HK1198181B - Nmr methods for monitoring blood clot formation - Google Patents

Description

NMR method for monitoring blood clot formation

Background

The invention features a method for monitoring rheological changes in an aqueous sample.

Blood is the circulating tissue of the organism, which carries oxygen and nutrients into the tissue and carries away carbon dioxide and various metabolites for excretion. Whole blood consists of pale yellow or gray-yellow fluid plasma in which red blood cells, white blood cells, and platelets are suspended.

Accurately measuring hemostasis, i.e., the ability of a patient's blood to coagulate and dissolve, in a timely and efficient manner is critical to certain surgical and medical procedures. Accelerating (rapid) accurate detection of abnormal hemostasis is also particularly important in terms of: patients suffering from a hemostatic disorder are given appropriate treatment and it is necessary to administer anticoagulants, antifibrinolytics, thrombolytics, antiplatelet agents or blood components to the patient in amounts that must be unambiguously determined after taking into account the abnormal components, cells or "factors" of the patient's blood that may contribute to the hemostatic disorder.

Hemostasis is a dynamic, extremely complex process involving many interacting factors, including coagulation and fibrinolysis proteins, activators, inhibitors, and cellular components, such as the platelet cytoskeleton, platelet cytoplast, and platelet cell surface. Thus, during activation, no factors remain static or behave in isolation. Thus, for comprehensive integrity, it is necessary to continuously measure the entire stage of a patient's hemostasis as the net product of the whole blood components in a non-isolated or static manner. Taking the results of measuring isolated fractions of hemostasis as an example, it is assumed that the patient exhibits fibrinolysis, which is caused by activation of plasminogen into plasmin (an enzyme that breaks down blood clots). In this case, the by-products of this process of fibrinogen degradation products act as anticoagulants. If a patient is tested for anticoagulation only and treated accordingly, the patient may remain at risk for treatment with an antifibrinolytic.

The end result of the hemostatic process is a three-dimensional network of polymerized fibrin fibrils (i.e., fibrin) that bind to platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptors to form the final clot. The unique property of this mesh structure is that it acts as a rigid elastomer, capable of resisting the deforming shear stresses of the circulating blood. The strength of the resulting clot against deforming shear stress is determined in part by the force exerted by the participating platelets.

Platelets have been shown to affect the mechanical strength of fibrin in at least two ways. First, by acting as nodal branching points, they significantly increase fibrin structural rigidity. Second, the "traction" force is applied to the fibers by the contractile force of platelet actomyosin, a muscle protein that is part of the cytoskeleton-mediated contractile apparatus. This force of contractility further increases the strength of the fibrin structure. The platelet receptor GPIIb/IIIa appears to be important in anchoring polymeric fibers in the basic cytoskeletal constrictor in activated platelets, thereby mediating the transfer of mechanical forces.

Thus, a blood clot that appears to adhere to the damaged vascular system as a result of activated hemostasis and resists the deforming shear stress of the circulating blood is essentially a mechanical device that forms during vascular recovery to provide a "temporary obstruction" against the shear stress of the circulating blood. The dynamics, strength and stability of the clot, which is its physical property against the deforming shear forces of the circulating blood, determines its ability to perform hemostatic tasks, i.e., stop bleeding without allowing undue thrombosis.

Platelets play a key role in mediating ischemic complications in thrombogenic (thrombogenic) patients. The use of GPIIb/IIIa inhibitors in thrombogenic patients or as an adjunct to Percutaneous Transluminal Coronary Angioplasty (PTCA) is rapidly becoming a standard of care. Inhibition of the GPIIb/IIIa receptor is an extremely effective form of antiplatelet therapy that can lead to death and reduced risk of myocardial infarction, but can also lead to extremely dangerous bleeding. The reason for bleeding potential or failure to achieve adequate therapeutic levels of platelet inhibition is the use of weight-adjusted platelet blocker therapy algorithms despite considerable interpersonal variability. This is a problem due in part to differences in platelet counts and variability in the number of GPIIb/IIIa receptors per platelet and their ligand binding functions. For clinical usefulness, platelet inhibition assays must provide clinically rapid and reliable information about receptor blockade, thus allowing dose modification to achieve the desired antiplatelet effect.

There is a need for a rapid, reliable, quantitative, point-of-care testing method and apparatus to continuously monitor therapeutic platelet blockade and measure the efficacy of antiplatelet agents throughout hemostasis from initial clot formation to dissolution.

Summary of The Invention

The invention features a method of monitoring a rheological change in an aqueous sample by: (i) measuring a signal characteristic of the NMR relaxation rate of water in the sample to obtain NMR relaxation data; (ii) determining from the NMR relaxation data a value or set of values of the magnetic resonance parameter characteristic of rheological changes in the sample; and (iii) comparing the result of step (ii) with a predetermined threshold.

In a related aspect, the invention features a method of monitoring a rheological change in an aqueous sample, the method including: (i) performing a series of magnetic resonance relaxation rate measurements on water in the sample; (ii) applying an algorithmic transformation measurement that distinguishes two or more observable water populations (water contributions) in a sample, wherein each observable water population has a distinct relaxation rate and distinct signal intensity at one or more time points during a rheology change; and (iii) monitoring the rheological change of the sample based on the relaxation rate or signal intensity of at least one of the two or more observable water populations.

The invention also features a method of monitoring a rheological change in an aqueous sample, the method comprising: (i) performing a series of measurements on the water in the sample, wherein the measurements distinguish two or more observable water populations within the sample, and each observable water population has a distinct signal and/or signal intensity at one or more time points during the rheology change; and (ii) monitoring the rheological change of the sample as a function of at least one observed signal and/or signal intensity of two or more observable water populations. The measurement may be a nuclear magnetic resonance measurement, electron paramagnetic resonance, microwave spectroscopy, or any other technique known in the art for measuring properties of water. In particular embodiments, the water in the aqueous sample is radiolabeled (e.g., with deuterium or tritium).

In one aspect, the invention features a method of monitoring a clotting or dissolution process in a first blood sample, the method including: (i) performing a series of magnetic resonance relaxation rate measurements on water in a first blood sample; (ii) transforming the measurement using an algorithm that distinguishes two or more separate water populations within the first blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (iii) monitoring the process based on the results of step (ii). The blood sample may be a plasma sample, a platelet poor plasma sample, a platelet rich plasma sample, a blood sample including separated and washed platelets, a whole blood sample, a blood clotting sample, or any of the types of blood samples described herein. In certain embodiments of the method, fibrinogen (e.g., 1 + -0.25, 2 + -0.5, 3 + -0.75, 4 + -1, 6 + -2, or 8 + -2 mg/mL) is added to the first blood sample prior to step (i). In a particular embodiment of the method, a clotting initiator or a clotting inhibitor is added to the first blood sample prior to step (i). The clotting initiator/inhibitor may be selected from RF (snake venom thrombin and factor XIII), AA (arachidonic acid), ADP (adenosine diphosphate), CK (kaolin), TRAP (thrombin receptor activating peptide), thrombin, platelet aggregation inhibitor or any clotting initiator or clotting inhibitor described herein. In still other embodiments of the method, prior to step (i), Tissue Plasminogen Activator (TPA) is added to the first blood sample. The method may further comprise the steps of: (iv) making a series of second relaxation rate measurements of water in a second blood sample from the subject; (v) transforming the second relaxation rate measurements using an algorithm that distinguishes two or more separate water populations within the second blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters, wherein each magnetic resonance parameter has one or more values; and (vi) monitoring the process based on the results of steps (ii) and (v). For example, the first blood sample can be a plasma sample, and the second blood sample can be a whole blood sample; the first blood sample may be a platelet rich plasma sample and the second blood sample may be a whole blood sample; the first blood sample may be a platelet poor plasma sample and the second blood sample may be a whole blood sample; the first blood sample may include separated and washed platelets, and the second blood sample may be a whole blood sample or any other type of comparative sample described herein. In particular embodiments, prior to step (i), platelet inhibitor is added to the first blood sample and no platelet inhibitor is added to the second blood sample; (ii) prior to step (i), adding platelet activator to the first blood sample and no platelet activator to the second blood sample; or prior to step (i), adding a clotting initiator selected from RF, AA, and CK to the first blood sample; and prior to step (iv), adding to the second blood sample a clotting initiator selected from the group consisting of ADP and thrombin. In certain embodiments of the method, prior to step (i), fibrinogen (e.g., 1 ± 0.25, 2 ± 0.5, 3 ± 0.75, 4 ± 1, 6 ± 2, or 8 ± 2 mg/mL) is added to the first blood sample; and adding fibrinogen (e.g. 1 + -0.25, 2 + -0.5, 3 + -0.75, 4 + -1, 6 + -2 or 8 + -2 mg/mL) to the second blood sample prior to step (iv).

In the above method, the magnetic resonance parameter value may be indicative of a characteristic of functional fibrinogen-associated water molecules in the blood sample. In particular embodiments, at least one of the two or more independent aqueous populations is positively correlated with platelet activation, platelet inhibition, clotting time, platelet-associated clot strength, hematocrit, or fibrinogen-associated clot strength. In still other embodiments, the magnetic resonance parameter value indicates low platelet activity, high functional fibrinogen activity, or low functional fibrinogen activity.

In any of the above methods, the algorithm may comprise an algorithm selected from the group consisting of: a multi-exponential algorithm, a bi-exponential algorithm, a tri-exponential algorithm, an exponential decay algorithm, a Laplace transform (Laplace transform), a goodness of fit algorithm, an SSE algorithm, a least squares algorithm, a non-negative least squares algorithm, or any algorithm described herein. In a specific embodiment, the algorithm is an inverse Laplace transform.

In any of the above methods, the relaxation rate may be selected from the group consisting of T1, T2, T1/T2 promiscuous, T_1rho、T_2rhoAnd T₂ ^*. In a particular embodiment, the relaxation rate measurements include a T2 measurement, which provides a decay curve.

In another specific embodiment, the two or more water populations comprise a water population having a serum-associated T2 signal and a water population having a clot-associated T2 signal. The method can include (i) calculating a T2 value for serum-associated water prior to initiating clot formation in a blood sample including red blood cells, and (ii) determining the hematocrit of the blood sample based on the T2 value. The method may further comprise (a) calculating the difference between the serum-associated T2 signal and the clot-associated T2 signal for the blood sample undergoing the clotting process; and (b) determining the intensity of the clot formed in the blood sample based on the difference. The method may comprise (a) calculating the difference between the serum-associated T2 signal and the clot-associated T2 signal for a blood sample that includes platelets and is undergoing a clotting process; and (b) determining the activity of the platelets in the blood sample based on the difference. In one embodiment, the method further comprises (a) measuring a time period prior to an initial detection of a clot-associated T2 signal after initiation of a clotting process in the blood sample; and (b) determining the clotting time of the blood sample based on the time period. In other embodiments, the method further comprises (a) calculating a T2 time curve for serum-associated T2 signal after initiation of the clotting process in the blood sample; (b) calculating the maximum value of the second derivative of the T2 time curve; and (c) calculating a value representing the clotting time based on the results of step (b). In still other embodiments, the method comprises, after initiating a clotting process in a blood sample, determining whether the blood sample is hypercoagulable, hypocoagulable, or normal based on the serum-associated T2 signal and the clot-associated T2 signal.

In one embodiment of any of the above methods, the method further comprises calculating from the decay curve a T2 relaxation spectrum at a predetermined time point after initiation of the coagulation or dissolution process. The method may further include, after initiating a clotting or dissolution process in the blood sample, (a) making a plurality of decay curves from the blood sample during the process by taking a plurality of relaxation rate measurements of the blood sample, and (b) calculating a plurality of T2 relaxation spectra from the plurality of decay curves. The optional method further comprises calculating a three-dimensional data set from the plurality of T2 relaxation spectra that describes (a) T2 relaxation times and (b) T2 signal intensities of two or more water populations in the blood sample as a function of time after initiation of a clotting or dissolution process in the blood sample.

In particular embodiments, the method further comprises (i) partitioning the three-dimensional data set into stable data and transitional data and (ii) determining from the stable data and the transitional data whether the blood sample is hypercoagulable, hypocoagulable, or normal, or whether the blood sample exhibits low platelet activity, high functional fibrinogen activity, or low functional fibrinogen activity. In certain embodiments, the method further comprises (i) calculating from the three-dimensional data set a relative volume of signal observed for each of the two or more water populations in the blood sample over a predetermined time in the process, and (ii) determining from the relative volumes of signals whether the blood sample is hypercoagulable, hypocoagulable, or normal, or whether the blood sample exhibits low platelet activity, high functional fibrinogen activity, or low functional fibrinogen activity.

In another specific embodiment of any of the above methods, the algorithm is an inverse Laplace transform that includes a lower bound for the T2 time constant of 1-50 ms (e.g., 1-10, 1-20, 1-30, 5-50, 5-30, or 10-50 ms) and an upper bound for the T2 time constant of 1000-. For example, the blood sample can be plasma, platelet poor plasma, or platelet rich plasma, and the upper bound of the T2 time constant is 2500-. In other embodiments, the blood sample is a whole blood sample, a sample comprising red blood cells, or a sample comprising magnetic particles, and the upper bound of the T2 time constant is 1000-. In yet another specific embodiment of any of the above methods, the algorithm is an inverse laplacian transform comprising a regularization parameter (α) in a range from about 1.0e-10 to about 4.0e 0.

The invention also features a method for evaluating the hemostatic condition of a subject, the method comprising: (i) providing blood drawn from a subject to produce a blood sample; (ii) performing a series of magnetic resonance relaxation rate measurements on water in the blood sample; (iii) according to the method of the invention, one or more magnetic resonance parameter values are obtained by applying an algorithmic transformation measurement that distinguishes two or more independent water populations within a blood sample; and (iv) determining whether the subject is normal, has a bleeding condition, or has a pro-thrombotic condition based on the results of step (iii).

In a related aspect, the invention features a method of evaluating platelet activity, the method including: (i) providing isolated and washed platelets; (ii) mixing the separated and washed platelets with platelet poor plasma comprising a predetermined minimum level of fibrinogen to form a test sample; (iii) initiating the coagulation process by adding a coagulation initiator to the test sample; (iv) performing a series of magnetic resonance relaxation rate measurements on water in the test sample; (v) applying an algorithmic transformation measurement that distinguishes two or more independent water populations within the test sample, wherein each independent water population is characterized by one or more magnetic resonance parameters having one or more values; and (vi) evaluating platelet activity based on the results of step (v). In a particular embodiment, the coagulation initiator is a combination of RF and AA. In still other embodiments, the method further comprises (a) measuring the test sample in the presence of the platelet activator and in the absence of the platelet activator.

The invention also features a method of evaluating platelet activity in a whole blood sample, the method comprising: (i) providing a whole blood sample; (ii) mixing the separated and washed platelets with platelet poor plasma comprising a predetermined minimum level of fibrinogen to form a test sample; (iii) initiating a coagulation process by adding a coagulation initiator to the test sample; (iv) performing a series of magnetic resonance relaxation rate measurements on water in the test sample; (v) applying an algorithmic transformation measurement that distinguishes two or more independent water populations within the test sample, wherein each independent water population is characterized by one or more magnetic resonance parameters having one or more values; and (vi) evaluating platelet activity based on the results of step (v). In a particular embodiment, the coagulation initiator is a combination of RF and AA. In still other embodiments, the method further comprises (a) measuring the test sample in the presence of the platelet activator and in the absence of the platelet activator.

In another aspect, the invention features a method of monitoring a solidification or dissolution process by measuring a signal representative of an NMR relaxation rate characteristic of water in a sample undergoing solidification or dissolution to obtain NMR relaxation data and determining from the NMR relaxation data a value or set of magnetic resonance parameters representative of a solidification or dissolution process in the sample.

In a related aspect, the invention features a method of monitoring a clotting process or dissolution process by measuring a signal representative of an NMR relaxation signature of water in a blood sample having one or more water populations to obtain NMR relaxation data, and determining from the NMR relaxation data a magnetic resonance parameter value or set of values associated with at least one water population in the blood sample, wherein the magnetic resonance parameter value or set of values is characteristic of the clotting or dissolution process. The blood sample may comprise at least 2 water populations or at least 3 water populations. In a specific embodiment, the magnetic resonance parameter value is representative of a characteristic of platelet-associated water molecules in the blood sample. In still other embodiments, the magnetic resonance parameter value is representative of a characteristic of functional fibrinogen-associated water molecules in the blood sample. In particular embodiments of the claimed methods, the method comprises measuring the relative concentration of platelet-associated functional fibrinogen.

In certain embodiments, the method comprises evaluating the hemostatic condition of the subject based on the coagulation behavior of a single blood sample drawn from the subject (e.g., for coagulation management of a patient undergoing surgery, identifying a risk of thrombotic complications in the patient, identifying a patient resistant to anti-platelet therapy, monitoring an anti-platelet therapy in the patient, and/or monitoring a procoagulant therapy in the patient).

In still other embodiments of the invention, the method comprises assessing the hemostatic condition of the subject within 3 minutes, 5 minutes, 6 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes of the initial NMR relaxation rate signal collected from the sample.

In a related aspect, the invention features a method of monitoring a blood clotting process or a dissolution process by: (i) performing a series of relaxation rate measurements on the water in the blood sample; (ii) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (iii) monitoring the blood clotting process of the blood sample based on the results of step (ii).

The invention features a method of monitoring platelet activity, the method comprising: (i) performing a series of relaxation rate measurements on the water in the blood sample; (ii) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (iii) monitoring the platelet activity of the blood sample based on the results of step (ii).

The invention also features a method of monitoring platelet inhibition, the method comprising: (i) performing a series of relaxation rate measurements on the water in the blood sample; (ii) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (iii) monitoring the blood sample for platelet inhibition based on the results of step (ii). In particular embodiments, the method further comprises: (iv) performing a series of relaxation rate measurements on the water in the second blood sample in the presence of a platelet inhibitor; (v) transforming the measurement using an algorithm that distinguishes two or more separate water populations within the second blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (vi) monitoring the blood sample for platelet inhibition based on the results of steps (ii) and (v).

The invention also features methods of evaluating the hemostatic condition of a subject by: (i) performing a series of relaxation rate measurements of water in a blood sample from a subject; (ii) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (iii) evaluating the hemostatic condition of the subject based on the results of step (ii).

The algorithm may be selected from, but is not limited to, a multi-exponential algorithm, a bi-exponential algorithm, a tri-exponential algorithm, an exponential decay algorithm, a laplacian transform, a goodness-of-fit algorithm, an SSE algorithm, a least squares algorithm, a non-negative least squares algorithm, and any other algorithm described herein. In a specific embodiment, the algorithm is an inverse laplacian transform or an algorithm given by equation (1) (i.e., for a bi-exponential fit) or (2) (i.e., for a tri-exponential fit) below.

In equations (1) and (2), I is the intensity of the measured value T2; t is time; amp_ATo extract the coefficients, the exponential term exp is indicated^(-t/T2A)The degree of contribution to the measured T2 intensity; amp_BTo extract the coefficients, the exponential term exp is indicated^(-t/T2B)The degree of contribution to the measured T2 intensity; amp_CTo extract the coefficients, the exponential term exp is indicated^(-t/T2C)The degree of contribution to the measured T2 intensity; T2A is the extracted relaxation time (extracted relaxation time), indicating the contribution of water population a to the measured T2 intensity; T2B is the extraction relaxation time, indicating the contribution of water population B to the measured T2 intensity; T2C is the extraction relaxation time, indicating the contribution of water population C to the measured T2 intensity; o is an offset constant (offset constant). In any of the above methods, the relaxation rate measurements may include T2 measurements. The magnetic resonance parameter values may include a T2 parameter value and/or an amplitude parameter value.

For samples that undergo rheological transformations, the water population may vary with the composition and/or complexity of the sample. In heterogeneous samples (e.g. whole blood), there are different water populations, such as plasma water, compartmentalized water, i.e. cells (red blood cells, white blood cells and platelets), and water associated with the functional characteristics of whole blood processing such as coagulation (e.g. serum) or clot lysis. The methods of the invention allow for monitoring of changes in various water populations in a sample, whether changes occur in location, compartment, or within the sample. The method can be used to examine changes in the rheology of a given sample or to calculate one or more analytical values (aPPT, PT-INR, hematocrit, platelet activity) for a given sample.

In any of the above methods, the water in the sample can include a first water population and a second water population, and the NMR parameter associated with a given water population can be correlated to a rheological change. For example, the NMR parameter value of the second water population can be positively correlated with platelet activation. Similarly, the value of the T2 parameter for the first water population may be correlated to clotting time.

In any of the above methods, the magnetic resonance parameter value may comprise a T2 value for the first water population of between 250 and 500 milliseconds.

In any of the above methods, the magnetic resonance parameter value may comprise a T2 value for the second water population of between 100 and 300 milliseconds.

In any of the above methods, the sample can further comprise a third water population having a T2 value between 1 and 10 milliseconds.

In any of the above methods, the signal is generated from monitoring protons in the water or monitoring hydrogen atoms in the water. Alternatively, in any of the above methods, the signal is generated from monitoring protons in the water or monitoring oxygen atoms in the water.

In some embodiments of the invention, the blood sample contains a fourth water population and a fifth water population, wherein the fourth water population is associated with a contracted blood clot and the fifth water population is associated with serum surrounding the contracted blood clot. The presence of the fourth and/or fifth water population does not imply the presence of the first, second or third water population as defined herein or require the presence thereof.

In any of the above methods, the method may further comprise assessing whether the sample is hypercoagulable, hypocoagulable, or normal based on the magnetic resonance parameter value or value set.

In any of the above methods, the magnetic resonance parameter may be representative of a hemostatic condition (e.g., a hemorrhagic condition or a prothrombotic condition) of the subject. For example, the magnetic resonance parameter may indicate low platelet activity; indicating high platelet activity; indicating high functional fibrinogen activity; or indicating low functional fibrinogen activity.

In a particular embodiment of the invention, a paramagnetic agent (e.g. manganese, manganese complex, gadolinium complex or magnetic particles) is added to the blood sample before measuring the signal characteristic of the NMR relaxation parameter. In a preferred embodiment superparamagnetic particles. The paramagnetic agent may be superparamagnetic particles having an average diameter between 3 and 40 nanometers, between 30 and 70 nanometers, between 70 and 100 nanometers, between 100 and 500 nanometers, or between 500 and 1000 nanometers. In a specific embodiment, a blood sample from a single patient is subjected to the methods of the invention, both in the presence and absence of a paramagnetic agent.

In another embodiment of the method of the invention, the sample to be analyzed has a volume between 2 μ L and 400 μ L (e.g., 2 μ L-10 μ L, 10 μ L-20 μ L, 15 μ L-50 μ L, 50 μ L-100 μ L, 100 μ L-250 μ L, or 200 μ L-500 μ L). In still other embodiments, the sample to be analyzed has a volume between 400 μ L and 4000 μ L.

