WO2011159820A1

WO2011159820A1 - Device

Info

Publication number: WO2011159820A1
Application number: PCT/US2011/040561
Authority: WO
Inventors: Benny Sorensen; Jorgen Ingerslev
Original assignee: Bayer Healthcare Llc
Priority date: 2010-06-15
Filing date: 2011-06-15
Publication date: 2011-12-22

Abstract

The invention is directed to a method for measuring a coagulation event by the continuous recording of the coagulation process. This method may be used to optimize the dosage of a procoagulant or anticoagulant for on demand or prophylaxis treatment. The invention is also directed to a device which incorporates this method.

Description

DEVICE

FIELD OF THE INVENTION

[001 ] The invention is directed to a method for measuring a coagulation event by the continuous recording of the coagulation process. This method may be used to optimize the dosage of a procoagulant or anticoagulant for on demand or prophylaxis treatment. The invention is also directed to a device which incorporates this method.

BACKGROUND OF THE INVENTION

[002] Blood coagulation is a complex chemical and physical reaction that occurs when blood comes into contact with an activating agent. The blood coagulation process can be generally viewed as two main activities: primary haemostasis and secondary haemostasis. Establishment of a primary haemostatic plug is characterized by platelet adhesion, platelet activation and platelet aggregation. Secondary haemostasis is triggered by tissue factor or contact activation and is characterized by assembling of coagulation factors on the surface of activated platelets. Activation of coagulation factors lead to dynamic generation of thrombin and cleavage fibrinogen and spontaneous

polymerization to a sticky fibrin network. In vivo, platelets and coagulation factors flow through the blood vessels in an inactivated state. The blood vessel lining, the endothelium, prevents uncontrolled activation of platelets and coagulation factors. When a blood vessel is damaged, however, the endothelium loses its integrity and von willebrand factor and platelets are exposed to and activated by contact with extravascular tissue underlying the damaged site. Activation of the platelets causes them to become "sticky" and adhere together. This process continues until a platelet "plug" is formed. This platelet plug then serves as a matrix upon which blood clotting or coagulation proceeds.

[003] If the chemical balance of the blood is suitable, thrombin is then produced which leads to the conversion of fibrinogen to fibrin, which forms the major portion of the clot mass. During clotting, additional platelets are activated and trapped in the forming clot, further contributing to clot formation. As clotting proceeds, polymerization and cross-linking of fibrin results in the permanent clot. Clotting may be measured by triggering the intrinsic pathway, which involves the activation of Factor XII (FXII), or by the extrinsic pathway, which involves the release of tissue factor.

[004] A series of pro- and anticoagulant factors are involved in directing and regulating the formation of a blood clot. These factors include Factor I (fibrinogen), Factor II (prothrombin), Factor V (FV), Factor VII (FVII), Factor VIII (FVIII), Factor IX (FIX), Factor X (FX), Factor XI (FIX), and FXII. The extrinsic pathway is triggered when tissue factor binds and activate circulation coagulation factor VI VTIa. Tissue factor-FVIIa activates FIX and FX. Activated FX initiates conversion of prothrombin to thrombin. Thrombin activates platelets, FV, FVIII, FXI and FXIII. The enzyme FIXa in the presence of FVIIIa constitute the intrinsic tenase complex and in the presence of phospholipids activates FX. Activated FX form a complex with activated FV - the prothrombinase complex, that can convert large amounts of prothrombin to thrombin and thereby induce a propagation phase of thrombin generation. The intrinsic pathway is triggered when the proenzyme FXII is converted to its enzyme FXIIa which in turn converts the zymogen FXI to the enzyme FXIa, which then activates FIX in the presence of calcium.

[005] Dynamic thrombin generation and clot formation is the momentum of the haemostatic process. Thrombin generation is described via i) initiation phase (initial activation of factor X by tissue factor-FVIIa), ii) amplification phase (establishment of intrinsic tenase and prothrombinase complexes), and iii) propagation phase - excessive production of thrombin driven by the

prothrombinase complex.

