which are roughly 7–8 microns in diameter. Due to RBC dynamics, platelet function is influenced by hemodynamic processes in which RBCs exclude platelets from the center of flowing blood and push them toward the vessel wall (see, e.g., Sugihara-Seki, M. et al., Margination of Platelet-Sized Particles in the Red Blood Cell Suspension Flow through Square Microchannels, Micromachines, 12, 1175 (2021) (https: / / doi.org / 10.3390 / mi12101175)). This specifically increases platelet interaction with the vessel wall (or device microfluidic channel surfaces). Therefore, a device that could also provide fundamental blood measures of hematocrit and platelet count (as well as other measures) would be highly beneficial for understanding assay performance with respect to platelet and fibrin signals.The systems discussed herein could provide such measures by spectroscopic analysis of blood in the fluid path, using published methods for determining hematocrit, oxy / deoxyhemoglobin, and even platelet count (see, e.g., Lipowsky, H. et al., Hematocrit determination in small bore tubes by differential spectrophotometry, Microvascular Research, Vol. 24 (1), pp. 42-55 (1982); Mattley, Y. et al., Light scattering and absorption model for the quantitative interpretation of human blood platelet spectral data, Photochem. Photobiol., Vol. 71 (5), pp. 610-619 (2000); Kitamura, Y. et al., Spectrophotometric determination of platelet counts in platelet-rich plasma, International Journal of Implant Dentistry, Vol. 4 (29) (2018)).

[0036] Devices and Methods for Inducing Platelet and Fibrin Signals: Generally, blood should flow across the reaction zone at an initial wall shear rate that is physiological. This has a wide range based on vessel diameter and blood pressure, but is generally between 100 and 500 s for venous blood flow. -1 and much higher s for artificial blood flow -1Higher wall shear stresses affect a variety of biological parameters. For example, platelet binding at high arterial shear is directly affected by von Willebrand factor (vWF), which has little to no effect at venous shear. vWF is a critical component of hemostasis, and known diseases affect vWF function. Consequently, assay sensitivity to such factors can be "tuned" by altering the assay's operating shear rate. While vessel diameters span a wide size range (from 1 cm for large arteries to several microns for capillaries), there are practical limits to what can be achieved from an assay perspective in terms of creating a physiologically representative model of hemostasis. If the sample is too small, the assay will be easily clogged by particulate debris, difficult to manufacture, and / or will generate significant backpressure. If the sample is too large, the blood volume required for the assay will be impractical. For a typical blood draw, 1–3 mL of blood is common and reasonably comfortable for the patient. This translates to flow devices with miniature vasculature-like channels on the order of tens to hundreds of microns in cross-section.

[0037] While the vasculature in the body is cylindrical, platelet and fibrin behavior for blood flow through a high-aspect-ratio rectangular channel approximates planar Poiseuille flow through an infinite parallel plate. A channel of this size allows 500 μL of blood to provide clot formation times of up to 15 minutes or more, more than sufficient for analyzing platelet and fibrin behavior. The clot zone of the device can be numerous in size, from only a few microns wide to several millimeters long. From a practical standpoint, for example, 100 to 500 microns (inclusive), 100 to 450 microns (inclusive), 100 to 400 microns (inclusive), 100 to 350 microns (inclusive), 100 to 300 microns (inclusive), 100 to 250 microns (inclusive), 100 to 200 microns (inclusive), 100 to 150 microns (inclusive), 150 to 500 microns (inclusive), 200 to 500 microns (inclusive), 250 to 500 microns A thrombosis zone of 100-500 microns (inclusive), 300-500 microns (inclusive), 350-500 microns (inclusive), 400-500 microns (inclusive), 450-500 microns (inclusive), 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns, 400 microns, 450 microns, 500 microns, etc., allows for sufficient signal accumulation over a 15-minute assay period. The thrombosis zone can be applied to all sides of the flow channel (top, bottom, left, right), but if applied to only one side of a geometric flow channel (e.g., rectangular), it may result in a completely occluding clot. Thus, in some embodiments, the thrombosis zone can be applied to, for example, the top side of the flow channel, the bottom side of the flow channel, the left side of the flow channel, the right side of the flow channel, or a combination thereof. While rectangular flow channels are practical to manufacture, alternative flow channel cross-sectional shapes (e.g., semicircular) are also contemplated. The thrombus formation zone can include one reactive site, or it can include multiple reactive sites within the same flow area and in close proximity to one another. Detection of platelet and fibrin accumulation can be accomplished optically (e.g., direct fluorescence) or using any number of additional methods available to those skilled in the art.

[0038] In some embodiments, the device can include surface modifications of the flow channels / fluidics (e.g., biologics, proteins, silanes, chemicals, combinations thereof, etc.) to passivate the surface to avoid activation or inadvertent reaction with the blood sample. In other embodiments, the surface modifications of the flow channels / fluidics include surface modifications to better mimic the in vivo properties of the body (e.g., coating the surface with fatty acids to mimic cell membranes or to literally grow endothelial cells within the device to form pseudo-tissue-like flow channels).

[0039] It is possible to use the device to determine whether drugs that may affect platelet function or coagulation are present in the blood using a limited number of independent reactions. In addition, the concentration of drugs that affect platelet or fibrin function can also be determined using a limited number of independent reactions. In a preferred embodiment, even classes of drugs that affect platelet or fibrin function can be determined using a limited number of independent reactions.

[0040] Platelet and fibrin detection: To determine the status of platelet and fibrin function, platelet and fibrin labels are applied to the blood sample being processed. This allows for simultaneous detection of platelets and fibrin in the same thrombus formation zone of the device. Of course, this does not preclude using either label independently if desired, but to assess both fibrin and platelet function together, both labels are applied to the same sample at the same time. Only a short incubation period is required to label the platelets, and no incubation period is required for fibrinogen. For example, the platelet label could be an antibody against human CD61 (a platelet integrin) conjugated with Alexa 488. For example, the fibrin label could be human fibrinogen conjugated with Alex 594. Fibrinogen-594 is added to the blood sample as a small percentage of the existing native fibrinogen and subsequently incorporated into the forming clot as part of the total fibrin. Alternative detection methods are anticipated that utilize the fundamental functional biochemistry of clot formation. Alpha 2-antiplasmin (A2-APF) is a protein that interferes with fibrinolysis and is naturally cross-linked to fibrin during clot formation (see, e.g., Liu, Y et al., Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow, Res. Pract. Thromb. Haemost., 7;8(1):102291 (December 2023), doi: 10.1016 / j.rpth.2023.102291, PMID: 38222077, PMCID: PMC10787300). Fluorescently labeled A2-APF would be incorporated into the growing clot simultaneously with fibrin, providing an additional means for monitoring fibrin formation.

[0041] In some cases, plasma may be useful for analysis instead of whole blood. Plasma lacks the cellular components of whole blood, and the addition of thickeners, freeze-dried platelets, and other blood components to plasma can be added to create a material that can be analyzed by a device. In some cases, blood (or plasma) samples can be pretreated with chemicals to avoid complications of platelet function and coagulation caused by blood collection and handling, such as contact activation. For example, corn trypsin inhibitor (CTI) is a small protein found in the kernels of most corn species. CTI is not only an inhibitor of trypsin but also a specific inhibitor of human factor XIIa. The inhibitor forms a one-to-one complex with either trypsin or factor XIIa and, when added to blood or plasma, prolongs the activated partial thromboplastin time (ACTT) without affecting the PT assay. Its specificity for factor XIIa makes it useful for the isolation and study of tissue factor (TF)-dependent coagulation reactions.The use of CTI to study TF-dependent responses has been reported in the literature (e.g., Rand, MD et al., Blood clotting in minimally altered whole blood, Blood, Vol. 88 (9), pp. 3432-3445 (1996); Cawthern, KM. et al., Blood coagulation in hemophilia A and hemophilia C, Blood, Vol. 91 (12), pp. 4581-4592 (1998); Dargaud, Y. et al., Platelet-dependent thrombography: a method for diagnostic laboratories, British Journal of Hematology, Vol. 134 (3), pp. 323-325 (2006); Mann, KG et al., Citrate anticoagulation and the dynamics of thrombin generation, Journal of Thrombosis and Hemostasis, Vol. 5 (10), pp. 2055-2061). (2007).

[0042] Studies have shown that inhibition of the contact pathway of coagulation is essential when attempting to perform TF-dependent assays in whole blood or plasma samples. Addition of CTI at the time of sample collection prevents activation of the contact pathway during subsequent sample processing steps, thus reducing in vitro artifacts. The most common use of CTI is associated with thrombin generation assays when attempting to work with low TF concentrations. Additional chemicals can be used to alter blood and plasma behavior prior to and during use within the device. Common examples of such chemicals are the rapid thrombin inhibitor chloromethylketone (FPRCK; commonly referred to as Phe-Pro-Arg-chloromethylketone; PPACK) and the rapid factor Xa inhibitor EGRCK (Glu-Gly-Arg-chloromethylketone; commonly referred to as GGACK). Both FPRCK and EGRCK are widely used during protein isolation procedures to inhibit serine protease activity and prevent further conversion of zymogens to activate enzymes.

[0043] FIG. 42 shows various possible configurations of exemplary device flow channels. It should be understood that the flow channels shown in FIG. 42 can be used in independent / separate devices, or multiple flow channels can be incorporated into a single device with any combination of single or multiple clot sites with the same or different TF concentrations. Single or combined devices can utilize any combination of clot sizes and TF concentrations. In some embodiments, the flow channels can include a single reaction chamber and a single independent flow channel (see FIG. 42, flow channels 1-3). For example, flow channel 1 in FIG. 42 includes one independent flow channel with one small clot site and one TF concentration. Flow channel 2 in FIG. 42 includes two independent flow channels with one large clot site and one TF concentration. Flow channel 3 in FIG. 42 includes three independent flow channels with three small clot sites and one TF concentration. In some embodiments, the flow channels can utilize more than one concentration of TF. For example, flow channel 4 in FIG. 42 includes one independent flow channel with clot sites with different TF concentrations located in series.

[0044] In other embodiments, a flow path can have two or more flow paths containing independent reactions (see Figure 42, Flow Paths 5-6). Flow Path 5 in Figure 42 contains two separate flow paths and two different TF concentrations in parallel. Flow Path 6 in Figure 42 contains two separate flow paths with two reaction zones on separate planes (z-axis) containing two different non-overlapping (z-axis) regions with different TF concentrations in parallel. Each flow path depicted in Figure 42 contains a reaction chamber fluidly connected to a reaction zone via a flow path, and the reaction zone contains one or more clot sites. The clot sites can have the same or different TF concentrations. The flow paths lead from the reaction zone to waste. Flow Path 5 in Figure 42 contains two separate, parallel flow paths with reaction zones on the same plane (along the z-axis) but with clot sites in overlapping positions. Flow Path 6 in Figure 42 contains two separate, parallel flow paths with reaction zones on different planes (along the z-axis) but with clot sites in a non-overlapping configuration.

[0045] In some embodiments, the independent flow paths can be connected to a common sample entry point to simplify the addition of the blood sample. In some embodiments, this common entry point can also include a common reagent chamber to facilitate interaction of the blood sample with reagents common to all reactions (e.g., CTI, fibrin label, platelet label, combinations thereof, etc.). In some embodiments, each independent flow path can include a separate reagent and / or mixing chamber where reagents specific to each flow path can be added, stored, and / or mixed with the blood sample. In some embodiments, the flow paths can be simultaneously connected to a priming circuit, whereby pressure applied to the priming circuit can force fluid into the device's flow paths, blocking surfaces and removing air (avoiding trapped air bubbles). The blood sample to be tested can be first mixed with CTI, subsequently labeled with platelet label and fluorescent fibrinogen in the sample chamber, and mixed with specific drugs or reagents in the same or additional reagent / mixing chambers. This mixture can then be drawn through the device (under vacuum or pressure) and across the reaction zone. A single thrombus formation site can provide both platelet and fibrin signals.

