Patent Application
20230166255
The coagulation system is a delicate balance between hemorrhage and thrombosis. There are many disease states, including cancer, auto-immune disease, infection, trauma, surgery, heart disease, and drugs, that can cause a disruption of this balance and result in a patient having severe, even life-threatening, bleeding or clotting events. Anticoagulant medications are commonly prescribed for thrombotic disorders. Conventional anticoagulant medications, such as Heparin, will indirectly inhibit multiple factors of the clotting cascade. The more recent introduction of direct oral anticoagulants (DOACs) allows for targeted inhibition of the coagulation pathway.
The biggest risk of anticoagulation therapy is the increased risk of bleeding, and thus, traditionally, patients taking anticoagulant medications are carefully monitored to ensure that they are receiving an appropriate dose. Current clinical tests available to evaluate a patient’s bleeding and clotting are either rudimentary and provide very vague information, such as prothrombin time (PT) and activated thromboplastin time (aPTT), or are more detailed but require expensive machines, lengthy training, and careful handling. Included in the latter category are thromboelastography (TEG), thromboelastometry (TEM), rotational thromboelastometry (ROTEM), platelet aggregometry and flow cytometry. Currently, specific tests for the DOACs are not available. Most of the DOAC assays that have been proposed are pharmacokinetic assays that measure the absolute concentration of the drug itself and, therefore, provide limited functional information to support clinical decision-making.
Coagulation tests are needed that can detect, characterize, and/or quantify impairments in coagulation, including detection of DOACs in patient samples, to better manage patients at high risk of severe bleeding or clotting, including, but not limited to, the urgent care setting.
Methods and devices for evaluating coagulation are described, including methods and devices for detecting an anticoagulation agent or a coagulation abnormality. Coagulation abnormality includes abnormality of clot formation (e.g., thrombosis) and abnormality of clot degradation (e.g., fibrinolysis). In various embodiments, the methods and devices of the invention measure coagulation of a sample in response to a gradient of one or more coagulation factors. These responses can be evaluated to accurately profile coagulation impairments of the sample, including the presence of a DOAC or traditional anticoagulant medication. In various embodiments, the invention provides point-of-care or bedside testing with a convenient, microfluidic device that can be used by minimally trained personnel.
In some aspects, the invention provides methods for assessing coagulation in a blood sample. The method comprises adding a coagulation factor to plural portions (e.g., aliquots) of the blood sample, each portion receiving the coagulation factor at a different concentration, and measuring clot formation or clot formation times in response to the different concentrations. By assessing coagulation in response to the different concentrations of one or more coagulation factors, blood clotting function can be accurately profiled, including the impact of DOACs or other drugs on coagulation. In some embodiments, the presence or absence of a genetic clotting abnormality is determined. The methods as described herein may be performed using a microfluidic device as described, where one or more of the channels can be configured to trigger formation and localization of a clot.
As used herein, unless described otherwise, a “blood sample” refers to a whole blood sample or a plasma sample. The term plasma includes both platelet-rich-plasma (PRP) and platelet-poor-plasma (PPP).
The term “coagulation factor” as used herein means any factor implicated in the coagulation cascade (intrinsic, extrinsic and common pathways), including Factors I to XIII, von Willebrand factor, prekallikrein (Fletcher factor), high-molecular-weight kininogen (HMWK) (Fitzgerald factor), fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, Protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), Tissue Factor Pathway Inhibitor (TFPI), and cancer procoagulant. The coagulation factor(s) can be in activated form or inactivated (e.g., precursor) form. For example, for detecting the presence of a coagulation factor inhibitor in a sample, the coagulation factor should be in activated form (e.g., Factor Xa or Factor IIa). In other embodiments, for detection of a genetic clotting abnormality, the coagulation factor may be in inactivated form (e.g., Factor X or Factor II). Further, the coagulation factor(s) can be from a human, an animal (such as bovine, porcine or other), or can be a synthesized or recombinant protein.
In some embodiments, the invention provides a method of detecting an anticoagulation agent. Anticoagulation agents are substances that prevent or reduce coagulation of blood, prolonging clotting time. Anticoagulation agents include, but are not limited to, Factor-specific inhibitors (such as FXa inhibitors, FIIa inhibitors, FXIa inhibitors, FXIIa inhibitors), heparins, and vitamin K antagonists (e.g., warfarin). In some embodiment, they include Direct Oral Anticoagulants (DOACs), also known as Novel Oral Anticoagulants (NOACs), such as XARELTO (Rivaroxaban) by Janssen Pharmaceuticals, Inc., ELIQUIS (Apixaban) by Bristol-Myers Squibb and Pfizer Inc., SAVAYSA (Edoxaban) by Daiichi Sankyo, Inc., PRADAXA (Dabigatran) by Boehringer Ingelheim, and BEVYXXA (Betrixaban) by Portola Pharmaceuticals, Inc.
