Method for analyzing a blood sample with respect to a hemostatic regulator

Abstract

Disclosed herein is a method, in particular an in vitro method, for analyzing a blood sample with respect to a hemostatic regulator, the method comprising the steps: Providing a blood sample to be analyzed, wherein the blood sample comprises a chelating agent being configured to chelate calcium ions; Selectively reducing the concentration of the hemostatic regulator in the blood sample with respect to which the blood sample shall be analyzed by the addition of a dilution plasma reagent which is in particular free from the analyte to be assayed and provides all other hemostatic components in excess; Adding a coagulation trigger reagent comprising tissue factor; Adding calcium ions at starting time t0 in at least such an amount that saturation of the chelating agent in the blood sample is achieved; Measuring the clotting time Δt between starting time t0 and end time te.

Description

[0001] METHOD FOR ANALYZING A BLOOD SAMPLE WITH RESPECT TO A HEMOSTATIC REGULATOR

[0002] Field of disclosure

[0003] The present invention lies in the field of hemostatic regulator analysis and relates to a method for analyzing a blood sample with respect to a hemostatic regulator.

[0004] Background, prior art

[0005] The cascade of secondary hemostasis (the coagulation cascade) comprises two different initial pathways namely the intrinsic and the extrinsic pathway, which both lead to thrombin formation and ultimately to fibrin production resulting in coagulation. Both pathways include a series of components (so-called coagulation components), in particular coagulation factors (abbreviated by “F”) which are involved in a series of cascading reactions leading to the formation of thrombin from prothrombin. Other coagulation components are in general also control proteins of the coagulation cascade. For example, in the extrinsic pathway, tissue factor (TF) from subendothelial sites or activated monocytes gets exposed to the circulation and forms a complex with FVII or traces of FVIIa present in blood. This activated form (TF-FVIla) then induces the activation of FX to FXa. FXa in turn together with FVa forms the prothrombinase complex which induces thrombin formation from prothrombin. Subsequently, thrombin cleaves fibrinogen (i.e Fl) to fibrin and further cleaves FXIII to FXIIIa which then binds calcium ions to form fibrin crosslinks and thereby the clot. TF-FVIla can also activate FIX to FIXa.

[0006] There is a plethora of components involved in the coagulation cascade and any over- or underrepresentation of such components in a patient may lead to severe blood coagulation disorders. As an example, tissue factor pathway inhibitor (TFPI) is a 40-kDa Kunitz-type protease inhibitor and is the endogenous protein that inhibits coagulation by bonding with activated factor X (FXa) (see for example J. Thromb. Haemost. 2022, 20, 1290-1300; DOI: 10.1111 / jth.15697). The circulating concentration of TFPI, mostly bound to lipoproteins, is normally 70 -150 ng / mL (1.8 -3.8 nM). At the endothelium surface, TFPI stays bound to glycosaminoglycans (GAGs), and the TFPI concentration can be increased 3 - 6-fold by heparin injection.

[0007] The TFPI / FXa complex then subsequently binds with TF / activated factor VII (FVIIa) complex, ultimately inhibiting thrombin generation. TFPI can also inhibit FXa in the prothrombinase complex. Protein S enhances the interaction between TFPI and FXa, thereby accelerating the feedback inhibition of the extrinsic TF / FVIla pathway by TFPI. Consequently, TFPI is one of the main hemostatic regulators of the initiation of the primary phase of the extrinsic pathway. Elevated levels of TFPI (e. g. in FV-Short / East Texas bleeding disorder, FV Amsterdam, FV Atlanta) lead to bleeding episodes and can be also linked to atherosclerosis. In contrast, low TFPI levels are a risk for venous and arterial thrombosis. TFPI is also the primary physiological regulator of bleeding in hemophilia.

[0008] Protein Z (PZ) is a 62 kDa vitamin K-dependent plasma glycoprotein. In contrast to several other vitamin K-dependent coagulation factors, however, protein Z is not a zymogen of a serine protease. Mean plasma protein Z levels range from 1.16 to 2.71 pg / mL and are influenced by genetic and non-genetic factors, which include chronic liver diseases, age, gender, vitamin K levels, concurrent use of vitamin K antagonist anticoagulants, and pregnancy. As demonstrated in in-vitro and in-vivo studies, protein Z plays an important role in inhibiting coagulation, serving as a cofactor for the inactivation of activated factor X (FXa) by plasma protein Z dependent protease inhibitor (ZPI). While the inhibition of FXa is performed by ZPI, PZ accelerates this reaction 1000-fold. Deficiency in protein Z secretion and / or function is linked with a pro-coagulant state and several thrombotic disorders, including arterial and venous thrombosis. In addition, a role for the PZ-ZPI complex in the regulation of pregnancy has been suggested, and an association between low plasma PZ levels and adverse pregnancy outcomes has been reported. Association of PZ with prediabetes and high-risk precursor for type 2 diabetes mellitus was also reported, that could serve as a biomarker for the early detection.

