Patent Application
20220283190
The coagulation system is a delicate balance between hemorrhage and thrombosis. There are many disease conditions and clinical situations (such as, for example, cancer, auto-immune disease, infection, trauma, surgery, heart disease, and drug treatments), that can cause a disruption of this balance and result in a patient having severe, and in some cases even life-threatening, bleeding or clotting events. In some cases, a patient may suffer from coagulopathy, which can be congenital and/or hereditary, or acquired. Coagulopathy may result from a deficiency in a coagulation factor; examples of coagulation factor deficiencies that may result in a coagulopathy include a deficiency in Factor VIII, deficiency in Factor IX, and deficiency in Factor XI (e.g., Hemophilia A, B, and C, respectively). While hemophilia is most commonly known as a congenital and/or hereditary disease, there are also acquired causes of hemophilia, such as the development of anti-factor autoantibodies (e.g., Acquired Hemophilia A or AHA). It is possible to have coagulation factor deficiencies and/or inhibitors (such as autoantibodies) to any of the coagulation factors, although deficiencies of and inhibitors to FVII, FXII, and FXIII tend to be less common than are deficiencies of and inhibitors to other coagulation factors. Deficiencies of or inhibitors to coagulation factors may lead to prolongation in bleeding times and adverse bleeding events; whether such consequences occur often depends on the level of deficiency or on the inhibitor concentration. While the most common treatment for deficiency of any given factor is factor replacement (via administration of the specific factor that is deficient), there are new treatments being pursued that trigger the body to produce the factor on its own. Such treatments include bypassing agents (e.g., emicizumab) and gene therapies (including DNA- and RNA-based therapies).
While congenital factor deficiencies range from mild to severe, the development of auto-antibodies to coagulation factors can rapidly lead to severe, life-threatening bleeding events. Anti-factor antibodies occur in up to 30% of all hemophilia patients treated with factor replacement and can also occur spontaneously in adults who have no factor deficiency and are not being treated with factor replacement. Clinical presentation for anti-factor antibody development is usually in the emergency room, where a patient may present with moderate to severe spontaneous bleeding. The treatment for anti-factor antibodies (a type of factor inhibition) can be different from the treatment for factor deficiency, due to the supra-physiologic levels of that factor that are required to overcome the presence of the factor inhibitor, depending on the concentration and inhibition activity of the inhibitor (in this case, the anti-factor antibody); this requirement for supra-physiologic factor levels usually necessitates the administration of high levels of factor replacement or of bypassing agents, where inactivated or activated coagulation factors downstream of the point of inhibition are administered in the event of adverse bleeding. Such inactivated or activated coagulation factors include, for example, activated Factor VII (FVIIa) or anti-inhibitor Coagulant Complex (FEIBA®), which contains Factors II, VIII, IX, and X, as well as FVIIa. It is imperative for a clinician to appropriately diagnose whether a patient is factor deficient and/or has factor inhibitors (such as anti-factor antibodies), as the treatment for factor inhibitors (e.g., administration of FVIIa or FEIBA®) involves activated bypassing factors that could result in embolic and thromboembolic events if not administered appropriately. The risk of embolic and thromboembolic events also makes the dosing and duration of administration of these bypassing agents very important clinical considerations.
In addition, an individual may suffer from a coagulation factor abnormality, such as an abnormality that results from a genetic mutation. An abnormality may or may not result in changes to coagulation factor function. In cases where an abnormality results in a decrease in coagulation factor function, such decrease in factor function may also result in a prolongation in bleeding time and/or adverse bleeding events. In such cases where an abnormality results in a decrease in coagulation factor activity, the abnormality causes a factor deficiency. In other cases, an abnormality to a coagulation factor, such as the abnormality caused by a mutation in Factor V in patients with Factor V Leiden (FVL) thrombophilia, results in an increase in blood clotting; in the case of FVL, the mutation results in resistance to Activated Protein C (APC), a circulating anticoagulant and inhibitor to Factor Va and VIIIa, and thereby causes an increase in blood clotting.
There are also conditions that result in transient changes in coagulation factor concentrations. Many coagulation factors are acute phase proteins, meaning that their concentration may increase or decrease during an inflammatory response. For example, Factor I (fibrinogen) and Factor VIII are positive acute phase proteins and their concentrations increase with inflammation, while antithrombin III (AT or ATIII) is a negative acute phase protein, as its concentration decreases with inflammation. These dynamic changes in coagulation factor levels that can happen during, by way of example, inflammation, stress, or pregnancy, are important considerations when assessing an individual's coagulation phenotype and selecting or monitoring patient treatment.
Current clinical tests used to detect and identify specific coagulation factor deficiencies or inhibition require a series of specialty coagulation tests, including, but not limited to, activated partial thromboplastin time (aPTT) assay, complex dilute plasma tests for identifying factor deficiency, and Bethesda assays (including modifications thereof) for detecting and quantifying anti-factor auto-antibodies. For a patient with an undiagnosed factor deficiency or inhibition, diagnosis usually requires multiple specialty tests (which are typically performed by specialty coagulation laboratories) and the simultaneous interpretation of these multiple test results. For patients with anti-factor antibody development, there are no rapid, non-specialty or point-of-care tests currently available to diagnose these patients and to differentiate factor deficiency from factor inhibition, such as inhibition caused by anti-factor antibodies. Consequently, time-delays in delivering appropriate treatment to these patients can increase morbidity and mortality. In particular, while the typical treatment for factor deficiency is factor replacement, patients with elevated levels of factor inhibitors (such as anti-factor antibodies) may require supra-physiologic levels of factors to overcome the inhibitor or may require bypassing agents to achieve rapid hemostasis. To further complicate the clinical picture, if a bypassing agent is given to a patient with a coagulation factor deficiency but with no factor inhibitors (such as anti-factor antibodies), such patient may be at an increased risk of an embolic or thromboembolic event due to the inappropriate administration of activated bypassing coagulation factors.
Two primary classes of coagulation tests are used to evaluate coagulation factor deficiency and inhibition: “activity” tests and “functional” tests. In addition to these tests, genetic analysis may be used to evaluate whether a patient has a coagulation factor mutation, and mass-spectrometry, western blots, and ELISAs can be used to quantify coagulation factor concentrations (although none of these quantitative tests is commonly used for patient treatment monitoring, as they are not functional- or activity-based tests and, therefore, have limited clinical value).
Currently available coagulation factor activity tests are indirect-activity tests that utilize a chromogenic read-out and require platelet-poor plasma (PPP). For example, one way to perform a Factor IX (FIX) chromogenic activity assay is to take a patient's plasma sample (FIX deficient) and add in Factor XIa, Factor VIIIa, calcium, and phospholipids. The resultant Factor Xa, which is generated through the complexing of Factor IXa (present at a sub-physiologic level or with sub-physiologic activity in a FIX-deficient sample) and Factor VIIIa, is then quantified using a chromogenic substrate that binds to and detects Factor Xa. Similarly, one way to perform the Factor VIII (FVIII) chromogenic activity assay is to take a patient's plasma sample (FVIII deficient), add in Factor XIa to activate coagulation, and add Factor X in excess, Factor IIa, calcium, and phospholipids. Factor Xa is then generated and quantified with the chromogenic substrate. These protocols, which use the Factor Xa chromogenic assay as an indirect method of detecting deficiencies in FIX or FVIII, are cumbersome and require the simultaneous addition of multiple coagulation factors in order to create optimal assay conditions. In essence, these tests attempt to isolate the coagulation factor in question (in these two examples, FIX or FVIII) by adding multiple exogenous coagulation factors to a single plasma sample in order to identify a deficiency in that factor. However, these tests are not able to assess how the patient's other endogenous coagulation factors affect the generation of Factor Xa. For example, a variety of comorbidities may result in changes to various endogenous factor levels and may play a role in the coagulation phenotype of a patient. See, e.g., Brummel-Ziedins et al., PLoS One 7:e29178 (2012). But because the chromogenic tests require the addition of coagulation factors to the patient's sample, the tests are not designed to and cannot determine how a patient's elevated level of another factor affects the patient's coagulation phenotype.