[0046] To provide averaging of platelet and fibrin signals, two or more reaction zones or thrombus formation sites can be included in the device (see, e.g., Figure 42). The thrombus formation sites can be of different sizes to facilitate larger or smaller reactive surface areas. For example, a single flow channel can split into two or more independent thrombus formation sites with independent or converging outlets. In some embodiments, the device can include independent flow channels and thrombus formation sites on different layers of the device, thereby creating multi-dimensional flow channels (in the x, y, and z planes).

[0047] Figure 51 shows additional diagrammatic representations of exemplary microfluidic device configurations. Pressure can be applied to the inlet to push blood through the device, vacuum can be applied to the outlet to draw blood through the device, or a combination of pressure and vacuum can be applied to move / oscillate blood back and forth through the device. The device can include a blood entry chamber (which in some embodiments includes a CTI) that serves as an inlet for the blood. The blood entry chamber is fluidly connected to the sample chamber by a flow path. Each device or flow path includes a reagent and mixing zone located upstream of a reaction zone with a clotting site. One or more clotting sites can be present in the reaction zone. Downstream of the reaction zone, the device / system includes a priming pump configured to apply positive pressure to the flow path. Each flow path can be independent and leads to downstream waste, which can optionally be under negative pressure (vacuum). By way of example, the device of Figure 51 can have patient unspiked blood, patient blood with Andexxa, patient blood with Praxbind, and patient blood with excess rivaroxaban, with the reagents located in the reagent and mixing zones.

[0048] Figures 52 and 53 show additional graphical representations of exemplary microfluidic device channel configurations. In particular, Figure 52 is a diagram of a microfluidic device channel including a multilayer structure with two unique reaction zones, and Figure 53 is a diagram of a microfluidic device channel including a single-layer structure with two unique reaction zones. While it is technically possible to have two reaction zones in the same channel, there may be technical difficulties in being able to do so (e.g., upstream and downstream reactive zones that conflict with each other). The channels in Figures 52 and 53 can provide parallel flow paths in three or two dimensions to allow for multiple reaction zones within the same device. For example, the device in Figure 52 includes layer 300 with channel 302 and reaction zone 304, and vertically offset layer 306 with channel 308 and reaction zone 310. The three-dimensional configuration allows for two reaction zones offset in the z-axis direction on separate planes. As a further example, the device of FIG. 53 includes a single layer 320 with channels 322, 324 separated in a two-dimensional manner, each channel 322, 324 containing its respective reaction zone 326, 328.

[0049] Multilevel drug sensitivity: While fluidic devices can be fabricated with a single reaction zone that has a specific level of reactivity toward platelet function and coagulation, it may be advantageous to have a device with two or more reaction zones, each with a different level of reactivity. For example, tissue factor (TF) is a primary activator of the coagulation process, such that a given amount of TF bound within a reaction zone will produce a specific coagulation response to any given sample. Collagen concentration can similarly be varied to different levels to alter platelet binding and activation behavior. This means that creating a second reaction zone containing different levels of TF and / or collagen will naturally result in different levels of reactivity to a given sample. This is useful, for example, when attempting to detect drugs that inhibit one or more reactions in the coagulation cascade. Different levels of tissue factor in the reaction zone can cause different rates of production of the molecular target of a particular anticoagulant, which in turn can alter the apparent activity of a given dose of the drug. For DOACs, two TF concentrations can be selected so that one TF concentration in the reaction zone is sensitive to low levels of the DOAC drug (e.g., <100 ng / mL or <50 ng / mL) and the second TF concentration in the second reaction zone is more sensitive to mid- to high-dose DOAC drugs (>100 ng / mL or >200 ng / mL). This is useful, for example, in overcoming limitations in assay linearity or sensitivity, where the reaction kinetics at a given TF concentration simply do not yield measurable differences between similar drug levels. By having two distinct reaction zones with different TF concentrations, linearity and sensitivity can be maintained over a wide range of biologically relevant drug concentrations.

[0050] Multiple reaction zones within the same device can be utilized to provide different degrees of reactivity to different biological contexts, such as different drug levels. For example, these different reaction zones can be arranged all within the same device, consecutively within the same blood stream, side-by-side within the same plane where the same blood sample is split between two separate flow paths each intersecting a unique reaction zone, or even dimensionally separated where one flow path / reaction zone is located on a separate fluidic layer compared to the first (see Figure 42).

[0051] To determine the presence of a single drug using a reversing agent, the device would require at least two independent reaction chambers (where the blood and reagents are mixed together), two independent reaction zones, and two independent clot formation sites. One independent flow path with an independent clot formation zone contains a blood sample containing platelets and fibrinogen markers. A second independent flow path with an independent clot formation zone contains a blood sample containing platelets and fibrinogen markers and a reversing agent for the drug in question. Figure 42 provides a diagram of such a microfluidic device for determining the presence of a single drug using a reversing agent. The device includes reaction chambers fluidically connected to each reaction zone and to a waste. The first reaction zone would identify the state of platelets and coagulation function. The second reaction zone would identify the blood sample's response to the reversing agent via fibrin and platelet signals at the second clot formation site. In all cases, both flow paths would produce both fibrin and platelet signals.

[0052] To determine the concentration of a drug, the device would include at least three independent flow paths. One independent flow path, comprising the thrombus formation zone, would contain a blood sample containing platelets and fibrinogen markers. The second flow path, comprising the thrombus formation zone, would contain a blood sample containing platelets and fibrinogen markers and a reversal agent for the drug in question. The third flow path, comprising the thrombus formation zone, would contain a blood sample containing platelets and fibrinogen markers and a supratherapeutic dose of the drug in question that completely attenuates the fibrin signal. The first flow path provides a general assessment of the sample's platelet and fibrin function; the second flow path identifies the presence of the drug and provides a maximum signal (either fibrin or platelets) without the effect of an inhibitor drug; and the third flow path provides a minimum signal (fibrin or platelets) with the saturable effect of an inhibitor drug. Generally, since the majority of drugs target either the coagulation pathway or platelet function, but not both, it would be expected that either the platelet signal or the fibrin signal would be most affected. However, in all cases, because the device provides both fibrin and platelet signals, both data points from each reaction zone can be used for analysis, and therefore patients receiving both types of drugs could be evaluated.

[0053] If drug class also needs to be determined, the device can include a minimum of four independent reaction zones and flow paths. One independent flow path with an independent thrombus formation zone contains a blood sample containing platelets and fibrinogen markers. A second independent flow path with an independent thrombus formation zone contains a blood sample containing platelets and fibrinogen markers and a reversing agent for drug A of interest. A third independent flow path with an independent thrombus formation zone contains a blood sample containing platelets and fibrinogen markers and a reversing agent for drug B of interest. A fourth independent flow path with an independent thrombus formation zone contains a blood sample containing platelets and fibrinogen markers and a supratherapeutic dose (molar excess) of one of the drugs of interest. The results would be similar to those discussed above for the three-flow path device, except that an additional data point would be present: a comparison of the effects of the two reversing agents to identify which class of drug was present (thus identifying the drug class for which there is a response to one reversing agent). This methodology is broadly applicable to any drug that affects platelet function and / or coagulation and has a specific reversing agent for that drug that can be used ex vivo.

[0054] Direct Oral Anticoagulant (DOAC) Specific Testing In some embodiments, the methodology for the system or device can include a comparison of the patient's blood to the same blood interactions with: (1) the patient's blood behavior in the presence of a reversal agent for the drug in question; or (2) the patient's blood behavior in the presence of a molar excess of the suspected drug, where behavior refers to the characteristic platelet and coagulation response of the sample (although other responses are possible).

[0055] For example, a drug that affects coagulation would be expected to attenuate a patient's fibrin signal from "normal." Adding a reversing agent for that drug to a patient's blood sample prior to analysis would be expected to increase the patient's fibrin signal into the "normal" range, provided no other complicating factors are present. This simple process, using simultaneously the patient's unaltered blood and patient blood treated ex vivo with a reversing agent and a molar excess of the same drug, will identify the presence and relative activity of the drug for each particular patient, as described above. That is, the patient's own blood is used as a control in the device to determine behavior for different drugs.

[0056] Detection of DOAC Drugs as an Exemplary Diagnostic Device: In particular, the method provides a means for detailed and accurate assessment of whether a patient is taking a DOAC to determine whether clinical therapy to reverse DOAC activity is necessary for the patient's appropriate treatment. The method provides rapid and more accurate hemostasis testing (compared to conventional systems), particularly during the critical 10-20 minute triage window, and can be used to improve early bleeding risk assessment, stabilization, and transitions of care, promoting evidence-based transfusion, blood product utilization, and costly DOAC reversal. The method provides results that can be used by medical professionals to reduce the overall cost of patient care while adhering to existing industry quality and patient safety guidelines.

[0057] According to an embodiment of the present disclosure, an exemplary system for direct-acting oral anticoagulant (DOAC) detection and quantification is provided. The system includes a first inlet, a second inlet, a third inlet, and a fourth inlet, each configured to receive a fluid sample. In some embodiments, the system can include more than four inlets, such as eight inlets. The system includes an outlet and a microfluidic channel fluidically connecting each of the first, second, third, and fourth inlets to the outlet. The unmodified sample can be labeled on or off the device prior to use. The labeling consists of a platelet-specific label and a fibrin-specific label. Introduction of the labeled sample into each of the first, second, third, and fourth inlets generates fibrin and platelet signals. An additional reversal drug is used specifically and independently to identify the class of DOAC drug (either Xai or DTi) present in the sample by reversing the action of the DOAC drug and thus generating a fully restored fibrin signal (e.g., including an Xai reversal drug at inlet 2 and a DTi reversal drug at inlet 3). A molar excess of inhibitors (either Xai, DTi, or both) provides a completely attenuated fibrin signal at inlet 4. Although DOAC drugs do not directly target platelet function, it is expected that platelet signaling at each inlet will also be affected. This is because platelets are affected by the activity of thrombin, which is affected by DOAC drugs either directly (DTi) or indirectly (Xai). Thus, there is an indirect effect on platelets via the coagulation pathway, specifically, the formation of thrombin.

[0058] The fluid sample may be, for example, a raw blood sample, a citrated blood sample that is recalcified, a heparinized blood sample that is treated with protamine, etc. If a DOAC is present in the patient blood sample, the fibrin signal, the fully reversed fibrin signal (by class), and the corresponding simultaneous platelet signal at each inlet can be used to determine the presence and concentration of the DOAC in the sample. The fibrin signal, the fully reversed fibrin signal, the fully attenuated fibrin signal, and the corresponding simultaneous platelet signal can be used to determine the DOAC concentration or "level" in the fluid sample by multiple mathematical methods. In some embodiments, the first inlet can be configured to receive the fluid sample in an unmodified form, the second inlet can be configured to receive the fluid sample and a Xai reversing agent, the third inlet can be configured to receive the fluid sample and a DTi reversing agent, and the fourth inlet can be configured to receive a fluid sample containing a molar excess of a Xai inhibitor (fully suppressed signal). The drug and sample can be combined either externally or internally.

[0059] The system can include a processing instrument configured to manipulate the sample within the device and simultaneously monitor the direct response of the fluid sample to the reversal agent to identify the DOAC class present in the fluid sample. In some embodiments, the DOAC class can be factor Xai or factor IIa (DT1). In some embodiments, the instrument can include a light source for monitoring clot development in the microfluidic channel. A green fluorescent signal can indicate platelet formation during clot development, and a red fluorescent signal can indicate fibrin formation during clot development. The processing instrument can be configured to measure the fluorescence intensity of the monitored clots as they develop and correlate the measured fluorescence intensity with platelet and fibrin accumulation in the microfluidic channel. The processing instrument can be configured to compare the fibrin signal with a fully reversed / restored fibrin signal and a fully attenuated fibrin signal, and to compare the simultaneous platelet signal for all reactions, to determine the DOAC concentration level in the fluid sample.