[0009] Plasma protein Z dependent protease inhibitor was first identified and purified from plasma as a PZ-dependent inhibitor of active factor X (FXa), and later identified as a member of the serpin superfamily (serpinAIO). ZPI circulates in plasma as a tight complex with the PZ. In human plasma, ZPI is present in about ~ 59 ± 8 nM, and with a modest excess over PZ levels. ZPI / PZ functions as an anticoagulant by specifically inhibiting membrane associated FXa. ZPI also inhibits active factor XI (FXIa). Plasma deficiencies in ZPI or PZ were found to be a risk factor for vascular thrombosis by several studies but an uncertain risk factor by others. ZPI / PZ comprises one of four major anticoagulant systems in plasma. The other three include: antithrombin III (ATIII) which inhibits FXa, active factor IX (FIXa), FVIIa and thrombin; activated protein C (APC) which proteolytically degrades active factor V (FVa) and active factor VIII (FVIIIa); and TFPI which inhibits the extrinsic FXase complex (TF- Vlla-FXa) and FX.

[0010] For the assessment and diagnosis of blood disorders as well as the monitoring of the effects of certain therapies (e.g. the use of anti-TFPI mAb), methods to analyze hemostatic regulators are crucial. TFPI is also the primary physiological regulator of bleeding in Haemophilia A (FVIII-deficiency) and B (FIX-deficiency), or other Rare Bleeding Disorders (RBD) such as FXI deficiency. Several products targeting TFPI are currently under development for potential therapeutic use in patients with Haemophilia A and B, including 3 monoclonal antibodies (mAb): Concizumab and Marstacimab. It is desirable to be able to provide reliable and fast functional test methods that can preferably be easily adapted to automated coagulometers used in the laboratories. Furthermore, the functional quantification of the hemostatic regulators is highly desirable. Additionally, it is desirable to provide efficient, in particular fast and easy to perform test methods. Current methods which investigate the extrinsic pathway cannot detect inhibitory effects of various different hemostatic regulators, such as antithrombin, TFPI, protein C, protein S, protein Z or ZPI. This is caused by the fact that the effects of these hemostatic regulators are not visible in assays such as the prothrombin time assay.

[0011] Currently available commercial assays for certain analytes mentioned are often complex, time consuming, do not detect the function of the protein, are difficult to automate, unreliable and / or inaccurate. Common approaches include immunochemical methods, which measure merely the concentration of the analyte in a blood sample. In the case of TFPI as an analyte, this may for example be the free TFPI or the total TFPI which includes also the lipoprotein bound fraction. Such assays may in certain cases be unreliable, because they are not able to detect dysfunctional variants that are often the result of mutations or of chemical reactions, such as oxidation or citrullination. Thus, the results and interpretation of such an assay can be dramatically falsified. Alternatively, chromogenic assays are commonly used as well. They measure the function of TFPI in the inhibition of factor Xa and the tissue factor (TF) complex with FVIIa, however in a diluted manner and therefore not under physiological conditions. Such chromogenic assays work with fixed long incubation times that does not consider the kinetics of the inhibition of both FXa and FVIIa / TF. The time to result in such assays is commonly about one hour. Therefore, it is very likely that mutations of TFPI that could be associated with slower inhibition of their targets are not reliably measurable with such assays.

[0012] Summary of disclosure

[0013] It is the general object of the present invention to advance the state of the art in the field of hemostatic regulator analysis and preferably to overcome the disadvantages of the prior art fully or at least partly. In advantageous embodiments, methods for analyzing a blood sample with respect to a hemostatic regulator is provided which are more specific and / or more selective. In further advantageous embodiments, methods for analyzing a blood sample with respect to a hemostatic regulator is provided, which are easier to perform. Further, methods are provided, which are operationally simple and can be automated to a large extent. In addition, advantageous embodiments of the invention provide methods which allow to determine the activity of the hemostatic regulator in question.

[0014] The general object is achieved by the subject-matter of the independent claims. Further advantageous embodiments follow from the dependent claims and the overall disclosure.