Coagulation factor functional assays are also cumbersome and cannot assess a patient's full coagulation phenotype. For example, in a 2-stage FVIII assay, the patient's plasma sample (FVIII deficient) is adsorbed prior to testing (this adsorption removes endogenous Factors II, VII, IX, and X from the patient's sample), exogenous Factor XIa is added to activate coagulation, and excess Factor X and Factor V are added, resulting in activation of Factor X. Next, part of this plasma mixture is added to normal plasma (which is plasma having physiologic levels of coagulation factor activity) and the time to clot is recorded, wherein the clotting time is presumed to depend on the level of Factor Xa generated during the first part of the test. In this assay, not only are multiple exogenous factors added to the patient sample, but the normal plasma also provides about 50% of all of the coagulation factors present in the total sample being tested at step two. The addition of normal plasma is imperative in this clotting assay due to the adsorption treatment of the patient's plasma to remove Factor II (in other words, the plasma would not be able to clot without the addition of the normal plasma). These 2-stage tests remove several of the patient's key coagulation factors via adsorption and then add them back via the addition of the normal plasma to enable clot formation. These tests are therefore optimized to isolate an aberrancy in the factor being tested (in this example, FVIII) and do not take into account any other endogenous factor levels, changes, or abnormalities.
There are also functional tests based on the modification of the prothrombin time (PT) or activated partial thromboplastin time (aPTT) assays. In the 1-step PT or aPTT assay, a patient's plasma, at various dilutions, is added to plasma that is deficient in a single factor (the reference plasma). First, a set of serial dilutions is performed for both the reference plasma and the patient's plasma to obtain a baseline of the clotting time at various dilutions. Second, these clotting time curves are plotted on the same graph, and the factor activity is derived by evaluating where the reference plasma curve intercepts the patient's plasma curve using a series of vectors drawings. This assay, however, does not assess the degree to which the factor in question contributes to changes in clotting time, as the assay simply compares clotting times to a reference. Additionally, because the aPTT assay is typically phospholipid-based, plasma samples from patients with lupus anticoagulant may record prolongations in the aPTT clotting time (depending on the aPTT reagents used), indicating a clotting factor deficiency that may not, in fact, be present.
There are also tests that are used for the detection and quantification of anti-factor antibodies (inhibitors), but these tests are similarly cumbersome and utilize the simultaneous addition of multiple exogenous factors. The most basic screening test for the presence of inhibitors entails mixing the patient's plasma with normal plasma and then comparing the resultant uncorrected, prolonged aPTT to both the normal plasma's aPTT and the patient's baseline aPTT. Once again, depending on the aPTT reagents used, if the patient's sample has lupus anticoagulant, this assay may mislead a clinician to suspect the presence of a factor inhibitor when the result instead may be caused by the presence of lupus anticoagulant.
The Bethesda Assay (BA) is the most widely used test to quantify inhibitor presence and multiple variations have been developed in an attempt to increase the assay's sensitivity and specificity. In the BA protocol for FVIII deficiency, for example, two assays are performed in parallel: diluted patient plasma is mixed with an aliquot of normal plasma in a first test, while an aliquot of normal plasma is mixed with buffer diluent in a second test. Both of these mixtures are incubated for 120 minutes and the residual FVIII activity is then calculated using a chromogenic or clot-based approach, as described above, or using the one-step aPTT assay. In the Nijmegen modification of the BA, diluted patient plasma is mixed with an aliquot of normal plasma for one test and FVIII-deficient control plasma is mixed with an aliquot of normal plasma for another test. Both of these mixtures are then incubated for 120 minutes and residual factor activity is then calculated using a chromogenic or clot-based approach, such as the one-step aPTT. In patients receiving FVIII replacement therapy, heat treatment is sometimes used in order to separate the inhibitor-factor complex to allow for complete inhibitor quantification; however, this heat treatment often results in a decrease in inhibitor activity due to heat inactivation. Furthermore, in all of these approaches, normal plasma is added to the patient plasma, replacing and/or overcoming any other factor impairment that the patient may have. While a FVIII inhibitor ELISA is available, as mentioned above, this assay is not a functional test.
Accordingly, because the testing approaches described above require the addition of exogenous factors via the addition of normal plasma or, in some cases, the simultaneous addition of multiple purified exogenous factors, such testing approaches do not capture the patient's endogenous clotting phenotype. Further, all of the testing approaches currently require platelet-poor plasma and therefore do not account for the role of platelets, red blood cells, and white blood cells in the clotting time and clotting phenotype. Accurate monitoring of coagulation phenotype is important for making treatment and dosing decisions for patients, especially for patients suffering conditions that result in acute or transient coagulopathies, such as trauma or infection. For example, if a patient is determined to have FIX deficiency, but is in an acute inflammatory state where levels and therefore activity of FVIII and FI are increased, the addition of exogenous FIX (for the treatment of the deficiency) may lead to a faster clotting time than it would in a patient in a non-inflammatory state. In addition, if a patient is deficient in fibrinogen due to a consumptive coagulopathy, such fibrinogen deficiency potentially would be missed with the addition of normal plasma to the patient plasma, as the normal plasma would provide fibrinogen and thereby mask the fibrinogen deficiency in the patient's plasma.
Thus there is a need in the clinic for coagulation tests that can detect, characterize, and/or quantify factor-specific impairments in coagulation to better manage patients at high risk of severe bleeding or clotting, including, but not limited to, patients with factor deficiency and/or factor inhibition (e.g., patients with anti-factor auto-antibodies and/or who have taken drug inhibitors). Additionally, there is a need for tests that assess the degree to which a factor deficiency or inhibition affects clotting time, and that further account for a patient's endogenous coagulation profile, such that treatment decisions are based on all factors contributing to a patient's coagulation phenotype rather than on only one or two specific factors.
Embodiments of the present invention provide methods and devices for evaluating coagulation, including methods and devices for detecting an impairment in coagulation factor function, such as impairments caused by coagulation factor deficiency and/or inhibition, or that are due to resistance to natural anticoagulants. An impairment in coagulation factor function may result in abnormal or impaired clot formation (e.g., thrombosis), or in abnormal or impaired 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 coagulation measurements can be evaluated to accurately profile coagulation factor impairments, including the presence of a factor deficiency or inhibition, or of resistance to anticoagulants, of the sample. 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 present invention provides methods for assessing coagulation or coagulation factor function in a sample of bodily fluid (e.g., a blood sample, or a sample of cerebral spinal fluid (CSF), peritoneal fluid, thoracic fluid, pleural fluid, or pericardial fluid). The method comprises adding a coagulation factor to two or more portions (e.g., aliquots) of the sample, each portion receiving the coagulation factor at a different concentration, and measuring clot formation (e.g., clot formation times) in response to the different factor concentrations. By assessing coagulation in response to the different concentrations of one or more coagulation factors, blood clotting function, including the presence and impact of Hemophilia A, B, or C or other coagulopathies, can be accurately profiled. In some embodiments, the presence or absence of a congenital and/or hereditary clotting abnormality is determined. In other embodiments, the presence or absence of an acquired clotting abnormality (e.g., anti-factor auto-antibodies or iatrogenic inhibition) is determined. In certain embodiments, changes in the concentration, activity, or inhibition of endogenous coagulation factors (including co-factors) in response to injury, infection, or inflammation (as some examples), and the effects of such changes on coagulation may be detected using the devices and methods described herein. The methods of the present invention may be performed using a microfluidic device as described herein, where one or more of the channels can be configured to trigger formation and/or localization of a clot.