[006] Deficiencies in blood coagulation factors can result in severe hematological disorders. For example, hemophilia A is a congenital bleeding disorder resulting from an X-chromosome -linked deficiency of FVIII, occurring with a frequency of 1 in 5000 males. It is caused by either a quantitative or a qualitative deficiency in FVIII, a critical component of the amplification and propagation of thrombin formation and clot formation. Currently, hemophilic patients are treated by intravenous administration of plasma-derived or recombinant FVIII on demand or as a prophylactic therapy administered two or more times a week; and the appropriate dosage for effective treatment depends on numerous factors such as body weight, residual coagulation factor levels, individual pharmacokinetics, individual pharmacodynamics and the type of bleeding. Thus, accurate measurement of blood coagulation would be important in treating hematological disorders.

[007] Venous thromboembolism (VTE) is a condition in which a blood clot (thrombus) forms in a vein. Blood flow through the affected vein can be limited by the clot, and may cause swelling and pain. Venous thrombosis occurs most commonly in the deep veins of the leg or pelvis; this is known as a deep vein thrombosis (DVT). An embolism occurs if all or a part of the clot breaks off from the site where it forms and travels through the venous system. If the clot lodges in the lung a potentially serious and sometimes fatal condition, pulmonary embolism (PE) occurs. Venous thrombosis can occur in any part of the venous system. However, DVT and PE are the commonest manifestations of venous thrombosis. The term VTE embraces both the acute conditions of DVT and PE, and also the chronic conditions which may arise after acute VTE-such as post thrombotic syndrome and pulmonary hypertension-both problems being associated with significant ill-health and disability. Thus, accurate measurement of blood coagulation, would be important in the diagnosis of VTE.

[008] Precise measurement of blood coagulation and blood coagulation factor activity is critical for both preclinical and clinical applications. Quantification of blood coagulation factor plasma levels is crucial for accurate measurement of pharmacokinetics in preclinical models and disease severity in clinical applications. For example, accurate determination of the level of procoagulant FVIII is necessary in order to assess severity in hemophilia A patients and to correlate clinical phenotype with circulating FVIII levels. Several different methods have been developed to quantify FVIII concentration in plasma including clotting methods, chromogenic assays, thrombin generation assays, and activated partial thromboplastin time (aPTT) clot waveform analysis. Since the current assays involve bioassays, they can exhibit problems of poor reproducibility due to complex reaction kinetics. In addition, these assays measure a specific timepoint in the clotting process. For example, measurements based on aPTT methods reflect only the initial phase of clot formation.

[009] For a more accurate assessment of the clotting process, a method that records and visualizes the rate specific and dynamic properties of the entire coagulation process (e.g., continued generation of thrombin and formation of a platelet-fibrin clot) may provide a more effective means to monitor the efficacy of treatment. In addition, such a method could provide an individually tailored treatment protocol for the patient as well as an optimal cost benefit for patients.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method for measuring a coagulation event in a biological sample comprising the steps of collecting a biological sample; recording a coagulation event in the biological sample, deriving dynamic parameter of the coagulation event; and comparing the dynamic parameter of the biological sample with the dynamic parameter of a control sample. A change in the dynamic parameter of the biological sample compared to the dynamic parameter of a control sample indicates the hemostatic response or hemostatic capacity. In one embodiment, the coagulation event may be based on triggering the coagulation process with tissue factor (prothrombin time) or by contact activation (aPTT or a continuous aPTT profile). In another embodiment, the dynamic parameter may be clot initiation, clot propagation or clot termination or clotting velocity such as maximum clotting velocity. In a further embodiment, the biological sample is whole blood, plasma, platelet poor plasma, or platelet rich plasma.

[0011 ] The method of the present invention may be used to measure a coagulation event after administration of a procoagulant or anticoagulant to a patient or after addition of a procoagulant or anticoagulant to a sample. In one embodiment, the method may also be used to optimize the dosage of a procoagulant or anticoagulant for on demand or prophylaxis treatment. In another embodiment, the method may be used to optimize the dosage of a procoagulant or anticoagulant based on the measurement of a coagulation event after administration of a procoagulant or anticoagulant to a patient or after addition of a procoagulant or anticoagulant to a sample. In one embodiment, the procoagulant or anticoagulant may have a defined activity. In a further embodiment, the procoagulant may be FVIII, Factor IX (FIX), Factor VII (FVII), factor Vila, anti-Tissue Factor Pathway Inhibitors (TFPI), and Tissue Factor (TF) or any anticoagulant directly or indirectly targeting Factor Xa, factor IXa, or thrombin or other coagulation associated factor. In another embodiment, the patient has a hematological disorder selected from coagulation factor deficiency (hemophilia), hypercoagulation, or arterial or venous thromboembolism.