By measuring clot formation (e.g., clot formation times) in response to increasing concentrations of exogenously added coagulation factors, the presence and/or point of inhibition by a therapeutic agent can be determined. For example, a sample that is positive for a coagulation inhibitor will show a concentration-dependent decrease in clotting time as the coagulation factor that is targeted by the inhibitor is added to the sample. Meanwhile, when a coagulation factor upstream from the point of inhibition is added (in increasing amounts), the clotting time will remain prolonged, as compared to the clotting time upon the addition of a coagulation factor downstream of the point of inhibition. See
In some embodiments, results for a patient sample can be compared to reference standards, including standards for normal and/or abnormal clotting, or reference standards corresponding to anticoagulant therapy with particular agents. In some embodiments, reference standards are personalized for the patient.
In various embodiments, clotting curves can be constructed to characterize the response of clot formation to the addition of various coagulation factors in increasing concentrations or amounts. These clotting curves allow for the identity and amount of coagulation inhibitors to be determined, to thereby guide patient care. In some embodiments, the appropriate coagulation inhibitor reversal agent is then administered to the patient to reverse the therapeutic intervention as needed.
In some aspects, the invention provides a microfluidic device for evaluating coagulation in a sample. The device includes a series of channels in a substrate, each channel having an area with a geometry to trigger and/or localize formation of a clot, to allow for evaluation of clot formation in response to one or more reagents, such as the amount or concentration of an exogenously added coagulation factor. The channels in the series each have the same geometry, so as to trigger identical clot formation properties (when exposed to the same sample and reagents). By evaluating clot formation in the presence of a gradient of one or more coagulation factors, the invention allows for sensitive and specific detection of coagulation abnormalities or impairments, including the presence or activity of a DOAC in the sample.
In one embodiment, the microfluidic device for detecting coagulation includes plural channels formed in a substrate, each channel including a clot forming area having a geometry configured to trigger and/or localize formation of a clot. The clot forming areas of the plural channels may be arranged in a central region of the substrate in some embodiments, such that the clotting properties can be simultaneously imaged or analyzed across the channels. See
The term “central region” as used herein means a region that is located in the center of a substrate relative to a periphery of the substrate and can include a region that is positioned off-center. For example, depending upon the configuration, the central region might be off-center and the areas in the microfluidic channels in which clots begin can be controlled by the flow patterns in the channels.
In some embodiments, the clot forming areas of the plural channels are arranged in a region of the substrate which is not central, such as, but not limited to, the periphery. See
Each channel may further comprise one or more additional input ports to receive reagents, such as coagulation factor(s) and/or calcium. In some embodiments, there is more than one input port (e.g., for introducing sample and one or more reagents) per output port. For example, in one embodiment, there can be one input port for the sample and 1 to 2 input ports for the reagents (e.g., coagulation factor and, optionally, calcium). See
In the microfluidic device, each clot forming area can be configured to create an area of stasis or disruption in fluid flow to trigger and/or localize formation of a clot. In some embodiments, each clot forming area can be configured to create an area of flow disturbance to trigger and/or localize clot formation. Exemplary geometries for triggering formation of and localizing a clot are illustrated in
Channels of the microfluidic device can be coated with, contain or otherwise include a coagulation factor at a different amount or concentration. For example, a first group or series of the plural channels can be coated with, contain or otherwise include a first coagulation factor, and a second group or series of the plural channels can be coated with, contain or otherwise include a second coagulation factor. Further, in some embodiments, one of the plural channels is a negative control channel, e.g., may not be coated with and may not include a coagulation factor. In other embodiments, the device does not comprise such a negative control channel.
In the case where one or more channels include the coagulation factor(s), the coagulation factor(s) may be in suspension or solution, or lyophilized and not surface-bound. The coagulation factor(s) can be pre-included in the channel(s) (e.g., at the time of manufacturing the device), can be added prior to placing the sample into the device, or can be entered into the device through an input port (or multiple input ports) simultaneously with the sample or after the sample.