The methods and devices described herein permit evaluation of the effect of the specific factor in question in a targeted fashion, while also allowing for the other coagulation factors present in a patient sample to affect clotting time. This approach provides the ability to not only detect a specific factor deficiency or inhibitor, but also to evaluate how the addition of that factor affects clotting time in a concentration-dependent fashion. For example, if a patient has decreased Factor VIII levels but increased Factor XI levels, this approach would identify the decrease in Factor VIII, as long as it results in a prolonged clotting time; and if the patient's increased Factor XI level resulted in a relative shortening of the clotting time, as Factor XIa above certain concentrations has been shown to bypass Factor VIII (see Kluft et al., Thrombosis Res. 135:198-204 (2015)), the testing approach described herein would take this Factor XI effect into account. This type of information would permit a precision-medicine approach to coagulation management, guiding clinicians to potentially giving lower levels of replacement Factor VIII to patients with very high Factor XI levels in order to avoid pushing them toward a pro-thrombotic state. This type of coagulation management is not possible using the traditional factor deficiency and inhibitor assays described above because, in an attempt to be so specific for the factor in question, the contributions of the other endogenous coagulation factors to the patient's clotting phenotype are masked by the plasma adsorption treatment and/or the addition of multiple exogenous coagulation factors in the form of purified factor mixtures or normal plasma. In other words, a clinician using the coagulation assays currently available may not be able to determine how a patient's other endogenous coagulation factors (factors other than the factor being tested) affect the patient's clotting time, as the assays require the addition of exogenous factors that may overcome any other factor deficiencies and would mask any compensatory changes in factor levels.
There are also situations where currently available tests may result in false positive identification of factor deficiency or inhibitor presence, such as when there is anticoagulant contamination in the sample. For example, Factor Xa inhibitors may result in a dose-dependent prolongation of the PT and/or aPTT as well as a decrease in Factor Xa chromogenic activity. Factor IIa inhibitors may also result in a dose-dependent prolongation of the PT and/or aPTT. Anticoagulant contamination can be due to patient drug administration or due to intravenous or intra-arterial catheter contamination. In addition, as discussed above, in the aPTT-based testing approaches the presence of lupus anticoagulant in some circumstances (depending on the particular aPTT reagent used) could also result in a false-positive result for inhibitors or deficiency.
In embodiments of the novel coagulation testing approach described herein, one or more coagulation factors upstream, and one or more coagulation factors downstream of the factor in question are each added to their own respective portions of the patient's sample (e.g., blood sample). The methods and devices described herein allow clinicians to identify other causes of prolongation of clotting time, aside from causes due to a deficiency or inhibition of the factor in question, and thereby provide a higher level of specificity by better pinpointing where in the entire cascade there may be a deficiency or an inhibitor acts. For example, the addition of Factor Xa as the downstream coagulation factor (in a test for FVIII impairment, for example) can serve as a positive control, such that the presence of any Factor Xa inhibitor would be detected, and likewise any cause of prolongation of clotting time at or downstream of Factor Xa, such as afibrinogenemia, would be detected. The addition of Factor IIa could serve as another downstream positive control, and although Factor IIa addition would identify the presence of thrombin inhibitors and certain heparins, it would miss specific Factor Xa inhibitors (as FIIa acts downstream of FXa). However, adding Factor Xa to one portion of the sample and adding Factor IIa to another, separate portion of the sample at appropriate concentrations allows for the identification of both Factor Xa and Factor IIa anticoagulants and provides a more comprehensive positive control. Additionally, as long as specific phospholipid reagents are not used as the clotting activator in this assay (e.g., a contact activator that does not interact with antiphospholipid antibodies is instead used), the presence of lupus anticoagulant in the sample would not result in a false positive identification of factor inhibition or deficiencies.
Further, because the current traditional testing protocols involve the addition of normal plasma and, in some cases, also require depleting endogenous factors from the patient's plasma sample, these approaches are unable to differentiate between inhibitors to the inactive (e.g., precursor) form of the factor and inhibitors to the activated form of the factor. For example, while a patient's sample may have inhibitors targeting Factor IXa, these current testing approaches will be able to assess only that there is an inhibitor to Factor IX/IXa, without assessing whether the inhibitor may be specific to FIX and not to FIXa or vice versa. In contrast, the methods of the present invention can differentiate between these two classes of inhibitors, as the methods involve adding the inactive or the active form of specific factors to separate portions of a sample. Determining whether inhibitors target inactive versus active factors would be useful for pre-clinical and clinical drug development, as well as for the study of spontaneous inhibitor development (e.g., anti-factor auto-antibody development).
The methods and systems described herein apply an upstream-downstream logic-based approach to coagulation phenotype evaluation where the addition of specific factor(s) allows for the identification of coagulation factor deficiency and/or the presence of inhibitors, including factor-specific drug inhibitors and/or anti-factor auto-antibodies. Drugs here include both natural and synthetic drugs, peptides, proteins, aptamers, and antibodies (whole immunoglobulins or fragments thereof). For example, using the methods described herein, a clinician can detect and differentiate between the presence of Factor IIa inhibitors and Factor Xa inhibitors, as can occur via multiple anticoagulant/antithrombotic drugs. The methods and systems described herein can be customized for the identification, screening, and monitoring of various types of drugs, including, but not limited to, Factor XI inhibitors, Factor XIa inhibitors, Factor XII inhibitors, and Factor XIIa inhibitors. For example, this testing methodology can be used to identify, differentiate among, and quantify the antithrombotic effects of FXa, FIIa, and FXI and/or FXIa inhibitors in one comprehensive assay that tests the effects of factors that act upstream, downstream, and at each point of potential inhibition. See, for example,
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 (including any co-factor) implicated in the coagulation cascade (intrinsic, extrinsic, and common pathways), including, e.g., 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. A coagulation factor can be in activated form or inactivated (e.g., precursor) form. For example, the difference in coagulation in response to the addition of the activated versus inactivated form of a coagulation factor can help pinpoint the exact deficiency or inhibition present in the sample. In other embodiments, e.g., for detection of a genetic clotting abnormality, the coagulation factor may be added in its inactivated form (e.g., Factor VIII, Factor IX, or Factor XI). Further, a coagulation factor can be from a human, from an animal (such as cow, pig, or other), or can be a synthesized or recombinant protein.