In any of the above methods, the NMR relaxation data is selected from T1, T2, T1/T2 promiscuous, T_1rho、T_2rhoAnd T₂ ^*And (4) data. In addition, the Apparent Diffusion Coefficient (ADC) can be determined and evaluated (see Vidmar et al, NMR in Biomedicine, 2009; and Vidmar et al, Eur J Biophys J. 2008). In addition, the method of the present invention may utilize pulsed field gradients (i.e., a measure of echo attenuation as a function of the square of the gradient strength), Hahn echo sequences, spin echo sequences, and/or FID signal ratios.

The invention features a magnetic resonance device for monitoring a blood clotting process, wherein the device includes a microprocessor having an algorithm to distinguish two or more separate water populations within a blood sample, wherein each separate water population is characterized by a magnetic resonance parameter value or set of values. The algorithm may be selected from, but is not limited to, a multi-exponential algorithm, a bi-exponential algorithm, a tri-exponential algorithm, an exponential decay algorithm, a laplacian transform, a goodness-of-fit algorithm, an SSE algorithm, a least squares algorithm, a non-negative least squares algorithm, and any other algorithm described herein.

In a related aspect, the invention features a method for monitoring clot lysis, the method including: (i) providing a coagulation sample; (ii) mixing the coagulation sample with tissue plasminogen factor (TPA); (iii) performing a series of relaxivity measurements on water in the coagulation sample; (iv) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (v) monitoring the lysis of the coagulation based on the results of step (iv).

In certain embodiments, the hemostatic condition of a subject is assessed based on the blood clotting behavior of a blood sample drawn from the subject. Examples of clotting behavior include clotting time (R), clot strength (MA), platelet-associated clot strength (MA)_{Blood platelet}) Functional fibrinogen-related clot strength (MA)_FF) And percent dissolution 30 minutes after MA (LY 30). Examples of hemostatic conditions include prothrombotic conditions, hemorrhagic conditions, and normal conditions. The clotting behavior or hemostasis condition may be established within a certain time frame (e.g., within 10, 6, or 3 minutes) after the initial NMR relaxation data is collected. Furthermore, one or more additives (e.g. fibrinogen or TPA) may be added to the sample when setting behaviour is established.

In other embodiments, the blood clotting process of blood drawn from a subject is monitored by: (i) performing a series of first relaxation rate measurements on water from a first blood sample (e.g., a sample treated with an additive) of a subject; (ii) transforming the first relaxation rate measurement using an algorithm that distinguishes two or more independent water populations within the first blood sample; (iii) performing a series of second relaxation rate measurements on water from a second blood sample (e.g., an untreated sample) of the subject; (iv) transforming the second relaxation rate measurements using an algorithm that distinguishes two or more separate water populations within the second blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters, wherein each magnetic resonance parameter has one or more values; and (v) monitoring the subject's blood for clotting processes based on the results of step (iv). One of the samples (e.g., the first sample) may contain an additive, such as fibrinogen or tissue plasminogen activator. In some embodiments, the hemostatic condition of the subject is assessed or diagnosed based on the results of the monitoring process (e.g., the hemostatic condition is assessed within 10, 6, or 3 minutes).

In other embodiments, the invention features methods of diagnosing a hemostatic condition in a subject by: (i) providing a blood sample from a subject; (ii) measuring a signal representative of an NMR relaxation rate characteristic of water in the blood sample to obtain one or more values of a magnetic resonance parameter; and (iii) diagnosing the subject based on the results of step (ii). The diagnosis may be based on a comparison of the magnetic resonance parameter value(s) with a predetermined threshold value(s). The predetermined threshold may represent a certain hemostatic condition (e.g., normal, pro-thrombotic, or hemorrhagic). The hemostatic condition may be assessed over a certain time (e.g. within 10, 6 or 3 minutes) during which the initial NMR relaxation rate signal is acquired.

In certain embodiments, the coagulation behavior is evaluated using well-defined data extraction methods. The clotting time (R) of the sample can be evaluated from the acquired NMR relaxation rate data set by: (i) calculating a T2 time curve for the first water population; (ii) calculating the maximum value of the second derivative of the T2 time curve; and (iii) calculating a value (R) characteristic of the clotting time based on the results of step (ii). The platelet-associated clot strength (MA) of a sample can be evaluated by_{Blood platelet}): (i) calculating a T2 time curve for the first water population; (ii) calculating the slope of a line connecting the start of T2 on the T2 curve to the maximum of T2 on the T2 curve; and (iii) calculating platelet-associated clot strength (MA) representative of the sample based on the results of step (ii)_{Blood platelet}) The value of the feature. The functional fibrinogen-associated clot strength (MA) of a sample can be evaluated by_FF): (i) calculating a first amplitude time curve for a first water population in a first sample that has been treated with fibrinogen; (ii) calculating a second amplitude time curve for the first water population in the second untreated sample; (iii) calculating a difference between the first amplitude-time curve and the second amplitude-time curve; and (iv) results from step (iii)Calculating the functional fibrinogen-associated clot strength (MA) representing the sample_FF) The value of the feature. The percent dissolution 30 minutes after MA of the sample (LY30) can be evaluated as follows: (i) calculating a first T2 time curve for a second water population in the first sample that has been treated with TPA; (ii) calculating a second T2 time curve for a second water population in a second untreated sample; (iii) calculating a difference between the first T2 time curve and the second T2 time curve; and (iv) calculating a value characteristic of the percent dissolution 30 minutes after MA of the sample (LY30) based on the results of step (iii).

In other embodiments, the subject is assessed or diagnosed for hemostatic condition using a T2 signature (signature) curve by: (i) measuring a signal representative of an NMR relaxation signature of water in a blood sample drawn from the subject to obtain NMR relaxation data; and (ii) determining from the NMR relaxation data a T2 signature characteristic of the hemostatic condition of the subject. The T2 signature can be compared to a standard curve or set of standard curves to establish the hemostatic condition of the subject.

In other embodiments, the invention features methods of monitoring an aqueous material by: (i) providing an aqueous material capable of being converted from a liquid fluid state to a gel state; (ii) measuring a series of signals representative of NMR relaxation characteristics of water in the substance to obtain NMR relaxation data; and (iii) determining a time curve from the NMR relaxation data that is characteristic of the aqueous material. In some embodiments, the time curve is a relaxation curve other than a T2 relaxation curve, such as a T1 time curve, a T2 time curve, or a hybrid T1/T2 time curve. In related embodiments, the aqueous material is whole blood, a polyacrylamide hydrogel, a polyvinylpyrrolidone hydrogel, a polyethylene glycol hydrogel, a polyvinyl alcohol hydrogel, a polyacrylic acid hydrogel, carrageenan, alginate gel, or gelatin. In a preferred embodiment, the aqueous material is an acrylamide hydrogel.

In another aspect, the invention features a method of evaluating a calibration status of a blood monitoring device by: (i) providing an aqueous material capable of being converted from a liquid fluid state to a gel state; (ii) measuring a characteristic of the hydrated material with a blood monitoring device; and (iii) assessing the calibration status of the blood monitoring device by comparing the characteristics obtained in step (ii) with a predetermined threshold. In a preferred embodiment, the aqueous material is an acrylamide gel and the blood monitoring device is a T2reader (T2reader) or a Thromboelastography (TEG) analyzer. In a particular embodiment, a paramagnetic agent (e.g., manganese complex, gadolinium complex, or superparamagnetic particles) is added to the sample prior to analysis. The paramagnetic agent may be superparamagnetic particles having an average diameter between 3 and 40 nanometers, between 30 and 70 nanometers, between 70 and 100 nanometers, between 100 and 500 nanometers, or between 500 and 1000 nanometers.

In certain embodiments, a series of NMR relaxation rate measurements from a blood sample are used to generate a series of magnetic resonance parameter values (e.g., T2 or amplitude values) that are characteristic of a certain water population in the blood sample. A series of magnetic resonance parameter values may be plotted as a function of time to produce a time curve that is also characteristic of the water population. For example, a T2 time curve (or amplitude curve) for a certain water population in a blood sample can be plotted as the sample is coagulated. These time profiles may also be used to generate other profiles. For example, the first or second time derivative of the T2 time curve may be plotted. The time profile can be used as a basis for evaluating the clotting behavior of a blood sample or the hemostatic condition of a subject from whom the blood sample is drawn. Similarly, the curves generated from two different samples (e.g., a treated sample and an untreated sample) obtained from the same subject can be used as a basis for evaluating the clotting behavior of the subject's blood or the hemostatic condition of the subject.

In other embodiments, the coagulation behavior of the sample or the hemostatic condition of the subject may be evaluated based on comparing the value of the magnetic resonance parameter or time curve associated with the sample or subject to a predetermined threshold value. The predetermined threshold may be established in a number of different ways. For example, the predetermined threshold may be representative of a hemostatic condition (e.g., normal, pro-thrombotic, or hemorrhagic condition). The threshold may be determined from the mean or range of values observed for blood drawn from normal and abnormal subjects. Alternatively, the threshold value may be determined from a standard sample that consistently provides the same parameter or curve when used in the NMR-based method of the invention (e.g., a blood sample or acrylamide gel treated to coagulate in a particular manner).

In another aspect, the invention features a method of evaluating the hemostatic condition of a subject by: (i) performing a series of relaxation rate measurements (e.g., T2 relaxation rate measurements) on water in a blood sample drawn from a subject, wherein the blood sample is undergoing a clotting process or a dissolution process, and wherein the measurements provide two or more decay curves, wherein each decay curve represents a time point characteristic of the process; (ii) applying a mathematical transform (e.g., inverse laplace transform) to the two or more decay curves to identify two or more water populations (e.g., a water population having a serum-associated T2 signal and a water population having a clot-associated T2 signal) in the blood sample at two or more time points in the process, resulting in two or more magnetic resonance parameter values having two or more signal intensities, wherein each water population has a characteristic magnetic resonance parameter value and a concentration characteristic of the signal intensity of the magnetic resonance parameter value; (iii) from (a) two or more time points; (b) two or more magnetic resonance parameter values; and (c) two or more signal strengths, generating a 3D data set; (iv) extracting from the 3D data set one or more coagulation behaviors characteristic of the hemostatic condition of the subject (e.g., coagulation time (R), fibrinolysis behavior, clot intensity (MA), clot kinetic behavior, platelet-associated clot intensity (MA)_{Blood platelet}) Functional fibrinogen-related clot strength (MA)_FF) Percent lysis 30 minutes post MA (LY30) or hematocrit); and (v) evaluating the hemostatic condition of the subject based on the one or more clotting behaviors.

In certain embodiments, the invention features the addition of an antiplatelet antibody or Fab fragment (e.g., abciximab) to a sample undergoing a clotting process or a lysis process (e.g., a whole blood sample). In other embodiments, the invention features the addition of a clotting activator or platelet inhibitor to a sample (e.g., a whole blood sample) undergoing a clotting process or a lysis process.

In another aspect, the invention features a method of evaluating the strength of a blood clot or the platelet activity of a blood clot by: (i) making a T2 relaxation rate measurement of the water in the blood clot, wherein the measurement provides a decay curve; (ii) applying a mathematical transformation (e.g., inverse laplace transform) to the attenuation curve to identify the signal intensity of a water population in the blood clot, wherein the water population corresponds to a contracted blood clot aqueous environment or a serum aqueous environment; and (iii) evaluating the intensity of the blood clot or the platelet activity of the blood clot based on the signal intensity of the water population.

In another aspect, the invention features a method of evaluating the hemostatic condition of a subject by: (i) providing a blood sample from a subject; (ii) making a T2 relaxation rate measurement of the water in the sample, wherein the measurement provides a decay curve; (iii) applying a mathematical transform (e.g., an inverse laplace transform) to the attenuation curve to identify serum-associated T2 signals and clot-associated T2 signals; and (iv) assessing the hemostatic condition of the subject based on the difference between the serum-associated T2 signal and the clot-associated T2 signal or the appearance of the clot-associated T2 signal.

In another aspect, the invention features a method of diagnosing a hemostatic condition in a subject by: (i) performing a series of NMR relaxation rate measurements on water in a blood sample drawn from the subject; (ii) transmitting data that is or is characteristic of the NMR relaxation rate measurements for processing, including any of the methods described herein; and (iii) receiving the results of step (ii) and diagnosing the subject based on the results.

The invention also features a method for reducing the risk of bleeding or clotting in a subject fitted with a heart assist device, the method comprising: (i) evaluating the hemostatic condition of the subject using the method described above; and (ii) adjusting an operating parameter (e.g., speed, intensity, pressure, flow rate, pump capacity, or fill capacity) of the heart assist device to reduce the risk of bleeding or clotting in the subject according to step (i). In particular embodiments, the cardiac assist device is selected from the group consisting of extracorporeal heart shunts (extracorporeal cardiac bypass machines) and implantable blood pumps, such as left ventricular assist devices.

The invention also features a method for reducing the risk of bleeding or clotting in a subject fitted with a heart assist device, the method comprising: (i) evaluating the hemostatic condition of the subject using the methods described herein; and (ii) administering an anticoagulant therapy, an antiplatelet therapy and/or a procoagulant therapy to the subject according to step (i) to reduce the risk of bleeding or clotting in the subject. In particular embodiments, the cardiac assist device is selected from an extracorporeal heart shunt and an implantable blood pump, such as a left ventricular assist device.

In another aspect, the invention features a method of comparing the coagulation or dissolution behavior of a sample measured using the NMR-based techniques of the invention with the rheological change or coagulation or dissolution measured in an equivalent sample using systems known in the art.

The methods of the invention can be used to simultaneously measure multiple parameters of a sample (e.g., simultaneously measure parameters associated with coagulation of the sample or the hemostatic condition of the subject).

In any of the above methods, the method may comprise the steps of: (i) adding whole blood or components thereof to a tube filled with one or more additives (e.g., heparin, citrate, nanoparticle formulation (nanoparticie formation), paramagnetic agent, fibrinogen, Tissue Plasminogen Activator (TPA), an antithrombotic agent such as abciximab, or any other additive described herein), and (ii) mixing the contents to initiate the clotting process. In particular embodiments, the one or more additives are dry additives that are reconstitutable in whole blood or a component thereof. For example, small particles of collagen at the bottom of a sample tube can be used to cause coagulation of whole blood or components thereof in situ. In a specific embodiment, at least a portion of the sample tube is coated with collagen to initiate coagulation. In still other embodiments, a localized region of the sample tube is coated with a clotting initiator (e.g., collagen or another clotting initiator described herein) to allow spatial control of clot formation.

In any of the above methods, the method may comprise the step of measuring the T2 signal of the sample using a T2 reader. For example, quantitative T2 measurements of blood (whole blood, diluted blood, PRP, etc.) may be made using a CPMG pulse sequence with a long total echo time, which is about 5 × T2 (e.g., τ is typically greater than 62.5 μ s and less than 1000 μ s). In particular embodiments, the sample is pre-incubated to the desired temperature using a T2reader prior to initiating the clotting process.

For samples containing uncoagulated blood, it is understood that uncoagulated blood can precipitate to produce samples with T2 values greater than 1, but the timescale of such precipitation is typically longer than the timescale of clot formation.

The method of the invention involves analysis of raw NMR data to generate information about two or more water populations of a sample undergoing a coagulation or dissolution process. An example is the inverse laplace transform of the data to identify signal intensities generated from different water populations observed simultaneously within the sample. Alternatively, information about two or more water populations in the sample can be obtained using data acquisition and data manipulation techniques known in the art, such as (i) relaxation measurements using a Hahn echo pulse sequence; (ii) a difference measurement based on the difference between the FID signal amplitude at a fixed time delay after the initial 90 degree pulse and the amplitude at a fixed delay after the subsequent 180 degree pulse; (iii) measurement of T2 values and echo attenuation in the presence of a pulsed field gradient configured according to its relaxation properties to attenuate certain water populations, thereby highlighting other water populations; and (iv) a difference measurement (i.e., the difference between signal intensities at two or more different points in time for (a) FID after a single 90 degree pulse, (b) CPMG relaxation curve, (c) relaxation curve obtained by a series of Hahn echoes or spin echoes, or (d) CP relaxation curve). Alternative methods may be employed in any of the methods described herein.

The term "3D data set" as used herein refers to a collection of measured and/or derived data points that can be compiled into a 3D map representing the characteristics of changes in a sample undergoing a coagulation or dissolution process over a period of time. A 3D map derived from the 3D data set may describe the appearance and/or disappearance of different water populations within the sample and quantify the intensity and relaxation times (e.g., T2 relaxation times) of these water populations at a particular point in time or within a range of times.

The term "first aqueous population" as used herein refers to an aqueous population of a whole blood sample characterized by a decrease in the initial amplitude of uncoagulated blood as a function of coagulation. The first water population may also be referred to as the water population of group a elsewhere in this application. The amplitude and T2 data extracted from the first water population are referred to as Amp, respectively_AAnd T2A.

The term "second aqueous population" as used herein refers to an aqueous population of a whole blood sample characterized by an initial amplitude of uncoagulated blood that increases with coagulation. The second water population may be related to the platelet concentration of the blood clot. The second water population may also be referred to as the water population of group B elsewhere in this application. The amplitude and T2 data extracted from the second water population are referred to as Amp, respectively_BAnd T2B.

The term "third aqueous population" as used herein refers to an aqueous population of a whole blood sample characterized by a substantially constant amplitude during coagulation. The third water population may be associated with water that binds biomolecules within the red blood cells. The third water population may also be referred to as the water population of group C elsewhere in this application. The amplitude and T2 data extracted from the third water population are referred to as Amp, respectively_CAnd T2C.

The term "fourth aqueous population" as used herein refers to an aqueous population of whole blood samples characterized by a broad distribution of T2 values ranging from about 400 milliseconds to about 2,200 milliseconds. The range of T2 values associated with the fourth water population may depend on the hardware and materials used to collect the data (e.g., sample tubes). The fourth water population may be associated with serum surrounding the contracted blood clot and may be combined with the fifth water population upon addition of an anti-platelet aggregation drug (e.g., abciximab) to the blood sample. The fourth water population may be characterized by platelet activity and/or clot strength.

The term "serum aqueous environment" as used herein refers to the aqueous environment present in an unsolidified blood sample, or the portion of a blood sample that remains unsolidified. "serum-associated T2 signal" refers to a signal arising from the aqueous environment of serum (e.g., the corresponding T2 signal within a coagulated blood sample associated with serum surrounding a contracted blood clot). The serum-associated T2 signal is typically present in both the sample containing the contracted blood clot and the coagulated sample from which the contracted blood clot has been removed. The serum-associated T2 signal contained an analytical peak with a T2 value higher than the T2 value of the clot-associated T2 signal. The serum aqueous environment may be associated with the fourth aqueous population described above.

The term "fifth water population" as used herein refers to the water population of the whole blood sample characterized by a distribution of T2 values ranging from about 80 milliseconds to about 500 milliseconds. The range of T2 values associated with the fifth water population may depend on the hardware and materials used to collect the data (e.g., sample tubes). The fifth water population may be associated with a contracted blood clot. The fifth water population may be characterized by coagulation time and/or fibrinolytic activity.

The term "contracting clot aqueous environment" as used herein refers to an aqueous environment that exists in a contracting clot and is characteristic of clot formation. The "clot-associated T2 signal" refers to a signal resulting from contracting clot aqueous environment (whether or not the clot is contracted at the time of measurement). The clot-associated T2 signal was present in both the coagulated whole blood sample containing the contracted clot and the sample containing the contracted clot from which the surrounding serum had been removed. The clot-associated T2 signal contained an resolved peak with a T2 value lower than the T2 value of the serum-associated T2 signal. The contracted clot aqueous environment may be associated with the fifth aqueous population described above.

The term "algorithm" as used herein refers to a mathematical procedure for processing or transforming data.

The term "assay" as used herein refers to a method of monitoring the coagulation behavior of blood.

The term "clotting behavior" as used herein refers to parameters related to a clot, the formation of a clot or a clot that undergoes lysis (e.g., clotting time (R), fibrinolysis behavior, clot strength (MA), clot kinetic behavior, platelet-associated clot strength (MA)_{Blood platelet}) Functional fibrinogen-related clot strength (MA)_FF) Percent dissolved 30 minutes after MA (LY 3)0) Etc.).

The term "solidification process" as used herein refers to a process in a liquid that results in a local spatial change of solvent water molecules within a sample and is characterized by a change in the NMR relaxation rate of solvent water molecules within an aqueous liquid. The aqueous liquid may have more than one population of solvent water molecules, each population being characterized by NMR relaxation parameters that vary as the aqueous sample undergoes the solidification process. The method of the present invention may be used to monitor the process of coagulation in aqueous solutions containing gel-forming components including, without limitation, proteinaceous solutions (such as blood, plasma or gelatin, among others) and non-proteinaceous hydrogels.

The term "dissolution process" as used herein refers to a process in a liquid that results in a local spatial change in solvent water molecules within the sample and is characterized by a change in the NMR relaxation rate of solvent water molecules within the aqueous liquid. The aqueous liquid may have more than one population of solvent water molecules, each population being characterized by NMR relaxation parameters that vary as the aqueous sample undergoes the dissolution process. The method of the present invention may be used to monitor the dissolution process of aqueous solutions containing gel-forming components including, without limitation, proteinaceous solutions (such as blood, plasma or gelatin, among others) and non-proteinaceous hydrogels.

The term "functional fibrinogen" as used herein refers to fibrinogen in a blood clot that contributes to the strength of the blood clot.

As used herein, the term "gel state" refers to a dispersion comprising water and solids, wherein the mobility of water molecules is reduced compared to the mobility of water molecules in the liquid flow state. The gel state may be formed from a polymer and/or protein (e.g., from coagulation, gelatin, or any of the gel-forming materials described herein).

The term "given time after the start of the measurement" as used herein refers to the time at which the coagulation behavior can be measured after the start of the measurement involving monitoring of the coagulation behavior of blood. Examples of the given time after the start of the measurement include 60 minutes, 35 minutes, 45 minutes, 10 minutes, 5 minutes, 2 minutes, and 1 minute.

The term "heart assist device" as used herein includes, but is not limited to, extracorporeal heart shunts and implantable blood pumps, such as left ventricular assist devices.

The term "hematocrit" as used herein refers to the percentage by volume of red blood cells in a whole blood sample.

The term "hemostatic condition" as used herein refers to a condition of a subject characterized by the coagulation behavior of the subject's blood. The hemostatic condition may be prothrombotic (increased risk of clot formation), hemorrhagic (increased risk of spontaneous bleeding), or normal (neither prothrombotic nor hemorrhagic). Hemostatic conditions may also refer to specific thrombotic disorders (e.g., protein C deficiency, protein S deficiency, protein Z deficiency, antithrombin deficiency, antiphospholipid antibody syndrome, anticoagulant therapy resistance or hyperhomocysteinemia). In other instances, the hemostatic condition may result from an anticoagulant administered to the subject in response to or in order to prevent the occurrence of the physiological condition of the subject. Such physiological conditions include atrial fibrillation, myocardial infarction, unstable angina, deep vein thrombosis, pulmonary embolism, and acute ischemic stroke. Likewise, a hemostatic condition may be caused by administration of an anticoagulant drug to a subject undergoing invasive surgery (e.g., joint replacement, surgical replacement of a mechanical heart valve, or other device implanted into the body). The hemostatic condition of a subject may be assessed by measuring one or more clotting behaviors of one or more blood samples drawn from the subject.