[0060] According to an embodiment of the present disclosure, an exemplary method for direct-acting oral anticoagulant (DOAC) detection and quantification is provided. The method includes adding a fluid sample to a first inlet, a second inlet, a third inlet, and a fourth inlet of an assay device. The assay device includes an outlet and a microfluidic channel fluidically connecting each of the first, second, third, and fourth inlets to the outlet. In some embodiments, the assay device may include a separate priming inlet for prefilling the device, or priming could be accomplished via the outlet. The method includes generating fibrin and platelet signals for each of the inlets. The method includes identifying the overall state of coagulation and platelet function from an unaltered sample (inlet 1) and the class of DOAC drug (either Xai or DTi) present in the sample by evaluating the fibrin signal using Xai and IIai reversing agents at inlets 2 and 3, respectively. A molar excess of Xa or IIa inhibitors provides a fully attenuated fibrin signal at inlet 4. In all cases, a simultaneous platelet signal is also generated for each inlet.

[0061] It should be noted that patients are typically not prescribed both Xai and DTi drugs at the same time, so even if a patient is administered a DOAC, only one reversal agent is expected to produce a response. However, the assay will still be able to generate a response even if both classes of drugs are present. The presence and class of DOAC can be detected from the same data.

[0062] The method can include providing a light source to a microfluidic channel to monitor clot development in the microfluidic channel. The method can include an instrument receiving measured fluorescence intensities of monitored clot development as input to a processing device. The method can include correlating the measured fluorescence intensities with platelet and fibrin accumulation in the microfluidic channel using the instrument. The method can include comparing the fibrin signal with a fully reversed fibrin signal, a fully inhibited fibrin signal, and a simultaneous platelet signal using the processing instrument. The fibrin and platelet signals provide information about the general state of platelet function and fibrin activity in an unmodified sample, as well as specific information about the sample's responsiveness to Xai and DTi reversing agents and Xa and / or DTi inhibitors. In some embodiments, platelet and fibrin function can be determined by comparing it to a population average. DOAC class and concentration can be determined by comparing unmodified sample function with chemically or drug-modified sample function.

[0063] According to embodiments of the present disclosure, an exemplary system for direct-acting oral anticoagulant (DOAC) detection and quantification is provided. The system includes an optical instrument with computing capabilities and an assay device capable of receiving a biological sample and further capable of receiving one or more chemical reagents. Introduction of the biological sample and one or more chemical reagents into the assay device results in a biological process in which fibrin and platelets accumulate in a reaction zone of the assay device. The system includes a fluorescent assembly capable of detecting the biological process by fluorescent labeling and detecting the resulting accumulating fluorescent signal. The accumulated fibrin and platelets in the reaction zone of the assay device and the accumulating fluorescent signal can be used to determine at least one of the presence, class, level, or concentration of a DOAC in the biological sample. In some embodiments, the system can be used to detect and quantify any type of drug that affects platelet and fibrin behavior, thereby determining at least one of the presence, class, level, or concentration of a drug in the biological sample. In some embodiments, the system can be used specifically for the detection and quantification of DOACs.

[0064] In some embodiments, the assay device can include a first inlet, a second inlet, a third inlet, and a fourth inlet, each configured to receive a biological sample. The biological sample is an unmodified sample containing a platelet-specific label and a fibrin-specific label. The assay device can include an outlet and a microfluidic channel fluidically connecting each of the first, second, third, and fourth inlets to the outlet. Introduction of the biological sample into each of the first, second, third, and fourth inlets generates a fibrin signal and a platelet signal.

[0065] At least one of the first, second, third, and fourth inlets can be configured to receive a reversal drug to identify the class of DOAC present in the biological sample. The reversal drug inhibits, antagonizes, or attenuates the activity of the Xai or DTi DOAC. The reversal drug reverses the action of the DOAC drug and generates a fully restored fibrin signal. In some embodiments, at least one of the first, second, third, and fourth inlets can be configured to receive a concentration of either the Xai or DTi drug that completely inhibits the fibrin signal, resulting in a completely attenuated fibrin signal.

[0066] In some embodiments, the system can include a processing device configured to receive the fibrin signal, the platelet signal, the fully recovered fibrin signal, and the fully recovered platelet signal as inputs and determine the concentration or level of a DOAC in the biological sample. In some embodiments, the first inlet can be configured to receive the biological sample in an unmodified form, the second inlet can be configured to receive the biological sample and a Xai reversing agent, the third inlet can be configured to receive the biological sample and a DTi reversing agent, and the fourth inlet can be configured to receive the biological sample containing a molar excess of an Xa or DT inhibitor.

[0067] The biological sample can include raw blood samples, citrated blood samples, or heparinized blood samples. In some embodiments, the system can include a processing device configured to process the biological sample and monitor the direct response of the biological sample to one or more reversal drugs to identify the DOAC class present in the biological sample. In some embodiments, the DOAC class can be factor Xai or factor IIai (DTi).

[0068] In some embodiments, the system can include a light source for monitoring clot development in the microfluidic channel and detecting a fluorescent reaction of one or more reagents. A green fluorescent signal can indicate platelet formation during clot development. A red fluorescent signal can indicate fibrin formation during clot development. In some embodiments, the system can include a processing device configured to receive as input the measured fluorescent intensity of the monitored clot development and further configured to correlate the measured fluorescent intensity with platelet and fibrin accumulation in the microfluidic channel. In some embodiments, the system can include a processing device configured to compare the fibrin signal with a fully restored fibrin signal, compare the fibrin signal with a fully attenuated fibrin signal, and compare simultaneous platelet signals for all reactions to determine the DOAC concentration or level in the fluid sample.

[0069] According to embodiments of the present disclosure, an exemplary method for direct-acting oral anticoagulant (DOAC) detection and quantification is provided. In some embodiments, the method can be used for the detection and quantification (e.g., drug presence, drug class, drug level relative to a threshold, drug concentration, etc.) of any drug that affects clot formation and has a reversal agent. Thus, the steps described herein can be used in a non-DOAC-specific manner. The method includes adding a biological sample to an assay device, adding one or more chemical reagents to the assay device to cause a biological process whereby fibrin and platelets accumulate in a reaction zone of the assay device, detecting the biological process using a fluorescent assembly by fluorescent labeling and detecting the resulting accumulating fluorescent signal, and using the accumulated fibrin and platelets in the reaction zone of the assay device and the accumulating fluorescent signal to determine at least one of the presence of the drug, drug class, drug level relative to a threshold, or drug concentration.

[0070] The method can include adding a biological sample to a first inlet, a second inlet, a third inlet, and a fourth inlet of an assay device. The assay device can include an outlet and a microfluidic channel fluidly connecting each of the first, second, third, and fourth inlets with the outlet. The biological sample can be an unmodified sample containing a platelet-specific label and a fibrin-specific label. The method can include generating a fibrin signal and a platelet signal from the biological sample for each of the first, second, third, and fourth inlets. The method can include determining a drug class and a drug concentration from the fibrin signal and the platelet signal.

[0071] In some embodiments, the method can include determining the overall state of coagulation from an unmodified sample by evaluating the fibrin signal and a fully restored fibrin signal obtained using a reversing agent. In some embodiments, the method can include determining platelet function from an unmodified sample by evaluating the platelet signal and a fully restored platelet signal obtained using a reversing agent. In some embodiments, the method can include using a Xai reversing agent at a second inlet and a DTi reversing agent at a third inlet. A molar excess of Xai or DTi DOAC at a fourth inlet can provide a fully attenuated fibrin signal. For example, the method can include receiving a concentration of the drug up to a point that does not result in further attenuation of the fibrin signal to obtain a fully attenuated fibrin signal.

[0072] In some embodiments, the method can include generating a fibrin signal and a simultaneous platelet signal for each of the first, second, third, and fourth inlets. In some embodiments, an unmodified sample fibrin and unmodified sample platelet signal can be generated from a microfluidic channel associated with the first inlet, a fully reversed fibrin signal and a simultaneous platelet signal can be generated from a microfluidic channel associated with either the second or third inlet by interaction with a Xai or DTi reversing agent, and a fully attenuated fibrin signal and a simultaneous platelet signal can be generated from a microfluidic channel associated with the fourth inlet.

[0073] In some embodiments, the method can include providing a light source to a microfluidic channel to monitor clot development in the microfluidic channel based on the fluorescent response of one or more reagents, receiving as input to a processing device the measured fluorescent intensity of the monitored clot development, and correlating the measured fluorescent intensity with platelet and fibrin accumulation in the microfluidic channel using the processing device. In some embodiments, the method can include comparing the fibrin signal, along with a simultaneous platelet signal, to a fully reversed fibrin signal and a fully inhibited fibrin signal to determine the DOAC concentration in the biological sample.

[0074] According to an embodiment of the present disclosure, an exemplary system for detecting and quantifying drug and / or chemical interactions with a biological sample is provided. The system includes a detection instrument having computing capabilities and an assay device capable of receiving a biological sample. Introduction of the biological sample into the assay device results in a biological process that may result in the accumulation of fibrin and platelets in a reaction zone of the assay device (e.g., both fibrin and platelets may accumulate in the reaction zone). The assay device is capable of receiving one or more chemical reagents that are compatible with the biological sample and usable to detect the accumulation of fibrin and platelets in the reaction zone. The assay device is capable of receiving one or more drug reagents that are compatible with the biological sample and usable to alter the accumulation of fibrin and platelets in the reaction zone. The accumulated fibrin and platelets in the reaction zone of the assay device and their associated signals can be used to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration in the biological sample.

[0075] The system can include a fluorescent assembly capable of detecting a biological process by fluorescent labeling and detecting the resulting accumulating fluorescent signal. The system can include a processing device configured to receive as input measurements of fibrin and platelets to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration within the biological sample, and the processing device can be configured to correlate the measured fluorescent intensity with the accumulation of fibrin and platelets in the microfluidic channels of the assay device.

[0076] The one or more chemical reagents or the one or more drug reagents can be fluorescent reagents capable of labeling fibrin and platelets from the biological sample, resulting in a fluorescent assembly that reports the accumulation of fibrin and platelets. The system can include a light source for monitoring clot development in the microfluidic channels of the assay device and for detecting the fluorescent response of the one or more chemical reagents or the one or more drug reagents.

[0077] The assay device can include a first inlet, a second inlet, a third inlet, and a fourth inlet, each configured to receive a biological sample. The biological sample can be an unmodified sample containing a platelet-specific label and a fibrin-specific label. The assay device can include an outlet and a microfluidic channel fluidically connecting each of the first, second, third, and fourth inlets to the outlet. Introduction of the biological sample into each of the first, second, third, and fourth inlets generates a fibrin signal and a platelet signal. At least one of the first, second, third, and fourth inlets can be configured to receive the unmodified biological sample. At least one of the first, second, third, and fourth inlets can be configured to receive a molar excess of a drug to provide a fully attenuated fibrin or platelet signal.

[0078] At least one of the first, second, third, and fourth inlets can be configured to receive a first reversal drug to identify a first class of drug present in the biological sample. At least one of the first, second, third, and fourth inlets can be configured to receive a second reversal drug to identify a second class of drug present in the biological sample. The first or second reversal drug reverses the action of a drug or chemical that attenuates fibrin or platelet signals in the biological sample to generate a fully restored fibrin or platelet signal. The drug can be a direct-acting oral anticoagulant (DOAC), and the first and / or second reversal drug inhibits, antagonizes, or attenuates the activity of a Xai or DTi class DOAC. The drug can be an antiplatelet medication, and the first and / or second reversal drug inhibits, antagonizes, or attenuates the activity of the antiplatelet medication.