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 deficiency and/or inhibition (such as by an anti-factor antibody or a drug inhibitor) can be determined. For example, a sample that has a coagulation factor deficiency will show a decrease in clotting time as the deficient coagulation factor is added to the sample. When the activated form of the deficient coagulation factor is added, the clotting time should decrease to achieve a clotting time that is normal because the point of deficiency (the inactivated form of the coagulation factor) has been bypassed. Meanwhile, when a coagulation factor (activated or inactivated) upstream from the point of deficiency is added (in increasing amounts), the clotting time should remain prolonged (relative to a normal clotting time). These clotting times are compared to the clotting time when a coagulation factor downstream to the point of deficiency is added; when the inactivated form of the downstream coagulation factor is added, clotting time does not achieve a normal clotting time, whereas when the activated form of the downstream coagulation factor is added, the clotting time decreases to a clotting time within a normal range. See, e.g.,
Furthermore, by evaluating clot formation (e.g., clot time) in response to the addition of coagulation factors as described herein, factor deficiency can be differentiated from factor inhibition. In the case of factor deficiency, the addition of physiological levels of the deficient coagulation factor decrease clotting time and result in a normalization in clotting time. In the case of factor inhibition (e.g., by an anti-factor auto-antibody), depending on the concentration and activity level of the factor inhibitor, a supra-physiological level of the factor will be necessary to decrease the clotting time and reach a clotting time within a normal clot time range. Additionally, the level of factor inhibition can be determined by evaluating the concentration-dependent response in clotting time. For example, a high level of anti-factor autoantibodies and inhibitory activity will result in a different clotting time response (clotting curve) to various concentrations of exogenously added factor, due to the high level of factor required to shorten clotting time and result in a normalization of the clotting time compared to the factor levels required to shorten and normalize the clotting time in a sample with a moderate level of anti-factor autoantibodies and inhibitory activity. See, for example,
As used herein and unless otherwise specified, a physiological level of a factor may refer to the level or concentration of that factor that is normally present in the species (e.g., human); such level or concentration can be represented as a range (see, e.g.,
The methods and devices described herein can thus be used to identify, differentiate among, and quantify the effects of multiple coagulation factors simultaneously by providing a matrix wherein multiple coagulation factors are added in a strategic upstream-downstream logic-based approach, each factor added to a separate portion of a sample, to pin-point the exact coagulation factor deficiency or inhibition in the sample with one single test. For example, a clinician could identify the presence of, and differentiate the effects of FIIa inhibitors, FXa inhibitors, FXI inhibitors, and FXIa inhibitors using one single assay. See, for example,
In some embodiments, results for a patient sample can be compared to one or more reference standards. Such reference standards include reference standards for normal clotting, reference standards abnormal clotting, reference standards corresponding to anticoagulant therapy with particular agents, and reference standards based on patient samples that have the same coagulation impairment that is being tested for. In some embodiments, reference standards are personalized for a patient and can be used for monitoring the patient's response to therapy, such as with longitudinal testing.
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 identification of, and quantification of the amount of coagulation inhibitors present in the sample, to thereby guide patient care. The change in the clotting times, and therefore in the clotting curve, after the administration of any treatment can be used to assess the patient's coagulation response to the treatment. The change in the clotting curve can also be used to monitor long-term patient treatment, such as for patients being administered replacement coagulation factors or for patients receiving a gene therapy (or other long-term therapy) that alters their coagulation phenotype.
In some aspects, the invention provides a microfluidic device for evaluating coagulation in a sample. The device may include 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 a coagulation factor added at a specific amount or concentration. The channels in the series may all have the same geometry, so as to provide identical clot formation environments and thereby trigger the same 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, such a device allows for sensitive and specific detection of coagulation impairments, including coagulation factor deficiency or inhibition. While various embodiments are discussed with reference to “channels,” other terms (such as, e.g., lanes, compartments, partitions) can describe spaces that are physically separated from each other, and that in some embodiments have the same geometry as each other.
In aspects of the invention, the microfluidic device for evaluating coagulation includes a plurality of 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 plurality of channels are arranged in a central region of the substrate, such that clotting can be simultaneously imaged or analyzed across the channels. E.g., 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 clot formation in the areas in the channels in which clot formation begins can be controlled by the flow patterns in the channels.
In some embodiments, the clot forming areas in each of the plurality of channels are arranged in a region of the substrate that is not central, such as, for example, the periphery of the substrate. E.g., 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 certain embodiments, there can be one input port for the sample and one or two input ports for the reagents (e.g., coagulation factor and, optionally, calcium). E.g., see
In some embodiments of the microfluidic device, each clot forming area can be configured to create an area of stasis or an area of 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.
Channels of the microfluidic device can be coated with, contain, or otherwise include a coagulation factor at different amounts or concentrations. For example, a first group or series of the plurality of channels can be coated with, contain, or otherwise include a first coagulation factor (with each channel in the first group or series including the first coagulation factor at a concentration that is different from each of the concentrations in the other channels of the group or series), and a second group or series of the plurality of channels can be coated with, contain, or otherwise include a second coagulation factor (with each channel in the second group or series including the second coagulation factor at a concentration that is different from each of the concentrations in the other channels of the group or series). Further, in some embodiments, one or more of the plurality of channels is a negative control channel, e.g., the channel is not coated with and otherwise does not include a coagulation factor. In other embodiments, the device does not comprise such a negative control channel. In some embodiments, there may be more than one coagulation factor in each channel. In other embodiments, the device may comprise multiple channels, each channel having a different coagulation factor, but all factors are included at a single (and in some instances, the same) concentration.
Coagulation factor(s) that may be included in one or more channels may be in suspension or solution, may be surface-bound (such as by dry-spotting), or may be 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 added into the device through an input port (or through multiple input ports) concurrently with the sample or after the sample has been added.
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 channels can be coated with, contain, or otherwise include a first coagulation factor at a unique amount or concentration, and each channel in the second group of channels can be coated with, contain, or otherwise include a second coagulation factor at a unique amount or concentration. In some embodiments, the two groups of channels may contain the same coagulation factor, but one group includes the inactivated form of the factor and the second group includes the activated form of the factor. In some embodiments, the microfluidic device may contain more than two groups or series of channels, such as three, four, five, or more groups, wherein each group or series of channels is coated with, contains, or otherwise includes a different coagulation factor at an increasing concentration across the channels in the group or series (e.g., a microfluidic device may contain four groups of channels, each group having channels coated with, containing, or otherwise including a coagulation factor selected from Factors XI, XIa, VIII, VIIIa, IX, IXa, Xa and/or IIa, wherein no two groups include the same factor, and with respect to each group's channels, the channels in a group contain the factor at increasing concentrations (a concentration gradient); Factor VIII may or may not include Factor VIII in the complexed form with von Willebrand Factor (vWF)). By measuring clot formation (e.g., clotting time) as a function of coagulation factor concentration gradients, the sample's clotting properties can be profiled at several specific points of the coagulation pathway(s), providing a clinician with detailed and specific information concerning the patient's clotting physiology and/or the status of any therapeutic intervention.
For some of the embodiments with two or more groups of channels as described above, the second coagulation factor can be upstream in the coagulation cascade from the first coagulation factor. In addition, the first coagulation factor can be in active or inactive form, and the second coagulation factor can be in active or inactive form. In certain embodiments, the downstream factor is in active form.
The microfluidic device may further include a detection device configured to measure clot formation times in each of the channels. The microfluidic device may further report the clot formation times measured, to assess coagulation based on the clot formation times. For example, the detection device can be configured to simultaneously image the clot forming areas 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 at fixed 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 methodologies 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, electrical capacitance, or electrical resistance; detection based on changes in flow dynamics, such as by measuring flow velocity (e.g., by the addition of beads (e.g., magnetic beads) and quantifying bead flow rate and/or number of moving beads), and detection based on changes in viscoelasticity; detection based on changes in pressure at and/or around the site of clot formation; detection based on rheological assessments; fluorescence detection (such as with fluorescent fibrinogen); detection based on changes in turbidity; infrared light detection; infrared spectroscopy; detection using acoustic and/or photonic sensors; detection based on flow cytometry; and visual clotting detection.
In some embodiments, the methods of the invention do not employ a microfluidic device and instead use 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 assessed. For example, properties of the clot such as size, strength, density, and composition can be analyzed, in addition to the time to form, retract, or dissolve a clot. Such properties may be assessed using the same modality that is used to detect clot formation times or may be assessed using a different detection modality. 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.