The term "magnetic resonance parameter" as used herein refers to the relaxation rate or amplitude extracted from the NMR relaxation rate measurement.

The term "NMR relaxation rate" as used herein refers to any of the following in a sample: t1, T2, T_1rho、T_2rhoAnd T₂ ^*. NMR relaxation rates may be measured and/or expressed using T1/T2 hybrid detection methods. In addition, the Apparent Diffusion Coefficient (ADC) can be measured or evaluated (Vidmar et al NMR in BioMedicine, 2009; and Vidmar et al Eur J Biophys J. 2008).

The term "platelet" as used herein refers to a cellular component that contributes to the formation of a blood clot.

The term "predetermined threshold value" as used herein refers to a standard parameter value or set of values, a standard time curve or a standard characteristic curve derived by the method of the invention and representing a characteristic of a particular rheological state or representing a characteristic of a normal or abnormal outcome (e.g. representing a characteristic of blood of a normal subject, or representing a characteristic of blood of a subject suffering from an abnormal hemostatic condition). The predetermined threshold may be obtained by measuring the NMR parameter value of, for example, a blood sample taken from a normal and/or abnormal population of subjects. The predetermined threshold may be selected to distinguish between two or more different possible rheological states of the sample. For example, when the sample is a blood sample, a predetermined threshold value can be used to diagnose the hemostatic condition of the subject.

The term "reader" or "T2 reader" as used herein refers to a device for detecting coagulation-related activation, including coagulation and fibrinolysis of a sample. A T2reader can generally be used to characterize the properties of a sample (e.g., a biological sample such as blood or a non-biological sample such as an acrylamide gel). Such devices are described, for example, in international publication No. WO2010/051362, which is incorporated herein by reference.

The term "relative concentration" as used herein refers to a comparative concentration or volume fraction of one water population relative to another (e.g., second or third) water population. For example, the relative concentration of water population a may be 2 times (or 5 times or 10 times) the concentration of water population B.

As used herein, the term "signal intensity of a water population" refers to the intensity measured for the relaxation rate of a particular water population in a sample, which is measured as the integral of (i) the peak height, or (ii) one or more peaks, representative of the characteristics of the particular water population.

The term "treated sample" as used herein refers to a blood sample containing an additive at a concentration greater than that necessary to prevent normal sample coagulation in the absence of a coagulation initiator (e.g., calcium chloride).

The term "untreated sample" as used herein refers to a blood sample that does not contain additives at concentrations greater than necessary to prevent normal sample coagulation in the absence of a coagulation initiator (e.g., calcium chloride).

The term "whole blood" as used herein refers to the blood of a subject, including red blood cells. Whole blood includes blood modified by a processing step or by the addition of additives such as heparin, citrate, nanoparticle formulations, fibrinogen, Tissue Plasminogen Activator (TPA), collagen, antithrombotic agents such as abciximab, or other additives.

The term "pooled whole blood platelets" as used herein refers to platelet-rich blood or blood products (e.g., plasma). Pooled whole blood platelets include samples modified by processing steps or by the addition of additives.

The term "T1/T2 scrambling" as used herein refers to any detection method that combines the T1 and T2 measurements. For example, the T1/T2 hybrid value may be a composite signal obtained by a combination of ratios or differences between two or more different T1 and T2 measurements. T1/T2 scrambling can be achieved, for example, by employing a pulse sequence in which T1 and T2 are alternately measured or acquired in an interleaved fashion. In addition, the T1/T2 confounding signal may be acquired with a pulse sequence that measures the relaxation rate consisting of both the T1 and T2 relaxation rates or mechanisms.

The term "T2 feature" as used herein refers to a curve established by applying a mathematical transform (e.g., laplace transform or inverse laplace transform) to a decay curve relating relaxation rate parameters at discrete points in time or over a specified duration during a rheological event. The T2 profile provides information about the relative abundance of multiple water populations in a blood clot. The T2 profile reflects changes in the blood clot as coagulation or fibrinolysis proceeds. The T2 signature can be advantageously utilized to assess in real time the differentiated hemostatic condition of a subject. Additionally, the T2 features may be two-dimensional images (intensity versus T2 values or T2 values versus time) or three-dimensional images (intensity versus T2 values versus time). Two or three dimensionsThe T2 values in the dimensional image may be contaminated by other NMR signals (e.g., T1, T1/T2, etc.)_1rho、T_2rhoAnd T₂ ^*) Either as an alternative or in comparison with other NMR signals.

Other features and advantages of the invention will be apparent from the following detailed description, the accompanying drawings, and the appended claims.

Brief Description of Drawings

FIGS. 1A-1C show diagrams and equations for identifying NMR relaxation rates of multiple water populations within a sample from a single FID and methods for extracting features from relaxation rates that are characteristic of the coagulation state of a sample. FIG. 1A is a graph illustrating cubic exponential curve fitting (using equation 2) of a CK sample. FIG. 1B shows a graph illustrating a quadratic exponential curve fit (using equation 1) of a CK sample after an initial rapid phase cut. FIG. 1C is a pictorial depiction of an algorithm used to identify blood coagulation behavior from NMR relaxation rate measurements.

Fig. 2 shows two graphs showing the observed changes in T2 relaxation values (T2A and T2B) for different water populations in a single CK blood sample undergoing coagulation. The graph illustrates that the relaxation rates of the individual water populations can be monitored as a function of time. During the solidification process, the T2 value for water population a initially decreased, then increased, and then reached a plateau; during the solidification process, the T2 value for water population B initially rises, reaches a peak, and then falls. A change in the value of the water population T2 in the blood sample undergoing the clotting or lysing process can be indicative of either too much clotting or too little clotting in the blood sample.

FIG. 3 is a series of graphs showing the variation of amplitude values (AmpA and AmpB) for different water populations in a CK blood sample undergoing coagulation. Blood samples were taken from 4 different healthy subjects who met the standard for blood donation and did not receive anticoagulants. The change in amplitude of the water population in the blood sample undergoing the clotting or lysing process can be indicative of either too much clotting or too little clotting in the blood sample.

Fig. 4 is a series of graphs of AmpA and AmpB of CK (kaolin) versus ADP (adenosine diphosphate) + RF (snake venom thrombin and factor XIII) versus a activation samples from 2 different patients (see example 1 and table 1 for a description of different activation pathways). Of the 2 samples, the kaolin activated sample showed a significant increase in AmpB during clot formation. Of the 2 subject samples, the a activation sample did not show a significant increase in AmpB during clot formation. The activator RF activates fibrin formation. When an ADP + RF activator is used, samples from one subject do not show significant changes in AmpB during clot formation, while samples from another subject show significant changes in AmpB during clot formation. Variability in the NMR parameter values of the water population in a blood sample subjected to a clotting or dissolution process under different conditions (i.e., in the presence of different clotting initiators or clotting inhibitors) may be indicative of hypercoagulability or hypocoagulability of the blood sample and/or indicative of the hemostatic condition of the subject from which the sample was taken.

Fig. 5 is an exemplary set of processed T2 coagulation curves for a single patient. The top row shows the original T2A, T2B, and AmpB curves. The middle row represents the first derivative of the curve and the bottom row represents the second derivative of the curve.

FIG. 6 shows the manner in which the T2A + pT2B curve is calculated: (a) a T2A plot showing how the addition of pT2B will affect the shape of the T2A curve; (b) showing how to calculate a T2B graph of pT2B by taking the difference between the maximum T2B value and the actual T2B value at all time points after the maximum T2B value is reached; and (c) a T2A + pT2B graph formed by adding a shaded portion in the T2B curve to the T2A curve with the same timer.

Fig. 7 is a T2 coagulation curve in which the extracted data is data shifted and mirrored to simulate a TEG trace. The figure illustrates that water detection reveals early changes in the microscopic order, which may allow additional information to be collected before the clotting time R is observed.

FIGS. 8a and 8b show graphs showing the inverse variation of the T2 signal with hematocrit level for uncoagulated blood. The T2 signal of uncoagulated blood varies inversely with Hematocrit (HCT) level. As shown in fig. 8a, different patient samples spanning a wide range of HCT reference values generally confirmed this. Application of the calibration method to a single patient sample across a range of HCT dilutions, represented in figure 8b, shows a linear dependence on HCT.

FIG. 9 shows the correlation between T2R and TEG R for CK operation; the solid line represents a regression line calculated from the correlation diagram.

FIG. 10 shows the T2 coagulation profile "T2A onset slope" and equivalent platelet-associated clot strength (MA)_{Blood platelet}) TEG term (MA) of_Thrombin– MA_A) The relationship (2) of (c).

Figure 11 shows the results of T2 coagulation titrated from functional fibrinogen in 50% citrate whole blood. Two T2 coagulation amplitude curves show AmpA and AmpB on the same intensity scale; (a) is 50% citrate whole blood without added fibrinogen; (b) is 50% citrate whole blood with 1.25 mg/mL fibrinogen added; and (c) a correlation plot showing the fibrinogen titration performed on the patient sample.

FIG. 12 shows the sensitivity of T2 to clotting to fibrinolysis. (a) And (b) shows the T2A curves for 2 different patients; (c) the T2B curves for the same 2 different patients are shown in (a) and (b). The solid line shows the kaolin curve in the absence of fibrinolysis and the dashed line shows the kaolin curve in the presence of fibrinolysis.

FIG. 13 shows a correlation between T2 coagulation and TEG for healthy and fibrinolytic samples. The data points in the solid circle are healthy K runs. The data points within the dashed circle are the fibrinolytic K runs. The data points outside the two circles are partial fibrinolysis runs.

FIGS. 14A-D show a correlation diagram comparing the method of the present invention with a prior art analysis. Preliminary correlations with Stago for PT/INR were demonstrated using thromboplastin reagent (Thrombotest, Axis Shield) for T2MR and 1:5 diluted whole blood and standard reagent and plasma protocols for the Stago Start system (see fig. 14A). Correlations with TEG clotting time were obtained through >40 normal patient samples with non-optimized R2 correlations > 0.8. (see FIG. 14B). Correlations of PT time with Stago for plasma (see fig. 14D) and with Hemochron for whole blood (see fig. 14C) were also found. See example 7.

Fig. 15 shows the T2 decay curve and the corresponding T2 characteristic, the characteristics of which on the T2 characteristic may vary with the sample composition.

Fig. 16 shows the inverse laplace transform of T2 data corresponding to patient sample KP 29476 and the respective inverse laplace transforms of T2 data obtained from post-isolation sera and contracted blood clots. The graph shows that the formation of a firm clot results in two distinct water populations (i.e., the water population in the clot and the water population in the serum). The longer T2 time signal corresponds to the serum-associated T2 signal and the shorter T2 time signal corresponds to the clot-associated T2 signal.

Figure 17 shows 2 overlapping T2 relaxation spectra and TEG clot intensities (MA) of two different samples taken from the same patient. Samples with weaker clots (MA = 19.2) showed lower differential values between clot-associated and serum-associated signals (182 ms) compared to samples with stronger clots (MA = 65; 258 ms). The spectra shown in figure 17 were collected on a T2 reader.

Fig. 18 shows graphs of two patients relating clot intensity (MA value) to the difference (Δ T2) between a clot-related signal and a serum-related signal. The data show that increasing clot intensity is positively correlated with Δ T2.

Figure 19 shows 2T 2 relaxation spectra of patient samples collected on a T2reader at 2 different time points. The spectrum shows an initial effective peak at time 0 corresponding to blood in the sample. At 20 minutes, two effective peaks were evident. The peak with the shorter T2 time (about 200 and 300 milliseconds) corresponds to the T2 clot aqueous environment and the peak with the longer T2 time (about 450 and 580 milliseconds) corresponds to the T2 serum aqueous environment.

FIG. 20 shows a 3D map of patient samples 29328 and 29350. The TEG MA values measured for samples 29328 and 29350 were 68.4 and 61.9, respectively.

Figure 21 shows a 3D plot of 4 samples containing 4 different concentrations of abciximab as described in example 14. Data were collected in a Bruker minispec.

FIG. 22 shows the T2 relaxation rate spectra of 5 blood samples containing varying concentrations of abciximab at 0.1 min as described in example 14. Data were collected in a Bruker minispec.

FIG. 23 shows T2 relaxation spectra of 4 blood samples containing varying concentrations of abciximab at 20 minutes as described in example 14. The difference between the 4T 2 relaxation spectra illustrates the effect of antiplatelet drugs on the distribution of water into discrete populations within the sample during coagulation. These differences are consistent with differences in clot strength as measured by TEG, which decreases when water populations are pooled at higher concentrations of abciximab. Data were collected in a Bruker minispec.

Figure 24 shows a 3D map made from a 3D data set collected using a T2 reader. The 3D plot shows the initial effective water population with a rapid drop in intensity. Within a period of between 8 and 10 minutes, two distinct water populations appeared. The appearance of the water population with the lower T2 value (population corresponding to clot-associated T2 signal) is consistent with clotting time.

FIG. 25 shows 2 3D plots made from a 3D data set collected using a T2reader for (a) natural samples not loaded with Reopro and (b) samples having platelets inhibited with 8 μ g/mL Reopro ® platelets. The 3D plot shows the initial effective water population with a rapid drop in intensity. The natural sample in (a) appeared to have two distinct water populations in the time between 6.3 and 8.5 minutes. Platelet inhibition decreases the signal intensity of the clot-associated signal and shifts the serum-associated signal to a lower T2 value.

FIGS. 26A-C show the change in T2 relaxation curves for blood samples with and without different size paramagnetic agents. The samples were run without magnetic particles (see FIG. 26A), with CLIO nanoparticles (30nm size; about 0.05ng) (see FIG. 26B), and with Seramag superparamagnetic particles (730nm size; about 0.05ng) (see FIG. 26C). The addition of 30nm nanoparticles in a typical citrated kaolin experiment eliminated the clot signal (at T2=200 ms), with only 1 peak at T2 of about 100 ms. The addition of 730nm superparamagnetic particles does not hinder the ability to observe 2 peaks. See example 4.

FIGS. 27A-C show that T2MR surfaces were sensitive to whole blood fibrinolysis. To evaluate this, healthy donor samples were spiked with Tissue Plasminogen Activator (TPA). High sensitivity and fast time to fibrinolysis results were demonstrated. See example 19.

Detailed Description

The methods and devices of the invention may be used to assess the risk and occurrence of thrombotic events, including myocardial ischemic events in patients having or suspected of having vascular disease, particularly patients who have undergone percutaneous intervention and may be at acute risk, for example, for stent thrombosis, vascular restenosis, myocardial infarction or stroke. For example, the methods and devices of the invention can be used to evaluate platelet reactivity (i.e., the relative concentration of platelet-associated water molecules in the clot), coagulation kinetics, clot strength, clot stability, and time to fibrin formation (i.e., R) as an indicator of risk of thrombotic events (e.g., myocardial ischemia) independent of responsiveness to drug therapy (e.g., by changes in platelet reactivity following administration of an antiplatelet agent such as clopidogrel). These indicators can also be used to prevent complications such as stent thrombosis or restenosis resulting from surgery and percutaneous vascular procedures such as stent placement or balloon angioplasty. In addition, the methods and devices of the present invention can be employed to identify safe and effective therapies (e.g., dose, regimen, antiplatelet therapy, among others) for patients at risk of thrombotic events or undergoing surgical procedures.

Complex samples having water in more than one location or compartment (i.e., samples having more than one water population) can be monitored using the methods and devices of the invention. For example, heterogeneous samples (i.e., whole blood) have various water populations such as plasma water, compartmentalized water, i.e., cells (red blood cells, white blood cells, and platelets), and water associated with functional characteristics of whole blood processes such as coagulation (e.g., serum, clot) or clot lysis. Similarly, in samples undergoing rheological transformation, there is generally more than one population of water; for example, water associated with the gel state, water not associated with the gel state, and in some cases, water associated with a particular compartment of the sample (i.e., cellular water). The method of the invention allows for monitoring changes in a sample by simultaneously observing changes in the various water populations present in the sample. Changes may include the formation of new water populations or changes in the relative signals of existing water populations, both of which may represent the underlying physical property characteristics of the sample (e.g., ordering or disordering of structures in the sample) before, during, or after the rheological change.

Initiation of coagulation

For performing the method of the present invention, various techniques may be employed to initiate coagulation. Citrated Kaolin (CK) is a common initiator for aPTT (activated partial thromboplastin time) and whole blood clotting time. To begin the clotting process, calcium chloride and kaolin were mixed with a citrate blood sample. CK-activated samples are characterized by clot formation, where platelets and fibrin contribute to clot production. Alternatively, the activator RF may be used to initiate coagulation with or without the addition of a platelet activator (e.g., TRAP, AA, or ADP). The activated sample is characterized by clot formation, where fibrin, but not platelets, primarily contributes to clot production. Or ADP can be used to activate blood clots. ADP-activated samples are characterized by clot formation, with fibrin primarily contributing to clot production and platelets producing a lesser degree of action. The signal response observed under different activation conditions can diagnose the hemostatic condition of the subject.

Other blood coagulation activators that may be used in the methods of the invention include collagen, epinephrine, ristocetin, thrombin, calcium, tissue factor, thromboplastin, kaolin, 5-hydroxytryptamine, Platelet Activating Factor (PAF), thromboxane A2(TXA2), fibrinogen, von Willebrand factor (VFW), elastin, fibronectin (fibrinonectin), laminin, vitronectin, thrombospondin, and lanthanide ions (e.g., lanthanum, europium, ytterbium, etc.). For example, a combination of activators can be used to help identify a underlying hemostatic condition that causes a blood sample of a subject to be too low in coagulability.

Signal acquisition and processing

Standard radio frequency pulse sequences for determining nuclear resonance parameters are known in the art, for example, if the relaxation constant T is to be determined₂Carr-Purcell-Meiboom-Gill (CPMG) is traditionally used. The optimization of the sequence of rf pulses, including the selection of the frequency of the rf pulses in the sequence, the pulse power and the pulse width, depends on the system under study and is performed using procedures known in the art.

Nuclear magnetic resonance parameters obtainable with the method of the invention include, but are not limited to, T1, T2, T1/T2 promiscuous, T_1rho、T_2rhoAnd T₂ ^*. Typically, at least one of the one or more nuclear resonance parameters obtained with the method of the present invention is the spin-spin relaxation constant T2.

As with other diagnostic agents and analytical instruments, the purpose of NMR-based diagnostic agents is to extract information from a sample and communicate high-confidence results to a user. As information flows from the sample to the user, several transformations are typically performed to make the information meet the requirements of the particular user. Diagnostic information regarding the hemostatic condition of a subject may be obtained using the methods and devices of the present invention. This is achieved by processing the NMR relaxation signals into one or more series of component signals representing different populations of water molecules present in, for example, a coagulating or coagulated blood sample. For example, NMR relaxation data, such as T2, may be fitted to an exponential decay curve defined by the following equation:

　　(3)，

where f (t) is the signal strength over time t, A_iAmplitude coefficient of the i-th component (T)_iIs the decay constant of the ith component (e.g., T2).For the relaxation phenomena discussed herein, the detected signal is the sum of discrete numbers of components (i =1, 2, 3, 4 … n). This function is called exponential, bi-exponential, tri-exponential, quad-exponential or multi-exponential, respectively. Due to the extensive need in science and engineering for analyzing multi-exponential processes, there are several ways to obtain A rapidly for each coefficient_iAnd (T)_iEstablished mathematical methods of estimation. Methods that have been successfully applied and can be applied to process raw data acquired using the method of the present invention include laplace transform, algebraic solution, image analysis, nonlinear least squares (where there are many mirrors), differential methods, methods of mode functions, integration methods, moment methods, rational function approximation, padder-laplace transform, and maximum entropy method (see Istratov, a. and Vyvenko, o.f. rev. sci. inst. 70:1233 (1999)). Other methods that have been clearly demonstrated for low-field NMR include singular value decomposition (Lupu, M. and Todor, D. Chemometrics and Intelligent Laboratory Systems 29:11 (1995)) and factor analysis.

There are several software programs and algorithms available that utilize one or more of these exponential fitting methods. One of the most widely cited sources of the index fitting program are those written and provided by Stephen Provencher referred to as "discrite" and "CONTIN" (Provencher, s. w. and Vogel, r.h. math. biosci. 50:251 (1980); Provencher, s.w. comp. phys. comm. 27:213 (1982)). Discrete is an algorithm that resolves up to 9 Discrete elements in a multi-element exponential curve. CONTIN is an algorithm for analyzing a sample having a relaxation time distribution by using inverse laplace transform. Commercial applications utilizing multi-exponential analysis employ these or similar algorithms. Indeed, for some of their analyses, Bruker minispec uses the publicly available CONTIN algorithm. For the invention described herein, it is expected that the relaxation times are discrete values that are unique to each sample and not a connected distribution, and thus do not require a CONTIN-like procedure, although they may be employed. The code for many other exponential fitting methods is generally available (Istratov, a.&Vyvenko, o.f. rev. sci. inst. 70:1233 (1999)) and can be used to obtain medical diagnostic information according to the method of the present invention. Can obtainInformation is obtained on how the signal-to-noise ratio and total sampling time relate to the maximum number of terms that can be determined, the maximum resolution that can be achieved, and the range of decay constants that can be fitted. For about 10⁴Is the theoretical limit value with respect to the resolution of the measured 2 attenuation constants independent of the analysis method is>Resolution of 1.2 = (T)_i/T_i+1) (Istratov, A. A and Vyvenko, O.F. Rev. Sci. Inst. 70:1233 (1999)). It is therefore believed that the difference between resolvable attenuation constants can be measured by their magnitude, which is not entirely intuitive and differs from the resolution with optical detection. Knowledge of the maximum resolution and the dependence of the resolution on the signal-to-noise ratio will help to evaluate the performance of the fitting algorithm.

The method of the present invention may be compared to systems and devices known in the art, for example, TEG ®, ROTEM or SONOCLOT or other devices measuring rheological changes. The method of the invention may also be used in desktop NMR relaxometers, desktop time domain Systems or NMR Analyzers (e.g., ACT, Bruker, CEM Corporation, Exstrom Laboratories, Quantum Magnetics, GE Security division, Halliburton, HTS-111 Magnetics Solutions, MR Resources, NanoMR, NMR Petrophysics, Oxford Instruments, Process NMRAssociates, Qualization NMR Analyzers, SPINLOCK Magnetic Resonance Solutions or Stelar, Resonance Systems).