In embodiments of the microfluidic device that include first and second groups of channels (whether or not such embodiments may also include a negative control channel in addition to the first and second groups of channels), each channel in the first group of the plural channels can be coated with, contain or otherwise include a first coagulation factor at a different amount or concentration, and each channel in the second group of the plural channels can be coated with, contain or otherwise include a second coagulation factor at a different amount or concentration. In some embodiments, the microfluidic device may contain more than two groups or series of plural channels, such as three, four, five or more groups, wherein each group or series of plural channels is coated with, contains or otherwise includes a different coagulation factor at an increasing amount across the group or series (e.g., a microfluidic device containing four groups of channels, each group of the plural channels can be coated with, contain or otherwise include a different coagulation factor selected from Factors IIa, Xa, XI, XIa, XII, and XIIa). By measuring clot formation or clotting time as a function of coagulation factor gradients, the sample’s clotting properties can be profiled at several specific points of the coagulation pathway(s) (illustrated in
The second coagulation factor can be upstream in the coagulation cascade from the first coagulation factor. For example, the first coagulation factor can be, e.g., prothrombin (Factor II), thrombin (Factor IIa), or both. The second coagulation factor can be, e.g., Factor X, Factor Xa, or both.
The microfluidic device can further include a detection device configured to measure clot formation times in each of the channels to assess coagulation based on the clot formation times measured. For example, the detection device can be configured to image the clot forming areas simultaneously to measure clot formation times. In some embodiments, the degree of clot formation in each of the channels is quantified at a fixed time or times. For example, the detection device in connection with the methods and devices described herein can include a microscope and an image sensor. Imaging the clot forming areas can include bright-field imaging. For the devices and assays described herein, clotting times can also be measured with other methodologies such as detection based on light absorbance, fluorescence measurements, ultrasound, etc., and the detection device can be configured to employ one or more of these other methodologies. Ways to detect clotting also include, but are not limited to, detection based on electrical impedance, the addition of beads and quantifying bead flow rate/number, measurement of flow velocity and/or pressure before and/or after the site of clot formation, thromboelastography, fluorescence detection (such as with fluorescent fibrinogen), turbidity, magnetic, flow dynamics (pressure or flow velocity), infrared light detection, infrared spectroscopy, detection using acoustic and/or photonic sensors, flow cytometry, and visual clotting detection.
In some embodiments, the method described herein does not employ a microfluidic device, but uses wells or containers suitable for inducing and measuring formation of a clot.
In addition to clot formation times, other characteristics of clot formation can be considered. It is contemplated that a qualitative measure of clot formation, in addition to clot formation times, can be useful, e.g., to determine the most sensitive detection mode for coagulation. For example, properties of the clot such as size, strength, density and composition can be assessed in addition to time to form a clot. Such properties may be assessed using the same or a different detection modality than is used to detect clot formation times.
In some embodiments, clot lysis can be assessed in addition to clot formation. For example, if a patient is on a fibrinolytic or thrombolytic agent, one can evaluate the clot when it is being formed as well as its breakdown over time. In one embodiment, the same methods described herein and known in the art to detect clot formation can be used to assess clot lysis over time.
As described herein regarding the use of thromboelastography (TEG), one can evaluate both clot formation and fibrinolysis. This would be useful for detecting clotting abnormalities in patients that are hypocoagulable due to problems with fibrinolysis or iatrogenic administration of fibrinolytic and thrombolytic drugs. See, for example, C. Mauffrey, et al., “Strategies for the management of haemorrhage following pelvic fractures and associated trauma-induced coagulopathy,” Bone Joint J. 2014; 96-B:1143-54, the relevant teachings of which are incorporated herein by reference.
In any of the devices and methods described herein, the blood sample can be a whole blood sample or a plasma sample. Using whole blood can be particularly useful for certain applications, such as those implemented at the bedside of a patient.
The disclosed devices and methods can be applied to all individuals, including mammals (e.g., humans, such as human patients, as well as non-human mammals), reptiles, birds, and fish, among others, and can be useful for research and veterinary medicine. An individual can be, for example, mature (e.g., adult) or immature (e.g., child, infant, neonate, or pre-term infant).
The disclosed devices and methods can be used not just for diagnostic purposes but also for research and discovery to explore the coagulation cascade in a research setting. For example, this can be useful for basic drug discovery, understanding disease or disorder pathophysiology, for example, in the context of hemorrhagic diseases (Dengue virus, Zika virus, Ebola virus, etc.), and also to monitor for adverse events of experimental treatments.