In some embodiments, clot lysis (which may also be called fibrinolysis or clot degradation) can be assessed in addition to clot formation. For example, if a patient is on a fibrinolytic or thrombolytic agent, a clinician may wish to evaluate the clot when it is being formed as well as evaluate its breakdown over time. In certain embodiments, the same methods described herein and known in the art to detect clot formation can be used to assess clot lysis over time. Assessing clot lysis may be useful in cases where there are multiple derangements to the coagulation system, where a treatment is administered, or where there are multiple targets or effects of the inhibitors detected. The methodology used for evaluating fibrinolysis may be the same as or different from the methodology used for clot detection (e.g., for measuring clotting time). For example, viscoelastometry is able to evaluate fibrinolysis, independent of clotting time.
Regarding the use of viscoelastic testing, such method can be used to evaluate both clot formation and fibrinolysis. Evaluating both processes would be useful for detecting clotting abnormalities in patients that are hypocoagulable due to problems with fibrinolysis or due to iatrogenic administration of fibrinolytic and thrombolytic drugs. See, for example, C. Mauffrey, et al., Bone Joint J. 96-B:1143-54 (2014).
For any of the devices and methods described herein, the sample may be any bodily fluid sample in which coagulation may occur, or that may or is known to contain coagulation factors. Such bodily fluids include blood (including, e.g., whole blood or plasma (which can be platelet-rich-plasma (PRP) or platelet-poor-plasma (PPP)); accordingly, a blood sample can be a whole blood sample or a plasma sample. Using whole blood can be particularly beneficial or convenient for certain applications, such as those implemented at the bedside of a patient or when a complete assessment of physiologic coagulation status is required (such complete assessment would include platelets, red blood cells, and white blood cells in the evaluation). Other bodily fluids that can be used in the devices and methods described herein include, for example, cerebral spinal fluid (CSF), amniotic fluid, peritoneal fluid, pericardial fluid, and pleural fluid.
The 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 devices and methods described herein can be used for diagnostic purposes as well as for research and discovery into the coagulation cascade. For example, the devices and methods described herein can be useful for basic drug discovery, for understanding disease or disorder pathophysiology, for example, in the context of infectious diseases that result in pathological bleeding or clotting (SARS-CoV-2 virus, Dengue virus, Zika virus, Ebola virus, etc.), and for clinical and pre-clinical assessment of new drugs/compounds/treatments.
In addition, the devices and methods described herein can be used to guide therapy of a patient. For example, the results of a factor deficiency identification as described herein can be used to determine optimal therapy, and the assays and devices may be further used to monitor response to treatment. Similarly, physicians can use the results of a factor inhibitor identification to guide their selection of appropriate treatment and to monitor the patient's response to that treatment. The methods and devices described herein can also be used to monitor a patient for the development of, or increase/decrease in the activity of, factor inhibitors. The detection and identification of factor inhibitor development can be used in the emergency setting, as well as for the monitoring of new therapies such as gene therapies. The devices and methods of the present invention can also be used for the rapid screening of the cause of coagulopathies, such as in the testing of an infant with spontaneous bruising or prior to an invasive procedure, such as emergency surgery or circumcision. In some cases, for patients who receive factor-replacement only at specific times, such as prior to an invasive procedure or high-risk injury activity, the methods and devices provided herein can be used to obtain a pre-factor replacement clotting time and post-factor clotting time to confirm therapeutic levels are achieved.
The devices and methods also can be used to detect and evaluate response to treatment of various coagulation factor inhibitors. For example, the devices and methods can be used to detect the presence of, and to differentiate between Factor Xa inhibitors, Factor IIa inhibitors, Factor XI inhibitors, and Factor XIa inhibitors. Such assessment could be important as new anticoagulants and antithrombotic drugs are developed and the need to identify and quantify the effects of these drugs in emergency settings grows increasingly critical and complex. For example, if a clinician does not have access to a patient's medical record and the patient is suspected to be on an anticoagulant or antithrombotic agent, the clinician could use a factor inhibitor screening test as provided herein to detect whether the patient is on an anticoagulant/antithrombotic drug, identify the class of the inhibitor (i.e., which coagulation factor is inhibited), and quantify the effect of the inhibitor on the patient's clotting time. This same test can be used to monitor patient response to therapy (e.g., if a patient is receiving a reversal or bypassing agent, or an anticoagulant). For example, the devices and methods described herein can be used to detect response to bypassing agent therapy, such as in the case of administration of FVIIa to a patient anticoagulated with a FXa inhibitor, or to monitor a patient receiving a reversal agent, such as a patient receiving protamine to reverse the anticoagulant effects of heparin. As an additional example, the devices and methods described herein can be used to detect resistance to an anticoagulation treatment, such as in the case of a patient with low antithrombin III activity, which can result in reduced anticoagulant activity of antithrombin III-dependent drugs, such as heparin.
Other aspects and embodiments of the invention will be apparent from the following detailed description.
The patent or application file contains at least one drawing or photograph executed in color. Copies of this patent or patent application publication with color drawings or photograms will be provided by the Office upon request and payment of the necessary fee.
The invention generally relates to methods and devices for the detection of coagulation factor deficiency and/or inhibition.
Congenital and/or hereditary and acquired coagulopathies are commonly complex and time-consuming to diagnose. Deficiencies (which may result from decreased factor level, and/or from factor abnormalities that decrease function, even if level is not decreased) may present during childhood or upon the first surgery or injury of the affected individual when bleeding times are prolonged. The diagnosis of a factor deficiency usually requires specialty training, referral to a specialist, and multiple sequential complex coagulation tests. Acquired coagulopathies, such as the development of anti-factor auto-antibodies, require the same level of specialty involvement and testing, although these cases commonly present as severe, life-threatening bleeding events in an acute setting. Currently, rapid (e.g., <30 minute turn-around time), point-of-care tests for the identification of and differentiation between factor deficiency (whether a result of decreased factor level or of an abnormality that decreases factor function) and factor inhibition are not available. Having a test available that not only can identify which factor is impaired, but that can also differentiate between deficiency and inhibition and quantify the impairment's effect on clotting time, would provide physicians with easily accessible, interpretable, and actionable information in treatment decision-making and monitoring.
For patients that have factor inhibitors, such as anti-factor antibodies, clinical presentation is usually adverse, uncontrolled bleeding events. In these cases, providing the positive identification of the presence of a factor inhibitor would provide valuable functional clinical information to the doctor to help personalize the treatment and dosage, and would be of great benefit to the patient and possibly decrease subsequent, related adverse events, such as in the case where a patient with hereditary Hemophilia A presents with Acquired Hemophilia A in adulthood and is treated with Factor VIII and yet continues to have bleeding. The information provided by the devices and methods described herein would allow the clinician to determine that the patient's bleeding is inhibitor-dependent bleeding, and would allow the clinician to provide the correct amount of replacement factor or provide another bypassing reagent to aid in hemostasis. Embodiments of the invention also provide clotting panels that evaluate the coagulation, fibrinolysis, and platelet function within an individual. The microfluidic technology and advanced assays described herein in some embodiments further provide 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.
While the devices and methods of the invention provide rapid and easy-to-interpret assays, they also provide customizable devices and methods, allowing for the selection of clinically-relevant coagulation and platelet function testing for each customer and/or end-user segment. Because embodiments of the devices and methods can be used in a bedside platform, they can also be utilized for trend-monitoring in patients on various treatments (including at the hospital, at coagulation clinics, and at home). For example, in certain aspects of the invention, a concentration gradient of a factor is added to a group of channels (or, e.g., to a group of wells or containers), each channel containing a portion of the sample, after the sample has been subdivided into and/or distributed among the channels in the multiple groups of channels; similarly, a concentration gradient of a second factor is added to the channels of a second group of channels. Such method permits evaluation of coagulation function/inhibition and identification and differentiation between various coagulation abnormalities within a sample. Such embodiments of the invention (e.g., clotting panels, assays, etc.) are useful for assessing coagulation in patients that have poor medical compliance, in patients whose medication dosage and/or time of medication administration are unknown, and in patients who are unconscious and the doctor, surgeon, or other healthcare provider must determine whether the patient has any coagulation impairment. Further, embodiments of the devices and methods described herein can help clinicians monitor patient treatment, such as factor replacement, gene therapy, or bypassing agents, and help guide the administration of various hemostatic treatments in the case of active bleeding.