[0079] The system can include a processing device configured to manipulate the biological sample and monitor the direct response of the biological sample to a first and / or second reversal agent to identify a drug class present in the biological sample.The system can include a processing device configured to manipulate the biological sample and monitor the direct response of the biological sample to a molar excess of a fibrin or platelet-depleting drug to identify a drug or chemical level or concentration present in the biological sample.

[0080] The biological sample can include a raw blood sample, a processed blood sample, a blood sample treated with an anticoagulant to prevent intrinsic pathway coagulation activation, a citrated blood sample that is recalcified, a heparinized blood sample that is treated with protamine, or a blood sample that is treated with an antiplatelet drug. The system can include a processing device configured to compare the fibrin or platelet signal to a fully restored fibrin or platelet signal, to a fully attenuated fibrin or platelet signal, and to compare all other simultaneous signals for all reactions to determine the presence, drug class, drug level relative to a threshold, or drug concentration in the biological sample.

[0081] In some embodiments, the reaction zone can include a single flow channel with separate clot sites in a serial configuration with different tissue factor (TF) concentrations. In some embodiments, the reaction zone can include two flow channels in parallel along the same plane, each with a different tissue factor (TF) concentration. In some embodiments, the reaction zone can include two flow channels on separate planes of the assay device, each with a clot site in a non-overlapping configuration with respect to each other and with a different tissue factor (TF) concentration. Increasing the reaction temperature in the reaction zone decreases the initiation time, increases the reaction rate, and provides a higher signal of fibrin and platelet accumulation within the reaction zone.

[0082] According to an embodiment of the present disclosure, an exemplary method for drug or chemical detection and quantification is provided. The method includes adding a biological sample to an assay device and adding one or more chemical reagents to the assay device to generate a biological process whereby fibrin and platelets may accumulate in a reaction zone of the assay device. The method includes detecting the biological process using a fluorescent assembly by fluorescent labeling and detecting the resulting accumulating fluorescent signal, and using the accumulated fibrin and platelets in the reaction zone of the assay device and the accumulating fluorescent signal to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration.

[0083] The method can include adding a biological sample to a first inlet, a second inlet, a third inlet, and a fourth inlet of an assay device. The assay device includes an outlet and a microfluidic channel fluidly connecting each of the first, second, third, and fourth inlets with the outlet. The biological sample can be an unmodified sample containing a platelet-specific label and a fibrin-specific label. The method can include generating a fibrin signal and a platelet signal from the biological sample for each of the first, second, third, and fourth inlets, and determining the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration from the fibrin signal and the platelet signal.

[0084] Biological samples include raw blood samples, processed blood samples, blood samples treated with anticoagulants to prevent intrinsic pathway coagulation activation, citrated blood samples that are recalcified, heparinized blood samples treated with protamine sulfate, or blood samples treated with antiplatelet drugs. In some embodiments, unmodified whole blood samples may be preferred, although other blood samples may be equally valuable. Considering common materials used for blood collection, citrated vacutainers and lithium or sodium heparin vacutainers may all provide useful samples, provided that the existing vacutainer anticoagulation is reversed prior to use in the assay. For example, citrate-anticoagulated blood can be reversed to near-normal clotting levels by the addition of calcium. Heparin-anticoagulated blood can be reversed to near-normal clotting levels through the addition of protamine sulfate (see, e.g., Figures 48A-48B). As long as the anticoagulation can be rapidly reversed to a state similar to that of whole blood, most reversibly anticoagulated samples can be utilized. By using a reversibly anticoagulated sample, useful results are still possible with modified blood samples, which helps solve the problem of inadvertent collection of blood samples into the "wrong" blood collection device and increases turnaround time by allowing additional time for testing the sample since clot formation is halted in the anticoagulated sample.

[0085] The method can include determining an overall state of coagulation from an unmodified sample by evaluating the fibrin signal and a fully recovered fibrin signal obtained using a reversal agent.The method can include determining an overall state of platelet function from an unmodified sample by evaluating the platelet signal and a fully recovered platelet signal obtained using a reversal agent.

[0086] The method can include using a Xai reversing agent at the second inlet and a DTi reversing agent at the third inlet. The method can include receiving a fibrin-attenuating drug at a concentration up to a point that does not result in further attenuation of the fibrin signal to obtain a fully attenuated fibrin signal. The method can include receiving a platelet-attenuating drug at a concentration up to a point that does not result in further attenuation of the platelet signal to obtain a fully attenuated platelet signal. The method can include generating a fibrin signal and a simultaneous platelet signal for each of the first, second, third, and fourth inlets. The unmodified sample fibrin signal and the unmodified sample platelet signal can be generated from a microfluidic channel associated with the first inlet, the fully reversed fibrin signal and the simultaneous platelet signal can be generated from either the microfluidic channel associated with the second or third inlet by interaction with the Xai or DTi reversing agent, and the fully attenuated fibrin signal and the simultaneous platelet signal can be generated from a microfluidic channel associated with the fourth inlet.

[0087] The method can include providing a light source to the microfluidic channel to monitor clot development in the microfluidic channel based on a fluorescent response of one or more reagents, receiving as input at a processing device a measured fluorescent intensity of the monitored clot development, and correlating the measured fluorescent intensity with platelet and fibrin accumulation in the microfluidic channel using the processing device. The method can include comparing the fibrin signal, along with a simultaneous platelet signal, to a fully reversed fibrin signal and a fully inhibited fibrin signal to determine a drug class or drug concentration in the biological sample.

[0088] Any combination and / or rearrangement of the embodiments is contemplated. Other objects and features will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the drawings are designed by way of illustration only and not as a definition of the limits of the present disclosure.

[0089] To assist those skilled in the art in making and using systems for DOAC-related data collection and analysis, reference is made to the accompanying drawings. [Brief explanation of the drawings]

[0090] [Figure 1A] FIG. 1 is an illustration of vasoconstriction following injury exposing collagen and tissue factor. [Figure 1B] FIG. 1 is an illustration of platelet plug formation accompanied by platelet aggregation. [Figure 1C] 1 is an illustration of clot formation in the form of coagulation. FIG. [Figure 2] FIG. 1 is a perspective view of a microfluidic device for monitoring blood biology under flow in an exemplary system for DOAC-related data collection and analysis. [Figure 3] FIG. 1 is a diagram of a microfluidic device for monitoring blood biology under flow in an exemplary system for DOAC-related data collection and analysis. [Figure 4] 1 is a flowchart illustrating the process of implementation of an exemplary system for DOAC-related data collection and analysis. [Figure 5A] FIG. 1 is a diagram of a graphical user interface of an exemplary system for DOAC-related data collection and analysis, including system results. [Figure 5B] FIG. 1 is a diagram of a graphical user interface of an exemplary system for DOAC-related data collection and analysis, including system results. [Figure 6] 1 is a table of sample data sets related to clinical decision points of interest. [Figure 7]1 is a table of sample detection for the 30 ng / mL decision point. [Figure 8] 1 is a table of sample detection for the 50 ng / mL decision point. [Figure 9] 10 is a table of performance characteristics for binary logistic regression and CART classification models. [Figure 10] 1 is a graph of the binary logistic regression for detecting samples greater than or equal to 30 ng / mL. [Figure 11] 1 is a graph of CART classification for sample detection of 50 ng / mL or greater. [Figure 12] 1 is a table of DOAC concentration predictions based solely on fibrin signals. [Figure 13] 1 is a table of DOAC concentration predictions based on both fibrin and platelet signals. [Figure 14] 1 is a graph of DOAC concentration predictions based on both fibrin and platelet signals using a CART regression model verses LC-MS / MS measured data, specifically, CART regression model predictions using fibrin, platelets, fully reversed fibrin, and fully reversed platelets, as compared to LC-MS / MS measured concentrations of apixaban or rivaroxaban. [Figure 15] Graph of DOAC concentration prediction based on both fibrin and platelet signals using Pearson correlation model verses LC-MS / MS measured data. [Figure 16] 1 is a table of patient data for apixaban and rivaroxaban. [Figure 17] 1 is a table of calibrators and controls for the LC-MS / MS assays for apixaban and rivaroxaban. [Figure 18] 10 is a graph of a fitted line plot of fibrin signal versus time of maximum difference between patient sample conditions and fully reversed patient sample conditions. [Figure 19]10 is a graph of a fitted line plot for the platelet signal versus the time of maximum difference between the patient sample condition and a fully inverted patient sample. [Figure 20] 1 is a graph of the receiver operating characteristic (ROC) curve for binary logistic regression for DOAC≧30 ng / mL including fibrin signal. [Figure 21] 1 is a graph of the receiver operating characteristic (ROC) curve for the binary logistic regression for DOAC≧30 ng / mL including fibrin signal, platelet signal, fully reversed fibrin signal, and fully reversed platelet signal. [Figure 22] 1 is a graph of the receiver operating characteristic (ROC) curve for CART regression for DOAC≧30 ng / mL including fibrin signal. [Figure 23] 10 is a table of the confusion matrix for fibrin signals only. [Figure 24] 1 is a graph of the receiver operating characteristic (ROC) curve for platelet signals only for DOAC≧30 ng / mL. [Figure 25] 10 is a table of the confusion matrix for platelet signals only. [Figure 26] 1 is a graph of the receiver operating characteristic (ROC) curve for fibrin signal only for DOAC≧50 ng / mL. [Figure 27] 10 is a table of the confusion matrix for fibrin signals only. [Figure 28] 1 is a graph of the receiver operating characteristic (ROC) curve for platelet signals only for DOAC≧50 ng / mL. [Figure 29] 10 is a table of the confusion matrix for platelet signals only. [Figure 30] 1 is a graph of the receiver operating characteristic (ROC) curves for both fibrin and platelet signals for DOAC≧50 ng / mL. [Figure 31] 10 is a table of confusion matrices for both fibrin and platelet signals. [Figure 32]1 is a graph of receiver operating characteristic (ROC) curves for fibrin and platelet signals for DOAC≧50 ng / mL and for fully reversed fibrin and platelet signals. [Figure 33] 10 is a table of confusion matrices for fibrin and platelet signals and fully inverted fibrin and platelet signals. [Figure 34] 1 is a graph of a matrix plot of LC-MS / MS and predicted fibrin concentrations. [Figure 35] 1 is a graph of regression model predictions using fibrin, platelets, fully reversed fibrin, and fully reversed platelets compared with LC-MS / MS measured apixaban or rivaroxaban concentrations. [Figure 36] 1 is a graph of a matrix plot for LC-MS / MS predictions based on a regression model. [Figure 37] 1 is a graph of regression model predictions using fibrin, platelets, fully reversed fibrin, and fully reversed platelets compared to LC-MS / MS measured apixaban or rivaroxaban using log10. [Figure 38] 1 is a table of constants and coefficients for LC-MS / MS. [Figure 39] 1 is a table of LC-MS / MS-measured apixaban or rivaroxaban concentrations from the distribution of bins and LC-MS / MS results. [Figure 40] 1 is a histogram of LC-MS / MS-determined apixaban or rivaroxaban concentrations from a distribution of LC-MS / MS results. [Figure 41] IC50 curve for determining drug concentration. [Figure 42] FIG. 1 is a diagram of a microfluidic device channel configuration for determining the presence of a single drug using a reversal agent, according to an embodiment of the present disclosure. [Figure 43] 1 is a graph of "normal" platelet accumulation over time. [Figure 44] 1 is a graph of "normal" fibrin accumulation over time. [Figure 45] Graphs of subject results and population dose response (IC50 curves). [Figure 46] FIG. 1 is a diagram of the coagulation pathway in relation to the use of the terms DTi and IIai herein. [Figure 47] FIG. 47 is a diagram of a simplified version of the coagulation pathway of FIG. 46. [Figure 48] 48A and 48B are charts of platelet signal change over time for protamine reversal of heparinized blood (FIG. 48A) and fibrin signal change over time (FIG. 48B), respectively, with reversal of heparinized blood with protamine demonstrating restoration of fibrin and platelet signals to near-normal levels using 0.1875 mg / mL protamine. [Figure 49A-B] 49A and 49B are charts demonstrating changes in fibrin and platelet signal development in the presence of DOAC at two temperatures (FIG. 49A shows the platelet signal at 37° C., and FIG. 49B shows the platelet signal at 21° C.). [Fig. 49C-D] 49C shows the fibrin signal at 37° C. and FIG. 49D shows the fibrin signal at 21° C. [Figure 50] Regression model charts: Figure 50A shows individual regression models for the known drugs Apixaban (A), Dabigitran (D), and Rivaroxaban (R); Figure 50B shows a regression model with unknown drugs but known drug classes as categorical input; and Figure 50C shows a CART regression model with unknown drugs but known drug classes. [Figure 51] FIG. 1 is a diagram of a microfluidic device for determining the presence of a drug using a reversal agent, according to an embodiment of the present disclosure. [Figure 52] FIG. 1 is a diagram of a microfluidic device pathway including a multilayer structure with two unique reaction zones. [Figure 53]FIG. 1 is a diagram of a microfluidic device pathway including a single layer structure with two unique reaction zones. DETAILED DESCRIPTION OF THE INVENTION