[0015] A first aspect of the invention relates to a method for analyzing a blood sample with respect to a hemostatic regulator, in particular a hemostatic regulator of the coagulation cascade, , preferably of the extrinsic pathway, of the coagulation cascade. The method may comprise the following steps: a. Providing a blood sample to be analyzed, respectively in which the hemostatic regulator shall be analyzed. The blood sample may comprise a chelating agent which is configured to chelate calcium ions. Thereby, the chelating agent prevents coagulation of the blood sample as such and particularly before step d.; b. Selectively reducing the concentration of the hemostatic regulator in the blood sample with respect to which the blood sample shall be analyzed by the addition of a dilution plasma reagent. Thereby a mixture of blood sample and dilution plasma reagent is obtained. For example, the dilution plasma reagent may be a dilution plasma reagent as described in any of the embodiment herein; c. Adding a coagulation trigger reagent comprising tissue factor; d. Adding calcium ions, at starting time to in at least such an amount that saturation of the chelating agent in the blood sample is achieved, in particular such that at least some calcium ions are not chelated by the chelating agent; e. Measuring the clotting time it between starting time to and end time te. End time temay for example be a predefined end time, e.g. at which clotting begins or at which thrombin formation begins.

[0016] In some embodiments, step c. and step d. are performed sequentially. In some embodiments, step b. and step c. are performed sequentially. In some embodiments step d. is performed after step c. In some embodiments, step c. is performed after step b. In some embodiments, calcium ions are only added in step d. and / or no calcium ions are added in steps b. and c. Such embodiments allow a more accurate measurement and avoid falsifications. In general, the blood sample obtained in step b. may be referred to as a diluted blood sample.

[0017] In some embodiments, during steps b. and / or c. and / or d. and / or e., no additional hemostatic regulator with respect to which the blood sample shall be analyzed is added, e.g. directly added. In step b. the concentration of the hemostatic regulator with respect to which the blood sample shall be analyzed is reduced in the blood sample, but thereafter, no additional such hemostatic regulator with respect to which the blood sample shall be analyzed is added. The hemostatic regulator with respect to which the sample shall be analyzed originates in some embodiments only from the blood sample provided in step a.

[0018] In some embodiments the dilution plasma reagent is configured such that by its addition to the blood sample, the concentration of the hemostatic regulator in the blood sample is selectively reduced over other coagulation components in the blood sample, in particular over all other coagulation components in the blood sample, preferably down to 5% or less, in particular 2% or less, preferably down to 0%, activity as compared to normal plasma (e.g. normal pooled plasma) having by definition an activity of 100%. These coagulation components may for example be components, respectively factors, or regulatory proteins, i.e. control proteins, of the coagulation cascade, in particular the intrinsic or extrinsic pathway.

[0019] Selectively reducing the concentration of the hemostatic regulator in the blood sample means that the concentration of the hemostatic regulator is reduced as compared to at least one or more or even all other components of the blood sample, in particular as compared to other coagulation components of the blood sample, such as coagulation components or other regulatory proteins of the coagulation cascade. This is for example achieved in that the dilution plasma sample to be added to the blood sample contains such other coagulation components and no or only a reduced amount (as compared to the other coagulation components) of the hemostatic regulator with respect to which the blood sample shall be analyzed. In some embodiments, it may generally be possible that the concentration of only one, i.e. a single, hemostatic regulator in the blood sample is reduced during step b (that is the hemostatic regulator with respect to which the blood sample shall be analyzed).

[0020] In some embodiments, step b. (i.e. selectively reducing the concentration of the hemostatic regulator in the blood sample (such as a plasma sample) with respect to which the blood sample (such as plasma sample) shall be analyzed by the addition of the dilution plasma reagent) is preceded by a dilution plasma reagent preparation step. The dilution plasma reagent preparation step may comprise depleting a sample, in particular a blood sample, of the hemostatic regulator to be analyzed, in particular by immunodepletion. Immunodepletion may for example be done by exposing the sample, in particular the blood sample, to an antibody which selectively binds to the hemostatic regulator to be analyzed. For example, this antibody may be bound to a solid support, such as gel forming particles. By transporting the sample, in particular blood sample, over the solid support being bound to this antibody, the dilution plasma reagent is prepared. The sample, in particular the blood sample, may in some embodiments be a platelet poor plasma sample or a platelet rich plasma sample, or it may be a whole blood sample. The blood sample being used in the dilution plasma reagent preparation step may preferably be the same blood sample, or a portion thereof, as used in step a. In particular, the blood sample may originate from the same source, such as from the same patient. In some embodiments, the sample, in particular the blood sample, being used in the dilution plasma reagent preparation step may preferably be a pooled plasma sample having been obtained from a plurality of subjects, in particular a plurality of healthy subjects. This allows to provide a typical mixture of all other coagulation components. Such a pooled plasma sample may then be depleted of the hemostatic regulator to be analyzed, in particular be immunodepletion to provide the dilution plasma reagent. The dilution plasma reagent having been prepared by any of the embodiments described herein with respect to the dilution plasma reagent preparation step may generally be used as dilution plasma reagent in the method according to any embodiment of the invention, in particular in step b. After the dilution plasma reagent has been prepared, it may in step b. be added to the blood sample with respect to which the hemostatic regulator shall be analyzed. By mixing the sample with an excess of the deficient plasma the other components that may be variable are added in excess. Therefore, the assay becomes dependent of the respective protein in the sample.