[010] The present invention is also directed to a device for measuring a coagulation event in a biological sample comprising a sample receiving area; a means for recording a coagulation event; a means for deriving a dynamic parameter of the coagulation event; and a means for generating a clotting profile and response pattern. In a further embodiment, the device comprises a means to transmit data to care center or physician. In one embodiment, the device may be individually calibrated for each patient. In another embodiment, the individual calibration is performed using dynamic parameters. In other embodiments, the individual calibration may be performed using a dynamic parameter of the coagulation event measured after the administration of a procoagulant or anticoagulant to a patient or after addition of a procoagulant or anticoagulant to a sample. The device of the present invention may be used by a patient to optimize the dosage of a procoagulant or anticoagulant for on demand or prophylaxis treatment. In one embodiment, the procoagulant may be FVIII, FIX, FVII or FVIIa, anti-TFPI, or TF or any anticoagulant directly or indirectly targeting Factor Xa, factor IXa, or thrombin or other coagulation associated factor.

[011 ] The device of the present invention may be a hand-held portable device. In one embodiment, the sample receiving area comprises a receiving area for test strips. In another embodiment, the biological sample is collected on a test strip. In a further embodiment, the test strip comprises one or more coagulation activators such as FXII activator or TF or TF and corn trypsin inhibitor.

[012] The present invention is directed to a kit comprising the device described herein, lancets, alcohol swabs, cotton pads, bandages, control samples, and calibration standards.

DESCRIPTION OF THE DRAWINGS

[013] Figure 1. The dynamic parameters of a continuous aPTT coagulation signal.

[014] Figure 2. Dynamic profiles of aPTT plasma clotting in patients with severe hemophilia.

[015] Figure 3. Dynamic profiles of aPTT-MaxVel plasma clotting in patients with severe hemophilia.

[016] Figure 4. FVIII titration study on platelet-poor plasma from patients with severe hemophilia.

[017] Figure 5. Vector-ratio analysis of FVIII concentrations.

[018] Figure 6. Individual patient ex vivo titration profiles. [019] Figure 7. Dynamic fibrin polymerization and continuous thrombin generation.

[020] Figure 8. Dynamic aPTT plasma clotting profiles in patients versus healthy individuals.

[021 ] Figure 9. Dynamic aPTT plasma clotting profiles (aPTT, aPTT-MaxVel, aPTT-t, MaxVel, aPTT-MCF) in patients versus healthy individuals.

[022] Figure 10. Dynamic PT plasma clotting profiles (PT, PT-MaxVel, PT-t, MaxVel, PT-MCF) in patients, patients with a positive biochemical risk profile for venous thromboembolism, patients with a negative biochemical risk profile for venous thromboembolism versus a control group of healthy individuals

DESCRIPTION OF THE INVENTION

[023] It is to be understood that this invention is not limited to the particular device or parts described and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[024] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" is a reference to one or more samples and includes equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[025] As used herein, the term "coagulation event" refers to the stages of clot formation during hemostasis.

[026] The term "biological sample" refers to a sample obtained from an organism. The sample may be any biological tissue or fluid. For example, the sample may be whole blood, plasma, platelet-poor plasma, or platelet-rich plasma.

[027] The term "dynamic parameter" refers to description of rate specific characteristics of thrombin or clot formation. Examples of dynamic parameters include, but are not limited to, clot initiation, time to clot initiation, clot propagation, clotting velocity, maximum velocity, time to maximum velocity of clot formation, time to peak and clot termination.

[028] The term "hemostatic response" refers to the effect of a particular agent on coagulation. The agent may be, for example, a procoagulant or anticoagulant. [029] The term "hemostatic capacity" refers to the efficacy of a particular agent on coagulation. The agent may be, for example, a procoagulant or anticoagulant.

[030] The term "maximum clotting velocity" refers to maximum rate of clot formation, thus the maximum speed by which blood changes in physical condition from being fluid to becoming a coagulum.