[0091] The system and method for DOAC-related data collection and analysis provides rapid and accurate assessment of whether a patient is taking a DOAC in a testing environment that mimics the blood coagulation physiology found in the human body. The system performs hemostatic assays that can be read from a portable benchtop instrument (analyzer) with an integrated computer, preformatted disposable cartridges, and accompanying software. The system can be used to determine early risk stratification, targeted therapy, and patient monitoring. The assay can mimic the microenvironment of clot formation and incorporate hemodynamic flow and individual clot activation. The system can provide real-time (or substantially real-time) characterization of blood coagulation physiology under flow. The system can provide dynamic, multiplexed signaling of platelets and fibrin in a single test. The system can provide functional response and (putative) drug levels. The system can provide rapid results in real time or substantially real time (e.g., over a time course of 15 minutes or less) and comprehensive determination of hemostatic function and anticoagulation levels in a single test within 30 minutes. Such determinations may be essential in critical care settings, as well as other patient care settings, and support reversal and monitoring strategies. Additionally, the system, if analyzed over longer periods of time, could provide additional information regarding thrombus formation behavior (e.g., fibrinolysis and platelet inhibition).

[0092] The system simulates in vivo conditions and is therefore sensitive to the patient's thrombus formation behavior. The information / results provided by the system can include independent functional fibrin and platelet responses, drug class, levels, and / or concentrations, and thrombus formation behavior over time. These results can distinguish between platelet and coagulation dysfunction and the drug levels and / or concentrations associated with dysfunction. The system can facilitate understanding of the effects of blood product delivery, such as transfusions, fresh frozen plasma (FFP), cryoprecipitate, and platelets, as well as real-time monitoring of drug action. The system can specifically fill the DOAC information gap by detecting and classifying DOACs, predicting DOAC levels and / or concentrations, and whether a need for reversal exists, evaluating targeted reversal strategies, and assisting with blood product management.

[0093] The system can use the same drug for which the patient is prescribed or being treated to determine the presence and class of drug using multiple reversal agents. For a drug and its reversal agent, the system can simultaneously use the patient's own blood as an internal control to determine DOAC drug activity, avoiding the calibration requirements of some conventional assays (e.g., the patient's own blood is used to determine the response to the reversal agent and its response to complete inhibition, avoiding the need for a calibration curve for each device run). Specific targeting elements in the reaction zone promote clot formation, including both platelet accumulation and fibrin formation simultaneously, regardless of the detection reagent. The combination of fibrin and platelet signals has been shown to be both meaningful for calculating drug presence and concentration. Additional targeting elements can be added to the reaction zone to target additional thrombus-forming elements, hemostatic elements, and / or other blood-born factors. In some embodiments, clot formation can occur in the presence of whole blood, which contains all of the essential components for hemostasis, a direct reflection of an individual patient's in vivo processes.

[0094] In some embodiments, the reaction zone can be "tuned" to various levels of sensitivity to platelet and fibrin accumulation by varying the specific targeting element (e.g., tissue factor) and concentration, changing the shear rate, changing the reaction temperature, or changing the fluid dimensions, thereby enabling specific levels of sensitivity to drug levels. The reaction zone contains immobilized reagents that bind to and activate platelets and activate coagulation. In some embodiments, two or more reaction zones with two or more levels of sensitivity can be placed on the same assay device, thereby expanding the device's ability to measure a wider range of drugs. Blood can be analyzed simultaneously under physiological flow, thereby demonstrating the realistic behavior of a patient's blood constituents (platelets and fibrin) in vivo. In some embodiments, blood flow (blow food) can be driven through the device by an external source (e.g., a pump) to achieve a specific range of physiological shear conditions. For example, biological sample flow can be driven through the assay device from an inlet to an outlet through the reaction zone via the application of a pressure gradient using an external source.

[0095] Temperature Control and Impact on Assay Results: For kinetic / time-based assays, temperature can play a large role in the behavior of individual blood components (such as the serine protease thrombin). Reactions performed at room temperature, approximately 17 degrees Celsius below body temperature, can have substantially reduced activity (reaction kinetics) and increased blood viscosity. A consistent reaction temperature is crucial for comparing results across time, locations, and samples to avoid variations caused by differences in the system's environment. While it is possible to analyze blood at temperatures below body temperature, blood behavior becomes less active as the analysis temperature decreases. This can be advantageous for designing systems that utilize this effect, as temperature can be used to control and alter both platelet and coagulation behavior (e.g., reducing or increasing the overall signal or rate of signal development, reducing or extending the overall assay time, and / or altering assay sensitivity).

[0096] Adding temperature control to an assay can be most simply achieved through controlled heating of the imaging location of the system. For example, a heating element can be applied to the floor of the imaging region to allow (indirect) convective heat transfer to control the temperature of the space in which the fluidic device is being imaged. Enclosure and insulation of the imaging / reaction space can provide reduced temperature fluctuations. Alternatively, a heating element in the imaging / reaction space can contact the bottom of the fluidic device when inserted into the system to directly heat the device. In some embodiments, a combination of direct and indirect heat transfer can be used. A thermocouple or similar type of temperature sensor can be used as a feedback mechanism to control the temperature to a specific set point at, above, or below body temperature. In some embodiments, one or more heating elements can be applied directly to or within the fluidic device, such that a connection to the instrument's electrical system can provide power to the heating element in the fluidic cartridge when placed in the instrument. Multiple methods of delivering and applying heat to a fluidic device within an instrument (e.g., direct contact, air, liquid, combinations thereof, etc.) and its control (PID, hysteresis, etc.) are contemplated. In some embodiments, cooling of the imaging / reaction chamber can be applied, for example using a Peltier element, to reduce the reaction temperature (and therefore the assay activity) if necessary.

[0097] Applying a constant temperature to an assay can result in either an increased reactivity / signal, typically with an increase in temperature, or a decreased reactivity / signal, typically with a decrease in temperature. This is advantageous in that sample reactivity, and potentially even sample sensitivity, can be easily altered by changing the reaction temperature. For example, increasing the reaction temperature can decrease the assay time, thereby shortening the time to reach a clinical result, which is crucial in critical care decision-making. Increasing the reaction temperature can increase the reaction kinetics for less reactive samples. For example, for patients taking very high doses of DOAC drugs, determining the drug concentration can be more easily identified if the reaction kinetics is increased by increasing the temperature. This can result in improved assay sensitivity to higher drug levels; or, conversely, using a lower temperature can make the assay more sensitive to lower drug levels. In addition, reducing the temperature can aid in the evaluation of overly reactive samples (e.g., hypercoagulation, excessive platelet activity, etc.). An overly reactive sample (coagulation or platelet function) can cause excessive clot formation within the device, which can lead to fluid blockage and erroneous assay results (or no results). For example, critically ill patients (e.g., COVID-19, sepsis, cancer, etc.) may present with problems of hypercoagulation. Reducing the reaction temperature of the patient's sample can sufficiently reduce the reaction kinetics to avoid premature device blockage and enable useful clinical results.

[0098] Figures 49A-49D demonstrate that the same overall dose-specific fibrin behavior is observed in blood samples treated with various doses of rivaroxaban, but with a decreased onset time, increased reaction rate, and higher signal when the reaction temperature is increased compared to body temperature. In particular, Figures 49A-49D demonstrate changes in fibrin signal development in the presence of a DOAC at two temperatures. As expected, the fibrin signal decreases as the rivaroxaban® dose increases compared to the HBS control. With increasing temperature, the fibrin reaction initiates earlier, the maximum rate of change of fibrin and platelets increases (steeper slope), and the fibrin and platelet signal increase overall at the same time point. The dose effect of the DOAC drug seen at higher temperatures follows a similar dose behavior seen at lower temperatures. As illustrated in Figures 49A-49B, an approximately two-fold increase in platelet signal at 400 s is observed, and a substantial increase in the maximum rate of change in fluorescence is also observed. As illustrated in Figures 49C-49D, there is an approximately 5-fold increase in fibrin signal at 400 s, with a more than 3-fold faster onset and a substantial increase in the rate of signal development.

[0099] The assay device can use flow channels (microfluidics) that mimic the physiological structure, size, function, flow, and distribution of cellular and subcellular components and / or platelet margination behavior under flow. The assay can measure the biological process of hemostasis in real time, thereby allowing evaluation of the process at all time points during the assay (e.g., kinetic assays). The system is rapid, providing a clear picture of hemostatic function within 15 minutes (in the majority of cases). The system has the long-term potential to measure clot lysis, clot strength, hematocrit, and other parameters of interest that affect hemostasis simultaneously with platelet and fibrin function. The system can detect classes of drugs that affect both platelets and fibrin.

[0100] FIG. 2 is a perspective view of a microfluidic device 100 for monitoring blood biology under flow in an exemplary system, and FIG. 3 is a diagram of the microfluidic device 100. Accordingly, like reference numerals are used to represent like structures. The device 100 generally includes a microfluidic circuit including multiple inlets 102 (e.g., eight inlets), microfluidic channels 104 in fluid communication with each of the inlets 102 and leading to a single outlet 106. In some embodiments, the device 100 may include a single dedicated inlet for each of the inlets 102. In some embodiments, the device 100 may include a dedicated outlet for each of the inlets 102. The channels 104 may all converge to create an imaging zone 108, after which a channel 110 extends to the outlet 106. In some embodiments, the channel 110 may be substantially straight. In some embodiments, the channel 110 may define a serpentine or continuously curved configuration. Of critical importance, all channels should be the same length between the inlet and the imaging region (and from the imaging region to the outlet) to ensure that resistance to flow is consistent between channels, and therefore, for a given applied pressure, the flow rate is similarly the same. It should be noted that channels of different lengths may be utilized, so long as the resistance to flow is balanced between channels. This can be done by several means, including reducing the overall size of the channels in certain regions to increase the resistance to flow, or by enlarging the channel size in certain regions to decrease the resistance to flow.

[0101] The device 100 can include a collagen / tissue factor reaction zone 112 located at or near the imaging zone 108 of the device 100. Having the channels in close proximity to one another simplifies imaging by allowing imaging of all channels / reaction zones within the same field of view (although in some cases imaging using multiple fields of view with higher magnification is also beneficial). Additional reactants specific to clot formation are also expected to be useful in the reaction zone (e.g., Factor XIa, kaolin, collagen-related peptides, etc.). The imaging zone can also include fiducial marks (both fluorescent and non-fluorescent) that allow for automatic detection and alignment of the reaction zone via the instrument's software.