Examples of potential users for products and services based on embodiments of the present invention can range from healthcare workers, e.g., clinicians and veterinarians, to researchers in pharmaceutical research and development.
Embodiments of the present invention can be used for patient care in various settings. In some embodiments, a 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 for preparing the patient for the procedure to minimize bleeding risks. In additional embodiments, a patient is administered a drug that affects 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 further embodiments, a patient receives a drug or blood product, and methods and devices of the invention can be used to monitor and guide administration and dose. In other embodiments, a patient with a familial history of coagulation abnormalities or spontaneous bleeding/bruising is screened for the presence of coagulation factor deficiency (e.g., abnormality) and/or inhibition. In some embodiments, the patient is an infant or neonate, where only small volumes of blood are available for evaluating coagulation (including for monitoring anticoagulant therapy or for detecting a congenital coagulation abnormality). In some embodiments, the patient is pregnant, 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 acquired Hemophilia A.
In some embodiments, the patient or subject is non-human (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 associated with 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. The novel coagulation testing platforms (e.g., assay, microfluidic device, and/or combination thereof) described herein satisfy such need.
In some embodiments, a patient is receiving an anticoagulant therapy, such as a heparin (such as low molecular weight or unfractionated heparin) (heparin is an indirect inhibitor of Factor IIa and Factor Xa), a Direct Oral Anticoagulant (DOAC) inhibiting Factor Xa (such as XARELTO® (Rivaroxaban), ELIQUIS® (Apixaban), SAVAYSA® (Edoxaban), or BEVYXXA® (Betrixaban)) or inhibiting Factor IIa (such as PRADAXA® (Dabigatran)), or an injectable Factor IIa or Factor Xa inhibitor (such as fondaparinux, bivalirudin, or argotraban). In certain embodiments, a patient is undergoing therapy with a different factor-specific inhibitor, such as an inhibitor targeting Factor XI and/or Factor XIa, Factor XII and/or Factor XIIa, Factor V and/or Factor Va, or Factor VII and/or Factor VIIa. In some embodiments, a patient is undergoing coagulation therapy for the prevention of fibrinolysis, such as with an inhibitor or antibody against plasmin or plasminogen, or for the promotion of fibrinolysis, such as with tPA. In addition, a patient may receive natural or synthetic coagulation factors for the manipulation of the coagulation system; for example, a patient may receive Factor VIIa, (activated) prothrombin complex concentrates (PCC or aPCC), or anti-inhibitor coagulant complex (FEIBA®).
Anticoagulant drugs are commonly used in many medical settings, including emergency and critical care, surgery, cardiology, and cancer. Several new anticoagulants have been introduced, but currently there are no tests that can reliably determine if a patient is taking the most optimal dose. Similarly, there are no reliable tests to monitor a patient's response to reversal or bypassing therapy. Too much anticoagulation can cause life-threatening bleeding, and too little anticoagulation can lead to an increased risk of stroke and heart attacks. Additionally, the inappropriate administration of specific agents may predispose a patient towards development of pathological blood clots, such as in the case of the administration of FEIBA® in some hemophilia patients without inhibitors (see FEIBA® product labelling).
In some embodiments, a patient may have pre-existing coagulation factor deficiency (e.g., abnormality), such as in hemophilia, and the detection of anti-factor antibodies is necessary. In certain embodiments, the quantification of the effect of anti-factor antibodies on coagulation must be determined and monitored before and after therapeutic treatment. Embodiments of the invention can be used as or incorporated into a bedside test that can accurately monitor therapeutic treatment, such as factor replacement or bypassing agents, and improve the safety for patients undergoing such treatment. The devices described herein can be used, and the methods provided herein can be performed, with minimal training and in an easy-to-interpret format. For example, the methods can be performed using a device requiring less than about 1 mL, less than about 500 μL, less than about 100 μL, or less than about 50 μL, or about 5 μL or less (e.g., in some embodiments, about one drop) of fresh or citrated whole blood, with the results being available within 10 minutes of starting the method.
Coagulation factor deficiency is most commonly treated with factor replacement. Depending on the severity of the deficiency and the patient's propensity for spontaneous or uncontrolled bleeding, the patient may receive regular infusions of coagulation factors, or the patient may receive the factors prophylactically, such as in the case of a planned invasive procedure. New treatments for coagulation factor deficiencies include long-term treatments, such as gene therapy. Because these treatments are still new and experimental, the long-term monitoring of these patients during clinical trials to determine factor levels over time and to confirm the lack of development of anti-factor antibodies is required. While patients with coagulation factor inhibitors may be treated with factor replacement, if inhibitors are present and active above low levels (mild based on Bethesda Assay results), they may require treatment with bypassing agents, such as activated coagulation factor concentrates, that may result in a pro-thrombotic state if the dose is too high. The diagnosis and management of coagulopathies is often complex and requires precise tests in order to optimally manage patients in the chronic and acute setting.
In some embodiments, methods of the present invention allow clinicians to detect a coagulation deficiency in a blood sample, and to pinpoint where the deficiency occurs within the coagulation cascade. Such methods may comprise comparing the clot formation times measured for the sample, 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 other 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 the treatment. A reference range can also be established using a sample obtained from a relative (e.g., parent, sibling, or offspring) of the individual from whom the test sample is obtained. The reference range(s) may be tailored to or dependent on a particular assay configuration, including microfluidic device configuration. For example, the clotting time of a patient's sample can be compared to the clotting time of a normal sample at the testing time, or to a previously-determined reference range clotting time (which can be a clotting time or clotting time range that represents normal clotting time, or that corresponds to an impairment in a specific coagulation factor or combination of factors). In some embodiments, the methods involve the establishment and/or verification of reference ranges.
In some embodiments, reference ranges may be established from clotting time standards, such as standards based on the clotting times of samples from individuals who do not suffer from a coagulation cascade impairment. In some embodiments, the reference ranges are established from samples spiked with known concentrations of various anticoagulants or from samples depleted of specific factors; such samples may be commercially available.
It should be understood that clot formation times can also be compared to reference ranges from individuals who do suffer from a coagulation impairment. For example, it is common with reference intervals to have a “normal” interval range for people who do not suffer from a deficiency and an “abnormal” interval range for people confirmed to have that deficiency. Sometimes, there is a gray zone in between the normal and abnormal zones, which is indicative that further in-depth testing is required on that patient sample for a definitive diagnosis.
In some embodiments, methods of the invention do not comprise comparison of a sample's clotting time to a reference range or standard clotting time. In certain embodiments, the methods provide internal controls by evaluating the clotting response when coagulation factors upstream and downstream of a suspected point of impairment in the coagulation pathway(s) are added to the sample.
Descriptions of example embodiments are provided herein.
Embodiments described herein provide 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 meets an unmet need within various coagulation testing environments by providing rapid, bedside diagnostics and drug and other therapy monitoring capabilities.