[0021] The coagulation trigger reagent comprises in some embodiments tissue factor, in particular in an amount that is selected such that the coagulation cascade and in particular the extrinsic pathway is triggered and clotting occurs. Thus, the amount of tissue factor may be such that the coagulation cascade is activated via the extrinsic pathway.

[0022] In some embodiments, the coagulation trigger reagent is free of calcium ions. In particular embodiments, additional calcium ions are only added in step d..

[0023] In some embodiments, step b. comprises or consists of adding the dilution plasma reagent to the blood sample in excess with respect to the blood sample, wherein the dilution plasma reagent is depleted of, or even devoid of, the hemostatic regulator to be analyzed. In certain embodiments, the dilution plasma reagent comprises all other coagulation components which are necessary for coagulation. Therefore, the method according to the invention may in another aspect be a method for analyzing a blood sample with respect to a hemostatic regulator, which comprises the steps of: a. Providing a blood sample to be analyzed, respectively in which the hemostatic regulator shall be analyzed. The blood sample may comprise a chelating agent which is configured to chelate calcium ions. Thereby, the chelating agent prevents coagulation of the blood sample as such and particularly before step d.; b. Adding a dilution plasma reagent to the blood sample provided in step a. in excess, in particular in volumetric excess, with respect to, i.e. as compared to, the blood sample. Thereby a mixture of blood sample and dilution plasma reagent is obtained. The dilution plasma reagent may be depleted of the hemostatic regulator to be analyzed as compared to the blood sample or it may particularly even be devoid of the hemostatic regulator to be analyzed. The dilution plasma reagent may in some embodiments comprise all other coagulation components which are necessary for coagulation; c. Adding a coagulation trigger reagent comprising tissue factor; d. Adding calcium ions at starting time to in at least such an amount that saturation of the chelating agent in the blood sample is achieved, in particular such that at least some calcium ions are not chelated by the chelating agent; e. Measuring the clotting timeAt between starting time to and end time te. End time temay for example be a predefined end time, e.g. at which clotting begins or at which thrombin formation begins.

[0024] The reference numerals as used herein a., b., c., d. etc. have the purpose of differentiating and clearly indicating the specific steps of the methods. Although the order of steps can in some embodiments be first step a., followed by step, b., followed by step c. followed by step d., this does not have to be the case in other embodiments as long as it does not result in an unworkable embodiment. For example, it may be possible that steps c. and b. are performed at the same time. Step e can also be extended by measuring the kinetics of the entire thrombin generation and its decay curve (for example its concentration over time). This may provide additional information of the hemostatic regulator to be analyzed.

[0025] In some embodiments, step c. and step d. are performed sequentially. In some embodiments, step b. and step c. are performed sequentially. In some embodiments step d. is performed after step c. In some embodiments, step c. is performed after step b. In some embodiments, calcium ions are only added in step d. and / or no calcium ions are added in steps b. and c.

[0026] In some embodiments, the coagulation trigger reagent contains tissue factor as the only coagulation component, e.g. coagulation component of the coagulation cascade, and / or hemostatic regulator. It is generally understood herein that the term “comprising” is interpreted as meaning that it includes those features following this term, but that it does not exclude the presence of other features, as long as they do not render the claim unworkable. On the other hand, if the wording "consist of" is used, then no further features are present apart from the ones following said wording.

[0027] As used herein, a “hemostatic regulator” refers to a component which is involved, e.g. directly or indirectly involved, in the regulation of thrombin generation and / or components of the coagulation cascade, such as the extrinsic. The methods according to the invention are methods for analyzing a blood sample with respect to a hemostatic regulator. That is, the methods may for example assess the presence or absence of the hemostatic regulator, and / or its concentration and / or its activity, particularly with respect to coagulation. The activity may for example be referenced to reference data, such as a reference value, as described in some embodiments herein. For example, the activity of the hemostatic regulator in healthy subjects may be set to 100% and the determined activity is determined relative to the activity of the hemostatic regulator in the healthy subjects. Thereby, it is not only possible to determine the presence or absence or the concentration of a certain regulator, but it may also be possible to detect a reduced or increased activity of the hemostatic regulator at issue, for example caused by a point mutation or the presence of an antibody directed against the coagulation regulator or the like, or oxidation or other chemical modification of the hemostatic regulator. In some embodiments, the hemostatic regulator with respect to which the blood sample is analyzed is TFPI. In particular, TFPI is the only hemostatic regulator with respect to which the blood sample is analyzed in the method.