The disclosed devices and methods can be used to guide therapy of a patient. For example, physicians can use the results to determine subsequent treatments with both drugs and procedural interventions (both invasive and non-invasive). For example, if a patient tests positive for Factor IIa inhibition due to dabigatran administration, then the healthcare provider may choose to administer the reversal agent (idarucizumab) for this inhibitor prior to surgery or other invasive procedures. Likewise, if the patient tests positive for Factor Xa inhibition, then the healthcare provider may choose to administer the appropriate reversal agent (coagulation factor Xa (recombinant), inactivated-zhzo) for this inhibitor. The healthcare provider may choose to administer other agents that overcome the effects of these inhibitors as well, such as 4-factor prothrombin complex concentrates or activated prothrombin complex concentrates.
Other aspects and embodiments of the invention will be apparent from the following Drawings and Detailed Description.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.
The invention generally relates to methods and devices for the detection of coagulation, including detection of coagulation abnormalities and detection of anticoagulants and platelet inhibitors in plasma and/or whole blood.
Acquired coagulopathies are a major component of morbidity and mortality in a number of medical settings. Individuals may have increased risk of internal bleeding secondary to drugs (e.g., clopidogrel, heparin, warfarin or other vitamin K antagonists, dabigatran or other Direct Oral Anticoagulants, etc.), trauma, surgery, sepsis, cancer, organ dysfunction (e.g., liver), or congenital abnormality (e.g., hemophilia). On the other end of the spectrum, increased propensity for clotting can be due to autoimmune disease, cancer, atherosclerosis, early trauma and sepsis, organ dysfunction (e.g., kidney), immobility, inflammation, foreign body (e.g., stent or prosthesis), or congenital abnormality (e.g., Factor V Leidin thrombophilia). With recent innovations in drug development (e.g., anticoagulants, including Direct Oral Anticoagulants, or DOACs), innovation is now needed for hemostasis/coagulation analyzers, to fully realize benefits for patients, including in the urgent care setting. Specifically, current clinical tests available to evaluate a patient’s bleeding and clotting are either rudimentary and provide very vague information, such as prothrombin time (PT) and activated thromboplastin time (aPTT), or are more detailed but require expensive machines, lengthy training, and careful handling, such as thromboelastography (TEG), thromboelastometry (TEM), rotational thromboelastometry (ROTEM), platelet aggregometry and flow cytometry. Currently, specific tests for DOACs are not available. Most of the DOAC assays that have been proposed are pharmacokinetic assays that measure the absolute concentration of the drug itself and, therefore, provide limited functional information for clinical decision-making.
With the increased use of DOACs, studies and reviews are finding that, although these new drugs pose less risk for acute, life-threatening bleeding events, they are potentially linked to higher rates of gastrointestinal (GI) bleeding. Additionally, these new drugs are found to have different pharmacokinetic properties in patients with decreased liver and/or kidney function or in patients that are on multiple drugs at the same time, as is common in the geriatric population. In these cases, providing functional clinical information to the doctor to help personalize the anticoagulant combination and dosage would be of great benefit to the patient and possibly decrease subsequent, related adverse events. Embodiments of the invention can be used in clotting panels that evaluate the coagulation, fibrinolysis, and platelet function within an individual. The microfluidic technology and advanced assays described herein in some embodiments provide for custom clotting panels, whereby clinicians can determine a patient’s coagulation function bedside. These embodiments provide for vast improvements in patient care, including in the urgent care setting.
In addition to these assays being rapid and easy-to-interpret, they can also be customizable, allowing for the selection of clinically-relevant coagulation and platelet function testing for each customer and/or end-user segment. Because embodiments of the assay can be applied in a bedside platform, it can also be utilized for trend-monitoring in patients on various treatments (including at the hospital, at anticoagulation clinics and at home). In an aspect of the invention, a gradient of the factor(s) is added to the sample after it is subdivided into and/or distributed among the multiple groups of plural channels, wells, or containers, which method permits evaluation of coagulation function/inhibition and identification and differentiation between various coagulation abnormalities within a sample. This means that embodiments of the invention (e.g., clotting panels, assays, etc.) are potentially useful for assessing coagulation in patients that have poor medical compliance, where dosage/time taken is unknown, or in patients that are unconscious, where the doctor, surgeon or other healthcare provider needs to know whether the patient has any of these drugs in their system. Further, embodiments can help in monitoring anticoagulation and guiding the administration of reversal reagents that are now becoming available.