In certain embodiments, the methods include an assay wherein a specific coagulation factor suspected of being impaired (e.g., deficient (such as by having an abnormality) or inhibited, or by being resistant to an anticoagulant) is added to 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 from each other by a factor of 2 up to a factor of 100. In some embodiments, a coagulation factor is added to divided portions of the sample at concentrations increasing by a factor of 5 up to a factor of 20 (e.g., about a factor of 10), across the divided portions of the sample. The concentration gradient used for each factor may be the same as or different from the concentration gradients used for other factor(s) in the methods and devices described herein. 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 pg/mL to 10 μg/mL. The addition of the coagulation factor at specific concentrations or amounts (e.g., a gradient, such that divided portions have different concentrations) allows the determination of:
The terms upstream and downstream are used herein to describe coagulation factors that are upstream or downstream in the coagulation pathway (including its associated arms, both fibrinolytic and anticoagulation) with respect to a given point in the pathway (e.g., the point of impairment). The exact pattern of the upstream-downstream combinations may change over time as new data and information are added to the coagulation literature. For example, although Factor XIa is depicted as upstream to FVIII/FVIIIa in the coagulation pathway schema, recently it has been shown that, above certain concentrations, Factor XIa (as well as Factor XI) is capable of bypassing Factor VIII/VIIIa via the direct activation of Factor X (especially in FVIII and FIX deficient plasma, see Kluft et al., Thrombosis Res. 135:198-204 (2015)). This bypassing is discussed in the context of the hemophilia screening assay embodiment in Example 25, where, for a sample deficient in FVIII, adding FXIa at a sufficiently high concentration will shorten the clotting time.
The terms upstream and downstream are also used to describe the activation state of a coagulation factor. For example, Factor IX (the inactive precursor form) would be considered upstream of Factor IXa, and Factor IXa would be considered downstream of Factor IX. Using both the inactive and active forms of a given coagulation factor can increase the specificity of the assay. To illustrate, a user of the devices and methods described herein may determine whether there is a deficiency in Factor IX by using (in the devices and methods) the addition of Factor XIa (which is directly upstream of Factor IX, and is the direct activator of Factor IX). In the case where there is a deficiency of Factor IX, the addition of Factor XIa will not shorten clotting time sufficiently to reach a normal clotting time, and clotting time will remain prolonged. The addition of FIX will shorten the clotting time (in a concentration-dependent fashion, as the exogenously added FIX supplies the factor or replaces any abnormal Factor IX), and the addition of Factor IXa (downstream) will result in an even shorter clotting time. Similarly, certain embodiments of the invention differentiate inhibition of the inactive form from inhibition of the active form of a coagulation factor. Such specific inhibition may be due to drug specificity, such as Factor XI versus Factor XIa inhibitors, or to auto-antibody specificity, such as Factor VIII versus Factor VIIIa auto-antibodies. Differentiating between inhibition of active and inactive forms of a factor may provide clinically relevant information as to the identification of the exact cause of a coagulation impairment and can help guide treatment decisions.
Designating the inactive and active forms of a factor as upstream and downstream relative to each other also applies to the various arms of the coagulation pathway, such as fibrinolysis (e.g., plasminogen is upstream of plasmin and plasmin is downstream of plasminogen) and anticoagulation (e.g., protein C is upstream of activated protein C, and activated protein C is downstream of protein C).
In some embodiments, activators of a specific coagulation factor may be included in addition to the upstream-downstream factors. For example, some embodiments may include a set of lanes that includes plasminogen (upstream of plasmin), plasmin (downstream of plasminogen), and tissue plasminogen activator or urokinase plasminogen activator (an activator of plasmin to plasminogen). In some embodiments, when Factor XIIa is used as an upstream factor, FXIIa can either be added exogenously or Factor XIIa can be generated within the test itself by activation of the contact activation pathway by known activators, such as, e.g., kaolin, celite, glass, certain metals, certain charged surfaces (negatively charged artificial or biological surfaces), and polyanions. This approach will not function if there is an impairment of the endogenous Factor XII in the sample. In such cases, exogenous Factor XII and/or Factor XIIa may be used as part of the upstream-downstream assay. Such cases include subjects with Factor XII deficiency (Hageman Factor Deficiency), which is a common congenital coagulopathy in cats, or in samples containing a Factor XII and/or Factor XIIa inhibitor. In some of the examples listed below, the negative control contains a Factor XII activator.
The positive control lane should include the addition of a downstream coagulation factor. Preferably, the downstream coagulation factor is in the active form, so as to serve as a coagulation activator itself. The use of an activated coagulation factor as the positive control helps eliminate any potential upstream abnormalities or inhibitions affecting the clotting time. For many of the examples described below and in the figures, Factor Xa and/or Factor IIa are frequently used as positive controls. However, the positive control can be any activated downstream coagulation factor. A benefit of using Factor IIa as a positive control includes the ability to verify that the sample is able to form a clot, as the only factor downstream of Factor IIa that would prevent clot formation is Factor I (fibrinogen). Although deficiencies in Factor XIII may result in aberrant or prolonged clotting times, they should not result in the complete absence of a blood clot in the presence of high levels of FIIa and adequate, functional fibrinogen levels. In some cases, a test can have multiple positive controls, such as a test for Factor XI including Factor IXa (downstream and activated) and Factor Xa and/or Factor IIa. If the factor being tested is restricted to activity in the upper portion of the intrinsic coagulation pathway, such as Factor XII, an activator of the extrinsic pathway, such as Factor III, could be used as a positive control, as Factor III would result in activation of the common pathway, downstream of Factor XII.
In some embodiments, a factor that prolongs rather than shortens clotting time may be selected as a positive control, such as in the case of testing for Activated Protein C resistance and using the addition of antithrombin III and/or heparin as a positive control for the appropriate response to an anticoagulant (prolongation of clotting time).
In some embodiments, the presence or absence of clot formation or clot properties may be used in place of, or in addition to the clotting time. For example, the level of tPA may be evaluated via panels that include factors such as plasmin and plasminogen; additionally or alternatively, clot degradation and/or clot integrity may be used as the endpoint. Clot retraction may also be used as an endpoint, for example in the evaluation of Factor XIII function. Another example includes the evaluation of Factor I impairment. In the case of Factor I deficiency (such as afibrinogenemia), or Factor I abnormality (such as dysfibrinogenemia), the clotting time may detect clot or no clot formation as a determination of the presence of a minimum threshold of functional fibrinogen. Quantification of fibrinogen function beyond this minimum threshold may comprise evaluating other clot properties in addition to clotting time.
Examples of the utility of the devices and methods described herein include:
Embodiments of the methods and devices described herein can be used to evaluate coagulation impairments (e.g., pro- or anti-thrombotic) using various coagulation detection technologies that can evaluate the multiple parts of the coagulation system, including coagulation cascade, anticoagulation, platelet activation, and fibrinolysis, such as those described herein. Detection methods include, for example, 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.
Whole blood or plasma can be used in the various embodiments described herein. In addition, the devices and methods described herein can be applied to other bodily fluids, including, for example, cerebral spinal fluid, amniotic fluid, peritoneal fluid, pericardial fluid, pleural fluid, and any other fluid sample that may contain coagulation factors.
Embodiments provided herein can be combined with ATP-luciferase assays in order to assess platelet and coagulation system function at the same time. Using the methods and devices described herein with ATP-luciferase assays 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 discussed herein, or by the addition of specific platelet agonists, such as, e.g., adenosine diphosphate (ADP), adenosine triphosphate (ATP), epinephrine, collagen, thrombin, and ristocetin. These agonists can be added as a concentration gradient in combination with the coagulation factors. This combined technique can be used to assess platelet function when patients are taking platelet inhibitors, such as aspirin or clopidogrel. Luciferase is typically measured by light absorbance. Platelet function can be measured via other detection methodologies, such as assessing platelet aggregometry by electrical impedance or light absorbance.
Coagulation impairments 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, a patient may have or be suspected to have a coagulation factor deficiency, which may be caused, for example, by the production of a deficient amount of the coagulation factor, or a mutation in the coagulation factor that decreases the factor's function of the factor, or both.