The CPMG pulse sequence used to collect data with a T2reader was designed to detect the intrinsic T2 relaxation time of the sample. Typically, this is specified by a value, but for samples containing complex mixing regimes (e.g. samples undergoing a coagulation process or a dissolution process) a distribution of T2 values can be observed. In this case, the signal obtained with the CPMG sequence is an exponential sum. One solution for extracting relaxation information from the output of the T2reader is to fit a sum of exponentials in a least squares fashion. In practice, this requires past information about how much function to fit. The second solution is to apply an Inverse Laplacian Transform (ILT) to solve the distribution of T2 values that make up the observed exponential signal. Furthermore, the result of the CPMG sequence s (t) is assumed to be the exponential sum:

　　(4)，

wherein A is_iIs corresponding to the relaxation time constant T2_iThe amplitude of (d). If instead of a discrete sum of exponents, the signal assumes a distribution of T2 values, the sum of the states can be expressed as:

　　(5)

this has the same functional form as the ILT:

　　(6)，

and can be processed as is. The ILT of the exponential function requires a constraint solution. Several methods that can be used to impose constraints are CONTIN, Finite Mixture Modeling (FMM), and Neural Networks (NN). The inverse laplacian transform may also be used to generate a 3D data set. The 3D data set may be generated as follows: a 3D data set is formed by collecting a time series of T2 attenuation curves and applying an inverse laplacian transform to each attenuation curve. Alternatively, an inverse 2D laplacian transform may be applied to the pre-assembled 3D data set to generate a transformed 3D data set describing the T2 temporal distribution.

In a heterogeneous environment containing two phases, several different exchange schemes may work. In such an environment with two water populations (a and b), r_aAnd r_bCorresponds to the relaxation rate of water in the two populations; f. of_aAnd f_bCorresponding to the fraction of nuclei in each phase; tau is_aAnd τ_bCorresponding to the residence time in the respective phase α = (1/τ)_a) + (1/τ_b) Corresponding to the chemical exchange rate. The switching scheme may be specified as: (1) slow exchange: if the relative relaxation rate r_aAnd r_bThe two populations are static or slowly switched, the signal containsWith two separate components, with time constant T_2aAnd T_2bAttenuation; (2) fast switching: if and r_aAnd r_bIn contrast, the rate of water molecule exchange between the two environments is fast, then the total population follows a single exponential decay, with the average relaxation rate (r) given by the weighted sum of the relaxation rates of the respective populations_av) (ii) a And (3) intermediate exchange: in general, there are two relaxation rates r₁And r₂At the slow exchange limit r_a<r_bMiddle r₁Is equal to r_a，Amp₁+ Amp₂=1, and in the fast exchange limit r_1,2Turning to the average relaxation rate, equations 7, 8, 9 and 10 may be applied:

　　(7)

　　8)

　　(9)

　　(10)

the invention also features the use of pulsed field gradients or fixed field gradients in the collection of relaxation rate data. The invention also features Diffusion Weighted Imaging (DWI) techniques as described by Vidmar et al (Vidmar et al, NMR biomed. 23: 34-40 (2010), incorporated herein by reference), or any method for porous media NMR (see, e.g., Bergman et al, Phys. Rev. E51: 3393-3400 (1995), incorporated herein by reference).

Alternatively, when the method of the invention is carried out using measurements of T2 or free induction decay rather than T2, off-resonance radiation (i.e. radiation not exactly at the larmor precession frequency) may be used to produce relaxation properties of a particular class of, for example, water protons in a sample. The output may be in the form of the height of a single echo obtained with a T2 measurement pulse sequence rather than a full echo sequence. In contrast, normal T2 measurements utilize many echoes to determine T2. The T2 method may include the steps of shifting the frequency or intensity of the applied magnetic field and measuring the width of the water proton absorption peak, where a wider peak or energy absorption is associated with a higher value of T2.

Paramagnetic agent

The method of the invention may be carried out in the presence of a paramagnetic agent (e.g. manganese, manganese complex, gadolinium complex or superparamagnetic particles) added to the blood, for example, prior to initiating coagulation. The paramagnetic agent may be free manganese, a manganese complex (e.g. EDTA complex of manganese), free gadolinium, a gadolinium complex (e.g. DTPA or DOTA complex of gadolinium) or a superparamagnetic particle. Paramagnetic agents can distinguish coagulated from unclotted samples at an earlier point in time after initiation of coagulation (see fig. 46).

Superparamagnetic particles useful in the methods of the present invention include, for example, those described in U.S. patent No. 7,564,245 and U.S. patent application publication No. 2003-0092029, each of which is incorporated herein by reference. Superparamagnetic particles are typically in the form of conjugates, that is, they are coated with a moiety that minimizes specific and non-specific binding of the particle to the components of the whole blood sample to be measured. The particles have a high relaxivity (relaxation) due to their superparamagnetism of iron, metal oxides or other ferromagnetic or ferrimagnetic nanomaterials. Iron, cobalt and nickel compounds and their alloys, rare earth elements (e.g., gadolinium) and certain intermetallic compounds (e.g., gold and vanadium) are ferromagnetic substances that can be used to produce superparamagnetic particles. The superparamagnetic particles may be monodisperse (one single crystal of magnetic material (e.g. a metal oxide, such as superparamagnetic iron oxide) per superparamagnetic particle) or polydisperse (e.g. multiple crystals per magnetic particle). The magnetic metal oxide may also comprise cobalt, magnesium, zinc or mixtures of these metals with iron. Superparamagnetic phaseThe particles typically comprise metal oxide crystals having a diameter of about 1 to 25 nm, for example about 3 to 10 nm or about 5 nm, per crystal. Superparamagnetic particles may also include a polymer component in the form of a core and/or coating, for example, about 5-20 nm or more in thickness. The total size of the superparamagnetic particles may be, for example, 20-50 nm, 50-200 nm, 100-300 nm, 250-500 nm, 400-600 nm, 500-750 nm, 700-1,200 nm, 1,000-1,500 nm or 1,500-2,000 nm. Superparamagnetic particle size can be controlled by adjusting the reaction conditions, for example by using low temperatures during neutralization of iron salts with alkali as described in us patent No. 5,262,176. The particles may also be classified using centrifugation, ultrafiltration, or gel filtration, as described, for example, in U.S. Pat. No. 5,492,814, to produce a uniform particle size material. Superparamagnetic particles may also be synthesized according to the method of Molday (Molday, R. S. and D. MacKenzie, "Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells)," J. Immunol. Methods, 52:353 (1982)), and treated with periodate to form aldehyde groups. The aldehyde-containing superparamagnetic particles may then be reacted with a diamine (e.g. ethylenediamine or hexamethylenediamine), which forms a Schiff base (Schiff base), followed by reduction with sodium borohydride or sodium cyanoborohydride. Superparamagnetic particles may be formed from a ferrofluid (i.e. a stable colloid suspension of superparamagnetic particles). For example, superparamagnetic particles may be a composite of a plurality of metal oxide crystals, of the order of tens of nanometers in size, and dispersed in a fluid containing a surfactant, which adsorbs to and stabilizes the particles, or precipitated in an alkaline medium by a solution of metal ions. Suitable ferrofluids are sold by liquid Research ltd, inc under the following reference numbers: WHKS1S9 (A, B or C), which is a water-based ferrofluid, containing magnets (Fe)₃O₄) Particles having a diameter of 10 nm; WHJS1 (A, B or C), an isoparaffinic-based ferrofluid, comprising magnets (Fe) with a diameter of 10 nm₃O₄) The particles of (a); and BKS25 dextran, which is a water-based ferrofluid stabilized with dextran, containing magnets (Fe) 9 nm in diameter₃O₄) The particles of (1). Other adaptations for the systems and methods of the inventionThe ferrofluid of (a) is an oleic acid stabilized ferrofluid available from Ademtech, which comprises about 70% by weight α -Fe₂O₃Particles (diameter about 10 nm), 15 wt% octane and 15 wt% oleic acid. Superparamagnetic particles are typically composite materials that include a plurality of metal oxide crystals and an organic matrix and have a surface modified with functional groups (i.e., amine or carboxyl groups) to attach binding moieties to the surface of the superparamagnetic particle. For example, superparamagnetic particles useful in the methods of the present invention include those commercially available from Dynal, Seradyn, Kisker, Miltenyi Biotec, Chemicell, Anvil, Biopal, Estapor, Genovis, ThermoFisher Scientific, JSR micro, Invitrogen, and Ademtech, as well as those described in U.S. patent nos. 4,101,435, 4,452,773, 5,204,457, 5,262,176, 5,424,419, 6,165,378, 6,866,838, 7,001,589, and 7,217,457 (each of which is incorporated herein by reference).

For certain assays requiring high sensitivity, analyte detection using the T2 relaxation assay may require selection of appropriate particles to enable sufficiently sensitive magnetic field-induced aggregation. Higher sensitivity can be obtained using particles containing multiple superparamagnetic iron oxide cores (5-15 nm diameter) within a single larger polymer matrix or ferrofluid assembly (particles having an overall diameter of 100 nm-1200 nm, e.g., an average diameter of 100 nm, 200 nm, 250 nm, 300 nm, 500 nm, 800 nm, or 1000 nm), or by using higher magnetic moment materials or particles having a higher density, and/or particles having a higher iron content. Without being bound by theory, it is hypothesized that these types of particles provide a sensitivity gain in excess of 100 x due to the much higher number of iron atoms per particle, which is believed to result from the decreased number of particles present in the assay solution and the potentially larger amount of superparamagnetic iron affected by the respective field-induced aggregation.

For certain assays, it may be desirable to minimize field-assisted aggregation of superparamagnetic particles by using particles with diameters less than 40 nm in the assay.

Database of characteristic curves

In one embodiment, the invention features a data processing tool that transforms raw relaxation NMR data into a format that provides a characteristic curve characteristic of a hemostatic condition. Preferred transforms include the laplacian transform or the Inverse Laplacian Transform (ILT). The data for each T2 measurement may transform the time dimension, where signal intensity is plotted against time, into the "T2 relaxation" dimension. The ILT not only provides information about the different relaxivity rates present in the sample and their relative amplitudes, but also reports the width of the signal distribution.

Each obtained T2 relaxation curve has a corresponding two-dimensional signature that is plotted against all different water populations or different T2 relaxation environments encountered by water in a sample. These curves can be compiled to form a 3D data set by stacking the plots over the duration of the clotting time dimension. This can be used to generate a 3D surface showing how different water populations change over time.

In cases where the underlying pathology is not discerned by current techniques, the T2 signature may become clinically relevant. For example, additional studies are often performed on patients with abnormal PT or aPTT values, which include PT, aPTT or PT and aPTT assays using 1:1 mixtures of patient blood and normal plasma (to exclude factor deficiency), the results may indicate specific factor deficiency or von willebrand factor deficiency. However, patients with deficiencies in coagulation factors often suffer from more than one deficiency or from a dysregulated or unrestricted coagulation cascade. In these patients, a single trial of one factor deficiency will not reveal the entire dysfunction and the clinician must rely on clinical signs (excessive bleeding or clotting), unfortunately time may lead to deleterious results. The ability to detect T2 characteristics (for patients with normal or abnormal hemostatic conditions) would allow rapid understanding of complex pathophysiological coagulation cascade conditions and improve clinical outcomes.

Data acquired using the method of the present invention can be presented using 3D maps generated from different NMR parameters. Other dimensions may be added by looking at a particular patient type or coagulation curve typeAnd (4) degree. Data reduction methods can be employed to simplify the complex information available. Techniques such as Principal Component Analysis (PCA), automated feature extraction methods, or other data processing methods may be employed. Ideally, features, 2D maps and 3D galleries may be generated for various clinical conditions. For example, two-dimensional (intensity versus T2 values or T2 values versus time) or three-dimensional images (intensity versus T2 values versus time). The T2 value for a two-dimensional or three-dimensional image may be blended using, for example, T1, T1/T2, T_1rho、T_2rhoAnd T₂ ^*And other NMR signals are substituted or compared.

In a related embodiment, the coagulation or dissolution process within the sample is evaluated by NMR parameters extracted from one or more Free Induction Decay (FID) signals obtained from the sample. For example, the NMR parameters may be extracted from the signal-to-noise ratio of the FID, from a comparison of the FID to a predetermined threshold, from an integration of the FID, or from a ratio calculation using different FID points. The NMR parameters obtained from this method can be used to characterize various coagulation or dissolution processes. In a specific example, the values extracted from one or more FIDs of a blood sample may be used to calculate blood clotting behavior, such as clotting time (R), fibrinolysis behavior, clot strength (MA), clot kinetic behavior, platelet-associated clot strength (MA)_{Blood platelet}) Functional fibrinogen-related clot strength (MA)_FF) Or percent dissolution 30 minutes after MA (LY 30). Likewise, data extracted from the FID data can be used to compile a library of characteristics.

3D data map

The method of the present invention (e.g., the method described in example 18) can be used to generate 3D images of T2 data in a sample undergoing a coagulation process or a dissolution process. In certain embodiments, the dimensions of the generated 3D map correspond to a relaxation time (e.g., T2 or 1/T2) dimension, an intensity or amplitude dimension, and a time dimension. The time dimension represents the time of a coagulation process that has been or is in progress. The 3D plots obtained from the clotting samples show various 3D surface features consistent with individual water populations in different physical and/or chemical environments within the sample. The 3D map and the indices relating to the blood sample used to generate the data of the 3D map or the clotting behavior (e.g., hematocrit, clot strength (MA), clotting time (R), platelet activity, fibrinogen activity, fibrinolysis, etc.) may be mined. The 3D map or the data used to generate the 3D data map may also be used to find new metrics.

The information contained within the different 3D surface features and water populations evident in the 3D map may be associated with specific indicators and coagulation behavior. The 3D map may be used to generate qualitative, semi-qualitative, and/or qualitative results for a particular parameter or index. For example, the hematocrit of a blood sample can be calculated from the properties of the initial water population, the clotting time and fibrinolysis behavior can be calculated from the properties of the divergent (diverging) water population having a time of 80-400 milliseconds T2, and the platelet activity and clot strength can be calculated from the properties of the second divergent water population having a time of 400-2200 milliseconds T2. Various methods may be employed to extract the indicator or coagulation behavior from the 3D data set. For example, the slope or curvature of the 3D surface features of the 3D map may be correlated to the coagulation behavior. The coagulation behavior can also be calculated using the cross-section of the 3D map. Specifically, cross sections showing time-varying T2 intensity for a particular T2 time can be used to calculate clotting time (R) and/or fibrinolysis. The cross-sections of the 3D plot showing the T2 time as a function of intensity at a given time (T2 relaxation spectrum) represent the various water populations present in the sample at a given time. Extensive solidification behavior can be mined that characterizes the T2 relaxation spectrum. For example, the difference between two signals in the T2 relaxation spectrum can be used to assess the clot intensity. Integration of specific 3D surface features (e.g., volume of a particular feature), or curves from cross-sections of the 3D map, may also be used to establish coagulation behavior (e.g., clot strength). The coagulation behavior can also be extracted by integration of extensive T2 relaxation spectra collected at sequential time points or at different time points.

Alternatively, 3D maps may be used to identify features characteristic of clot behavior. The characteristic may be one that is not measured by 3D analysis, for example, by selectively monitoring a pulse sequence of a water population having an average T2 relaxation rate of about 400 milliseconds or 1,000 milliseconds at a particular time after initiation of coagulation. Optionally, the water population in the sample is measured to the exclusion of other water populations.

The characteristics of the T2 relaxation curve, including the range of T2 values associated with a particular signal, may vary with the instrument used to collect the data (e.g., a T2reader or Bruker minispec). Likewise, the range of T2 values for a given sample may depend on the material (e.g., plastic or glass) used to construct the tube containing the sample during the T2 measurement. The invention includes the use of any magnetic resonance instrument and any sample container in acquiring a 3D data set for blood sample analysis.

Management of patients

The methods and devices of the present invention can be used to provide a point-of-care assessment of a patient's hemostatic condition (e.g., for coagulation management of a patient undergoing surgery, identifying a patient at risk for thrombotic complications, identifying a patient resistant to anti-platelet therapy, monitoring a patient for anti-platelet therapy, and/or monitoring a patient for procoagulant therapy).

There are medical conditions for which a coagulation test is required, including: 1) finding the cause of abnormal bleeding or bruising, 2) in patients with autoimmune disease, 3) in patients with underlying cardiovascular disorders, 4) prior to surgery or surgery where bleeding too much may be a concern, 5) monitoring anticoagulant therapy, 6) monitoring perioperative and trauma patients, and 7) identifying patients with sepsis or septic shock.

Coagulation management in patients undergoing cardiac surgery is complicated by the balance between anticoagulation by cardiopulmonary bypass (CPB) and hemostasis after CPB. In addition, more and more patients suffer from impaired baseline platelet function due to the administration of antiplatelet drugs. During CPB, optimal anticoagulation indicates that coagulation is antagonized and platelets are protected from activation, rendering clot formation impossible. After surgery, coagulation abnormalities, platelet dysfunction and fibrinolysis may occur, creating a situation where hemostatic integrity must be restored. The complex processes of anticoagulation with heparin, antagonism with protamine and postoperative hemostasis can be guided by the method and apparatus of the present invention (on-site point-of-care test) which evaluates hemostatic function in a timely and accurate manner.

Problems associated with poor liver function (e.g., reduced coagulation factor synthesis and clearance and platelet defects) can lead to impaired hemostasis and hyperfibrinolysis. Systemic complications, such as sepsis and disseminated intravascular coagulation, further complicate pre-existing coagulopathies. In the liver-free stage and right after organ reperfusion, there is a clear change in hemostasis in orthotopic liver transplantation, mainly hyperfibrinolysis, resulting from tissue plasminogen activator accumulation due to insufficient liver clearance and release of exogenous heparin and endogenous heparin-like substances. Thus, patients undergoing liver surgery, particularly orthotopic liver transplantation, may develop large disorders in their coagulation, making the methods and devices of the present invention useful for monitoring this patient population.

The methods and devices of the invention can be used to guide heparin therapy, particularly anticoagulant therapy. For example, the method of the invention can be performed with heparinase to assess the coagulation status without anticoagulation by heparin. In addition, the methods of the invention can be used to evaluate protamine therapy, i.e., to monitor coagulation after protamine therapy and to treat heparin or protamine induced hemostatic conditions. Similarly, analysis can be performed pre-and post-operatively to determine the anticoagulation status or hemostasis status of the surgical patient.

The methods and devices of the present invention may also be used to guide anti-platelet therapy and identify resistance to anti-platelet therapy. Antiplatelet therapies are increasingly being prescribed for primary and secondary prevention of cardiovascular disease to reduce the incidence of acute cerebrovascular and cardiovascular events. Antiplatelet drugs are often targeted to inhibit cyclooxygenase 1/thromboxane a2 receptors (e.g., aspirin), adenosine diphosphate receptors (e.g., clopidogrel), or GPIIb/IIIa receptors (e.g., abciximab, tirofiban). Although anti-platelet drugs are thought to act primarily by reducing platelet aggregation, they have also been shown to act as anticoagulants. Since platelets play a critical role in whole body clotting, the assessment of platelet function (beyond their number) is crucial in the perioperative setting.

The methods and devices of the present invention may also be used to monitor and/or guide anticoagulant therapy. Anticoagulant therapy (such as rivaroxaban, dabigatran, among others) may be monitored for efficacy and compliance and to ensure that adverse side effects and/or adverse events (such as bleeding events) are avoided. Dose modulation of such therapies to control bleeding has been reported in a number of randomized studies. In particular, administration of anticoagulants, including direct factor Xa inhibitors, can be used to aid in the maintenance of the therapeutic window and result in a reduced risk of stroke in patients with atrial fibrillation and deep vein thrombosis.

The methods and devices of the invention can be used to identify patients who are resistant to anti-coagulation therapy. Anticoagulant therapy includes aspirin, boli and prasugrel, especially anticoagulants. The method comprises (i) administering anticoagulant therapy to a subject; (ii) evaluating the hemostatic condition of the subject using the methods of the invention; and (iii) identifying the subject as a non-responder to the anticoagulation therapy if the subject is found to be thrombogenic. Identification of non-responders may allow physicians to identify safe and effective anticoagulants to which patients respond, thus reducing the risk of adverse events (i.e., thrombosis and stroke).

Procoagulant therapy may be monitored using the methods and devices of the present invention. Modern practice of coagulation management is based on the concept of specific component therapy and requires rapid diagnosis and monitoring of procoagulant therapy. For example, platelet infusion during perioperative coronary artery bypass surgery has been shown to be associated with an increased risk of serious adverse events. Clinical judgment alone cannot predict who would benefit from platelet infusion in an acute perioperative setting. Thus, infusion of the clotting product should preferably be directed by a point-of-care test (e.g., the test provided by the methods and devices of the present invention).

The methods and devices of the invention can be used to provide companion diagnostic assays or tests to monitor the effects of therapeutic compounds in clinical or medical applications. Diagnostic analysis can include determining whether a test subject or patient is responding or not responding to therapy.

Perfusion by blood clots, hypercoagulability, or coagulation that are harmful in people such as stroke or cardiac arrest may be determined using the methods and apparatus of the present invention.

The method and apparatus of the present invention may be employed as part of a set of analyses. The set of assays may include (i) immunoassays for proteins involved in the coagulation cascade; (ii) immunoassays for detecting fibrin degradation products; (iii) an immunoassay to detect anti-phospholipid antibodies; (iv) assays that test for heparin or warfarin or other anticoagulants to assess therapeutic concentrations; (v) a PT or aPTT or PTT assay to monitor plasma prothrombin time; (vi) genetic test to evaluate the polymorphic differences of genes encoding proteins related to: (a) thrombin formation or dissolution, (b) the coagulation cascade, (c) heparin binding or (d) therapeutic activity.

Medical devices having an effect on coagulopathy may be administered using the methods and devices of the present invention. One example is ventricular assist devices that are often used as a bridge for patients awaiting heart transplantation. Because of the function of the device, patients carrying such implants may have blood clot formation inside and outside the device due to the function of the device, and these blood clots may cause a stroke or other thrombus related events. Infection and bleeding episodes may also result. One way to avoid these problems is to monitor a number of diagnostic markers that affect the success of the device. For example, routine testing of PT-INR may allow for closer monitoring of a patient's coagulation status, thus providing tight control over bleeding and coagulation events.

INR is the ratio of prothrombin time of the patient to the normal (control) sample, raised to the power of the international sensitivity index value of the assay system used. A high INR level (e.g., INR =5) indicates a high chance of bleeding, whereas if INR =0.5, the chance of having a clot is high. For healthy humans the normal INR ranges from 0.9 to 1.3. For people receiving warfarin therapy, INR ranges typically from 2.0 to 3.0. The target INR may be higher in special cases, such as where the warfarin is bridged with a low molecular weight heparin (e.g., enoxaparin (Lovenox)) for humans with mechanical heart valves or perioperative.