[0028] The dilution plasma reagent is a composition, typically a liquid composition, with which the blood sample is diluted. In particular embodiments, the activity of the hemostatic regulator with respect to which the sample is analyzed in the dilution plasma reagent is below 2%, in particular below 1 %, more particular below 0.05%, even more particular 0%, as compared to normal plasma having by definition an activity of 100%, respectively a blood sample of a healthy patient or a pool of healthy patients having by definition an activity of 100%. The dilution plasma reagent may in some embodiments at first be depleted of the hemostatic regulator to be analyzed, or even devoid thereof, and may preferably secondly comprise also other, e.g. all other, coagulation components, in particular all other coagulation components of (respectively being involved in) the coagulation cascade. This means that the dilution plasma reagent may comprise all other coagulation components being necessary for coagulation along the coagulation pathway with which the hemostatic regulator (with regard to which the blood sample is analyzed) is associated with. Depleted or devoid of the hemostatic regulator may mean for example that the hemostatic regulator is not present in the dilution plasma reagent or that it may be in an inactive form in which it cannot perform its regulating function in the coagulation cascade.

[0029] In preferred embodiments, the composition of the dilution plasma reagent and the blood sample to be analyzed is the same (either with respect to all components or at least with respect to the coagulation components) with the only exception that the dilution plasma reagent is depleted of, or even devoid of, the hemostatic regulator to be analyzed (e.g. as compared to the blood sample with respect to which the hemostatic regulator is analyzed).

[0030] Thus, it is clear to the skilled person that if a certain hemostatic regulator affects a certain factor of the extrinsic pathway, for example by activation or inhibition, then the coagulation components are present which are required that this factor involved, e.g. by activation or inactivation, in coagulation (see Fig. 1). For example, if TFPI is the hemostatic regulator with regard to which the blood sample is analyzed, then all other coagulation components, e.g. hemostasis proteins, may be comprised and in excess in the dilution plasma reagent, such as AT-III, PS, FV, FVII (respectively FVIIa), FX, prothrombin (Fll), fibrinogen and FXIII, or such as PS, FV, FVII (respectively FVIIa), FX, prothrombin (Fll), fibrinogen and FXIII, because these coagulation components are necessary for coagulation along the extrinsic pathway with which TFPI is associated (see Fig. 1 , which indicates that TFPI inactivates FVIIa). If for example active protein C is the hemostatic regulator with regard to which the blood sample is analyzed, all other coagulation components, e.g. hemostasis proteins, may be comprised and in excess in the dilution plasma reagent, such as FV, respectively FVa,, FVIII, FXa (or TF, Vila and FX), prothrombin, and fibrinogen, because these coagulation components are necessary for coagulation along the common pathway with which active protein C is associated. Furthermore, other protein C activators, such as thrombomodulin or glycoproteins being derived from the copperhead snake Agkistrodon contortrix, may be present in or added to the dilution plasma reagent.

[0031] Thus, the dilution plasma reagent reduces the concentration of the hemostatic regulator to be analyzed, in particular only the concentration of the hemostatic regulator to be analyzed, with respect to the other components of coagulation being present in the blood sample. In some embodiments, the concentration of the hemostatic regulator to be analyzed is selectively reduced by the dilution plasma reagent. The concentration of the other, i.e. remaining coagulation components, may however not or at least to a much lesser extent be reduced. Thereby, the method becomes highly dependent on the hemostatic regulator to be analyzed from which the dilution plasma reagent is depleted or even devoid of, respectively whose concentration has been selectively reduced. This has further the advantage that the influence of anticoagulant drugs being present in the blood sample (for example a sample taken from a patient being under the influence of such an anticoagulant drug) is drastically reduced due to the dilution of the blood sample, the addition of a heparin antagonist such as hexadimethrine bromide, and further due to the fact that the dilution plasma reagent adds additional coagulation components. Thereby, the coagulation components are provided in a relative excess as compared to the drug and its influence on the outcome is significantly reduced. Hence, falsifications of the result by anticoagulant drugs in the blood sample are reduced.

[0032] The methods according to the invention may particularly be in-vitro methods. The blood sample to be analyzed may originate from a subject, such as a patient suffering from a bleeding disorder or a hemostatic disorder or being suspected to suffer from a bleeding or thrombotic disorder.