[0102] The priming inlet 114 can be in fluid communication with a microfluidic priming channel 116, which fluidly couples with the outlet channel 110 at or near the imaging zone 108. In operation, when priming fluid is applied under pressure to the priming circuit, the priming fluid can flow through the microfluidic channel 104 to the inlet 102 due to the lower resistance to flow in the microfluidic channel 104 compared to the outlet 106. Other devices and methods of priming are contemplated, such as those described in International Patent Application No. PCT / US2019 / 022965, which is incorporated herein by reference in its entirety.

[0103] The device 100 may allow for simultaneous testing of up to eight unique conditions. Blood flow over localized areas 112 of collagen and tissue factor can induce clot formation (platelet and fibrin deposition), resulting in susceptibility to any disruption of the hemostatic / coagulation process. Assay testing using the device 100 provides the ability to detect DOAC type by using actual reversal drugs. Estimated drug levels are calculated using the IC 50The efficacy of DOACs can be determined through comparison of fully attenuated and fully reversed samples using drug-response curves, linear regression, CAR-T analysis, or other statistical methodologies. DOAC testing can use a factor Xa inhibitor at maximal inhibition, a factor Xai reversal agent (full reversal), a direct thrombin inhibitor (DTi) reversal agent (full reversal), and unmodified patient blood. For example, two inlets can receive maximal inhibition (high dose), two inlets can receive a factor Xai reversal agent, two inlets can receive a DTi reversal agent, and two inlets can receive unmodified patient blood. The utility of using multiple inlets containing the same reactants is to allow for averaging of results, which can attenuate the effects of variability in device performance and preparation. The reaction zone 112 can have one, two, or multiple collagen / tissue factor regions to assess thrombogenic function (see, for example, Figure 44). These reactants can be identical in concentration and composition (useful for statistical / averaging purposes), or different concentrations and compositions can be utilized to assess different sensitivity of samples to coagulation and platelet function. In some embodiments, one or more inlets could be used to determine other blood characteristics, such as hematocrit, oxyhemoglobin, platelet count, or similar results in the flow path, at a reaction zone, or some other location on the device, using the disclosed means.

[0104] The system can rely on direct fluorescent signals for analysis of results with collagen / tissue factor 112. Fresh whole blood (used within minutes of venous blood collection), AF594 fibrinogen label (human plasma-derived fibrinogen, Alexa Fluor TM 594 conjugate) and platelet MCA2588A488 conjugate (mouse anti-human CD61, Alexa Fluor TM488 conjugate) can be added to the device 100, and the direct fluorescent signal can be analyzed. Chemicals that inhibit specific coagulation pathways, such as corn trypsin inhibitor or PPACK, can also be added to the patient's blood (before or in conjunction with the labeled chemical) to prevent undesired reactions, such as contact activation of the intrinsic coagulation pathway. The result is the production of a direct fluorescent signal in green (platelets) and red (fibrin), with the measured fluorescence intensity (FI) directly correlating with platelet and fibrin accumulation. Fluorescent signal intensity can be measured directly at each clot site over time to generate clot response curves for both fibrin and platelets. Fluorescent signal (intensity) can be extracted for each clot and plotted for each assay condition.

[0105] The platelet and fibrin labels could be added to and mixed with the blood prior to addition to inlet 102, or could be added to the inlet prior to addition and mixing with the blood. The reagents could also be stored wet or dry / lyophilized in inlet 102 (or elsewhere in the device) prior to use. Addition of blood and mixing of reagents could be done manually or automated (pipetting robot) outside the device, or could be done within the device using macro- and microfluidic structures known to those skilled in the art.

[0106] DOAC class determination can be performed by comparing the change in biological response between an unmodified sample and a sample modified with a reversing agent. DOACs generally come in two classes: factor Xa inhibitors (Xai) and factor IIa (thrombin) inhibitors (IIai or DTi), both of which affect fibrin formation. By using small amounts (micrograms) of the actual reversing agent used in vivo to promote the reversal of Xai or IIai DOAC activity in blood (actual medically prescribed reversing agents are used with the assay device), the assay device directly identifies which DOAC class is present by the direct coagulation response of the patient's blood. For example, if factor Xai is present in the patient's blood, ANDEXXA® (a Xai reversing agent) will improve thrombogenic behavior (increase the fibrin signal), but PRAXBIND® will not. Similarly, if factor IIai is present in the patient's blood, PRAXBIND® (a IIai reversing agent) will improve thrombus formation behavior (increase fibrin signal), but ANDEXXA® will not. If neither DOAC class is present in the sample, neither reversing agent will have a significant effect on fibrin signal (neither a specific increase nor a decrease will be seen).

[0107] The determination of DOAC concentrations can be determined by comparison to a population-average dose-response that is specific to the drug being evaluated. Because DOACs specifically affect fibrin formation through their direct or indirect reduction in thrombin activity, the ratio of a patient's fibrin signal to a patient's fully attenuated fibrin signal and a fully reversed fibrin signal can be calculated using known IC values ​​generated using the device / system. 50It can be used to determine the approximate drug dose from the drug response curve. Each drug (or class) in question will have its own IC curve, and comparison of patient data will be specific to the drug identified in the sample. Data can be normalized to the patient's own clotting response (unmodified sample) (see, for example, Figure 45, which illustrates subject results and population dose-response using IC curves).

[0108] Exemplary systems / devices may also rely on statistical methods to derive drug concentrations from comparisons to results from quantitative analytical techniques such as LC / MS. Because derivation of results relies on the relationship between the device output and an independent quantitative measurement, this method does not necessarily require knowledge of the specific drug in question. In this case, only four signals (two for fibrin function and two for platelet function) are required to derive a result. For example, the device may include a first inlet to provide unaltered sample fibrin and platelet signals, and a second and third inlet to assess fibrin and platelet signal reversal in the presence of a DOAC. The fibrin and platelet signals at the fourth inlet, containing a fully DOAC-inhibited sample, may also provide additional analytical benefits, such as verifying that the patient's blood behaves as expected in the presence of a coagulation inhibitor. In some embodiments, the fourth inlet data can be used to assess acceptable system performance. If a DOAC is detected, the unaltered and reversed platelet and fibrin signals can be compared to determine the class of DOAC present and the concentration of the DOAC (e.g., quantification of the DOAC). Rather than relying solely on the fibrin signal, the system relies on both unaltered and fully reversed signals related to fibrin and platelet function to provide a more accurate classification and quantification output. These four signals are generated simultaneously (or substantially simultaneously) by the device / system. The class of DOAC present can be determined by the system through a simple comparison (ratio) between the unaltered sample and the fully reversed sample. Regression and / or classification and regression tree (CART) analysis can be performed by the system based on the four signals to determine the DOAC concentration. The detected DOAC level can be compared to industry thresholds to provide guidelines to medical professionals regarding the next step in patient treatment, such as reversal.

[0109] The system can include an assay device and accompanying software on a computing or processing device capable of controlling / monitoring and analyzing data generated by the assay device. In some embodiments, the computer is separate from the imaging device, while in other embodiments, the computer is integrated into the device. The software can control instrument functions such as camera gain, frame rate, light-emitting diode (LED) intensity, pump operation, and pressure control, as well as other electromechanical functions. Images of clot formation, as well as plotted data extracted from those images, can be displayed in real time or substantially real time.

[0110] Blood can be drawn from a patient and placed into a vacutainer or other suitable holding device, such as a syringe. Alternatively, blood could be drawn directly into the device through an appropriate connection (e.g., an intravenous line and a Leur connection). The vacutainer can be plugged into the device, and the device can be attached to an analyzer (e.g., an imaging instrument with computing and software for analyzing the device). Automated analysis can provide results to medical professionals in 15 minutes or less. Useful data from the sample analysis process can be discernible earlier, including as early as 30 seconds after the start of measurement. Readouts / reports can be generated from the data and provided to clinicians. A user interface on a computer or mobile device can be used to visualize real-time data or reports containing information related to assay device testing and analysis, such as fibrin, platelet, and DOAC information. Additional useful data from the process can be attained 30 minutes or more after the start of analysis (e.g., clot lysis).

[0111] FIG. 4 is a flowchart illustrating a process 150 for implementing an exemplary system discussed herein. In step 152, the system can be used to determine whether a DOAC is present in a patient blood sample. If no DOAC is detected, in steps 154 and 155, the system determines whether platelet and fibrin activity is abnormal. If platelet and fibrin activity are not abnormal (i.e., normal), normal hemostasis can be identified in step 157. If fibrin activity is abnormal, a medical professional is notified to inquire about a coagulation disorder or other anticoagulants present (step 156). If platelet activity is abnormal, a medical professional is notified to inquire about a platelet disorder or other antiplatelet medications present (step 158). In step 160, if a DOAC is detected, the system can be used to determine which class of DOAC is present.

[0112] In step 162, for the IIai class, the system can determine what level of DOAC is present. In step 164, the system can determine whether the concentration level is above or below a threshold. The system can instruct the medical professional to initiate IIai reversal (step 166) if the DOAC level is above the threshold concentration value or an alternative Tx procedure (step 168) if the DOAC concentration level is below the threshold. In step 170, for the Xai class, the system can determine what level of DOAC is present. In step 172, the system can determine whether the concentration level is above or below the threshold. The system can instruct the medical professional to initiate Xai reversal (step 174) if the DOAC concentration level is above the threshold or an alternative Tx procedure (step 176) if the DOAC concentration level is below the threshold. Based on the output results of the DOAC class and level, the medical professional / clinician can make decisions regarding reversal therapy and the next treatment plan. The assay device can verify whether the DOAC is the cause of delayed or poor clotting, or indicate whether the dysfunction may be arising from another source. Results from the system can be integrated with other blood tests (e.g., platelet count) to verify the patient's hemostatic and coagulation function.

[0113] 5A and 5B are diagrams of a graphical user interface 200 of an exemplary system for displaying results to a clinician based on the automated analysis and determination of the assay device and accompanying software. Interface 200 can display fibrin function in section 202, platelet function in section 204, detected DOAC class in section 206, and DOAC level in section 208. User interface 200 can thereby provide a concise readout of the detected (or not) DOAC class, estimated DOAC concentration (if present) in the patient's blood, the patient's fibrin response to the DOAC drug and reversal agent (if a DOAC was present), and overall platelet function compared to population normals.

[0114] Experiments were conducted using an exemplary assay device and system. Specifically, microfluidic testing was performed on samples from patients receiving DOACs using fresh whole blood. Data was collected and independently tested using LC-MS / MS by Pharmaron ((PH) - a commercial analytical services company) using plasma derived from these patient blood samples. The study included 13 patients receiving apixaban and 7 patients receiving rivaroxaban. The test configuration used an 8-channel microfluidic device (e.g., exemplary assay device) with four different conditions, each in duplicate. The four different conditions included: (i) unaltered patient samples; (ii) patient samples with 5000 nM DOAC clot formation inhibition; (iii) patient samples with partial DOAC reversal; and (iv) patient samples with fully reversed DOAC. In some cases, the amount of reversal agent used may result in incomplete reversal of the fibrin signal. Intermediate concentrations of reversal agent can be used to distinguish between intermediate concentrations of DOAC administered to patients due to incomplete reversal of the fibrin signal.

[0115] Two signals were generated during testing, representing fibrin and platelets accumulating in the region of interest on the device, as fluorescence intensity (FI). Duplicate replicates for each of the four channel conditions for fibrin and platelet signals were averaged, resulting in a total of eight signals available for data analysis. The optimal time for selecting data for analysis was determined to be the time of maximum difference between the unaltered patient blood signal and the fully reversed signal using fibrin FI. This is referred to as the "time to maximum Δ" and ranges from 300 to 1520 seconds (5 to 25 minutes). In some embodiments, the time range can be, for example, approximately 1 to 25 minutes (inclusive), 1 to 5 minutes (inclusive), 5 to 25 minutes (inclusive), 1 to 60 minutes (inclusive), 5 to 60 minutes (inclusive), 25 to 60 minutes (inclusive), etc. Comparison of exemplary assay device test results with LC-MS / MS results is provided on a ng / mL basis (consistent with clinical decision-making regarding DOAC reversal). Various analyses were performed. Exemplary assay device tests for best performance compared to LC-MS / MS are discussed herein.