Monitoring platelet function, fibrinolysis, clot strength, and other factors are also important in improving outcomes. Understanding the physiological concentration or activity of these factors is not only important for their interaction with the devices, but because they are regulated by many different therapies (particularly aspirin, rivaroxaban, borrelid, warfarin) often prescribed to patients on these devices. Another metric used with these types of devices is hematocrit, which is often used to adjust the function of the device (speed, strength, etc.) to maintain the function of the heart. The methods and devices of the present invention will likely provide all of these results (hematocrit, platelet, PT-INR, etc.) simultaneously, and may provide additional information about clot formation and lysis. The above-mentioned standard metrics may be incorporated into an index or characteristic that identifies the status of the patient and the efficacy of the device.

The method and apparatus of the present invention can be utilized and configured in a variety of ways. They may be used as laboratory devices, point-of-care systems or even implantable monitoring systems. For example, as an implantable monitoring system, the sample may consist of blood that is continuously monitored; a vacuum blood collection tube (vacutainer) containing whole blood, serum or plasma; or finger lancets (finger sticks) and other sample fluids.

For example, the methods and devices of the invention may be used to monitor perioperative and trauma patients (e.g., to provide measures or surrogate measures of PT/INR, aPTT, ACT, Hct, platelet activity, and fibrinolysis). These patient populations need a quick and efficient determination of the need for transfusion, as patients may exhibit about a 6-fold increase in mortality, ischemic events, infections, early onset complications, and ICU/hospitalization prolongation. In particular, the determination of the root cause of bleeding events (coagulation cascade and platelet activation) can lead to rapid and focused therapies.

Regardless of the situation in which the methods and devices of the present invention are used, small volumes can be rapidly measured using the methods of the present invention, which is particularly important for platelet function, which has previously been difficult to measure using other systems because clotting is initiated at the site of blood draw.

Mechanism of blood coagulation

In order for coagulation to occur, there must be activation of the coagulation cascade that peaks in fibrin deposition through the action of thrombin on fibrinogen. The coagulation system consists of a proteolytic cascade that amplifies the initial stimuli with a delicate feedback regulatory mechanism to keep the entire process in control and equilibrium. There are two interconnected pathways of coagulation activation: (i) contact activation (internal pathway); and (ii) tissue factor activation (external pathway). Both pathways rely on various coagulation factors. Prothrombin is factor II, thrombin is factor IIa, fibrinogen is factor I, and fibrin is factor Ia. In addition to coagulation factors, platelets are crucial for both induction and formation of adequate blood clots. Platelets act as phospholipid surfaces on which prothrombinase complexes are formed and act as physical scaffolds for the generation of blood clots.

The internal coagulation cascade is usually activated by contact with collagen from damaged blood vessels, but many negatively charged surfaces can stimulate this pathway. The internal pathway usually requires platelet activation to assemble the tenase complex including factors VIIIa, IXa and X. The activation process is linked to the inositol triphosphate (IP3) pathway and involves degranulation and myosin 1c kinase activation to alter platelet shape, ultimately allowing adhesion.

Alternatively, coagulation can be activated by an external coagulation cascade, which requires tissue factor from the surface of extravascular cells. The external pathway involves complex formation of coagulation factor V, VII and X. The major inducer of in vivo coagulation is Tissue Factor (TF), a 47 kDa glycoprotein. The only cells in the bloodstream that are capable of expressing TF are endothelial cells and monocytes. In contrast, many cells outside the blood stream (including adventitial fibroblasts) constitutively express TF, thus forming an "extravascular membrane" capable of initiating coagulation in the event of a disruption in vascular integrity.

The final stage of the cascade is common in both pathways, including the tenase complex, the activation complex. Tenase is a "ten" and suffix "enzyme"Indicates that the complex is activated by cleavage of its substrate (inactive factor X). The internal tenase complex contains activating factor ix (ixa), its cofactor factor viii (viiia), the substrate (factor X), and they are activated by negatively charged surfaces (e.g. glass, activated platelet membranes, sometimes the cell membranes of monocytes). The external tenase complex consists of tissue factor, factor VII, a substrate (factor X) and Ca as the activating ion²⁺And (4) forming.

Factor X is activated to factor Xa through either an external or internal pathway, resulting in proteolytic conversion of prothrombin to thrombin, which in turn is activated to initiate clot formation. Factor VIII then catalyzes the transglutaminase reaction to crosslink fibrin monomers into a cross-linked network.

Crosslinked fibrin polymers in blood clots are broken down by plasmin (a serine protease) into soluble polypeptides. Plasmin can be produced from its inactive precursor plasminogen and recruited to the site of a fibrin clot in two ways: by interacting with tissue plasminogen activator on the surface of fibrin clots, and by interacting with urokinase plasminogen activator on the surface of cells. The first mechanism appears to be the main mechanism responsible for intravascular clot lysis. The second mechanism, while capable of mediating clot lysis, can generally play a major role in tissue remodeling, cell migration, and inflammation.

Clot dissolution is regulated in two ways. First, efficient plasmin activation and fibrinolysis occur only in complexes formed on the surface of the clot or on the cell membrane; free proteins in the blood are inefficient catalysts and are rapidly deactivated. Second, both plasminogen activator and plasmin themselves are inactivated by specific serine protease inhibitory proteins (proteins that bind to serine proteases to form stable, enzymatically inactive complexes). Pharmacologically, Tissue Plasminogen Activator (TPA) and streptokinase or urokinase are used in the clot (clot buster) to activate this internal fibrinolytic mechanism.

Medical conditions

The methods and devices of the invention described herein can be used to detect rheological changes in various fluids, particularly blood samples, to diagnose coagulation, thrombotic disorders, and thrombotic disorders due to: such as sepsis and Disseminated Intravascular Coagulation (DIC), hemophilia a, hemophilia B, hemophilia C, congenital deficiencies of other coagulation factors, factor XIII deficiency, Von Willebrand's disease, internal anticoagulant-induced bleeding disorders, defibrination syndrome, acquired coagulation factor deficiency, other coagulation defects, purpura and other bleeding conditions, allergic purpura, noch-schennemia purpura (Henoch-Sch nlein purpura), thrombocytopenia, immune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, secondary thrombocytopenia, and nonspecific bleeding conditions.

The cardiovascular system requires tight regulation of hemostasis. Excessive coagulation can cause venous or arterial obstruction, while failure to coagulate can cause excessive bleeding; both conditions lead to adverse clinical conditions. In most human subjects, the coagulation balance is more or less static. However, there are many different clinical parameters (e.g. genetic disorders, disease states, therapeutic drugs or pharmacological stressors) that can alter hemostasis and lead to cardiovascular dysfunction.

There are many different known coagulation disorders caused by non-functional coagulation factors, such as hemophilia (factor VIII (hemophilia a), IX (hemophilia B), XI (hemophilia C)), Alexander disease (Alexander disease) (factor VII deficiency), prothrombin deficiency (factor II deficiency), olon's disease (Owren's disease) (factor V deficiency), Stuart-power deficiency (factor X deficiency), heuman factor deficiency (Hageman factor deficiency) (factor XII deficiency), fibrinogen deficiency (factor I deficiency) and von willebrand disease.

Activation of the coagulation cascade appears to be an essential component in the development of multiple organ failure that occurs in terminal sepsis. Current therapies for sepsis specifically target these cascades to regulate the progression of the terminal phase and prevent organ failure.

The method and apparatus of the present invention can be used to determine the hematocrit of a blood sample. Hematocrit is a measure of the percentage of the volume occupied by red blood cells in a subject's blood, with normal values for healthy women and men being about 36-44% and 41-50%, respectively. Hematocrit depends on the number of red blood cells in the sample and the size of the red blood cells. Measurements of hematocrit can be used to determine various physiological conditions of a subject. Thus, the methods of the invention can be used to diagnose any condition associated with a sub-normal hematocrit or a higher than normal hematocrit. A lower than normal hematocrit may indicate anemia, sickle cell anemia, internal bleeding, loss of red blood cells, malnutrition (e.g., iron, vitamin B12, or folate deficiency), or water toxicity. Higher than normal hematocrit may indicate congenital heart disease, dehydration, polycythemia, pulmonary fibrosis, polycythemia vera, or abuse of the drug erythropoietin.

The methods of the invention are useful for monitoring factors and associated coagulopathies associated with the following diseases, conditions or dysfunctions and risk factors: diseases, disorders or dysfunctions such as cancer, autoimmune disorders, lupus erythematosus, Crohn's disease, multiple sclerosis, amyotrophic lateral sclerosis, deep vein or artery thrombosis, obesity, rheumatoid arthritis, Alzheimer's disease, diabetes, cardiovascular disease, congestive heart failure, myocardial infarction, coronary artery disease, endocarditis, stroke, emboli, pneumonia, ulcerative colitis, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, infection, transplant recipient, liver disease, hepatitis, pancreatic disease and disorder, renal disease and disorder, endocrine disease and disorder, obesity, disease or disorder associated with thrombocytopenia, and medical (stents, implants, major surgery, joint replacement, pregnancy) or therapeutic (cancer chemotherapy) induced coagulopathy; risk factors such as high smoking, high alcohol consumption, sedentary lifestyle. The methods of the invention can also be used to evaluate genomic and proteomic changes that affect coagulation and blood properties.

The methods of the invention may also be used to monitor patients undergoing anticoagulant therapy and/or antiplatelet therapy. Examples of antithrombotic agents that can be monitored using the methods of the invention (e.g., thrombolytic, anticoagulant, and antiplatelet agents) include, without limitation, vitamin K antagonists such as acetocoumarin (acenocomarol), clidanedione, dicoumarin, benzindenone, coumarone acetate, coumarine, phenindedione, thiocoumarin, and warfarin; heparins (platelet aggregation inhibitors), such as antithrombin III, bemiparin, dalteparin, danaparoid, enoxaparin, heparin, nadroparin, parnaparin, heparin, sulodexide and tinzaparin; other platelet aggregation inhibitors, such as abciximab, acetylsalicylic acid (aspirin), alopril, beraprost, ditozole, carbapenem calcium, clocrolimus, clopidogrel, dipyridamole, epoprostenol, eptifibatide, indobufen, iloprost, picotamide, prasugrel, ticlopidine, tirofiban, treprostinil, and triflusal; enzymes such as alteplase, ancrod, anistreplase, plasmin, C protein (procein C), reteplase, sareprunose, streptokinase, tenecteplase and urokinase; direct thrombin inhibitors such as argatroban, bivalirudin, desipramine, lepirudin, melagatran and ximelagatran; other antithrombotic agents such as dabigatran, defibrotide, dermatan sulphate, fondaparinux and rivaroxaban; and others such as citrate, EDTA and oxalate.

Sepsis and disseminated intravascular coagulation

The methods and devices of the invention can be used to evaluate the hemostatic condition of a subject suffering from sepsis or disseminated intravascular coagulation.

In sepsis, an overwhelming inflammatory response causes extensive collateral damage to the host microcirculation. Damage to the endothelium exposes tissue factor, which can occur on a large scale in sepsis. The tissue factor in turn binds to activated factor VII. The resulting complex activates factors IX and X. Factor X converts prothrombin to thrombin, which cleaves fibrinogen to fibrin, inducing clot formation. At the same time, the fibrinolytic system is inhibited. Cytokines and thrombin stimulate the release of plasminogen activator inhibitor-1 (PAI-1) from platelets and endothelium. When formed in the human body, blood clots are ultimately broken down by plasmin activated by Tissue Plasminogen Activator (TPA). PAI-1 inhibits TPA. Thus, subjects with severe sepsis are treated with anticoagulants such as protein C (blood coagulation factor XIV).

Disseminated Intravascular Coagulation (DIC) is a complex systemic thrombotic hemorrhagic condition involving the formation of intravascular fibrin and the consumption of procoagulants and platelets. The resulting clinical condition is characterized by intravascular coagulation and bleeding. DIC is not a disease itself, but is the result of complications or other disease processes, estimated to be present in up to 1% of hospitalized patients. DIC is always secondary to the underlying disorder and is associated with a number of clinical conditions, generally involving activation of systemic inflammation. DIC has several components in agreement, including activation of intravascular coagulation, depletion of coagulation factors, and end organ damage. DIC is most commonly observed in severe sepsis and septic shock. Indeed, the development and severity of DIC correlates with mortality from severe sepsis. While bacteremia (including both gram-positive and gram-negative organisms) is most commonly associated with DIC, other infections, including viral, fungal and parasitic infections, can also cause DIC. Trauma, particularly nerve trauma, is also frequently associated with DIC. DIC is more commonly observed in trauma patients with systemic inflammatory response syndrome. There is evidence that inflammatory cytokines play a major role in DIC in both trauma and septic patients. In fact, the systemic cytokine profile is nearly identical in both septic and trauma patients.

DIC exists in both acute and chronic forms. DIC occurs acutely and intravascular coagulation occurs when sudden exposure of blood to procoagulants, including tissue factor (tissue thromboplastin), occurs. Compensatory haemostatic mechanisms rapidly dominate, and therefore, severe consumption coagulopathy leading to bleeding occurs. Abnormalities in blood coagulation parameters are easily identified and organ failure frequently occurs in acute DIC. In contrast, chronic DIC reflects a compensatory state that occurs when blood is continuously or intermittently exposed to small amounts of tissue factor. In chronic DIC, compensatory mechanisms in the liver and bone marrow are not overwhelming and may have few obvious clinical or laboratory indications of DIC presence. Chronic DIC is more commonly observed in solid tumors and large aortic tumors.

Tissue factor exposure to the circulation occurs through endothelial destruction, tissue damage or inflammatory or tumor cell expression of procoagulant molecules, including tissue factor. Tissue factor activates coagulation through an external pathway involving factor VIIa. Factor VIIa has been implicated as an important mediator of intravascular coagulation in sepsis. Blocking the factor VIIa pathway in sepsis is shown to prevent the occurrence of DIC, while interrupting the alternative pathway does not show any effect on coagulation. The tissue factor-VIIa complex thus provides for the activation of thrombin, which in turn cleaves fibrinogen to fibrin, causing platelet aggregation. Evidence suggests that internal (or contact) pathways are also activated in DIC, which produce more of a promoting effect on hemodynamic instability and hypotension than on activation of coagulation.

Decreased levels of antithrombin are associated with increased mortality in septic patients. Thrombin generation is often tightly regulated by a number of hemostatic mechanisms. Antithrombin function is one such mechanism responsible for regulating thrombin levels. However, due to a number of factors, antithrombin activity is reduced in septic patients. First, antithrombin is continuously consumed by the constant activation of coagulation. In addition, elastase produced by activated neutrophils degrades antithrombin and other proteins. More antithrombin is lost due to capillary leakage. Finally, antithrombin production is impaired, with subsequent liver damage resulting from perfusion and microvascular coagulation.

Depletion of Tissue Factor Pathway Inhibitor (TFPI) is evidence for DIC subjects. TFPI inhibits the tissue factor-VIIa complex. Although TFPI levels are normal in septic patients, a relative deficit in DIC is evident. Depletion of TFPI in animal models predisposes them to DIC, and TFPI blocks the procoagulant effects of endotoxin in humans. Intravascular fibrin produced by thrombin is typically cleared via a process known as fibrinolysis. An initial response to inflammation showing an increase in fibrinolysis; however, this reaction is soon reversed, because inhibitors of fibrinolysis are released. High levels of PAI-1 precede DIC and are indicative of poor clinical outcome. Fibrinolysis is not synchronized with increased fibrin formation, eventually leading to under-exposed fibrin deposition in the vascular system.

Protein C, as well as protein S, play a role in anticoagulant compensatory mechanisms. Under normal conditions, protein C is activated by thrombin and forms a complex with thrombomodulin on the endothelial cell surface. Activated protein C opposes coagulation by proteolytic cleavage of factors Va and VIIIa. However, cytokines produced in sepsis and other systemic inflammatory states, such as tumor necrosis factor alpha (TNF- α) and interleukin 1 (IL-1), primarily incapacitate the protein C pathway. Inflammatory cytokines down-regulate thrombomodulin expression on the surface of endothelial cells. The C protein levels are further reduced by depletion, extravascular leakage, reduced hepatic production and by reduction of free circulating S protein.

The inflammatory and coagulation pathways interact in a substantial manner. Many of the activated coagulation factors produced in DIC contribute to the spread of inflammation by stimulating the endothelial cell release of pro-inflammatory cytokines. Factor Xa, thrombin and tissue factor-VIIa complex are each shown to induce proinflammatory effects. Furthermore, given the anti-inflammatory effects of activated protein C, its damage in DIC further contributes to dysregulation of inflammation.

The components of DIC include: exposing blood to a procoagulant substance; fibrin deposition in the microvasculature; impaired fibrinolysis; coagulation factors and platelet depletion (wasting coagulopathy); organ damage and failure. DIC can occur in 30-50% of septic patients.

The methods and devices of the invention can be used to monitor subjects suffering from various DIC-related conditions, such as: sepsis/severe infection; trauma (nerve trauma); organ destruction; malignancies (solid malignancies and myeloproliferative malignancies); severe transfusion reaction; rheumatic diseases; obstetric complications (amniotic fluid embolism, placenta early detachment, hemolysis, retention dead fetus syndrome); vascular abnormalities (ka-mei syndrome, aneurysms); liver failure; toxic reactions, transfusion reactions and graft rejection. Similarly, the present invention may be used in subjects characterized by a hemostatic condition with acute DIC associated with: bacterial infections (e.g. gram negative sepsis, gram positive infection or rickettsia), viral infections (e.g. infections associated with HIV, cytomegalovirus, varicella or hepatitis), fungal infections, parasitic infections (e.g. malaria), malignancies (e.g. acute myeloid leukemia), obstetric conditions (e.g. eclampsia placental premature peeling or amniotic fluid embolism), wounds, burns, infusions, haemolysis or transplant rejection.

The NMR-based methods of the invention can be used to monitor any and all of the above blood-related conditions. Time domain relaxation measurements (relaxometric), in particular T2 relaxation measurements, can be used to measure changes in the coagulation status of a sample. This measurement relies on measuring the NMR parameters of hydrogen nuclei that are sensitive to changes in the macroscopic coagulation state of the sample. Most of the hydrogen nuclei are in the bulk (bulk) aqueous solvent, but a significant portion are in the biological macromolecules and cells and platelets of the sample. Thus, measurements of the average NMR signal from all hydrogen nuclei can be made such that when the coagulation state of the sample changes for any of the above clinical reasons, the signal changes in a considerable manner. The NMR measurement may be a T2 relaxation measurement, or a "valid" T2 relaxation measurement (e.g., a T2 relaxation measurement in which the parameters of the signal acquisition are such that they are set for an optimal readout of the coagulation event, rather than the most accurate measurement of the T2 relaxation value). Other "time domain" relaxation measurement methods may be applied to measure changes in coagulation behavior. These may include, among other measurements, time domain free induction decay analysis. Any of the NMR time domain measurements described herein can be acquired in a repetitive manner to obtain a dynamic readout of the NMR signal over time due to changes in the coagulation or dissolution properties of the sample.

Subjects with normal and abnormal hemostasis profiles

The methods of the invention can be used to distinguish subjects having normal and abnormal hemostasis profiles. For example, NMR relaxation parameter values and/or T2 characteristics characteristic of normal and abnormal hemostasis profiles can be determined and used for differential diagnosis of subjects. The abnormal hemostasis profile can include a profile of subjects sharing a common deficiency of one or more of: coagulation factors, coagulation cofactors and/or regulatory proteins (such as, inter alia, factor XII, factor XI, factor IX, factor VII, factor X, factor II, factor VIII, factor V, factor III (tissue factor), fibrinogen, factor I, factor XIII, von Willebrand factor, protein C, protein S, thrombomodulin and antithrombin III).

A deficiency in antithrombin is found in about 2% of patients with venous thromboembolic disease. Inheritance occurs as an autosomal dominant trait. The incidence of symptomatic antithrombin deficiency in the general population ranges from 1/2000-1/5000. Deficiencies are caused by mutations affecting the synthesis or stability of antithrombin or mutations affecting the protease and/or heparin binding sites of antithrombin. The methods of the invention can be used to distinguish between normal subjects and subjects with antithrombin deficiency.

Deficiency of factor XI confers a bleeding tendency associated with injury. This deficiency, originally called hemophilia C, was identified in 1953. Factor XI deficiency is very common in jewish people of the german line and is inherited as an autosomal disorder with homozygosity or heterozygosity of compounds. The methods of the invention can be used to distinguish between normal subjects and subjects with factor XI deficiency.

Von willebrand disease (vWD) results from a genetic von willebrand factor (vWF) deficiency. vWD is the most common hereditary bleeding disorder in humans. The lack of vWF leads to a defect in platelet adhesion and causes a secondary factor VIII deficiency. The result is vWF deficiency which can cause bleeding that appears similar to bleeding caused by platelet dysfunction or hemophilia. vWD is a very heterogeneous disease, which has been classified into several major subtypes. vWD type 1 is most common and inherited as an autosomal dominant trait. This variant is due to the simple lack of number of all vWF multimers. Type 2 vWD is further subdivided according to whether dysfunctional proteins have reduced or abnormally increased function in certain laboratory tests of platelet binding. Type 3 vWD is clinically severe and is characterized by recessive inheritance and an actual lack of vWF. The methods of the invention can be used to distinguish between normal subjects and subjects suffering from von willebrand factor deficiency.

Several cardiovascular risk factors are associated with abnormal conditions of fibrinogen. Elevated plasma fibrinogen levels are observed in patients with coronary artery disease, diabetes, hypertension, peripheral artery disease, hyperlipoproteinemia, and hypertriglyceridemia. In addition, pregnancy, menopause, hypercholesterolemia, use of oral contraceptives, and smoking result in elevated plasma fibrinogen levels. There are hereditary fibrinogen disorders including achromboemia (complete absence of fibrinogen), hypofibrinogenemia (reduced fibrinogen levels) and abnormal fibrinogen disease (presence of fibrinogen dysfunction). The fibrinogenemia is characterized by bleeding of the umbilical cord of the newborn, ecchymosis, mucosal bleeding, internal bleeding and habitual abortion. The disorder is inherited in an autosomal recessive manner. Hypofibrinogenemia is characterized by fibrinogen levels below 100mg/dL (normally 250-350mg/dL) and may be acquired or inherited. The methods of the invention can be used to distinguish between normal subjects and subjects with fibrinogen abnormalities.

Platelet monitoring

The methods and devices of the invention can be used to determine platelet function and compare with platelet aggregometry (see, e.g., Harris et al, Thrombosis Research 120:323 (2007)). There are currently two detection methods used in FDA-licensed instruments for platelet aggregometry: optical measurements and impedance measurements. For example, the methods of the invention can be used to identify any platelet activity or diagnose any platelet dysfunction in a subject that can be measured by a platelet aggregometry assay. Platelet aggregometry is a functional test performed in whole blood or platelet rich plasma samples. In general, platelet aggregometry methods involve adding a platelet activator to a sample and measuring the induced platelet aggregation. Platelet aggregometry can be determined by dipping the electrodes into a blood sample to be tested. Platelets adhering to the probe form a stable monolayer. Upon addition of the activator, platelet aggregates form on the electrodes and increase the impedance of the current applied across the electrodes. The instrument monitors changes in electrical impedance that reflect the platelet aggregation response. Aggregometry methods also include techniques based on the release of ATP from the aggregating platelets monitored by luminescence. Optical detection of platelet aggregation is based on the observation that there is an increase in light transmittance as platelets aggregate into large blood clots. Different aggregation inducers stimulate different activation pathways and different aggregation patterns are observed. The major drawback of optical methods is that they are typically performed on PRP, requiring separation of platelets from red blood cells and adjustment of platelet counts to nominal values.