[031 ] The term "procoagulant" refers to any agent that promotes blood coagulation. For example, a procoagulant may be any blood clotting factor zymogen which may be activated to form a clotting factor serine protease or a pro-cofactor which is needed for a clotting factor activity. The term procoagulant includes, for example, thrombin, Factor V (FV), FVII, FVIII, FIX, Factor X (FX), Factor XI (FXI), prothrombin, or fibrinogen which can be activated to form FVa, FVIIa, FVIIIa, FIXa, FXa, FXIa, thrombin, and fibrin, respectively.

[032] The term "anticoagulant" refers to any agent that prevents the formation of blood clots such as heparin, warfarin, antithrombin III, direct or indirect factor Xa inihibitor, or direct thrombin inhibitor.

[033] The term "defined activity" refers to the physiological response a drug produces. For example, a procoagulant promotes blood coagulation.

[034] Traditional plasma coagulation analyses, such as aPTT and prothrombin time (PT), only provide information on early events of initiation of clot formation. However, following the initiation of clot formation, there is a rate specific dynamic development of the clot. Continuous profiles of plasma clotting may be obtained. For example, by adopting simple signal processing including differentiation and filtration, dynamic profiles and parameters may be generated.

[035] The method of the present invention provides a means to measure whole blood clotting in a sample based on continuous recording of the clotting process. The information provided by this continuous recording may be used to derive additional data such as the maximum velocity (MaxVel) of clotting and the time to maximal velocity (t,maxvel) in a sample (e.g., a blood sample). These values illustrate more completely the dynamic properties of blood coagulation as compared to the clotting time (e.g., aPTT or PT).

[036] Using this strategy, the hemostatic response in an individual patient may be mapped at, for example, multiple concentrations of FVIII or FIX using a titration principle. Based on the ability of the patient' s blood to produce an increase in the MaxVel combined with reduced clotting time, calculations may be performed to estimate the correction required to sufficiently improve blood clotting. Measuring a coagulation event in whole blood may more completely illustrate the hemostatic capacity of blood clotting because a procoagulant intended to correct a patient' s hemostatic deficiency may only exerts its full activity in the environment of whole blood as compared to plasma.

[037] The clinical bleeding phenotype in patients with hemophilia A varies considerably. As such, the dynamic and rate-specific properties of thrombin generation and clot formation may provide more details in the biologic heterogeneity among patients with hemophilia. One feature of this method may be the ability to record a continuous and rate-specific profile of clot formation. These continuous dynamic profiles of whole blood clot formation reflect the pattern of dynamic thrombin generation.

[038] As an example, the dynamic parameters of the aPTT clotting pattern may be obtained by simple signal processing. The dynamic aPTT parameter aPTT-MaxVel has been shown to reflect more biological heterogeneity among patients with severe hemophilia A than the standard aPTT recording. This may partly reflect the ability of the dynamic aPTT parameters to detect and visualize differences among patients at functional levels of FVIII below one percent (1 %). In addition, the dynamic parameters of aPTT based plasma clotting correlated to the rate specific characteristics of thrombin generation. Furthermore, evaluation of the dynamic response patterns following in vivo infusion of rFVIII may be useful for estimating the functional importance of various dosages and formulations of rFVIII.

[039] As described herein, in vitro rFVIII titration experiments followed by response evaluation using the calculation of vector-based ratios were useful for estimating pharmacodynamic response characteristics in individual patients. The in vitro estimated response pattern correlates well with the response observed following infusion of rFVIII. As such, individualized in vitro rFVIII spiking experiments may serve as an additional tool for selecting appropriate dose regimens - both for patients on prophylaxis as well as patients treated on demand. The pharmacodynamic vector ratios also correlated with the recovery findings, providing information on the clotting function response of different concentrations of FVIII.

[040] The method of the present invention may be used to assess other hematological disorders such as hypercoagulation and arterial and venous thromboembolism (VTE). Risk factors for venous thromboembolism include surgery, immobilization, vessel damage, anti-phospholipid antibodies as well as inherited disorders such as FV Leiden and prothrombin polymorphisms and deficiencies in antithrombin or protein C or S. The aPTT-MaxVel of fibrin formation represents a stronger predictor of hypercoagulation than standard aPTT measures in patients with objectively documented VTE.