[0033] The term “blood sample” refers in particular to a sample which may originate from blood of a subject and / or which has been obtained from the blood of a subject. Thus, the blood sample may be derived, respectively originate from a whole blood sample of a subject. It may be possible that the blood sample used in the methods according to the invention may be manipulated before step b. or even before step a. of the methods according to the invention. It may be possible that auxiliary agents are added or that the sample is mechanically manipulated, for example by centrifugation or filtration. For example, the chelating agent may be added to the blood sample during or before such a manipulation, such as sodium citrate. Non-limiting examples of the blood sample may be a plasma sample, such as a blood plasma sample, e.g. a platelet poor plasma sample or a platelet rich plasma sample, or it may be a whole blood sample. In typical embodiments, the blood sample provided in step a. comprises the hemostatic regulator with respect to which the blood sample shall be analyzed.

[0034] Typically, the chelating agent is provided in such a concentration in the blood sample that the blood sample does no coagulate as such, respectively by itself. For example, the chelating agent may be present in such a concentration in the blood sample that the blood sample does no coagulate as such, respectively by itself.

[0035] In some embodiments, the dilution plasma reagent may comprise the same coagulation components, e.g. the same concentration of the coagulation components, as the blood sample provided in step a. with the exception of the hemostatic regulator to be analyzed, respectively its concentration. In certain embodiments, it may even be possible that the dilution plasma reagent has essentially the same composition as the blood sample provided in step a. with the exception of the hemostatic regulator to be analyzed. In other words, the dilution plasma reagent may be termed as a “coagulation composition”, “deficient plasma” or “hemostatic regulator depleted plasma”. Such a coagulation composition or reagent is depleted of or even devoid of the hemostatic regulator but comprises all other coagulation components which are necessary for coagulation.

[0036] In some embodiments, the dilution plasma reagent comprises the coagulation components which are necessary for coagulation in the same concentration or even in excess as compared to the blood sample provided in step a. The coagulation trigger reagent typically comprises water, e.g. distilled or deionized water.

[0037] In some embodiments, the coagulation trigger reagent comprises a buffer. The buffer may for example be selected from HEPES buffer (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid), Tris buffer (tris(hydroxymethyl)aminomethane), imidazole buffer and other buffer substances.

[0038] In some embodiments, the pH of the coagulation trigger reagent is between 7.0 and 8.0, in particular 7.4.

[0039] In some embodiments, the coagulation trigger reagent comprises NaCI, in particular in a concentration of 10 to 200 mM, more particular 125 to 175 mM, even more particular 150 to 175 mM, even more particular 150 mM.

[0040] In step d. calcium ions are added at starting time to in at least such an amount that saturation of the chelating agent in the blood sample is achieved. This means that a sufficiently high calcium ion concentration is added to induce the coagulation cascade, in particular the extrinsic pathway. As noted above, the chelating agent in the blood sample and also the dilution plasma reagent prevents coagulation of the blood sample as such, i.e. without adding other components to it and in particular prior to step d. This is achieved by chelation of calcium ions by the chelating agent. Due to the chelation, the calcium ions are not available to induce the coagulation cascade. However, since a chelating agent can only chelate a certain number of calcium ions and the blood sample contains only a certain amount of chelating agent, the addition of calcium ions in step d. saturates the chelating agent being present in the blood sample. When the saturation point is reached, additionally added calcium ions are not chelated anymore and are therefore available to induce the coagulation cascade. Since the nature of the chelating agent and its concentration in the blood sample is known (e.g. because a predetermined amount of the chelating agent has been added to the blood sample prior to step b., e.g. during blood sample preparation, or because the blood sample has been titrated before step b.), the minimum amount of calcium ions to be added in step d. and to induce the coagulation cascade is known. Furthermore, and as outlined herein, the start of the coagulation cascade can readily be observed, for example by observing the activation of factor VII to Vila or the formation of FXa from FX or any other occurring reaction along the coagulation cascade.

[0041] In some embodiments, the calcium ions being added in step d. are added as an aqueous solution with a calcium ion concentration of 10 to 200 mM, in particular 20 to 30 mM. As an example, it may be possible to add 1 to 200 mL, in particular 10 to 100 mL, more particular 25 to 100 mL of such an aqueous solution in step d.

[0042] In some embodiments, step d. is performed such that the calcium ion concentration after step d. is at least 2.5 mM, in particular at least 10 mM, more particular at least 25 mM. In some embodiments, step d. is performed such that the calcium ion concentration after step d. is about 2 to 10 mM, in particular about 4 to 8 mM. In some embodiments, step d. is performed such that the calcium ion concentration after step d. is equal or higher than in the blood sample to be analyzed (i.e. before step b. is performed).