In platelet aggregometry, the methods of the invention can be used to evaluate the platelet count of a subject's blood sample or to diagnose a condition of thrombocytopenia (platelet count <150,000/μ L) or thrombocytosis (platelet count >400,000/μ L) in a subject. Such a diagnosis may be used as a basis for deciding to provide a subject with platelet infusion or anticoagulant. Similarly, the methods of the invention can be used to evaluate a subject's response to platelet infusion or an anticoagulant.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are made, prepared, and evaluated, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Other uses

The NMR-based methods of the invention described herein can be used in a variety of applications where a substance or mixture of substances is undergoing or has undergone a solidification process or a dissolution process. The method of the invention can be used to obtain information about changes in the material undergoing a process or about the state of the material that has undergone a process.

In the field of petroleum products, the method of the invention can be used to monitor asphalt/bitumen, bitumen polymer production, boilers, crude oil, coal ash, coal slurry, cracking, distillers bottoms, engineering, diluent-added bitumen, high viscosity oils, furnace oil, lubricants, fuel blends, oil additives, lube clarifiers, blend oils, oil counters (pipeline terminals), oil attrition control, plastisol, mineral oil, petroleum additive production, petroleum products, pipeline counters, asphalt coatings, asphalt dilution, Erika failure pumping, quench oil, residue conversion in fuel, separation of water, sediment and oil, specialty oils, synthetic rubber, tar control prior to use, extra heavy oil, and water coal slurry.

In the paint and paint field, the method of the invention can be used for monitoring automotive paints, metallic paints, water-borne paints, specialty inks for scraping games, specialty inks for aluminum or plastic surfaces, water-borne inks, PTFE coatings, white paper coatings, specialty paper coatings, wallpaper, glue, varnish, automotive varnish, specialty paints for engines, ink production, gravure printing, dyes, printed circuit board varnish, magnetic inks, magnetic varnishes, gloss coatings, silver plating for mirrors, specialty varnishes for glasses and enamel powders.

In the food and beverage area, the method of the invention can be used to monitor white sauce production, bread production, chocolate production, dough control, fermentation control, fish sauce (evaporation control), fresh cheese production, gelatin food consistency, ice cream production, jam production, margarine production, mayonnaise production, melted cheese production, milk and cheese research, paraffin coating control, protein concentration control, protein for animal food, alginate gelatin, waste oil control, candied fruit, pan cooker (crystallization control), sugar mixer, surimi, aroma synthesis, tomato sauce, vegetable butter and oil, yeast, yogurt, beer/yeast control, bakery dough, food additives, gelatin (protein concentration), milk atomization, yogurt, processed cheese, sweetened juice, salad dressing, food thickeners, food additives, and the like, Enzyme concentration control, chilled hydraulic control, artificial food flavor, tobacco pulp, residual syrup, industrial soup (soup), pudding, milk powder, pet food, livestock food, baby food, condensed milk, starch gel, fruit puree, and fruit juice.

In the field of industrial chemistry, the method of the invention can be used to monitor base resins, polymers, polymer-asphalt production, polymerization control, polycarbonates, PVC production, two-component resins, fibers and polymers, cable resins, epoxy resins, polyamide resins, chloroaldehyde methyl resins, PVC, carboxymethylcellulose, hydrochloric acid, urethane gums, tolyl diisocyanate, MEK toluene, plastic recycling, silicone oils, pastes, gums, PBU, ethanol toluene, polycarbonates, polyester resin production, polyether polyol control, polyisobutylene, polymer resin production, polymerized vinyl + toluene, polymerization industry, resin polymerization, silicone oils, unsaturated polyester resins, urea-formaldehyde resins, gums, polyamide resins, nylons, polypropylene resins, polyethylenes, epoxy resins, polyepthine waxes, dimethyl acetate, polyethylene glycol, Phenolic resin, gypsum, melamine and methyl methacrylate.

The method of the invention can also be used for monitoring biochemical products, cellulose acetate, fabric softeners, enzymes, gel coats, pharmaceutical capsules, aerosols, chemical production (laundry base), cosmetic production and control, creams, cosmetic machine engineering, fermentation control, glasses for glasses, pharmaceuticals, photographic emulsions, shampoo production, toothpaste, uv-sensitive varnishes, emulsion viscosity control, vitamin a, photographic emulsions, videotapes, gels, emulsions, fine chemistry, fluorescent pastes for lighting, hydraulic oils, emulsion atomization, uv-gel, thermosol, drilling mud, plastisol, acid concentration, mercury, battery acid, detergents, ceramics, slurries, glues, viscous polymers, calcium carbonate, acrylic glues, lime milk, ammonia + MCB + oil, high viscosity fuels, crude oil counts, mixing of two oils, lubricating oils, animal fat boilers, Fuel oil, wastewater concentration, slurry concentration, yeast sludge, oil contamination, solvent contamination, oil distress control, quench oil, cutting oil, and processes involving settling towers.

The methods and devices of the invention are useful as lead compounds and compound validation discovery tools. The methods and devices of the invention can identify changes in the coagulation cascade that vary as a function of intervention in the coagulation cascade by one or more candidate compounds, or changes outside the coagulation cascade in response to a candidate compound (e.g., platelet morphology). The methods and devices of the invention can be used to screen compound libraries to identify active agents, and to pinpoint new mechanisms for disease and therapy. These may be in the coagulation cascade, but many are targets that are not ordinarily defined or identified in the cascade. The method can also be used to identify disease states that are distinguishable from known coagulation disorders.

The coagulation system is a very complex system and the available tools for studying the system are limited in the range of information that can be provided to scientists investing in drug development. Many existing drug developments rely on target-based screening, while others rely on phenotypic screening. Targeting methods include molecular methods that use effects similar to gene expression or using an assay to isolate RNAi of a target of interest, and then using the assay to search for compounds. Phenotypic screens identify biological changes that occur as a result of a compound or agent, and therefore characterize promising lead compounds because they have the desired effect in vivo. For the case of target inhibition, a wild-type construct of the target may be used. For example, activation of the target, i.e., a genetically modified form of the target, may be employed. This approach overcomes the limitations of some current screening methods that do not allow for the search for the activity of a target or a target in a substrate (e.g., whole blood). For targets in hemostasis and coagulation, this can be problematic due to the complex nature of the biological interactions that any given target may undergo in the blood. Potential screening targets include proteins, peptides, enzymes, fibrinogen, thrombin, platelets, platelet receptors, diseased states of platelets, coagulation factors, diseased states of any of these targets.

The methods and devices of the present invention may be generated from informative data sets generated by the coagulation of blood or plasma from patient samples resulting from the addition of specific initiators. Various initiators can be used to initiate blood coagulation at different starting points. These different initiators may be used to separate or highlight different parts of the coagulation cascade by activation of selected points or branches of the cascade and/or inhibition of selected points or branches of the cascade. For a given coagulation response, the method of the invention enables the generation of a 3D surface dataset capturing a plurality of parameters such as hematocrit level, coagulation time, platelet activity, fibrinolysis and many other physiological and biological parameters. Thus, specific initiators can be used to explore and isolate specific coagulation pathways.

Another method of selecting and finding (hone in) on specific parts of the coagulation pathway or the haemostatic system is to use a genetically modified system, such as a knockout mouse or rat. These systems/models allow for the generation of targets representative of a diseased state (e.g., diseased platelets) and the application of the methods of the invention to the blood of the system/model in the absence and presence of candidate compounds to evaluate the effectiveness of the compounds in ameliorating the disease state. The small sample volume requirement of T2MR may be advantageous for animal studies. Conventional platelet methods require 0.5-25 mL of blood, whereas the methods of the invention can be performed using much smaller volumes.

Another advantage of the screening method of the present invention is that multiple features are available to identify anchor points, as well as sensitivity features for measuring the effect of compounds on the hemostatic process.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein are made, prepared, and evaluated, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Example 1: monitoring blood clotting process using whole blood samples

The clotting process was monitored using fresh citrated whole blood or heparinized whole blood samples. Several different activation pathways were investigated.

For the kaolin activation pathway (CK pathway), 1 mL of citrate blood was added to a kaolin vial (pateletmapping assay kit, Haemonetics). The vial was inverted 5 times to mix the samples. Transfer 34 μ L of the blood/kaolin mixture to a 200 μ L PCR tube preheated at 37 ℃. Adding 2 mu L0.2M CaCl₂Added to the PCR tube and immediately started the T2 measurement.

For the activator pathway (A pathway), 1 μ L of freshly prepared activator solution (A-P1, PlateletMapping assay kit, Haemonetics, also referred to herein as "RF") was added to a 200 μ L PCR tube that was preheated at 37 ℃. 36 μ L of blood collected in heparinase vials was added to the PCR tube and the samples were mixed 3-4 times with a pipette tip. The T2 measurement was started immediately.

For the activator (RF) + ADP pathway, 1 μ L of a freshly prepared activator solution (A-P1, PlateletMapping assay kit, Haemonetics) and 1 μ L of a freshly prepared platelet agonist solution (ADP-P2, PlateletMapping assay kit, Haemonetics) were added to 200 μ L PCR tubes preheated at 37 ℃. 36 μ L of blood collected in heparinase vials was added to the PCR tube and the samples were mixed 3-4 times with a pipette tip. The T2 measurement was started immediately.

Figure 4 shows the difference in coagulation behavior associated with 3 different activation pathways.

Example 2: data extraction using an algorithm for interpreting blood coagulation in a sample

The data output from the T2reader of example 1 was processed using a three-step method of performing quadratic exponential fitting, mapping and verification, and feature extraction:

quadratic exponential fit

The read time is registered by NDXClient. The complex y data is converted to a magnitude and normalized. The initial 25 milliseconds of x and y are deleted. Each relaxation curve was fitted using a default non-linear least squares method. Curves were fitted to quadratic exponential equations using the starting points (e.g., seed values) of AmpA, AmpB, T2A, and T2B with fixed seed values for the first 5 time points. The seed value for the 6 th time point was obtained from the average of the previous 5 time points. In general, a time point determines a seed with the output of a previous time point. Negative values are not allowed for the parameters. Alternatively, the data may start fitting in the middle of the time series, similarly going to the end. The goodness-of-fit term is calculated by taking the sum of the squares of the fit residuals (called SSE), excluding non-negative values (see fig. 1A and 1C). The parameters AmpA, AmpB, T2A, and T2B are boxed into their respective categories to generate text files.

Drawing and verification

Marks and deletes fits that fail to meet SSE criteria. A simple smoothing function based on local regression applying weighted LLS and a polynomial of degree 1 is performed, removing outliers. Each fit parameter is plotted against time (smooth and non-smooth). Data shifts and mirroring were performed to simulate TEG traces and to generate T2 coagulation curves. A T2 coagulation curve can be made by applying a linear transformation to the extracted magnetic resonance parameters. Specifically, the T2 coagulation curve can be obtained from the T2A data by subtracting the minimum T2A value from each data point and taking a negative value for each resulting data point, effectively reflecting the curve about the x-axis. Likewise, the T2 coagulation curve was obtained from the T2B data by subtracting the maximum T2B value from each data point and taking negative values for each resulting data point, effectively reflecting the curve about the x-axis. The amplitude data AmpA and AmpB can be converted into a T2 coagulation curve by a similar method.

Extracting features

The plotted curve was measured to extract values related to coagulation behavior "R", "MA" and "angle". Data extraction may be performed using any of a variety of methods known in the art. For example, the metric may be obtained from the shape of the curve of the resulting data and/or calculated from the values of one or more NMR parameters.

Example 3: use of capillary method for measuring coagulation behavior

2-5 μ L blood samples were collected from the patient using a capillary tube. Blood samples were collected from the fingers using a needle, collected into heparinized capillaries, and capped with clay. Or blood was collected in a heat-resistant glass tube and capped with clay. Alternatively, blood samples were collected in glass Dagan capillaries, capped with clay, and without activator. Data for T2 NMR relaxation rates were recorded from capillary samples using a T2 reader. It was determined that the collected data was uni-exponential and this may reflect a different clot structure than that present in the standard CK curve. It is speculated that the surface of the capillary induces blood clotting.

Example 4: nanoparticle treatment for blood

The incorporation of superparamagnetic nanoparticles into blood results in a change in the magnetic susceptibility of the bulk water in the blood. A similar phenomenon has been used previously, which enables the exchange of measurements of water inside and outside the red blood cells by adding manganese to the blood to change the T2 value of the bulk water and to make chemical shifts possible. Blood samples from individual patients were used in the experiment according to the protocol provided in example 1. In addition, blood samples were treated with non-functionalized nanoparticles 800 nm in diameter. 2 different concentrations of nanoparticles were tested. Two different concentrations were obtained by adding 10 μ L nanoparticles or 20 μ L nanoparticles. T2 data was recorded using a T2 reader. The recorded T2 data was processed using the algorithm described in example 2. Based on a series of graphs of AmpA and AmpB for samples containing nanoparticles, noise was observed in the nanoparticle reduction curve allowing easier differentiation between AmpA and AmpB. From a series of graphs of T2A and T2B for samples containing nanoparticles, it was observed that the nanoparticles produced a greater change in T2A. Superparamagnetic nanoparticles may be used in the method of the present invention to: (i) increase the signal change (or change in the NMR parameter value) in response to a rheological change in the sample, and/or (ii) allow earlier detection of the rheological change in the sample undergoing the coagulation or dissolution process.

In a separate experiment, the effect of superparamagnetic particle size on T2MR in the coagulated blood was observed. Blood samples were prepared by mixing 1 mL of citrated whole blood with 1 tube of kaolin reagent from (TEG) gently by inverting 5 times. 34 μ L of the blood/kaolin mixture was placed in a PCR tube (200 μ L) and preheated at 37 ℃ for 1 minute. By adding 2 mu L0.2M CaCl₂To initiate coagulation, the tube is placed in a T2 reader. The samples were run without magnetic particles (see FIG. 26A), with CLIO nanoparticles (30nm size; about 0.05ng) (see FIG. 26B), and with Seramag superparamagnetic particles (730nm size; about 0.05ng) (see FIG. 26C).

In a typical citrated kaolin experiment, the addition of 30nm nanoparticles eliminated the clot signal (at T2=200 msec), with only 1 peak at about 100 msec T2. The addition of 730nm superparamagnetic particles does not hinder the ability to observe 2 peaks.

Example 5: determination of magnetic resonance parameter values of donor blood samples

Using blood samples obtained from donors, magnetic resonance parameters used for data extraction were obtained. Blood samples were evaluated using the TEG method and the T2 clotting method. Several different types of assays were run. T2 coagulation data was collected using one of a variety of T2 readers.

Whole blood coagulation assay

Donor blood samples were evaluated using the method of the invention and the TEG method. A variety of different whole blood coagulation assays were used as described in example 1. These assays are further summarized in table 1.

TABLE 1 Whole blood clotting assay and activators tested

The K assay forms blood clots by both platelet activation and the enzyme cascade leading to fibrin formation. This clot avoids any type of platelet inhibitor drug and provides an assessment of whole blood coagulation. TEG R values are thought to reflect the enzymatic pathway, while TEG MA values reflect clot strength, which is driven by both platelets and the product fibrin network of the enzymatic pathway.

RF, ADP, and AA assays use heparin to inhibit the thrombin pathway to isolate pathways activated by various specific agonists. ADP and AA activate platelets and additionally promote clot strength through the formation of fibrin network. RF operation does not activate platelets, but rather the fibrin network. Thus, when the RF run MA was subtracted from the K, ADP or AA runs, the platelet contribution to these respective run MAs was determined. It is noteworthy that this is an indirect measure of platelet function.

Running K and ADP blood coagulation assays on patients can be used to determine the amount of platelet inhibition in patients due to antiplatelet therapy (e.g., bolivitamin, Ticlid, and effect as ADP platelet inhibitors). The TEG MA values for the K and ADP runs were compared to determine the inhibition.

Running the K and AA blood coagulation assays can be used to determine the percentage of platelets inhibited by anti-platelet therapy (e.g., aspirin, ReoPro, Aggrastat, Integrilin, and non-steroidal anti-inflammatory drugs). These drugs all inhibit the GPIIb/IIIa platelet receptor, which is an agonist activated receptor in AA. This is done by comparing MA values for K and AA. When platelets are activated, the GPIIb/IIIa receptor is expressed on the surface of platelets (Corporation, H., TEG 5000 System Guide to plate mapping Assay 2010; enrique z, L.J. and L. Shore-Lesseson, Point-of-car clotting testing and transfusing algorithms Br J Anaesth, 2009.103 suppl 1: pages i 14-22; Kroll, M.H., Thromboelastograpy: Theory and Practice of Measuring hemostasis.) Clinical Laboratory News 2010: pages 8-10).

The FF test excludes the contribution of platelets to coagulation. Subtraction of FF MA from kma yielded the intensity of the clot presumably due to platelet contribution. This is an indirect measure of platelet function. Comparison of FF and K tested MA with the reference range may indicate whether the bleeding problem is from low platelet activity or low functional fibrinogen activity, thus instructing the clinician to perform the appropriate treatment. Similarly, patients with prothrombotic activity can be diagnosed with high platelet activity or high functional fibrinogen activity or both. Fibrinogen is an important component of both primary and secondary hemostasis, and is involved in both reversible and irreversible platelet aggregation. In secondary hemostasis, fibrinogen is cleaved and converted to fibrin to form a fibrin matrix (Corporation, h., Guide to Functional fibrin, 2009).

Example 6: processing of feature extraction

A great deal of work was done to determine the characteristic correlation between T2 coagulation and TEG curves. The two main features of the TEG curve for which correlation is sought are R and Maximum Amplitude (MA). All feature extractions were performed using the T2A, T2B, AmpA and AmpB curves smoothed at the point where the total curve 1/15 was referred to or every 40 data points.

The feature search was based on several time points of the T2 coagulation curve. All features of the curve were constructed from the first candidate for R, which was determined by simple numerical calculations, by the time at which the second derivative of the T2A curve reached a maximum. This point in time is called "PossibleR". Also used for feature extraction is the time at minimum or maximum of T2B and the time at minimum or maximum of T2A. Fig. 5 illustrates a T2 coagulation curve with first and second derivatives of the T2 coagulation curve for a single subject.

There may be false data points, particularly in the first and second derivatives. The minimum and maximum values are therefore defined as the extreme points closest to the first time T2A reaches the maximum divalent derivative. After identifying these time points on the T2 coagulation curve and on the first and second derivatives of the T2 coagulation curve, several different features were calculated from the time points, combinations of time points, numerical derivatives, and differences in the T2 coagulation curve. These features are related to TEG R, MA and MA_{Blood platelet}The values are compared.

Standard regression analysis was applied to determine the strength of feature correlation. Two terms are used to facilitate searching features, namely Pearson product-moment correlation (R) coefficient and measure (R)²) The coefficient of the term. The R term is used as a general guide in mining promising correlations, R²The term is used as a measure of how well the correlation is in fact good.

90 features were tested. Example characteristics include a value of a derivative of a first numerical value at PossibleR; the slope of T2A at maximum acceleration of T2A; the difference between the maximum and minimum values on the T2B curve; and the slope between the minimum T2A and maximum T2A values.

T2A + partial T2B Curve

In one example of feature extraction, the T2A and T2B curves can be combined to yield a T2A + partial T2B curve, which can be used to search for correlations between the T2 coagulation curve and TEG MA values. This combined curve, called "T2A + pT 2B", is calculated by identifying the point in time when T2B is maximum and generating a vector consisting of the absolute values of the differences between this T2B value and all subsequent T2B values. This vector is then added to T2A on the same time recorder. Fig. 6 shows an example of how the T2A + pT2B curve is calculated. The T2A + pT2B curve simulates the upper half of the TEG curve.

The T2A + pT2B curve generated as described above, yielded a potential correlation with TEG MA. The last 50 of the T2 and T2A + pT2B curves identified by T2RThe change in T2 signal between the mean T2 within a point. This feature follows an inverse correlation with MA, which is consistent with our initial impression of MA correlation (see fig. 7). That is, the maximum T2 change of the T2A + pT2B curve is inversely related to the TEG kaolin MA value. Through a large number of samples, the characteristic is obtained from R²The correlation of the values is between 0.39 and 0.55. These R²Values may be generated from T2 coagulation T2A curves sensitive to different aspects of coagulation, such as platelets, rather than to TEG.

Pre-solidification T2A value

The hematocrit of a blood sample can be calculated from a single initial water population in the sample prior to coagulation. This single water population corresponds to the T2A value established using the quadratic exponential fitting method described in example 2, prior to clot formation. The initial T2A value for the sample was found to have an approximately inverse linear correlation with hematocrit measured using standard methods known in the art. A calibration curve was established by using blood drawn from a single patient to generate a set of 4 standards at different dilutions. The hematocrit and initial relaxation rate observed for the water population a before initiation of coagulation were plotted against each other to obtain a calibration curve. Blood samples were drawn from 10 patients and queried according to the calibration curve. The T2 signal of uncoagulated blood was found to vary inversely with Hematocrit (HCT) levels. As shown in fig. 8a, different patient samples spanning a wide range of HCT reference values generally confirmed this. As described in figure 8b, the calibration method was applied to single patient samples diluted across the HCT range, showing a linear correlation to HCT. FIGS. 8a and 8b show that it is possible to calculate the hematocrit of a blood sample using the method of the present invention.

Example 7: correlation between extracted magnetic resonance parameters and coagulation behavior determined by TEG

Magnetic resonance parameters extracted from the measured average NMR relaxation rate data may be correlated to the coagulation behavior determined by the TEG. Table 2 illustrates the correlation between the extracted T2A values and the clotting time parameter "R" measured using a TEG hemostasis analyzer.

The TEG hemostasis analyzer provides quantitative and qualitative measurements of the physical properties of blood clots (Samara et al, Thromb. Res.115:89-94, 2005). In this example, the Maximum Amplitude (MA) and clotting time (R) were measured for a clot sample of thrombin formation. MA is an indicator of the viscoelastic properties of clot formation or clot strength and depends on platelet aggregation and fibrin formation and polymerization. R is the latency period until initial fibrin formation and correlates with the rate of thrombin formation.

From the plots of AmpA and AmpB measurements, T2A measurements and T2B measurements, it was found that extracting the early part of the data curve can provide an indication of the clotting behavior (e.g. fibrinolysis or LY30) that could not be obtained from the corresponding TEG curve.