[041 ] The management of patients with hemophilia, hypercoagulation, venous thromboembolism, or other hematological disorders may be improved by providing an individually calibrated point-of- care evaluation of the patient' s baseline clotting capacity and the patient's clotting capacity following treatment with a procoagulant or anticoagulant. Individualized dosing and continuous monitoring of clotting capacity may minimize the risks of hemorrhage or thromboembolism. Thus, the method of the present invention may be used to generate individually tailored treatment regimens for patients with hemophilia, hypercoagulation, venous thromboembolism, or other hematological disorders and as such may contribute to the improvement of each patient's quality of life as well as providing a favorable cost/benefit ratio.

Device

[042] The present invention is also directed to a device which incorporates the method described herein to monitor, for example, a patient's clotting capacity. For example, the device may be a handheld portable device that records continuous blood clotting parameters in a sample such as whole blood, platelet-poor plasma, or platelet-rich plasma. Samples may be deposited on a disposable test strip and inserted into the device for measurement of one or more dynamic parameters. The test strip may include a coagulation activator, for example, FXII activator, TF, or FXII activator and TF, or TF and corn trypsin inhibitor.

[043] The device may be individually calibrated according to the patient's baseline hemostatic capacity or clotting profile and/or the patient's individualized response to a given treatment.

Calibration of the device may be accomplished using algorithms that correct for a coagulation factor abnormality, for example, utilizing Pythagoras vector analysis to calculate a change in the clotting process.

[044] The device may also include a dynamic image of the clotting profile and the patient's response pattern. The device may also provide automated guidance on the patient's dosage requirement for a procoagulant or anticoagulant. In addition, the device may also transmit the patient's clotting profile and other data to the patient's physician or care center.

[045] In general, the device may include a sample receiving area, a means for recording a coagulation event, a means for deriving a dynamic parameter of the coagulation event, and a means for generating a clotting profile and response pattern.

Kits

[046] The invention further provides kits that may be used, for example, by patients or healthcare providers to monitor the effectiveness of treatment or optimize dosage for a more effective treatment. The kit may comprise a device as described herein. The kit may further comprise lancets, test strips, alcohol swabs, cotton pads, and bandages. The kit may also comprise control samples (e.g., human blood or plasma) and calibration standards.

[047] The devices, methods, and materials described herein are intended to be representative examples of the invention, and it will be understood that the scope of the invention is not limited by the scope of the examples. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed devices, methods, and materials, and such variations are regarded as within the ambit of the invention.

EXAMPLES

[048] In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.

Example 1. Patients

[049] Forty-eight males with severe hemophilia A (FVIII:C < 0.01 IU/mL) were entered into this study. The mean age was 30 years (range 1-66) and the mean body weight was 63 kg (range 10-106). These patients did not present with inhibitors (Bethesda: < 0.06 IU/mL). All patients had not received FVIII during at least 5 days prior blood sampling. Evaluation of the FVIII response (recovery) was performed in 34 of the 48 patients. The overall mean recovery was 116% (60-220). The demographic and laboratory data are listed in Table 1. All patients were treated with recombinant FVIII concentrate. All patients had not received FVIII during at least 5 days prior blood sampling.

Reference values were obtained from analyses of plasma in 42 healthy males with a mean age of 42 years (95%CI = 38 - 46).

M§3F5 |3¼Rg©} Example 2. Blood Sampling Procedure

[050] Blood samples were drawn following smooth venipuncture employing minimal stasis, discarding the first tube aspirated, and stored in siliconized glass tubes (3 mL) with EDTA

(VenoJect®, Terumo Europe, Leuven, Belgium), sodium fluoride/sodium heparin (Vacuette®, Greiner bio-one GmbH, Kremsmiinster, Austria), or 3.8% trisodium citrate (VenoJect®), as appropriate. Plasma for the routine coagulation analyzes were obtained after centrifugation at 2800 x g for 25 minutes and stored in 500 μΐ_^ aliquots at -80 °C until analysis.