Examples of potential users for product or services based on embodiments of the invention can range from healthcare workers, e.g., clinicians and veterinarians, to researchers in pharmaceutical research and development.
The invention can be applied to patient care in various settings. In some embodiments, the patient is scheduled for surgery or is in need of an invasive procedure, and the methods and devices of the invention can be used for clinical decision-making, including preparing the patient for the procedure to minimize bleeding risks. In some embodiments, the patient is administered a drug that impacts coagulation, and the methods and devices of the invention can be used for early evaluation of drug action and for selection of the appropriate therapy and dose. In some embodiments, the patient receives a drug or blood product, and methods and devices of the invention can be used to guide administration and dose. In some embodiments, the patient has or is suspected of having, or is at risk of acquiring, a hemorrhagic virus. In some embodiments, the patient is a neonate, where only small volumes of blood are available for evaluating coagulation (including for administering anticoagulant therapy or for detecting a congenital coagulation abnormality). In some embodiments, the patient is a pregnant mother, and the methods and devices allow for detecting a congenital coagulation abnormality, or for early diagnosis of a condition that results in a coagulation abnormality such as pre-eclampsia and eclampsia.
In some embodiments, the patient or subject is a veterinary or animal patient (e.g., such as a dog, cat, or horse). In some embodiments, the patient is a non-human mammal. The cost-restrictions and limited blood volume of veterinary patients and laboratory animal research result in a large need for coagulation diagnostics that are easy-to-use, require only microliters of blood, and have lower overhead costs.
Due to the immense interest in novel coagulation testing platforms, the blood-testing platform (e.g., assay, microfluidic device, and/or combination thereof) described herein offers tremendous potential for research and product development.
In some embodiments, the patient is receiving an anticoagulant therapy, such as a heparin or vitamin K antagonist (e.g., warfarin). In some embodiment, the patient is undergoing therapy with a Direct Oral Anticoagulant (DOAC), such as XARELTO (Rivaroxaban), ELIQUIS (Apixaban), SAVAYSA (Edoxaban), PRADAXA (Dabigatran), or BEVYXXA (Betrixaban). In some embodiments, the patient is undergoing therapy with an antibody against TFPI. Anticoagulant drugs are used commonly in many medical settings, including emergency and critical care, surgery, cardiology, and cancer. Several new anticoagulants have been introduced, but there are no current tests that can reliably determine if a patient is on the right dose. Too much anticoagulation can cause life-threatening bleeding and too little can lead to an increased risk of stroke and heart attacks. Embodiments of the invention can be used as or incorporated into a bedside test that can accurately monitor these new anticoagulants and improve the safety for these patients. This test can be performed with minimal training and in an easy to interpret format. In an embodiment, these assays can be performed in the lab in a device requiring less than about 1 mL, or less than about 500 µL, or less than about 100 µL, or less than about 50 µL (one drop) of fresh or citrated, whole blood, with the results being read within 10 minutes.
The Direct Oral Anticoagulant (DOAC) market currently consists of drugs that selectively target specific factors within the coagulation pathways, e.g., Factor IIa or Factor Xa. While these drugs are very potent, because of the dearth of reliable or easy-to-use diagnostic and monitoring tests, there is an increased risk associated with the use and administration of these drugs, especially in the critical care setting. One of the primary risks of DOAC use is gastrointestinal bleeding. These adverse events not only lead to morbidity and mortality but also result in increased medical costs and longer hospitalization times.
In some embodiments, the method involves detecting a coagulation abnormality in a blood sample, and pinpointing where it occurs within the coagulation cascade, by comparing the clot formation times determined to coagulation factor-specific clot formation reference ranges, e.g., from individual(s) who do not suffer from a coagulation cascade abnormality. In some embodiments, the reference ranges can be established using the detection method on a normal subject or subjects, e.g., individuals who do not suffer from a coagulation abnormality. In some embodiments, the reference range can be established based on the same individual from whom the test blood sample(s) is obtained. For example, the reference range can be established prior to commencement of a medical treatment of an individual, and the test sample can be obtained from the same individual after the commencement of a treatment. The sample can also be obtained from a relative (e.g., parent, sibling or offspring) of the individual from whom the test sample is obtained. The reference ranges may be tailored to or dependent on a particular assay configuration, including microfluidic device configuration. In some embodiments, each subject’s clotting can be compared to a “normal” control at the testing time or to previously-determined “normal” reference ranges for the specific coagulation factor or combination of factors. In some embodiments, the assay approach requires the establishment and/or verification of reference ranges.