TABLE 2 correlation between extracted magnetic resonance parameters and coagulation behavior determined by TEG (data in minutes)

T2 coagulation correlation of clotting time (R)

As described above, a method of correlating coagulation time (R) with T2 coagulation characteristics was developed based on obtaining the second numerical derivative (second numerical derivative) of T2A in an automated manner. Through 25 runs, an R (whole blood clotting time) value of 0.80 was observed between T2 and TEG²(FIG. 9). The T2 coagulation curve used for this correlation was a kaolin run, where SSE ≧ 2. The 3 outlier runs are highlighted in fig. 9, with the T2A curve shown on the right. In all 3 cases, the T2A curve reached maximum acceleration before the R TEG time (T2A = 0).

Platelet-associated clot strength (MA)_{Blood platelet}) T2 blood coagulation correlation of

Several characteristics were studied for correlating TEG MA time with T2 coagulation characteristics, run using kaolin. NeedleThe most significant correlation found for the kaolin runs was the T2 coagulation profile between time zero and the minimum T2 value (just before the T2R profile). This is typically the initial decrease in the T2A curve. The slope and TEGMA of the T2A signal between time zero and the minimum T2 value_{Blood platelet}And (6) associating the values. Because TEG cannot directly measure platelet activity, the MA term (also known as MA) was run from kaolin_Thrombin) Subtract MA term (MA) from activator or A run_A) To deduce the contribution of thrombin-induced platelets to clot strength. FIG. 10 shows TEG term MA for 14T 2 clotting runs and 28 TEG runs_Thrombin– MA_AOr to subtract the correlation between MA from CK run from MA from a run. As for the R correlation, SSE.gtoreq.2 was used. To obtain MA from TEG_{Blood platelet}In terms of terms, two different TEG curves must be obtained, and the user must perform manual calculations to determine the contribution of platelets to clot strength. However, for the T2 coagulation, this information was available within the first 6 minutes of a single T2 coagulation run, which is much easier to set up.

Functional fibrinogen-related clot Strength (MA)_FF) T2 blood coagulation correlation of

The clotting behavior was determined by including additives in the blood sample prior to collection of NRM relaxation data. Fibrinogen may be added to the sample to effectively titrate the range of MA values. Figure 11 shows clear evidence of the correlation between the T2 coagulation curve for functional fibrinogen titration and the MA and features from the T2 coagulation curves of the FF run. The T2 coagulation profile was calculated as the difference between the initial and final T2 coagulation AmpA values (Δ AmpA). Fibrinogen was added to the samples at 3 different concentrations (0.63, 1.25 and 2.5 mg/mL). As can be seen in FIG. 11(c), these Functional Fibrinogen (FF) (MA) compared to the sample without added fibrinogen_FF) There is a strong correlation between Δ AmpA and TEG MA running. Fibrinogen titrations were performed on 2 other patient samples, resulting in correlations above 0.9 and up to 0.98. In this protocol, citrate whole blood is diluted to 50% of its original concentration.

T2 coagulation association of percent lysis 30 min post MA (LY30)

A correlation was observed for item LY30 for detecting fibrinolysis. Samples of 5 different healthy patients were used to test whether T2 coagulation was sensitive to fibrinolysis. By adding two levels of Tissue Plasminogen Activator (TPA) to the blood of healthy patients, the addition was used to stimulate fibrinolysis. TPA cleaves fibrin when it is formed. It was observed that fibrinolysis could be detected within 10 minutes of T2 clotting, which was less than half the time required for detection on TEG. For all 5 patients there was a clear difference between healthy and fibrinolytic samples. Fig. 12 shows T2 coagulation data for T2A and T2B curves for non-fibrinolytic (solid line) and fibrinolytic samples (dashed line).

There was a strong correlation between the change in the signal for T2B and the item LY30 from the TEG, as shown in FIG. 13. There are several potential features that can be used for this correlation. The 3 features studied differed between: (i) a minimum T2B value and a maximum T2B value (fig. 13), (ii) an average of the T2B values over the last 25 minutes of the T2B curve, and (iii) a T2B value at 10 minutes. All have R²Correlation with a value between 0.72 and 0.79. The correlation plot shows that T2 coagulation has a higher sensitivity to fibrinolysis compared to TEG. This can be seen by categorizing the different levels of fibrinolysis in fig. 13. Healthy patient samples are circled with solid lines. Complete fibrinolytic samples (containing 118 ng/mL Cathflo @, recombinant alteplase produced by Genettech) are circled with dashed lines, and partial fibrinolytic samples (containing 60 ng/mL Cathflo @, recombinant alteplase produced by Genetch) are not circled with dashed lines. The T2 coagulation T2B item was detectable in only 10 minutes, which is much faster than the detectable TEG LY30 item, indicating the utility of the T2 coagulation signal for more rapid measurement of fibrinolysis.

Set time, PT/INR and aPTT

The sensitivity of T2MR to clot formation in blood and plasma can be used to measure clotting times, such as PT/INR, aPTT, and TEG R.

The clotting time is measured by T2MR by first establishing a time zero T2 value for the T2 time curve (i.e., by taking the first point or some average or linear fit to the first 5-10 data points), and second determining the time point of the T2 time curve (i.e., clotting time or PT time) when the T2 value changes by a predetermined amount (e.g., 5% or 10%) indicative of clotting in the sample. INR was calculated from the observed PT clotting time using the method described by Jan et al, Clin. chem. 35:840 (1989).

The correlation of PT/INR to Stago was confirmed using thromboplastin reagent (Thrombotest, Axis Shield) and 1:5 diluted whole blood for T2MR and standard reagents and plasma protocols for the Stago Start system (see FIG. 14A). The 18 normal samples and 24 abnormal samples were tested against the reference range established by the 20 normal samples. Abnormal samples with extended PT time were obtained by spiking increasing levels of rivaroxaban (Rivaroxiban), an anti-Xa inhibitor (10 ng/mL-1000 ng/mL). Correlations with TEG clotting time were obtained in samples involving >40 normal patients, with non-optimized R2 correlations > 0.8. (see FIG. 14B). A correlation of PT time for plasma and Stago (see fig. 14D) and whole blood and Hemochron (see fig. 14C) was also found.

Example 8: t1 relaxation measurement

In addition to being able to determine the T2 measurement, a T2reader may be configured to determine the T1 measurement. T1 measures different physical properties of the hydrogen atom spin system in a sample. Thus, the T1 data provides alternative and supplemental information about blood coagulation compared to the T2 data. While conventional T1 measurements are time consuming (2-3 minutes) due to the stepwise nature of acquiring the T1 signal, the z-refocused echo (ZRE) method is employed to acquire T1 in less than 5 seconds. Measurements of T1 relaxation curves of native and clotted whole blood were made and the sensitivity of T1 to blood clotting was observed (unclotted whole blood T1 = 787 ms; clotted whole blood T1 =860 ms).

Example 9: T1/T2 hybrid detection method

T1/T2 hybrid detection methods are known in the art (Edzes, J. Magn. Reson. 17: 301-313, 1975; Sezginer et al, J. Magn. Reson. 92: 504-527, 1991, incorporated by reference). These methods and related methods may be used in the present invention to evaluate magnetic resonance parameter values and/or to establish a T2 coagulation curve.

T1 is typically obtained by sampling with an inversion-recovery sequence. The inversion recovery sequence can take several minutes to acquire, depending on the accuracy of the measured relaxation time that the user needs to achieve, which is represented by the number of data measurements for the pulse sequence. The details of the inversion recovery sequence will not be described herein as it can be looked up in any standard NMR textbook.

The T1ZRE pulse sequence allows the measurement of T1 (about 3-5 XT 1) within the time required for the magnetization to fully relax. This is achieved by reversing the magnetization with a 180 pulse and then changing the magnetization to a pulsed signal while the magnetization returns to a plateau by measuring its magnitude and returning it to its original-z position with a series of pulses.

The time taken for the magnetization to become a pulse signal is called tau_cTime between pulse modulations is tau_r。τ_cThe time from the start to the actual measurement is τ_m. The resulting relaxation constant is R₂And R₁A combination of (1), referred to as R₁₂. The 3 terms are related by the equation:

　　(11)

wherein p = τ_c/τ_r. From this relationship, it can be found that when p goes to 0, R12 goes to R1.

τ_rWill depend on the number of points and the total duration of the measurement. For the T2 coagulation measurement, pairsAt τ of 100 milliseconds_rAnd 30 points in more than about 3 seconds. Tau is_cTerms can be calculated from the pulse sequence and are approximately equal to 3 × τ or 750 μ s. thus, the p term is equal to 0.0075, and the measured relaxation or hybrid T1/T2 (hT12) term should provide primarily a linear equation for T1. Sezginer et al to derive the T1 measurement from the hT12 signal, which is

　　(12)

Wherein T is₁ ^measIs hT12, T₁Is T1, T_bFor the duration between two measurements, t_cpHalf of the inter-echo attenuation or tau, T₂Is the measured T2 time.

The above description merely illustrates how the hybrid relaxation time can be measured. There are other pulse sequences that can be used to measure hybrid relaxation constants and to derive T1 in a fast manner.

Regardless of the pulse sequence used, the inventive concept relates to the rapid acquisition of magnetic resonance relaxation measurements for monitoring coagulation (i.e., blood coagulation). Other types of magnetic resonance pulse sequences may be used to monitor the subject (bulk) hydrogen signal in the sample during the coagulation process. Examples include T2, T1, T1/T2 promiscuous times, their inverse terms R2, R1, R12, and pulsed NMR measurements commonly used for material analysis in relaxometers, such as Free Induction Decay (FID) based analysis, fast fourier transform based analysis (FFT). FID analysis often distinguishes between fast and slow fading signals. The strength of the two signals can be compared, as can their decay constants. These pulse sequences have been commonly used for fat analysis, fat content and solid to liquid ratio, solid fat to liquid ratio, hydrogen content determination, oil content, solid content and total fat content determination, oil-water emulsions, and fat and moisture determination. Similar real-time or kinetic measurements can be made with those NMR parameters in the sample being subjected to the coagulation reaction.

Alternative relaxation measurements are attractive, which provide: (1) more information on the solidification process; (2) specific information not captured by the T2 measurement; and (3) normalization of the factor to which both T2 and the new parameter are sensitive. To describe point 3 more, for example, if the T1 measurement is sensitive to patient-to-patient variation, but T1 does not contain coagulation information, there would be an algorithm that uses the T1 curve to "subtract out" the portion of the T2 signal that results from the undesired sensitivity in the clinical sample. The undesired sensitivity may be a change in hematocrit, platelet count, or the like.

To confirm this concept, we measured the CK coagulation curve in Bruker minispec. The experiment was performed in a sample volume of 300. mu.L and in a glass NMR tube. The relaxometer has a pulse sequence that can measure both T2 and hybrid T12(hT12) relaxation times in a rapid continuous manner. T1 is derived by equation 12.

The resulting T2 coagulation curve showed a very similar shape and characteristics to the T2 coagulation curve obtained on the T2reader used in the present invention. This is a confirmation of the other T2 coagulation curves, as the Bruker minispec used has a detection coil, magnet and curve fitting algorithm that is very different from the T2 reader. Under the pulse sequence conditions, the T1/T2 hybrid is mainly T1. In this experiment, T1/T2 promiscuous was obtained in a crossed fashion. The T2 signal was obtained using the CPMG sequence, and then the T1/T2 signal was obtained using the T1ZRE sequence. The signals can be combined with each other to generate a new curve calculated by dividing the T1/T2 hybrid (mainly T2) by T2A and can be used in the method of the invention to evaluate blood samples for hypercoagulability and/or hypocoagulability.

Example 10: coagulation control for acrylamide synthesis

Acrylamide gel polymerization was used as a synthetic control for T2 clotting measurements. A set of acrylamide gels was prepared using deionized water, 40% acrylamide, 0.5M Tris pH 6.8 buffer, and 10% ammonium persulfate in the amounts listed in table 3 to form a set of mixtures. The mixture was incubated at room temperature for 30 minutes. 36 μ L of each mixture was stored in a separate set of PCR tubes and used as a control. mu.L of Tetramethylethylenediamine (TEMED) was added to each mixture. After 30 minutes, T2 measurements were made for each control and polymerization reaction.

TABLE 3 composition of acrylamide gels

Percent change in T2 was calculated by comparing T2 values for TEMED treated samples and corresponding control samples. The measured data are shown in Table 4.

TABLE 4 percent Change in T2 time for acrylamide gels

Sample (I)	Control, T2 ms	Polymerization, T2 ms	% T2 Change
				3%	1386.3	1351.16	3
6%	1339.31	1107.84	17
				10%	1326.36	828.29	38
15%	1355.67	604.56	55
				20%	1235.29	450.85	64
40%	1200.66	145.87	88

15%, 20% and 40% acrylamide gels indicate maximum% T2 change. These reactions were selected for time course T2 measurements. 15%, 20% and 40% acrylamide gels were prepared according to Table 3. Immediately upon addition of TEMED, each sample was placed in a T2T 2reader and measurement was started. The time course data shows that the overall change in T2 and the rate of reaction depend on the percent acrylamide. This reflects the different polymer densities of these different reactions. The sensitivity of T2 to polymer gel composition may be due to the sensitivity of T2 to changes in the mean length of water diffusion over the time course of a single T2 measurement.

Superparamagnetic nanoparticles may be used with synthetic coagulation reactions (e.g. acrylamide gel formation). The initial portion of the T2 time curve flattened when a 40% acrylamide gel was polymerized in the presence of approximately 800 nm carboxy Sera-mag nanoparticles (1. mu.g/mL nanoparticles). The CPMG parameters used for the above T2 measurements were as follows: pulse width = 6.8 μ s; radio frequency blanking width =1 μ β; 90-180 pitch = 249.6 μ s; num 180s = 7000; total excitation = 3494.902 ms; repetition (Tr) =1000 ms; receiver phase = 0; 90 phases = 0; and 180 phases = 90.

This example illustrates how the NMR parameters (e.g., T1 or T2) in a sample undergoing a phase change. Increasing polymer density resulted in a faster decrease in T2 and an overall greater decrease in T2, indicating the dependence of the T2 signal on polymer matrix density, pore size, and other parameters of the gel. Similar results can be obtained with other gels such as kappa carrageenan (potassium ion added gel), l carrageenan (calcium ion added gel), calcium sodium alginate, gelatin, especially food products.

Example 11: detection of bacterial endotoxins

The method and device of the invention are used for the detection of bacterial endotoxins, i.e. cell wall material from gram-negative bacteria. Endotoxin can cause high fever in human beings, and therefore, injectable drugs and medical devices that come into contact with blood are often tested for the presence of endotoxin. Coagulation-based assays for detecting endotoxin rely on reactions between bacterial endotoxin and the particular lysate used in the assay. The lysate of circulating amoebocyte cells derived from Limulus polyphemus is a specific lysate that can be used. In this assay, a lysate is introduced into a sample to be tested for the presence of endotoxin. If a gel is formed by the coagulation process, endotoxin is considered to be present. The formation and properties of such gels are monitored by any of the NMR-based methods described herein.

Example 12: characteristic curve of T2

The NMR data was processed to generate a T2 characteristic curve showing a clear signal (i.e., maximum) representative of each water population in the blood sample. The T2 profile is generated by applying a mathematical transform (e.g., laplace transform or inverse laplace transform) to the decay curve associated with T2 at a point in time during the coagulation event. Fig. 15 shows the T2 decay curve and the corresponding T2 characteristic curve. The 3 signals represent 3 water populations in the blood sample. Fig. 15 also shows one way in which the signal may vary over time.

The correlation of water populations a and B with the coagulation sample component observed in NMR relaxation data was confirmed by separating the contracted blood clot from the surrounding serum and measuring the T2 data for the two components separately (see figure 16, T2 characteristic curve after completion of coagulation). 2T 2 relaxation spectra of patient samples collected in a T2reader at 2 different time points (see fig. 18). The spectrum shows an initial effective peak at time 0 consistent with blood in the sample. At 20 minutes, two effective peaks were evident. The peak with the lower T2 time (about 200 and 300 milliseconds) corresponds to the aqueous environment of the T2 clot, and the peak with the higher T2 time (about 450 and 580 milliseconds) corresponds to the aqueous environment of the T2 serum.

The difference between T2A and T2B (△ T2) was observed to correlate with clot intensity fig. 17 shows 2 overlapping T2 relaxation spectra and TEG clot intensity (MA) for two different samples taken from the same patient a sample with a weaker clot (MA = 19.2) showed a lower difference in clot-related signal and serum-related signal (182 ms) than a sample with a stronger clot (MA = 65; 258 ms) data showed a positive correlation of increasing clot intensity with △ T2 (R = 65; 258 ms)²= 0.8771, see fig. 18).

Example 13: 3D map of water populations in blood clots

T2 relaxation rate data were collected from CK blood samples drawn from healthy patients. For each sample, a series of T2 decay curves were measured over a 50 minute period. The attenuation curves were each processed using an inverse laplace transform (ILT CONTIN) to provide a series of curves showing the intensity of T2 as a function of time at T2. These curves were compiled to generate a 3D data set by overlapping inverse laplace transform curves over the duration of the clotting time dimension to generate a 3D surface showing how different water populations within the sample vary over time. Figure 20 illustrates a 3D data set of patient samples 29328 and 29350 collected in a Bruker minispec. The 3D data set shows an initial water population with a time equivalent to 250-500 ms T2 for non-coagulated blood. Within a time period between 5 minutes and 30 minutes, the single water population is divided into two effective water populations. These two water populations correspond to the two components of the clot, the contracted clot and the serum surrounding the contracted clot. The first clot-associated water population had a T2 time of 80-400 milliseconds, corresponding to a contracted clot. The second clot-associated water population had a broader T2 time frame of 400-. For some samples, the two peaks cannot be fully resolved. The correlation between the components of the water population and the coagulated sample was confirmed by separating the contracted clot from the surrounding serum and measuring the T2 data for these two components separately.

Example 14: inhibition of platelet activity using abciximab

The antithrombotic drug abciximab (ReoPro) was used to interfere with the clotting of the whole blood samples. Abciximab is a Fab fragment of a human-murine monoclonal antibody that binds to the IIb/IIIa receptor of human platelets and inhibits platelet aggregation. Abciximab is used to inhibit the contraction of blood clots during clotting.

Samples containing abciximab were prepared according to the following procedure and with reference to the present application and the accompanying figures, with numbers corresponding to abciximab concentrations in units of μ g/mL.

1. Blood was drawn from normal donors into 6 citrate tubes. Normal donors will not take aspirin or any antiplatelet drugs for the past 2 weeks.

2. Combine 6 tubes of blood and mix gently by inverting 8-10 times. The samples were used within 2 hours of collection.

3. The abciximab solution (10 mg/5 mL) was diluted at a ratio of 1:4 using saline as the diluent to form "solution 1".

4. Blood was spiked with solution 1 and saline as per table 5.

5. After allowing the samples to sit for at least 5 minutes, any samples were run in a T2reader or Bruker minispec.

TABLE 5 preparation of blood samples containing abciximab

Mu l solution 1	Mu l saline water	Mu l blood	Reopro® (µg/ml)
				0	96	1904	0
2	62	1936	0.50
				4	60	1936	1
6	58	1936	1.5
				8	56	1936	2
10	54	1936	2.5
				12	52	1936	3
16	48	1936	4
				24	40	1936	6
32	32	1936	8

In the first experiment, 4 aliquots of patient sample 29488 were prepared with varying concentrations of abciximab (0, 2, 4, and 8 μ g/mL) prior to initiating the clotting process. During clot formation, the T2 relaxation times of the 4 samples were measured and the corresponding 3D maps were determined using the technique described in example 13. The clear trend is evident in the 4 3D plots, where higher concentrations of abciximab resulted in the contraction of the clot water population and the serum water population merging into a single water population. At the highest concentration of abciximab, there were no more visible two phases in the clot. This physical change was confirmed and quantified in the corresponding 3D plot (fig. 21), indicating a rapid exchange of water in the homogeneous sample at the highest concentration of abciximab.

In a second experiment, 5 aliquots of patient sample 29494 were prepared with different concentrations (0, 2, 4, 8, and 0 μ g/mL) of abciximab before initiating the clotting process. The T2 relaxation times of the samples were measured continuously, with each set of measurements lasting about 50 minutes. The first sample tested and the last sample tested did not contain added abciximab. The collected T2 attenuation curves were processed using the inverse laplace transform described in example 18. The data for 5 samples over a 0.1 minute period are shown in FIG. 22. The curve shows that in the presence of abciximab, a narrower distribution of T2 signal was observed in the initial water population present in the blood sample. The difference in the shape of the curve between the first and last sample runs (both without added abciximab) may indicate the effect of sample aging (sampleage) on the T2 time profile. The data for the first 4 samples over the 20 minute period are shown in FIG. 23. The MA values determined for the corresponding TEGs for these samples were 60.7, 61.4, 30.4, and 22.0, respectively. The two samples with the highest concentration of abciximab formed weaker clots (measured by TEG) and showed narrower, more resolved T2 peaks, compared to the other two samples that formed stronger clots and showed poorly resolved T2 peaks (indicating multiple water populations and environmental heterogeneity within these samples). This data demonstrates that the NMR-based method of the invention can be used to measure platelet activity within a coagulated sample. Figures 22 and 23 show that additives that modulate the clotting behavior of a sample (e.g., abciximab) can be advantageously used to establish a correlation between indicators of TEG determination (e.g., clot strength and platelet activity) and NMR derived data determined using the methods of the present invention.

Figure 24 shows how a 3D map made from a 3D data set can be used to determine the clotting time of a blood sample. Fig. 25 shows how platelet inhibition reduces the signal intensity of the clot-associated signal and shifts the serum-associated signal to a lower T2 value using a 3D plot made from the 3D data set.

Example 15: evaluating data quality and fitting quality

T2 coagulation data for a single sample the T2 CPMG relaxation curve observedLine S collection assemblyWhere j =0, 1,2, …, S is the point in time during the entire sample coagulation/clot formation process, where j =0 corresponds to coagulation initiation and j = S corresponds to the point after the sample has undergone the expected coagulation event the T2 coagulation algorithm transforms the collection of decay functions into the surface of decay constant potential (decay constant potential) in the T2 × coagulation time domain, and then extracts useful information about the sample from these potentials.

Equation (3) can be applied to the single T2 CPMG relaxation dataModeled as a finite sum of weighted exponential decay functions. Usually due to sample complexity, n is quite large (e.g., n>300). Many algorithms have been developed to estimate the parameters of the model (see Istratov et al, Rev. Sci. Instrum. 70:1233 (1999)). For example, the Tikhonov regularization method (see Tikhonov Sov. Math. Dokl. 4:1035 (1963)) may be performed in CONTIN (see Provencher, Compout. Phys. Commun. 27:229 (1982)) to evaluate the relaxation data collected by the present method. As is common (see Davies, Inversion schemes in Scattering and Imaging, edited by m. Bertero and e.r. Pike (Adam Hilfer, Bristol, 1992), page 393), Inversion methods utilizing this T2 coagulation relaxation data may require the application of specific modifications or settings, such as those provided below.