Example 3. Dynamic Plasma Clotting Parameters

[051 ] The aPTT was recorded using platelet-poor plasma (100 μυ), Platelin LS® (BioMerieux, Durham, France) as test reagent (100 μυ), and 25 mM calcium chloride (100 μυ). The analyses were performed using a BCT Coagulation Analyzer (Dade Behring, Marburg, Germany) and the aPTT results were given as the mean of two determinations. The continuous aPTT coagulation signal was exported as an ASCII data file from the computer connected to the BCT Coagulation Analyzer. The dynamic parameters of the aPTT clotting signal were calculated using DyCoDerivAn Platinum (AvordusoL, Risskov, Denmark) (Figure 1, panel A). To enhance the dynamic profile, the first time derivative is calculated. Since differentiation enhances high frequency components, the raw signal is initially filtered using a discrete centralized moving average filter (finite impulse response class) with symmetric coefficient ensuring a linear phase characteristic, hence causing no distortion of the signal in time domain (Figure 1, panel B). After filtering, the differentiation is performed according to Formula 1 :

where x_; is the * element of the n-sample long clotting data array, t is the time between each sample of the clotting data, and is the * element of the differentiated data. In the differentiated data row, the maximum value (aPTT-MaxVel) is located and the time at which it occurs is determined (aPTT-t, MaxVel) (Figure 1, panel B).

[052] As compared to healthy males, the dynamic aPTT clotting profile of patients with severe hemophilia A were characterized by a prolonged initiation phase, corresponding to the second value of the traditional aPTT, followed by a very slow clot development (aPTT-MaxVel), reaching a final maximum amplitude (aPTT-MCF) comparable to the amplitude of healthy individuals or even slightly higher (Table 1 and Figure 2).

[053] Patients with severe hemophilia A showed pronounced variability in the dynamic aPTT clotting profile (Figure 2). The standard aPTT result varied from a minimum of 62 seconds to a maximum of 117 seconds (ratio = 1.9), whereas the aPTT-MaxVel ranged from 14.4 to 49.3 (ratio = 3.4) (Figure 3). Hence, the dynamic parameters of the aPTT clotting signature appears to be more heterogeneous than the standard aPTT itself.

Example 4. Titration Analysis

[054] Various amounts of recombinant FVIII (0.005 U/mL - 3.00 U/mL) were added to platelet- poor plasma from patients with severe haemophilia A. These in vitro spiking experiments showed a dose-dependent reversal of the clotting profile by shortening of the aPTT and increasing the aPTT- MaxVel (Figure 4). Using the mathematical algorithm of Pythagoras (a² + b² = c²), it was possible to estimate the minimal concentration of FVIII needed to normalize the clotting pattern. Based on average values of the healthy males, a range of normal plasma aPTT-t, MaxVel and aPTT-MaxVel was established (Figure 5). Addition of FVIII to hemophilic plasma was considered effective if the result fell within the range of the controls. The expected changes were opposed to the observed changes. From these data, a vector-ratio was calculated (observed/expected) (Figure 5). When this vector-ratio reached the value 1, the changes observed equalled the expected changes. In plasma from eight different patients (6 patients with severe hemophilia A and 2 patients with severe hemophilia B), the final reaction concentration of FVIII inducing a vector-ratio of 1 ranged from 0.30 U/mL to 1.10 U/mL (Figure 6). Hence, a pronounced inter-individual variation was observed. The individual ex vivo titration response characteristics corresponded well with the response observed following infusion of rFVIII (Figure 6).

Example 5. Thrombin Generation

[055] The materials used in measurement of continuous thrombin generation included: 1) platelet- poor plasma (PPP) from a healthy male and a patient with severe haemophilia A; 2) Platelin® LS as test reagent; 3) calcium chloride; 4) microtiter plates; 5) stop reagent consisting of a protease inhibitor cocktail (Complete Mini®, Roche) with 50 mM EDTA in a HEPES buffer, pH 7.4; 6), a table centrifuge; and 7) thrombin-antithrombin (TAT) complex recording ELISA kits (Dade Behring, Marburg, Germany). The experiments were performed by mixing PPP, Platelin® LS, and calcium chloride in wells of microtiter plates. The clotting process was stopped at predetermined time intervals by quenching with the inhibitor cocktail. The reaction mixture from each test tube was centrifuged 10,000 rpm for 2 minutes and then the supernatant was pipetted off. The amount of thrombin generated was recorded as TAT complexes at each time point of sampling using the TAT ELISA kit and an ELISA reader.