[0116] During the experiment, the two clinical decision points of interest were 30 ng / mL and 50 ng / mL. Figure 6 is a table of sample datasets associated with the clinical decision points of interest. In particular, the number of samples within the 21 sample dataset that were either less than, equal to, or greater than these values ​​is shown in Figure 6. Two classification methods provided the best performance using four of the eight possible signals available. The four signals that provided the best performance were: (i) unmodified patient samples with DOAC, fibrin FI; (ii) unmodified patient samples with DOAC, platelet FI; (iii) fibrin FI of patient samples with DOAC and fully reversed samples; and (iv) platelet FI of patient samples with DOAC and fully reversed samples.

[0117] These four signals were used as inputs to different methods for categorical discrimination around the clinical decision points of 30 ng / mL and 50 ng / mL. One method was fitted to a binary logistic regression model, and the other method was fitted to a CART classification. Binary logistic regression provided the best performance for the 30 ng / mL decision point, and CART classification provided the best performance for the 50 ng / mL decision point.

[0118] Test performance and associated performance characteristics are provided in the tables (e.g., "truth tables") of Figures 7-9. In particular, Figure 7 is a table of sample detection for a 30 ng / mL decision point, Figure 8 is a table of sample detection for a 50 ng / mL decision point, and Figure 9 is a table of performance characteristics for the binary logistic regression and CART classification models. Figures 10 and 11 are graphs of performance for discrimination. In particular, Figure 10 is a graph of binary logistic regression for sample detection above 30 ng / mL, and Figure 11 is a graph of CART classification for sample detection above 50 ng / mL.

[0119] The exemplary device and system rely on analysis of both platelet and fibrin signals, as well as fully reversed platelet and fibrin signals, to provide accurate quantitative determination of DOACs. For comparison, experiments included quantitative analysis relying solely on the fibrin signal to predict DOAC concentrations. Figure 12 shows a table of DOAC concentration predictions based solely on the fibrin signal. Figure 13 shows a table of DOAC concentration predictions based on both the fibrin and platelet signals. Both fibrin and platelet signals, along with fully reversed fibrin and platelet signals, were used with time to maximum Δ (four input variables), and two different models were used to calculate the predictions. Figure 13 provides a comparison of predicted DOAC concentrations with liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0120] The best results were obtained from the CART regression model, which produced an 8-node (7-fold) decision tree for assigning concentrations to each sample. Figure 14 is a graph of DOAC concentration prediction based on both fibrin and platelet signals using the CART regression model, compared with LC-MS / MS measurement data. Figure 15 is a graph of DOAC concentration prediction based on both fibrin and platelet signals using the Pearson correlation model, compared with LC-MS / MS measurement data.

[0121] Based on the data, as a semi-quantitative method to provide discrimination around DOAC concentrations used in clinical decision-making, the exemplary device testing demonstrated the feasibility of achieving the desired target ROC AUC, sensitivity, specificity, PPV, and NPV. Based on the data, as a quantitative method, the exemplary device testing demonstrated the feasibility of using four different signals in the assay configuration and the ability to use different methods (e.g., binary logistic regression, CART regression, etc.) from which concentrations can be calculated.

[0122] The data used in the experiments and analysis are described below. The data included estimated DOAC concentrations (e.g., explicit concentrations) and (patient sample - complete inhibition) / (complete reversal - complete inhibition). In some cases, manual imaging analysis was used, which entailed modifying the region of interest to exclude aggregates reaching the clot zone from upstream. In an attempt to determine the feasibility of an exemplary test method / device without secondary manual intervention to generate data, a file that did not include modified ROIs was used. For the collected data, the time was the time of maximum Δ (full reversal condition - unaltered patient sample, using fibrin signal without platelet signal). Values ​​for fibrin and platelet FI were obtained from the raw data. Mean values ​​were used. Figure 16 is a table of patients (based on their assigned numbers) for apixaban and rivaroxaban. Subject 33 did not have a calculated value for the predicted DOAC based on the time to maximum Δ.

[0123] To compare the results of the described exemplary system in comparison to LC-MS / MS, Pharmaron was used to perform internal reference assays for apixaban and rivaroxaban for LC-MS / MS calibrator and control performance.

[0124] The PH LC-MS / MS assay calibration and control compositions are provided in the table in Figure 17 (provided by Pharmaron), also in ng / mL. The controls are overlapping concentrations of the calibrators. The calibrators and concentrations used by PH were relative to commercially available test standards.

[0125] During the experiment, different time points were evaluated for optimal sample analysis times. These were based on the fibrin signal. One was the time to 80 percent of the maximum response, and the other was the time to the maximum difference between the patient sample and 300 nM reversal reagent. All patient samples in this study contained a DOAC. The relationship between signal and DOAC concentration was improved using the time to maximum delta (see Figures 18 and 19). The relationship is nonlinear. Figures 18 and 19 use a log10 relationship without attempting more precise model fitting. Specifically, Figure 18 is a graph of a fitted line plot for the fibrin signal with the time to maximum difference between the patient sample condition and the fully reversed patient sample condition, and Figure 19 is a graph of a fitted line plot for the platelet signal with the time to maximum difference between the patient sample condition and the fully reversed patient sample.

[0126] The exemplary system offers the potential for multiplexed microfluidic hemodynamic assays that simulate physiological blood flow and measure functional components of blood during the clot formation process via fluorescent optical detection. The system can be used for in vitro diagnostic use by trained medical professionals at the point of care and by laboratory technicians in clinical laboratories. When used with a DOAC assay, the system provides semi-quantitative (and potentially quantitative) results for the detection of direct oral anticoagulants (DOACs) in whole blood, the identification of DOACs as either factor Xa or direct thrombin inhibitors, and the classification of anticoagulation relative to concentrations above or below the ISTH guidelines for appropriate levels for reversal of anticoagulation therapy and for ivtPA thrombolysis to treat stroke. The system also generally provides real-time assessment of coagulation and platelet function.

[0127] The system can be adapted for evaluating hemostatic status in patients suspected of or being treated with DOACs, and evaluation of DOAC use and anticoagulation status can assist in the assessment of bleeding and thrombosis risk and recovery of hemostasis. The system can be used for patients in various situations where DOAC evaluation can be useful as additional information, such as patients experiencing DOAC-associated bleeding episodes, patients at risk for major bleeding (patients undergoing emergency invasive surgery, trauma patients, stroke patients requiring tPA), patients requiring reversal, etc. The system could also be used to evaluate drug administration during initial DOAC administration and to evaluate hemostatic response to chronic DOAC use. In some cases, the assay can be used as a standalone test. In some embodiments, the assay can be used in conjunction with other clinical and laboratory findings.

[0128] In assessing the relationship of system-predicted DOAC concentrations with LC-MS / MS-measured concentrations, all comparisons were made in ng / mL, as this is the commonly used unit for clinical decision points for DOAC reversal. Data from patient samples were evaluated for their ability to provide discrimination near clinical decision points for DOAC reversal of 30 ng / mL and 50 ng / mL (e.g., the two decision points are used due to different clinical cutoff requirements for DOAC reversal in certain patient populations, which entails differences in assay sensitivity).

[0129] A discussion of binary logistic regression for DOAC ≥ 30 ng / mL is provided. Using the fibrin signal at time of maximum Δ to detect DOAC concentrations ≥ 30 ng / mL, the receiver operating characteristic (ROC) AUC was 0.9000, with 87% sensitivity and 83% specificity (see Figure 20). Adding the platelet signal to the model did not change the ability to detect concentrations ≥ 30 ng / mL. However, adding the fully reversed fibrin signal to the model improved discrimination, with an ROC AUC of 0.9011, with 80% sensitivity and 100% specificity. Using both the fibrin and platelet signals at time of maximum Δ in conjunction with the fibrin and platelet signals from fully recovered samples, performance had an ROC AUC of 0.9556, with 93% sensitivity and 100% specificity (see Figure 21).

[0130] A discussion of binary logistic regression for DOAC ≥ 50 ng / mL is provided. The fibrin signal alone did not provide acceptable discrimination for detecting DOAC concentrations above 50 ng / mL (ROC AUC 0.7182), and this was not improved by adding the platelet signal to the model. The platelet signal alone also did not demonstrate the ability to discriminate at this concentration. Using a four-factor model in binary logistic regression, the best performance was an ROC AUC of 0.7636. The assay discrimination at this concentration, the manner in which the regression model operated, and the limited data set (limited number of samples around the 50 ng / mL threshold) influenced the results described.

[0131] A discussion of the CART regression for DOAC > 30 ng / mL is provided. Evaluation yielded a 3-node classification tree with a ROC AUC of 0.9056 with a sensitivity of 87% and a specificity of 83% for the fibrin signal (see Figure 22).

[0132] Figure 23 is a table of the confusion matrix for the fibrin signal alone, and Figure 24 is a graph of the receiver operating characteristic (ROC) curve for the platelet signal alone, using the CART classification for DOAC > 30 ng / mL. The platelet signal alone produced a 5-node classification tree with an ROC AUC of 0.8611, with a sensitivity of 73% and a specificity of 83%. Figure 25 is a table of the confusion matrix for the platelet signal alone. This analysis was performed to assess the performance of the platelet signal alone. The data showed that the platelet signal alone provided performance results.

[0133] Figure 26 is a graph of the receiver operating characteristic (ROC) curve for the fibrin signal alone for DOAC >= 50 ng / mL, and Figure 27 is a table of the confusion matrix for the fibrin signal alone. The ability of the fibrin signal alone to detect DOAC concentrations >= 50 ng / mL had an ROC AUC of 0.7136 with a sensitivity of 70% and a specificity of 73%.

[0134] Figure 28 is a graph of the receiver operating characteristic (ROC) curve for the platelet signal alone for DOAC > 50 ng / mL, and Figure 29 is a table of the confusion matrix for the platelet signal alone. The platelet signal alone performed better than fibrin alone, resulting in a four-terminal node decision tree with a ROAC AUC of 0.7773 with a sensitivity of 70% and a specificity of 73%. This analysis was performed to demonstrate that measurement of the platelet signal had some performance on its own.

[0135] Figure 30 is a graph of the receiver operating characteristic (ROC) curves for both fibrin and platelet signals for DOAC > 50 ng / mL, and Figure 31 is a table of the confusion matrix for both fibrin and platelet signals. Using both signals at the time of maximum delta (based on fibrin) produced the highest ROC AUC of 0.8955 with a sensitivity of 60% and a specificity of 100%.

[0136] Figure 32 is a graph of the receiver operating characteristic (ROC) curves for the fibrin and platelet signals for DOAC > 50 ng / mL and the fibrin and platelet signals from fully reversed samples, and Figure 33 is a table of the confusion matrix for the fibrin and platelet signals and the fibrin and platelet signals from fully reversed samples. Using the same four factors that gave the best results in binary logistic regression, fibrin and platelet patient signals and the fibrin and platelet signals from fully reversed samples, the ROC AUC was 0.9091, with a sensitivity of 90% and a specificity of 82%.

[0137] For comparison, quantitative performance was determined based on fibrin signal alone. Results are provided using DOAC concentrations as estimated using calculations and equations based solely on fibrin signal at time of maximum Δ. All relationships are based on ng / mL concentrations for ease of interpretation relative to clinical decision points.

[0138] FIG. 34 is a graph of a matrix plot of LC-MS / MS and predicted fibrin concentrations as predicted using IC50 curves.