The inverse laplacian transform is known to be an ill-defined computation, which means that many equally accurate solutions can be generated from a given complex attenuation data set. This is true for the purest data, which deteriorates when the composite decay curve becomes noisier or moves significantly from the sum of the first exponential decay functions.

CPMG data quality

To overcome this problem, prior to inversion, each T2 CPMG data set may be tested to determine the quality of both the data and the overall likelihood that the inversion will be successful. The signal-to-noise ratio (SNR) is estimated from the CPGM intensity at T =0 compared to the variation observed later in the relaxation data (i.e. T is close to T). The low SNR values caused by the reduction of the start signal are related to instrumental and operator errors that render the data disregarded. Low SNR can also produce increased degradation late in the CPMG data, or when the duration of the observation of the decay process is too short. The latter case indicates that the decay constant in a given data set is greater than expected. Since the Tikhonov regularization method requires knowledge of the expected maximum T2 value, the T2 algorithm caters for the low SNR case due to the linear trend in the later stages of the CPMG curve by increasing the maximum expected T2 value (a parameter in the CONTIN).

The increase in degradation is also caused when a few extreme points (relative to the complex attenuation hypothesis) may be present at the tail of the CPMG curve. After fitting the segment of the CPMG curve with a first order exponential decay function (0< T1< T < T2< T), the points present in the positive or negative tail of the fitted residue distribution were deleted prior to inversion.

After the T2 CPMG data was judged, in the case when the decay process was observed to be long enough to allow the fitted Jackknife test to be applied, the CPMG data was randomly split into two subsets for training and testing, such that the training had no more than 8,000 data points, with the remaining points assigned to the test set. The training data is inverted and the resulting model parameters (which define a multi-exponential decay function) are used to evaluate the quality of the fit to the test data. The quality of the fit of the test data gives an independent measure of the fit and is used in the feature extraction phase of the T2 coagulation algorithm.

CONTIN parameter value

Several continue parameters significantly affect the quality of the inverse laplacian transform. Table 6 sets forth these parameters and the values taken by the T2 coagulation algorithm to best fit the T2 CPMG data. The regularization value (α) plays a major role in the ability to resolve two or more T2 peaks. The T2 coagulation algorithm requires a narrow peak to distinguish when a subpopulation of water molecules begins to distinguish itself from other populations. The specific range of alpha values specified in table 6 forces the CONTIN to search for solutions with regularized weights that allow discrimination of this subpopulation.

TABLE 6 CONTIN parameters and values for the T2 clotting algorithm

The minimum T2 value is typically set in the range of 1-50 ms. This accounts for the CPMG time constant generated from the plastic in the sample container. When the minimum increases beyond 1 ms, the CPMG curve data for which ILT is performed must be truncated to exclude those short time constants that do not allow inversion. For example, if the minimum T2 is set to 40ms, CPMG data collected before 40ms should be deleted before performing ILT.

The maximum T2 value varies with sample/assay type and mode of use in the following manner. Platelet rich and platelet poor plasma samples containing small numbers of RBCs require larger maximum T2 values. The PPP and PRP maximum T2 values are set in the range of 2500-3500 ms. RBC-containing samples have maximum T2 values in the range of 1000-. The maximum T2 value may also decrease when magnetic particles are included in the sample. For the case where there is no predicted T2 value, the maximum T2 value may be set in the 2500-. False peaks may occur at the T2 fit boundary because the ILT fit attempts to account for the entire CPMG curve, but only over a limited range of T2 values. To accommodate this, an additional 0.5-1 second was added to the maximum T2 value used. The ranges provided above include this additional time.

The Tikhonov regularization method includes controlling the parameters (α) of inversion smoothing across the T2 domain. Too small and broad with the potential to generate compound peaks, too large and narrow with the potential to divide a single peak into several closely located peaks. Upon execution of the Tikhonov algorithm, the CONTIN can search for regularization terms that seek a compromise between these two extremes. This is accomplished by performing a CONTIN that has no course alpha grid search, but is enhanced with a fine grid search (12 iterations) in the range of alpha values of about 1.0e-10 and about 4.0e 0.

Construction of T2 decay constant latent surface

Once each ILT is performed, the collection of curvesJust replace original CPMG curve set. For a given CPMG curveEstimating the attenuation constantMake itPromote multi-exponential decomposition (see equation 13):

　　(13)，

wherein for a number of k's,may be zero. The non-zero decay constant and its promotion of the above described decomposition over time correspond to changes in the sample, and the T2 coagulation algorithm accounts for these values and changes in it to derive information about the rheological state of the sample or to derive information about the hemostatic condition of the subject. Prior to extracting these features, the pseudopeaks and the multiple peaks are resolved (as described below).

False peak identification and deletion

In general, for a given j, ILT amplitude valueSmall in which the value is non-zero (<6) The T2 fields around the area are clustered.Ideally, these islands of non-zero intensity are unimodal and far from the boundary of the range of T2 values for which the inversion is performed. This is not always the case, and care is taken to explain correctly

Application toIs deleted for non-zero values, wherein the T2 value does not correspond to a past expectation based on the type of sample to be analyzed. Examples include T2<50ms, which results from the plastic containing the sample; if the sample is expected to have a high concentration of protein, then T2<100 ms; t2 at or near the maximum T2 range for inversion (the presence of which indicates a larger maximum T2) was applied to the CPMG data of the inverted samples.

In addition, in estimating eachCalculation by Tikhonov regularization methodStandard error of. When in useNear zero time, it is commonIt means that the edges of most non-zero islands are blurred, although averaged over this set of non-zero intensitiesA nearby strong signal. TheseAre not deleted. Or when inIs locally largestToThe entire non-zero island is considered unreliable and set to zero.

Example 16: use of dried or frozen reagents

Dried or frozen reagents may be used in the methods of the invention. The results below show that drying and freezing the reagents had no discernible effect on the clotting process observed by T2 relaxation.

Tube-dried collagen as platelet activator

Collagen is a potent activator of platelets, and recognition of exposed collagen from the cellular matrix is one of the in vivo signals of platelet activation. We demonstrate that a small aliquot of collagen dried at the bottom of the microtube can effectively form the T2MR coagulation peak in both whole blood and RBC-depleted plasma. To prepare the tubes, 2 micrograms (2 μ L of 1mg/ml solution) were deposited on the bottom of the microtubes, placing the tubes at 37 ℃ for 2-5 hours. Tubes prepared in this way can be kept at room temperature or at 37 ℃ for several weeks. To measure the T2MR signature, 34 μ L of citrate whole blood (or RBC depleted blood) was added to the tube at 37 ℃; after 1 minute, 2 μ L of 0.2MCaCl was added₂And the sample is placed in a magnet for relaxation measurement and analysis.

Frozen one-pot (one-pot) preparation for platelet ADP activation

We have developed a standard formulation for ADP activation of platelets in citrated whole blood that uses heparin addition to control thrombin activation, thus minimizing variability in platelet activation by thrombin. Coagulation in such systems is initiated using a combination of calcium (to overcome citrate) and snake venom thrombin/factor XIIIa (to replace thrombin). The complete one-pot activation mixture, which activates platelets through ADP and thus can be used to probe the status of the P2Y12 ADP receptor, contains heparin, snake venom thrombin, factor XIIIa, calcium and ADP.

We demonstrate that such mixtures can be prepared in small batches and frozen in single use aliquots (at-20 ℃) for the T2MR measurement of ADP-induced platelet activity measurements. The use of this mixture included melting a pre-prepared microtube with the appropriate amount of reagent mixture at room temperature, adding 34 μ L of citrate whole blood, mixing, and placing the sample into a reader for relaxation measurement analysis.

Dry kaolin assay

The kaolin runs allowed for resolution of serum and clot signals. These runs may show deviations in absolute T2 values from user error between patient samples. The use of dry reagents avoids pipetting errors and reduces reagent addition bias commonly encountered in all wet reagent experiments. In addition, the use of dry reagents provides a much simpler assay protocol, where blood is added directly to the tube containing the dry reagents, without an additional pipetting step.

Preparation of a catalyst containing CaCl₂And a dry mixture of kaolin. Adding 24 mu L0.2M CaCl₂And 10.8 μ L kaolin (Haemonetics) and aliquots were distributed into 10 PCR tubes (2.9 μ L of prepared mixture into each tube). The contents of each tube were dried in an incubator at 37 ℃ for more than 2 hours with the tube lid open. The tubes were capped and the tubes were stored at 2-25 ℃. To the pre-heated PCR tube containing the dry CK reagent mixture was added 36 μ L of pre-heated citrate whole blood. The reagent and blood were mixed 3 times by pipetting up and down with a 100 μ L pipette tip. The mixture was capped and the sample was placed in a T2reader for relaxation measurement analysis. Wet reagents were also mixed with citrate whole blood as a control.

Example 17: effect of Aspirin

In this experiment, platelet inhibition was monitored by ex vivo addition of 600 μ M aspirin to heparin blood prior to activation by AA + RF (test aspirin-containing or aspirin-free samples). A significant reduction or absence of clot-associated T2 signal was observed in the presence of the aspirin-incorporated samples. These studies demonstrated that clot characteristics are dependent on platelet activity. The utility of the T2MR signature for measuring platelet inhibition was also demonstrated.

Example 18: effect of 2-ThiomethylAMP (MeSAMP)

In this experiment, inhibition of the P2Y12 pathway in platelets was tested by using ex vivo incorporated mesmp (2-thiomethyl AMP, an irreversible ADPP2Y12 receptor inhibitor) as a mimic of the inhibition of platelet activity phivitamin.

Standard ADP activation preparations including heparin, snake venom thrombin, factor XIIIa, calcium and ADP were used to activate platelets in citrate whole blood obtained from healthy donors. The samples were tested in the presence and absence of the platelet activation inhibitor mesmp. Again, the clot profile was observed to be dependent on platelet activity. A significant reduction or absence of clot-associated T2 signal was observed in the presence of the samples spiked with mesmp.

Example 19: effect of tissue plasminogen activator

T2MR surfaces were also sensitive to whole blood fibrinolysis. To evaluate this, healthy donor samples were spiked with Tissue Plasminogen Activator (TPA). High sensitivity and fast time to fibrinolysis results were demonstrated.

TPA was added at 100U/mL to blood from healthy donors. In the absence of TPA (see fig. 27a), two stable peaks were formed at the expected set time. In the presence of 100U/mL TPA (see FIG. 27b), blood clots formed at the expected time, but proved unstable after 20-30 minutes. Both the instability and the distance between the final T2 peaks indicate sensitivity to fibrinolysis induced by the presence of TPA.

T2MR on fibrinolysis was acted on as represented by AU values (arbitrary units; see FIG. 27c) obtained by calculating the difference between the maximum and minimum T2 values for the clot signal (or T2B) in the T2 time curve. This difference was found to be sensitive to the degree of fibrinolysis in the sample.

Fibrinolysis was monitored by T2 magnetic resonance (T2MR) in blood samples from 5 patients with or without TPA (100U/mL) (fig. 27 c). 1 sample 27790, showed significant fibrinolysis without the addition of TPA. All other samples required the addition of TPA to induce fibrinolysis as demonstrated by the reference method.

Other embodiments

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Other embodiments are within the claims.

Cross Reference to Related Applications

The present application claims the benefit of united states provisional application No. 61/507,307 filed on 7/13/2011, united states provisional application No. 61/537,396 filed on 9/21/2011, united states provisional application No. 61/538,257 filed on 9/23/2011, united states provisional application No. 61/560,920 filed on 11/17/2011, united states provisional application No. 61/596,445 filed on 2/8/2012, and united states provisional application No. 61/625,945 filed on 4/18/2012, all of which are incorporated by reference herein.

Claims (32)

1. A method of monitoring a clotting or dissolution process of a first blood sample, the method comprising:

(i) performing a series of magnetic resonance relaxation rate measurements of water in said first blood sample;

(ii) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the first blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and

(iii) (iii) monitoring the process based on the results of step (ii),

wherein the method is not disease diagnostic.

2. The method of claim 1, wherein said first blood sample is a plasma sample, a whole blood sample, or a coagulation sample; or comprises isolated and washed platelets.

3. The method of claim 2, wherein prior to step (i), fibrinogen is added to said first blood sample.

4. The method of any one of claims 1-3, wherein prior to step (i), a clotting initiator or clotting inhibitor is added to said first blood sample; optionally a coagulation initiator selected from RF, AA, ADP, CK, TRAP and thrombin; platelet aggregation inhibitors or Tissue Plasminogen Activator (TPA); or a coagulation activator optionally selected from the group consisting of collagen, epinephrine, ristocetin, calcium, tissue factor, thromboplastin, 5-hydroxytryptamine, Platelet Activating Factor (PAF), thromboxane A2(TXA2), fibrinogen, von Willebrand factor (VFW), elastin, fibronectin, laminin, vitronectin, thrombospondin, lanthanide ions, and heparinase; or optionally a coagulation inhibitor selected from heparin and citrate.

5. The method of claim 1, further comprising:

(iv) making a series of second relaxation rate measurements of water in a second blood sample from the subject;

(v) transforming the second relaxation rate measurements using an algorithm that distinguishes two or more separate water populations within a second blood sample, wherein each separate water population is characterized by one or more magnetic resonance parameters, wherein each magnetic resonance parameter has one or more values; and

(vi) (vi) monitoring the process based on the results of step (ii) and step (v).

6. The method of claim 5, wherein (a) said first blood sample is a plasma sample and said second blood sample is a whole blood sample; or (b) the first blood sample is a platelet rich plasma sample and the second blood sample is a whole blood sample; or (c) the first blood sample is a platelet poor plasma sample and the second blood sample is a whole blood sample; or (d) the first blood sample comprises separated and washed platelets and the second blood sample is a whole blood sample; or (e) wherein prior to step (i), platelet inhibitor is added to said first blood sample and no platelet inhibitor is added to said second blood sample; or (f) wherein prior to step (i), platelet activator is added to the first blood sample and no platelet activator is added to the second blood sample; or (g) wherein prior to step (i), a clotting initiator selected from RF, AA and CK is added to the first blood sample, and prior to step (iv), a clotting initiator selected from ADP and thrombin is added to the second blood sample; or (h) wherein prior to step (i), fibrinogen is added to the first blood sample and prior to step (iv), fibrinogen is added to the second blood sample.

7. The method of claim 1, wherein said magnetic resonance parameter value is representative of functional fibrinogen-associated water molecules in said blood sample; or wherein at least one of the two or more separate aqueous populations is positively correlated with platelet activation, platelet inhibition, clotting time, platelet-associated clot strength, hematocrit, or fibrinogen-associated clot strength; or wherein the magnetic resonance parameter value indicates low platelet activity, high functional fibrinogen activity or low functional fibrinogen activity; or wherein the algorithm comprises an algorithm selected from the group consisting of: a multi-exponential algorithm, a bi-exponential algorithm, a tri-exponential algorithm, an exponential decay algorithm, a laplacian transform, a goodness-of-fit algorithm, an SSE algorithm, a least squares algorithm, and a non-negative least squares algorithm; or wherein the relaxation rate is selected from the group consisting of T1, T2, T1/T2 promiscuous, T_1rho、T_2rhoAnd T₂ ^*。

8. The method of claim 1, wherein the relaxation rate measurements comprise a T2 measurement, and wherein the measurements provide a decay curve, and wherein the two or more water populations comprise a water population having a serum-associated T2 signal and a water population having a clot-associated T2 signal.

9. The method of claim 8, further comprising (a1) prior to initiating clot formation in a blood sample containing red blood cells, calculating a T2 value for serum-associated water and determining the hematocrit of said blood sample based on said T2 value; or (a2) calculating the difference between the serum-associated T2 signal and the clot-associated T2 signal for a blood sample undergoing a clotting process; and determining the intensity of a clot formed in said blood sample based on said difference; or (a3) calculating the difference between the serum-associated T2 signal and the clot-associated T2 signal for a blood sample that contains platelets and is undergoing a clotting process; and determining the activity of platelets in said blood sample based on said difference; or (a4) measuring the time period prior to initial detection of the clot-associated T2 signal after initiation of a clotting process in a blood sample; and determining the clotting time of said blood sample according to said time period; or (a5) after initiating a clotting process in a blood sample, calculating a T2 time curve for the serum-associated T2 signal, calculating a maximum of the second derivative of the T2 time curve, and calculating a value representing clotting time; or (a6) wherein after a clotting process is initiated in a blood sample, it is determined whether the blood sample is hypercoagulable, hypocoagulable, or normal based on the serum-associated T2 signal and the clot-associated T2 signal.

10. The method of claim 1, further comprising, after initiating a clotting or dissolution process in a blood sample, (a) making a plurality of relaxation rate measurements on said blood sample during said process to produce a plurality of decay curves, and (b) calculating a plurality of T2 relaxation spectra from said plurality of decay curves.

11. The method of claim 10, further comprising:

-calculating from the plurality of T2 relaxation spectra a three-dimensional data set describing: a change in (a) T2 relaxation time, and (b) a change in T2 signal intensity over time of two or more water populations in the blood sample after initiation of a clotting or lysis process in the blood sample; and optionally, dividing the three-dimensional data set into stable data and transitional data, and determining whether the blood sample is hypercoagulable, hypocoagulable, or normal based on the stable data and the transitional data; or

-determining whether the blood sample shows low platelet activity, high functional fibrinogen activity or low functional fibrinogen activity; or

-calculating from the three-dimensional data set the relative volume of signal observed for each of the two or more water populations in the blood sample over a predetermined time in the process, and determining from the relative volumes of the signals whether the blood sample is hypercoagulable, hypocoagulable or normal; or

-determining whether the blood sample shows low platelet activity, high functional fibrinogen activity or low functional fibrinogen activity.

12. The method of claim 10, wherein the method:

-for evaluating platelet activity, the method comprising: (ai) providing isolated and washed platelets; (aii) mixing the separated and washed platelets with platelet poor plasma comprising a predetermined minimum level of fibrinogen to form a test sample; (aiii) initiating a coagulation process by adding a coagulation initiator to the test sample; (aiv) performing a series of magnetic resonance relaxation rate measurements on the water in the test sample; (av) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the test sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (avi) evaluating said platelet activity based on the results of step (av); or

-for evaluating platelet activity in a whole blood sample, the method comprising: (bi) providing a whole blood sample; (bii) initiating a clotting process by adding a clotting initiator to the test sample; (biii) making a series of magnetic resonance relaxation rate measurements of the water in the test sample; (biv) transforming the measurements using an algorithm that distinguishes two or more separate water populations within the test sample, wherein each separate water population is characterized by one or more magnetic resonance parameters having one or more values; and (bv) evaluating the platelet activity based on the result of step (biv).

13. The method of claim 1, further comprising determining whether the whole blood is hypercoagulable, hypocoagulable, or normal.

14. The method of claim 4, wherein prior to step (i), a clotting initiator is added to said first blood sample, wherein said clotting initiator induces activation of clotting by a contact activation pathway or a tissue factor activation pathway.

15. The method of claim 14, wherein the coagulation initiator is tissue factor.

16. A method of evaluating platelet activity, the method comprising:

(i) providing isolated and washed platelets;

(ii) mixing the separated and washed platelets with platelet poor plasma comprising a predetermined minimum level of fibrinogen to form a test sample;

(iii) initiating a coagulation process by adding a coagulation initiator to the test sample;

(iv) performing a series of magnetic resonance relaxation rate measurements on the water in the test sample;

(v) transforming the measurements using an algorithm that distinguishes two or more independent water populations within the test sample, wherein each independent water population is characterized by one or more magnetic resonance parameters having one or more values; and

(vi) (vi) evaluating the platelet activity based on the results of step (v),

wherein the method is not disease diagnostic.

17. A method of assessing the intensity of a blood clot, the method comprising the steps of:

(i) making a T2 relaxation rate measurement of water in a blood clot, wherein the measurement provides a decay curve;

(ii) applying a mathematical transformation to the attenuation curve to identify a signal intensity of a water population in the blood clot, the water population being in a serum water environment or a contracted blood clot water environment; and

(iii) evaluating the intensity of the blood clot based on the signal intensity,

wherein the method is not disease diagnostic.

18. A method of evaluating platelet activity of a blood clot, the method comprising the steps of:

(i) making a T2 relaxation rate measurement of water in a blood clot, wherein the measurement provides a decay curve;

(ii) applying a mathematical transformation to the attenuation curve to identify signal intensities of an aqueous population of the blood clot, the aqueous population being in a serum aqueous environment or a contracted blood clot aqueous environment; and

(iii) evaluating the platelet activity of the blood clot based on the signal intensity,

wherein the method is not disease diagnostic.

19. A magnetic resonance device for monitoring a blood clotting process, the device comprising a blood sample containing a clotting process and a clotting initiator,

wherein after the coagulation process has begun, the device is configured to make a plurality of relaxation rate measurements of the blood sample during the coagulation process to produce a plurality of decay curves and calculate a plurality of T2 relaxation spectra from the plurality of decay curves, an

Wherein the device comprises a microprocessor with an algorithm to distinguish two or more separate water populations within the blood sample by the plurality of T2 relaxation spectra, wherein each separate water population is characterized by a magnetic resonance parameter value or set of values.

20. The device of claim 19, wherein the blood sample is a plasma sample, a whole blood sample, or a coagulation sample, or comprises separated and washed platelets.

21. The apparatus of claim 19, further comprising an algorithm for: (a) calculating the difference between the serum-associated T2 signal value and the clot-associated T2 signal value; and (b) determining the activity of the platelets in the blood sample based on the difference.

22. The apparatus of claim 19, further comprising an algorithm for determining a period of the clotting process prior to the beginning of detection of the clot-associated T2 signal.

23. The apparatus of claim 19, further comprising an algorithm for: (a) calculating a T2 time curve for the first water population; (b) calculating the maximum value of the second derivative of the T2 time curve; and (c) calculating a value representing the clotting time based on the results of step (b).

24. The apparatus of claim 19, further comprising an algorithm for: (a) calculating a T2 value for the blood sample prior to initiating clot formation, and (b) determining the hematocrit of the blood sample based on the T2 value.

25. The device of claim 19, said blood sample comprising added fibrinogen.

26. The device of claim 19, wherein the coagulation initiator is tissue factor.

27. The device of claim 19, wherein the blood sample has a volume between 2 μ L and 400 μ L.

28. The device of claim 27, wherein the blood sample has a volume between 2 μ L and 250 μ L.

29. The device of claim 27, wherein the blood sample has a volume between 2 μ L and 50 μ L.

30. The device of claim 19, wherein the blood sample has a volume between 200 μ L and 500 μ L.

31. The method of claim 2, wherein said first blood sample is a plasma sample that is a platelet poor plasma sample or a platelet rich plasma sample.

32. The device of claim 20, wherein the blood sample is a plasma sample that is a platelet poor plasma sample or a platelet rich plasma sample.