[056] The results of continuous thrombin generation are shown in Figure 7. In healthy individuals, an early initiation of aPTT plasma clotting, followed by a brisk clot development correlated with an early and rapid increase in the total amount of thrombin generated. In contrast, in severe hemophilia A where the dynamic aPTT plasma clotting profile was characterized by a prolonged clot initiation followed by a very slow clot development, thrombin generation also initiated late and increased comparably slowly. The dynamics of aPTT plasma clotting thereby paralleled the dynamics of thrombin generation. Noticeable, in the healthy individual, measurable thrombin generation appeared to concur second to early clot development.

[057] All statistic analyses were performed using Analyse-it™ version 1.62 (Analyse-it Software, Ltd., UK) a statistical add-in program for Excel® (Microsoft®, USA). Descriptive statistics of the dynamic aPTT parameters (aPTT, aPTT-MaxVel, aPTT-t, MaxVel and aPTT-MCF) were used to assess the type of statistical distribution for proper description of the data. Normal distribution of data was evaluated based on graphical and mathematical interpretation of histograms and Q-Q plots. Differences among independent groups were tested using unpaired students t-test, whereas differences of paired data were evaluated by a paired student's t-test. Threshold for stating statistical significance was set at a p-value < 0.05.

Example 6. Dynamic Clotting Profiles in Patients with VTE

[058] The dynamic aPTT clotting profiles of patients suffering from a previous episode of VTE were compared to healthy references and were characterized by an increased maximum rate of fibrin formation together with a shortened initiation of clot formation in some of patients (Figure 8).

Patients had a significantly higher mean aPTT-MaxVel (195.5 sec SD=57, 95 CI: 176.8-214.1) as compared with healthy controls (137.3 sec SD=31, 95% CI: 130.7-143.8) (Figure 9). Patients also had a significantly shorter mean aPTT (26.9 sec, SD=3.2, 95%CI: 25.9-28.0) than healthy controls (28.5 sec, SD=2.8, 95%CI: 27.9-29.0). As depicted in Figure 9, patients with a positive biochemical risk profile had a significantly (p<0.05) higher aPTT-MaxVel (221.5 sec^"1, SD=59, 95%CI: 193.9-

259.1) of clotting than patients with no known risk profile (167.8 sec SD=39, 95%CI: 147.8-187.9), whereas the aPTT was not significantly (p=0.46) different among the two groups (pos. risk profile: 26.6 sec, SD=3.1, 95%CI: 25.1-28.0; neg. risk profile: 27.3 sec, SD=3.5, 95%CI: 25.6-29.1).

Example 7. Dynamic visualization and derived data of tissue factor stimulated clotting time (prothrombin time)

[059] The prothrombin time (PT) was measured using Innovin® and a BCT Coagulation Analyzer (Dade Behring, Marburg, Germany). Dynamic parameters such as PT -MaxVel, PT-t, MaxVel, PT- MCF was derived using an in-house developed software program DyCoDerivAn Platinum

(AvordusoL, Risskov, Denmark).

[060] A study measuring PT was performed comprising 38 patients (17 males, 21 females) with verified venous thromboembolism, wherein 19 of 38 patients had a positive biochemical risk profile for venous thrombo-embolism (Factor V leiden mutation, increased FVIII, etc.), and a control group comprising 88 healthy individuals (42 males, 46 females). Comparing the study group with the control group, it was found that patients had a significantly higher PT-MaxVel (ref: 145,906 (95% CI: 139,263; 152,548) vs. pt: 188,978 (95% CI: 169,023; 208,933)); p=0.002. The PT-t,MaxVel was shorter in patients (ref: 8,551 (95% CI: 8,454; 8,648) vs. pt: 8,232 (95% CI: 8,063; 8,402));

p=0.00886. The MCF was higher in patients (ref: 704,761 (95% CI: 659,255; 750,267) vs. pt: 715,081 (95% CI: 663,062; 767,101)); p=0.0282. (See Figure 10). Patients with a positive biochemical risk diagnosis revealed even more pronounced differences.