[0139] Alternative methods for quantitative performance were considered. Analysis of different models to provide discrimination around specific DOAC concentrations used in clinical decision-making identified that the use of unaltered sample fibrin and platelet signals and fully inverted sample fibrin and platelet signals at the time of maximum Δ provided the best performance. Novel and different potential methods for estimating DOAC concentrations from these signals were evaluated. One method used a regression model including four continuous predictors compared with concentrations from LC-MS / MS (without any adjustment). The results provided a regression equation with an R-sq value of 63%. However, the intercept was high and the slope was low. Figure 35 shows a graph of the regression model predictions using unaltered sample fibrin and platelet signals and fully inverted sample fibrin and platelet signals compared with LC-MS / MS-measured concentrations of apixaban or rivaroxaban, and Figure 36 shows a matrix plot of the LC-MS / MS predictions based on the regression model (Pearson r-value correlation).

[0140] In the next iteration of the analysis, the four fibrin and platelet signals, along with the LC-MS / MS values, were transformed to log10. The regression equation was reapplied and the fit saved. The fit was then converted back to ng / mL and compared to the LC-MS / MS values. The r value was somewhat lower, but the slope and intercept were improved. Precision within clinical decision-making was similarly improved. Figure 37 is a graph of the regression model predictions using the unaltered and fully inverted sample fibrin and platelet signals compared to the LC-MS / MS-measured concentrations of apixaban or rivaroxaban using log10, and Figure 38 is a table of the constants and coefficients for LC-MS / MS. The data in Figure 37 can be used for LC-MS / MS predictions based on the regression model using log10 to obtain Pearson's r values. From the results, it was determined that the exemplary device testing methodology output from the four signals of interest could be used to generate predictions for concentrations using standard types of regression analysis. Of particular note from this analysis was the apparent accuracy within the concentration range where clinical decision-making occurs.

[0141] As an alternative to the standard regression model described above, CART regression was also used on the same set of four signals to predict drug concentrations from LC-MS / MS. Examination of the distribution of LC-MS / MS results determined that the values ​​were not normally distributed (A-sq p-value = 0.006) but rather skewed to the right (Figure 40). The histogram clusters the results into eight bins. Figure 39 is a table of bins and LC-MS / MS-measured apixaban or rivaroxaban concentrations from the distribution of LC-MS / MS results, and Figure 40 is a histogram of LC-MS / MS-measured apixaban or rivaroxaban concentrations from the distribution of LC-MS / MS results.

[0142] For CART regression, the selected option was to use six samples for splitting nodes and two samples for terminal nodes (similar to bins). This resulted in an eight-node (seven-fold) decision tree for assigning concentrations to each sample, which matched the distribution of actual LC-MS / MS results. Figures 14 and 15 show the results of using orthogonal regression and Pearson correlation compared to LC-MS / MS measurements. In particular, Figure 14 is a graph of CART regression model predictions using unaltered sample fibrin and platelet signals and fully inverted sample fibrin and platelet signals compared to LC-MS / MS-measured concentrations of apixaban or rivaroxaban, and Figure 15 is a graph of a matrix plot of LC-MS / MS predictions based on the CART regression model. The results demonstrate that the CART algorithm using four signals from the exemplary device test method can produce DOAC concentration determinations that approach agreement with those determined using LC-MS / MS.

[0143] While exemplary embodiments have been described herein, it is expressly noted that these embodiments should not be construed as limiting; rather, additions and modifications to those explicitly described herein are also included within the scope of the present invention. Furthermore, it should be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and rearrangements without departing from the spirit and scope of the present invention, even if such combinations or rearrangements are not expressed herein. [Explanation of symbols]

[0144] 100 Microfluidic Devices 102 Entrance 104 Microfluidic Channels 106 Exit 108 Imaging Zones 110 Flow path 112 Reaction Zone 114 Priming inlet 116 Microfluidic Priming Channel 200 User Interface 300 layers 302 Channel 304 Reaction Zone 306 layers 308 Channel 310 Reaction Zone 320 Single Layer 322, 324 flow path 326, 328 Reaction Zone

Claims

1. a detection device having computing power; and An assay device capable of receiving a biological sample, wherein introduction of the biological sample into the assay device results in a biological process whereby fibrin and platelets may accumulate in a reaction zone of the assay device.

1. A system for detecting and quantifying drug and / or chemical interactions with a biological sample, comprising: the assay device is capable of receiving one or more chemical reagents that are compatible with the biological sample and that can be used to detect fibrin and platelet accumulation within the reaction zone; the assay device is capable of receiving one or more drug reagents that are compatible with the biological sample and that can be used to alter the accumulation of fibrin and platelets in the reaction zone; and The fibrin and platelets accumulated in the reaction zone of the assay device and their associated signals can be used to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration in the biological sample. The above system.

2. 10. The system of claim 1, comprising a fluorescent assembly capable of detecting a biological process by fluorescent labeling and detecting a resulting accumulating fluorescent signal, and comprising a processing device configured to receive as input measurements of fibrin and platelets to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration within the biological sample, wherein the processing device is configured to correlate the measured fluorescent intensity with the accumulation of fibrin and platelets in the microfluidic channels of the assay device.

3. 10. The system of claim 1, wherein the one or more chemical reagents or the one or more drug reagents are fluorescent reagents capable of labeling fibrin and platelets from a biological sample, resulting in a fluorescent assembly that reports fibrin and platelet accumulation.

4. 10. The system of claim 1, comprising a light source for monitoring clot development in the microfluidic channels of the assay device and for detecting the fluorescent response of one or more chemical reagents or one or more drug reagents.

5. The assay device comprises: a first inlet, a second inlet, a third inlet, and a fourth inlet port, each configured to receive a biological sample, the biological sample being an unmodified sample comprising a platelet-specific label and a fibrin-specific label; Exit; and a microfluidic channel fluidly connecting each of the first, second, third and fourth inlets with an outlet; Including; 10. The system of claim 1, wherein introduction of a biological sample into each of the first, second, third and fourth inlets generates a fibrin signal and a platelet signal.

6. 6. The system of claim 5, wherein at least one of the first, second, third and fourth inlets is configured to receive an unmodified biological sample.

7. 6. The system of claim 5, wherein at least one of the first, second, third and fourth inlets is configured to receive a molar excess of drug to provide a fully attenuated fibrin or platelet signal.

8. 6. The system of claim 5, wherein at least one of the first, second, third, and fourth inlets is configured to receive a first reversal agent for identifying a first class of drug present in the biological sample, and at least one of the first, second, third, and fourth inlets is configured to receive a second reversal agent for identifying a second class of drug present in the biological sample.

9. 9. The system of claim 8, wherein the first reversal agent or the second reversal agent reverses the action of a drug or chemical that attenuates a fibrin or platelet signal in a biological sample to generate a fully restored fibrin or platelet signal.

10. 9. The system of claim 8, wherein the drug is a direct-acting oral anticoagulant (DOAC) and the first and / or second reversal drug inhibits, antagonizes, or attenuates the activity of a Xai or DTi class DOAC.

11. 9. The system of claim 8, wherein the drug is an antiplatelet medication and the first and / or second reversal drug inhibit, antagonize, or attenuate the activity of the antiplatelet medication.

12. 10. The system of claim 8, comprising a processing device configured to manipulate the biological sample and monitor the direct response of the biological sample to the first and / or second reversal drugs to identify a drug class present in the biological sample.

13. 10. The system of claim 8, comprising a processing device configured to manipulate a biological sample and monitor the direct response of the biological sample to a molar excess of fibrin or a platelet attenuating drug to identify a drug or chemical level or concentration present in the biological sample.

14. 10. The system of claim 1, wherein the biological sample comprises a raw blood sample, a processed blood sample, a blood sample treated with an anticoagulant to prevent intrinsic pathway coagulation activation, a citrated blood sample that is recalcified, or a blood sample treated with an antiplatelet drug to prevent platelet activation.

15. 6. The system of claim 5, comprising a processing device configured to compare the fibrin or platelet signal with a fully restored fibrin or platelet signal, to compare the fibrin or platelet signal with a fully attenuated fibrin or platelet signal, and to compare all other coincident signals for all reactions to determine the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration in the biological sample.

16. 10. The system of claim 1, wherein the reaction zone comprises a single flow channel with separate clotting sites in a serial configuration having different tissue factor (TF) concentrations.

17. 10. The system of claim 1, wherein the reaction zone comprises two parallel channels along the same plane, each channel having a different tissue factor (TF) concentration.

18. The system of claim 1, wherein the reaction zone comprises two flow paths on separate planes of the assay device, each of the two flow paths having a clot site in a non-overlapping configuration with respect to each other and having different tissue factor (TF) concentrations.

19. 10. The system of claim 1, wherein increasing the reaction temperature in the reaction zone decreases the initiation time, increases the reaction rate, and provides a higher signal of fibrin and platelet accumulation within the reaction zone.

20. adding a biological sample to the assay device; adding one or more chemical reagents to the assay device to effect a biological process whereby fibrin and platelets may accumulate in a reaction zone of the assay device; Detecting a biological process using the fluorescent assembly by fluorescent labeling and detecting the resulting accumulating fluorescent signal; and using the accumulated fibrin and platelets and the accumulating fluorescent signal in the reaction zone of the assay device to determine at least one of the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration.

1. A method for drug or chemical detection and quantification, comprising:

21. adding a biological sample to a first inlet, a second inlet, a third inlet, and a fourth inlet of an assay device comprising an outlet and a microfluidic channel fluidly connecting each of the first, second, third, and fourth inlets with the outlet, wherein the biological sample is an unmodified sample comprising a platelet-specific label and a fibrin-specific label; generating a fibrin signal and a platelet signal from the biological sample for each of the first, second, third, and fourth inlets; and determining the presence of a drug, a drug class, a drug level relative to a threshold, or a drug concentration from the fibrin signal and the platelet signal; 21. The method of claim 20, comprising:

22. 22. The method of claim 21, wherein the biological sample comprises an unprocessed blood sample, a processed blood sample, a blood sample treated with an anticoagulant to prevent intrinsic pathway coagulation activation, a citrated blood sample that is recalcified, or a blood sample treated with an antiplatelet agent to prevent platelet activation.

23. 22. The method of claim 21, comprising determining the overall state of coagulation from an unaltered sample by evaluating the fibrin signal and the fully restored fibrin signal obtained using a reversal agent.

24. 22. The method of claim 21, comprising determining the overall state of platelet function from an unaltered sample by evaluating the platelet signal and the fully restored platelet signal obtained using a reversal agent.

25. 22. The method of claim 21, comprising using a Xai reversing agent at a second inlet and a DTi reversing agent at a third inlet.

26. 22. The method of claim 21, comprising receiving a concentration of the fibrin-attenuating drug up to a point that does not result in further attenuation of the fibrin signal, to obtain a fully attenuated fibrin signal.

27. 22. The method of claim 21, comprising receiving a platelet-attenuating drug at a concentration up to a point that does not result in further attenuation of the platelet signal to obtain a fully attenuated platelet signal.

28. 22. The method of claim 21, comprising generating a platelet signal simultaneously with a fibrin signal for each of the first, second, third and fourth inlets.

29. 22. The method of claim 21, wherein an unmodified sample fibrin signal and an unmodified sample platelet signal are generated from a microfluidic channel associated with the first inlet, a fully reversed fibrin signal and simultaneous platelet signal are generated from either a microfluidic channel associated with the second or third inlet by interaction with the Xai or DTi reversing agent, and a fully attenuated fibrin signal and simultaneous platelet signal are generated from a microfluidic channel associated with the fourth inlet.

30. 22. The method of claim 21, comprising providing a light source to a microfluidic channel to monitor clot development in the microfluidic channel based on a fluorescent response of one or more reagents, receiving measured fluorescent intensities of the monitored clot development as input to a processing device, and correlating the measured fluorescent intensities with platelet and fibrin accumulation in the microfluidic channel using the processing device.

31. 22. The method of claim 21, comprising comparing the fibrin signal, along with a simultaneous platelet signal, with a fully reversed fibrin signal and a fully inhibited fibrin signal using a processing device to determine a drug class or drug concentration in a biological sample.

Images