[0043] The end time teis in particular a predefined point in time. That is, for each measurement, the end time is predefined. Further, in embodiments in which the measured clotting time is compared to reference data, it is understood that the reference data is obtained in regard to the same predefined end time. The end time te is indicative for the clotting time. The end time temay for example be the time at which clotting of the blood sample, respectively the mixture of blood sample and dilution plasma reagent and optionally the added calcium ions in step d., begins. This may for example be thrombin formation or fibrin formation or the occurrence of a clot. In embodiments in which the measured clotting time is compared to reference data, as outlined in some embodiments described herein, it is clear that the end time teis consistently defined in the same manner. That is, the end time te may be standardized. For example, the end time may be a point in time at which thrombin or fibrin begins to form, particularly at which the first molecule of thrombin or fibrin forms. It is also possible that teis the point in time at which the thrombin or fibrin concentration reaches a certain level. For example, fibrin formation may be detected by a coagulometer. Such a coagulometer may for example measure either viscoelastic properties by magnetic detection of a moving steel ball through the blood sample, sonometry or spectroscopic methods, in particular optical methods, e.g. the change of refractive index, which for example detects a change of turbidity. As discussed herein, a selective detecting agent may be present in a predefined concentration which undergoes a detectable reaction with thrombin or fibrin (such as forming a luminescent, in particular fluorescent entity) and te is the point in time at which this reaction is detected, respectively observed. Alternatively, it may also be possible that the end point refers to the point in time at which a clot reaches a certain size, volume or weight. In some embodiments, the end time te may be predefined in such a manner that it refers to the time at which a parameter (such as thrombin concentration, fibrin concentration, clot size, refractive index, conductivity, etc.) exceeds or falls below a predefined threshold value.

[0044] In some embodiments, the chelating agent may for example be citrate (i.e. citrate ions) or (2R,3S)-lsocitrate, ), preferably citrate.

[0045] In some embodiments, the measured clotting timeAt is in a subsequent step (e.g. after step e.) compared to reference data. Such a step may for example be considered as “step f.”. The reference data may typically be reference data with regard to the hemostatic regulator to be analyzed. In some embodiments, the reference data may be a calibration curve. Such a calibration curve may provide a dependence between the clotting timeAt and a certain property of the hemostatic regulator, such as its concentration or activity. The comparison with such a calibration curve allows based on the measured clotting time to determine this property of the hemostatic regulator with regard to which the blood sample is analyzed. It may additionally or alternatively be possible that the reference data is a reference value, e.g. the clotting time obtained for another subject, i.e. a blood sample obtained from another subject with a predetermined pathological state. For example, the reference data may be the clotting time obtained from a healthy subject or from a subject with a clinically confirmed pathological state of a bleeding disorder. Thereby, a relative comparison between the blood sample analyzed and the reference data, i.e. the predetermined pathological state of the subject from which the reference data has been obtained, can be made. It is understood that the reference data is typically obtained by measuring reference blood samples by the method according to the invention and thus in the same manner as the blood sample being analyzed. The comparison of the clotting time to reference data therefore provides a reliable and very fast analysis of the blood sample. For example, if the activity is used, it may be compared to a pooled sample of a plurality of healthy subjects which may for example be referenced to an activity of 100 or as 1 U / rnL. It also allows to easily determine a relevant property of the hemostatic regulator with regard to which the sample is analyzed. For example, if the reference data is a calibration curve associating the clotting time with an activity of the hemostatic regulator, it is also possible to rapidly detect that the hemostatic regulator at issue is behaving in a more active or less active manner than it should be, which may for example be caused by a point mutation or by an antibody directed against the protein that is measured.

[0046] In some embodiments phospholipids, e.g. additional phospholipids, are added during or prior to step d. For example, the phospholipids may be present in the coagulation trigger reagent added in step c. The addition of phospholipids (PL) minimizes the potential interference of anti-phospholipid antibodies and accelerates clotting while maintaining the dependency of the entire blood sample, respectively the entire mixture, on the hemostatic regulator with regard to which the blood sample is being analyzed.

[0047] In certain embodiments, the coagulation trigger reagent being added in step c. comprises phospholipids, in particular in a concentration of 0.01 to 200 pM, in particular 1 to 30 pM, more particular 15 to 25 pM, more particular 25 pM.

[0048] In some embodiments, the coagulation trigger reagent being added in step c. comprises phospholipids in particular in a concentration of 0.01 - 0.2 mg / mL, preferably 0.01 - 0.02 mg / mL.