Claims

CLAIMS We claim:

1. A method for measuring a coagulation event in a biological sample comprising:

collecting a biological sample;

recording a coagulation event in the biological sample;

deriving a dynamic parameter of the coagulation event; and

comparing the dynamic parameter of the biological sample with the dynamic parameter of a control sample;

where a change in the dynamic parameter of the biological sample compared to the dynamic parameter of a control sample indicates the hemostatic response or hemostatic capacity.

2. The method of claim 1 , wherein the coagulation event is aPTT.

3. The method of claim 2, wherein the coagulation event is a continuous aPTT profile.

4. The method of claim 1, wherein the coagulation event is prothrombin time (PT).

5. The method of claim 4, wherein the coagulation event is a continuous PT profile.

6. The method of claim 1, wherein the dynamic parameter is clot initiation, clot propagation, clot termination, or clotting velocity.

7. The method of claim 6, wherein the clotting velocity is the maximum clotting velocity.

8. The method of claim 1, wherein the biological sample is whole blood, plasma, platelet poor plasma, or platelet rich plasma.

9. The method of claim 1, wherein the method is used to measure a coagulation event after

administration of a procoagulant or anticoagulant to a patient.

10. The method of claim 1, wherein the patient has a hematological disorder selected from

hemophilia, hypercoagulation, or arterial or venous thromboembolism.

11. The method of claim 1 , wherein the method is used to measure a coagulation event after

addition of a procoagulant or anticoagulant to a sample.

12. The method of claims 9 and 11, wherein the procoagulant or anticoagulant has a defined

activity.

13. The method of claim 1, wherein the method is used to optimize the dosage of a procoagulant or anticoagulant for on demand or prophylaxis treatment.

14. The method of claim 1, wherein the method is used to optimize the dosage of a procoagulant or anticoagulant based on the measurement of a coagulation event after administration of a procoagulant or anticoagulant to a patient.

15. The method of claim 1, wherein the method is used to optimize the dosage of a procoagulant or anticoagulant based on the measurement of a coagulation event after addition of a procoagulant or anticoagulant to the sample.

16. The method of claims 14 and 15, wherein the procoagulant or anticoagulant has a defined

activity.

17. The method of claims 9 to 16, wherein the procoagulant is selected from FVIII, FIX, FVII, anti-TFPI, TF, and any anticoagulant directly or indirectly targeting Factor Xa, factor IXa, or thrombin or other coagulation associated factor..

18. A device for measuring a coagulation event in a biological sample comprising:

a sample receiving area;

a means for recording a coagulation event;

a means for deriving a dynamic parameter of the coagulation event; and

a means for generating a clotting profile and response pattern.

19. The device of claim 18, further comprising a means to transmit data to care center or physician.

20. The device of claim 18, wherein said device is individually calibrated for each patient.

21. The device of claim 20, wherein the individual calibration is performed using dynamic

parameters.

22. The device of the claim 18, wherein the individual calibration is performed using a dynamic parameter of the coagulation event measured after administration of a procoagulant or anticoagulant to a patient.

23. The device of claim 18, wherein the individual calibration is performed using a dynamic

parameter of the coagulation event measured after the addition of a procoagulant or anticoagulant to a sample.

24. The method of claims 22 and 23, wherein the procoagulant or anticoagulant has a defined

activity.

25. The device of claim 18, wherein the device is used by a patient to optimize the dosage of a procoagulant or anticoagulant for on demand or prophylaxis treatment.

26. The device of claims 22 to 25, wherein the procoagulant is selected from FVIII, FIX, FVII, TFPI, TF and any anticoagulant directly or indirectly targeting Factor Xa, factor IXa, or thrombin or other coagulation associated factor..

27. The device of claim 18, wherein the device is a hand-held portable device.

28. The device of claim 18, wherein the sample receiving area comprises a receiving area for test strips.

29. The device of claim 18, wherein the biological sample is collected on a test strip.

30. The device of claim 29, wherein the test strip comprises one or more coagulation activators.

31. The device of claim 30, wherein the coagulation activator is selected from FXII activator and TF.

32. A kit comprising one or more components selected from the device of claim 18, lancets, test strips, alcohol swabs, cotton pads, bandages, control samples, and calibration standards.