Examples of congenital 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., coronavirus, 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., pheochromocytoma, cushing syndrome, diabetes, etc.), chronic inflammatory disease (e.g., irritable bowel syndrome, irritable, bowel disease, colitis, etc.), and disseminated intravascular coagulation, and radiation.
Coagulopathies may also be iatrogenic (e.g., caused by medical treatment, such as some cancer treatments, bone marrow transplant, and certain drug treatments) or may be due to idiopathic causes.
In some embodiments, the invention employs a microfluidic approach. In certain embodiments, 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, polyimide, 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 some embodiments, calcium is added to the sample. Calcium may be added to the sample prior to inputting the sample into the device. In addition, calcium can be added within the device, e.g., through an additional port, or pre-loaded within the channel.
In some embodiments, 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 certain embodiments, 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 clot. The time that it takes for the clot to form can be quantified and recorded (e.g., reported to the user).
In various embodiments as described herein, the device is used to detect the presence and assess the effect of coagulation factor deficiencies or inhibition by assessing the time is takes to form a clot.
In some embodiments, a microfluidics device is used to detect the presence of and assess the effect of coagulation factor deficiencies or inhibition by assessing the time is takes to form a clot and/or by evaluation of other blood clot formation parameters, such as clot strength, clot retraction, or clot degradation.
In certain embodiments, a device as described herein provides a read-out (e.g., of clot formation time(s)) in a relatively short period of time, for example, in about 3-10 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×103/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×103/mL.
At a concentration of 500 ng/mL Rivaroxaban, clot formation detected<2.5 minutes down to 750 ng×103/mL.
At a concentration of 0 ng/mL Apixaban, clot formation detected in <2.5 minutes with agonist concentration down to 7.5 ng×103/mL.
At a concentration of 250 ng/mL Apixaban, clot formation detected in <2.5 minutes with agonist concentration down to 75 ng×103/mL.
At a concentration of 500 ng/mL Apixaban, clot formation detected in <2.5 minutes with agonist concentration down to 938 ng×103/mL.
At a concentration of <25 ng/mL Dabigatran, clot formation detected in <2.5 minutes with agonist concentration down to 71 ng×103/mL.
At a concentration of 250 ng/mL Dabigatran, get clot formation detected in <2.5 minutes with agonist concentration down to 710 ng×103/mL.
At a concentration of 500 ng/mL Dabigatran, clot formation detected in <2.5 minutes down to 710 ng×103/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 where a subject has a factor deficiency, the activated form of the factor that is deficient (or, alternatively, the activated form of another factor that is downstream of the deficient factor) can serve as the downstream factor in the assay; addition of the activated form of the deficient factor (or the addition of the activated form of another factor that is downstream of the deficient factor) would result in unaffected clotting time (clotting time that is not prolonged, such as clotting time that is or approaches the clotting time of normal plasma or the clotting time of a reference range). In such cases, addition of the factor that is deficient can be used to detect the deficiency, while the activated form of the deficient factor (or the activated form of a different downstream factor) can serve as the positive control to confirm the ability of the sample to form a clot.
There is a critical concentration of functional fibrinogen necessary for the formation of a cross-linked fibrin clot that can be detected. The absolute concentration of functional fibrinogen required for clot formation detection may depend on the methodology used for clot detection (e.g., a sensitive clot detection method may detect a weak, low-fibrinogen clot, whereas a less sensitive methodology may require a higher amount of functional fibrinogen to produce a larger and stronger clot that can be detected). If there is a low amount of functional fibrinogen in the test sample, adding exogenous fibrinogen (at an amount reaching the minimum concentration required by the detection methodology to detect a clot), should result in clot formation in the sample, and depending on clot detection sensitivity, the addition of fibrinogen may result in a concentration-dependent change in clotting time. Adding FIIa in conjunction with fibrinogen (FI) to a single test sample portion serves as the positive control for this test and controls for any other impairment that may be present upstream of fibrinogen, including the presence of a thrombin inhibitor or heparin. In a sample that has low functional fibrinogen or dysfunctional fibrinogen, testing for clot formation upon the addition of exogenous FI and FIIa to the sample will reveal whether there is a fibrinogen impairment. Unlike the dilute thrombin time test (dTT), which is used to detect thrombin inhibition and fibrinogen deficiency or abnormality but is unable to differentiate between thrombin and fibrinogen impairment, this testing approach provided herein allows for the detection of fibrinogen deficiency even in the presence of other inhibitors (such as thrombin inhibitors) that may prolong or prevent coagulation. In order to account for the inhibition due to thrombin inhibitors, a concentration of FIIa sufficient to overcome all FIIa inhibition is required.
FXIII deficiency/abnormality would result in changes in clot strength and clot stability and retraction over time with the addition of a factor upstream of FXIII, concentration-dependent changes in clot strength and stability over time with the addition of FXIII
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.
In the Factor-XI inhibitor example (
The methods and devices described herein can also be used to detect hyperfibrinolysis or hypofibrinolysis. Fibrinolysis refers to the degradation of a cross-linked fibrin clot.
In embodiments of this example, the fibrinolysis reaction can be performed at a temperature above room temperature, e.g., at a temperature that is within the range of about 37° C. to 45° C. Performing the assay at such temperatures can increase the fibrinolysis reaction rate and thereby reduce the time required to perform the assay (e.g., from 30-40 minutes to ≤15 minutes).
A sample with inhibition of FXIa or inhibition of FXI would exhibit a concentration-dependent prolongation of clotting time with the addition of FXIa or FXI (clotting time would change in a concentration-dependent manner, with higher concentrations of factor leading to further reductions in clotting time until a normal clotting time is reached). In the case of an inhibitor that specifically inhibits FXI (and does not inhibit FXIa), the addition of exogenous FXI would result in a concentration-dependent prolongation in clotting time, while the addition of exogenous FXIa would result in an unaffected clotting time (a clotting time that falls within a reference or normal clotting time range). In the case of sample containing an inhibitor that targets FXIa, the sample would exhibit a concentration-dependent prolongation or change in clotting time with the addition of exogenous FXIa at varying concentrations, while the addition of exogenous FXI would result in no response in clotting time (clotting time remains prolonged) or would result in a concentration-dependent prolongation or change in clotting time, depending on the concentration and inhibition activity of the FXIa inhibitor. The addition of exogenous FXII or FXIIa would result in no change in clotting time, such that clotting time remains prolonged, while the addition of FXa or FIIa would result in unaffected clotting times (clotting times would be unaffected relative to the clotting time of a normal sample or relative to a reference clotting time).
In the case of a sample with a FXa inhibitor, the sample would exhibit a prolongation of the clotting time upon the addition of exogenous FXII, FXIIa, FXI, or FXIa, would exhibit a concentration-dependent prolongation of clotting time with the addition of exogenous FXa (such that clotting time would decrease further upon the addition of higher concentrations of exogenous FXa), and would exhibit an unaffected clotting time (such as a normal clotting time) upon the addition of FIIa.
In the case of a sample with a FIIa inhibitor, the sample would exhibit a prolongation of clotting time upon the addition of exogenous FXIIa, FXII, FXIa, FXI, or FXa, while the addition of exogenous FIIa would result in a concentration-dependent prolongation in clotting time, as higher concentrations of exogenous FIIa would result in further reductions in clotting time. The asterisk in the figure is included to note that, in some cases of FIIa inhibition, the addition of exogenous FXa may similarly result in a concentration-dependent prolongation in clotting time. In such cases, the difference between a FXa-inhibitor sample and a FIIa-inhibitor sample is that, for the FXa-inhibitor sample, the addition of exogenous FIIa results in an unaffected clotting time (such as a normal clotting time), while for the FIIa-inhibitor sample the addition of exogenous FIIa results in a concentration-dependent change in clotting time.
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.