In some embodiments, reference ranges are from controls or standards of a specific coagulation cascade abnormality, such as from individuals who do not suffer from a coagulation cascade abnormality. In some embodiments, the reference ranges are from spiked or depleted samples/controls, which can be commercially available.
It should be understood that one can also compare clot formation times to reference ranges from someone who does suffer from a coagulation abnormality. For example, it is common with reference intervals to have a “normal” interval range for people who do not suffer from an abnormality and an “abnormal” interval range for people confirmed to have that abnormality. Sometimes, there is a gray zone in-between the normal and abnormal zones, that is indicative that further in-depth testing needs to be done on that patient sample for a definitive diagnosis.
In some embodiments, the invention does not require comparison to a reference range or standard and, instead, provides internal controls by evaluating coagulation factors upstream and downstream of a suspected point of inhibition in the coagulation pathway(s).
A description of example embodiments follows.
Embodiments described herein include rapid assays (e.g., <30 minutes, <20 minutes, <15 minutes, or <10 minutes in some embodiments) for the detection of anticoagulants and platelet inhibitors in whole blood or plasma and the assessment of patient coagulation status. The availability of these customizable coagulation panels fills an unmet need within various coagulation testing environments by providing rapid, bedside diagnostics and drug monitoring capabilities.
In an embodiment, the method includes an assay wherein a specific coagulation factor suspected of being inhibited is added into a blood sample (e.g., a whole blood or plasma sample), in various concentrations or amounts. For example, the coagulation factor can be added to divided portions of the sample in amounts that vary by a factor of 2 to a factor of 100. In some embodiments, coagulation factor is added to divided portions of the sample at concentrations increasing by a factor of 5 to a factor of 20 (e.g., about a factor of 10) across the divided portions. In some embodiments, the concentration of the coagulation factor added to the divided portions of the sample can be in the range of 0.1 ng/mL to 10 µg/mL. The addition of the coagulation factor at specific concentrations or amounts (e.g., a gradient or multiple samples with different concentrations) enables determination of:
Examples of the utility of this assay include:
Embodiments of methods and devices described herein can be used to evaluate coagulation abnormalities (e.g., pro- or anti-thrombotic) using various coagulation detection technologies, such as those described herein, including: electrical impedance, the addition of beads and quantifying bead flow rate/number, measurement of flow velocity and/or pressure before and/or after the site of clot formation, thromboelastography, fluorescence detection (such as with fluorescent fibrinogen), turbidity, magnetic, flow dynamics (pressure or flow velocity), infrared light detection, infrared spectroscopy, detection using acoustic and/or photonic sensors, flow cytometry, and visual clotting detection.
Whole blood and plasma can be used in various embodiments.
Embodiments of the assays can be combined with ATP-luciferase assays in order to measure platelet and coagulation system function at the same time. This can provide evaluation of the coagulation cascade, as well as platelet function, via the degranulation of the platelet upon sufficient activation. Activation of the platelet can occur via the addition of the coagulation factors listed herein, or by the addition of specific platelet agonists, such as, e.g., adenosine diphosphate (ADP), adenosine triphosphate (ATP), epinephrine, collagen, thrombin, and ristocetin. This combined technique can be used to assess platelet function when patients are taking platelet inhibitors, such as aspirin or clopidogrel. These agonists can be added as a concentration gradient in combination with the coagulation factors. Luciferase is typically measured by light absorbance.
Coagulation abnormalities that can be detected or analyzed include, but are not limited to, congenital or hereditary coagulopathies and acquired coagulopathies.
Congenital or hereditary coagulopathies include acquired mutations and hereditary coagulopathies, i.e., inherited from a parent.
Congenital coagulopathies are present at birth and are likely due to a developmental abnormality that occurred in utero. Congenital coagulopathies may or may not be genetic. In some embodiments, the patient may have or be suspected to have a coagulation factor deficiency, which may be caused by the production of a deficient amount of the clotting factor, or the clotting factor is encoded by a gene with a mutation that decreases the function of the clotting factor.
Examples of congenital and hereditary coagulopathies include, but are not limited to:
Causes of acquired coagulopathies include, but are not limited to: organ (e.g., liver) dysfunction or failure, bone marrow dysfunction or failure, trauma (e.g., automobile accident), surgery, infection (e.g., flavivirus, hemolytic uremic syndrome, sepsis, etc.), cancer, immobility, drugs (e.g., antibiotics, anticoagulation, fibrinolytics, thrombolytics, chemotherapy, fluids, etc.), neutraceuticals/pharmaceuticals, toxicities, envenomation (e.g., snake, spider, etc.), foods, auto-immune diseases (whether primary, acquired or idiopathic), implants (e.g., surgical), cardiovascular event(s) (e.g., a clot of blood anywhere in the body, including stroke, heart attack, etc.), vasculitis, transfusions (e.g., whole blood, packed red blood cells, plasma, platelets, etc.), transplants (e.g., bone marrow, kidney, liver, etc.), pregnancy (e.g., pre-eclampsia, eclampsia, diabetes, etc.), endocrine disease (e.g., pheochomocytoma, cushings, diabetes, etc.), chronic inflammatory disease (e.g., irritable bowel syndrome, irritable, bowel disease, colitis, etc.), disseminated intravascular coagulation, and infection.
Coagulopathies may also be iatrogenic (e.g., caused by medical treatment) or have idiopathic causes (e.g., cancer treatment, such as chemotherapy, or bone marrow transplant).
In some embodiments, the invention employs a microfluidic approach. The microfluidic device includes a series of channels in a substrate, each channel having an area with a geometry to trigger and/or localize formation of a clot, to allow for evaluation of clot formation in response to one or more reagents, such as the amount or concentration of an exogenously added coagulation factor. Each of the channels in the series has the same geometry, so as to trigger identical clot formation properties (when exposed to the same sample and reagents). By evaluating clot formation in the presence of a gradient of one or more coagulation factors, the invention allows for sensitive and specific detection of coagulation abnormalities or impairments, as described above.
Embodiments employing a microfluidic device, may involve the following procedures:
A microfluidic device for detecting coagulation can include plural channels formed in a substrate, each channel including a clot forming area having a geometry configured to trigger and/or localize formation of a clot. In some embodiments, the clot forming areas of the plural channels are arranged in a central region of the substrate. In some embodiments, the device further includes plural sample input ports, each sample input port connected to a first end of one of the plural channels. In some embodiments, the device comprises plural output ports, each output port connected to a second end of one of the plural channels. The input and output ports may be arranged in an alternating pattern at a periphery of the substrate. In some embodiments, the device comprises a common sample input port, in fluid connection with all channels or a series of channels.
A substrate can be, for example, any type of plastic, polydimethylsiloxane (PDMS), silicon, glass, or other material or combination of materials. In an embodiment, the device includes a substrate bound to glass, but other substrates can be used, such as glass on glass, PDMS on PDMS, silicon, any type of plastic, or combinations thereof. In one embodiment, the substrate is plastic. The substrate can be (but need not be) transparent to facilitate the detection of clot formation (vis-à-vis, e.g., imaging).
The device can include microfluidic channels with a diameter of about 50 µm, a height of about 11 µm, and a length of 100+ µm. Other channel dimensions can be employed.
One entry and one exit port for the sample input can be provided for each channel. Alternatively, devices can provide a single sample port for all channels or for one or more groups (or series) of channels.
In various embodiments, an agonist (e.g., a coagulation factor) is added to the sample prior to input into the device or the agonist is coated to, or otherwise pre-loaded within, the device prior to sample loading. In the case where one or more channels include the coagulation factor(s), the coagulation factor(s) may be in suspension, solution, or lyophilized, and may be surface-bound or not surface-bound. The coagulation factor(s) can be pre-included in the channel(s) (e.g., at the time of manufacturing the device), can be added prior to placing the sample into the device, or can be entered into the device through an input port (or multiple input ports) simultaneously with the sample or after the sample.
In an embodiment, calcium is added to the sample prior to input into the device. Calcium can be added within the device, through an additional port, or pre-loaded within the channel.
In an embodiment, 488-conjugated fibrinogen is added to the sample to detect the time it takes for a clot to form via the detection of cross-linking of the fibrinogen.
In bright-field, clot formation can also be detected by visualizing the cross-linking of fibrin and by the stopping of the flow of the sample through the microfluidic channel, which can be performed with or without an additional flushing step to flush out material not associated with a clot.
In an embodiment, the sample is loaded into the device or microfluidic cartridge via capillary action. The sample can also be forced to flow through the channel, e.g., through the use of a vacuum, syringe-pump, or other suitable means, including, in some embodiments, gravity. The sample can also be encouraged to load by capillary action or flow by using coating that alters the surface properties of the microfluidic device (e.g., substrate), such as by making it hydrophilic.
In an embodiment, the design of the microfluidic channel(s) includes one area of an altered geometry (including different angled bends and/or diameters) in order to create one area of flow separation and stasis to trigger and/or localize formation of the blood or fibrin clot. The time that it takes for the clot to form can be quantified and recorded.
In an embodiment, the device is used to detect the presence and assess the effect of anticoagulation agents, e.g., FXa inhibitors, FIIa inhibitors, heparin, and vitamin K antagonists (e.g., warfarin) by assessing the time is takes to form a clot.
The measured clot formation time is correlated to the amount of clotting inhibition that is resultant from an anticoagulant in the sample. This process can also be applied to a fibrinolytic drug. This process can also be applied to other pathologies, including acquired or congenital causes of abnormal clotting times, as described herein.
In an embodiment, the device provides a read-out in a relatively short period of time, for example, in about 3-10 minutes, and, in a particular example, in about 5 minutes.
Example microfluidic devices and assays are described below and illustrated in the figures.
As illustrated in
In some embodiments, the disruptor can include a concavity (e.g.,
A general protocol for performing the assay according to an embodiment of the invention is as follows:
In an example, the process of clot detection can include the following procedural steps:
The example in
Optionally, as illustrated in
The microfluidic device for use in the method of
At a concentration of 0 ng/mL Rivaroxaban, clot formation detected in < 2.5 minutes with agonist concentration down to 7.5 ng/mL.
At a concentration of 250 ng/mL Rivaroxaban, clot formation time is significantly longer than the negative control but lower than 500 ng/mL with agonist concentration down to 375 ng/mL.
At a concentration of 500 ng/mL Rivaroxaban, clot formation detected < 2.5 minutes down to 750 ng/mL.
At a concentration of 0 ng/mL Apixaban, clot formation detected in < 2.5 minutes with agonist concentration down to 7.5 ng/mL.
At a concentration of 250 ng/mL Apixaban, clot formation detected in < 2.5 minutes with agonist concentration down to 75 ng/mL.
At a concentration of 500 ng/mL Apixaban, clot formation detected in < 2.5 minutes with agonist concentration down to 938 ng/mL.
At a concentration of < 25 ng/mL Dabigatran, clot formation detected in < 2.5 minutes with agonist concentration down to 71 ng/mL.
At a concentration of 250 ng/mL Dabigatran, get clot formation detected in < 2.5 minutes with agonist concentration down to 710 ng/mL.
At a concentration of 500 ng/mL Dabigatran, clot formation detected in < 2.5 minutes down to 710 ng/mL.
Automation can be employed to reduce variation between samples and assays.
In addition to the detection of the presence of FXa inhibitors and estimation of their relative concentrations, the assay described here can differentiate FXa inhibitors from FIIa inhibitors by selecting appropriate upstream and downstream clotting factors to add to the samples.
For congenital disorders, embodiments can add non-activated factor(s) for detection, whereas non-activated factor(s) can serve as control.
In addition to identifying inhibition, as illustrated in the examples of
When a patient is at high-risk for a bleeding event or has an active bleed coagulation tests are ordered. These tests can include PT, INR, aPTT, ACT, TEG, or other currently available point-of-care tests. Abnormal clotting results on currently-available tests are non-specific for the presence of DOACs and leaves the healthcare worker guessing as to which treatment is the most appropriate for the patient. If the coagulation times are normal, due to the lack of sensitivity of these tests, the healthcare worker may miss the presence of a DOAC in the patient sample and proceed with treatment, putting the patient at an increased risk of bleeding.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.
This application is a divisional of U.S. Application No. 16/046,816, filed Jul. 28, 2018, which claims the benefit of U.S. Provisional Application No. 62/538,618, filed on Jul. 28, 2017, and U.S. Provisional Application No. 62/699,665, filed on Jul. 17, 2018, the entire contents of which are hereby incorporated by reference.
This invention was made with Government support under Grant Nos. P41 EB002503, P30 ES002109, and P50 GM021700 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62538618 | Jul 2017 | US | |
| 62699665 | Jul 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16046816 | Jul 2018 | US |
| Child | 18159281 | US |