[0049] In some embodiments, the phospholipids are phosphatidyl derivatives, such as phosphatidyl serine, phosphatidyl ethanolamine and / or phosphatidyl choline. In certain embodiments, the coagulation trigger reagent being added in step c. comprises phospholipids. In particular, the coagulation trigger reagent is configured and added such that the phospholipid concentration after step c. (i.e. in the mixture of blood sample, dilution plasma reagent and coagulation trigger reagent) or after step d. (i.e. in the mixture of blood sample, dilution plasma reagent, coagulation trigger reagent and calcium ions), is 0.01 - 0.5 mg / mL, preferably 0.01 - 0.2 mg / mL, more preferably 0.01 - 0.02 mg / mL.

[0050] In some embodiments, an antidote for anticoagulant agents, in particular anticoagulant drugs such as but not limited to heparin and heparin derived anticoagulant agents, is added during or prior to step d. In particular embodiments, the antidote is added to the blood sample, e.g. before step b. This further reduces the influence of and potential falsification caused by the presence of specific pharmaceutical anticoagulant agents in the blood sample. Antidotes to these anticoagulant agents are generally configured to specifically prevent the anticoagulating effect of a specific anticoagulant agent. The antidote may in some embodiments be an antibody binding the anticoagulant drug. Further, the antidote may be an inactive form of the coagulation component, e.g. factor, being able to bind the coagulation drug. Another example of a heparin antidote is hexadimethrine bromide or protamine sulfate (CAS 9009-65-8). In certain embodiments, the antidote is added in a concentration of 0.5 to 5.0 U / rnL. Another suitable antidote may for example by heparinase. In particular embodiments heparinase may be added to the blood sample in a range of 0.5 to 5.0 U / rnL.

[0051] In some embodiments, the dilution plasma reagent is provided in a volumetric excess with respect to the blood sample of at least 2-fold, in particular at least 3-fold, more particular at least 5-fold.

[0052] In some embodiments, the dilution plasma reagent is provided in a volumetric excess with respect to the blood sample of at most 50-fold, in particular at most 20- fold, more particular at most 10-fold. In some embodiments, the dilution plasma reagent is provided in a volumetric excess with respect to the blood sample of 2-fold to 50-fold, in particular 2-fold to 20-fold, more particular 3-fold to 10-fold.

[0053] In some embodiments, the end time te is reached when thrombin formation or fibrin formation is detected. Thrombin or fibrin formation may for example be detected by a clotting assay. Clotting assays are beneficial as they are easily automatable and operationally simple to perform. Furthermore, as compared to other techniques, such as chromogenic or fluorescent-based techniques, the conditions of a clotting assay are closer or even equal to physiological conditions. This is because no non-physiological entity is present, such as a chromophore. In addition, other techniques, such as fluorescent-based techniques are more prone to falsified results. For example, certain compounds being present in the blood sample, such as drugs, may show an intrinsic fluorescence, which can falsify the measurement results. In some embodiments, the method according to the disclosure may be free of chromophores being bound to, respectively interacting with, a component of the coagulation components. In certain embodiments, the method according to the disclosure may be free of fluorescent entities.

[0054] In some embodiments, fibrin formation is detected by optical methods, in particular measuring the change of transmission, turbidimetry or refractometry, or viscoelastic methods.

[0055] It may further be possible to detect thrombin formation by adding a selective detecting agent which is configured to selectively undergo a reaction with thrombin upon which a detectable signal or detectable entity is generated. The entity is typically a chemical entity, such as a small molecule (i.e. a molecule with a molecular mass of less than 1000 Da) or a large molecule (i.e. a molecule with a molecular mass 1000 Da or more), specifically a peptide with a thrombin selective structure. It is clear that the detectable entity and / or the detectable signal may typically be unique and / or differentiable over the selective detecting agent. In particular, the signal or entity may be detectable by spectroscopy, in particular luminescence, e.g. fluorescence or phosphorescence, LIV-VIS, IR or Raman spectroscopy. It may also be possible that the signal or entity is detected by a physical or an electrochemical method, such as amperometry. The signal or entity may particularly be detectable in real time. Thus, the method may in some embodiments further comprise the step of monitoring and detecting a detectable signal or entity being generated due to the reaction of thrombin with the selective detecting agent.

[0056] In certain embodiments, the selective detecting agent may be configured to form a leaving group as detectable entity which in contrast to the selective detecting agent has different optical properties or electrochemical properties. For example, it may be possible that the selective detecting agent as such is not fluorescent but the leaving group formed as detectable entity is fluorescent. It may also be possible that the selective detecting agent as such shows different optical properties, such as different light absorption properties, than the leaving group formed upon reaction of thrombin or fibrin with the selective detecting agent.

[0057] In some embodiments, the addition of activated protein C (APC) or protein C activators is excluded in step b., step c. and step d. and optionally also in steps a. and / or d.. In some embodiments, no activated protein C (APC) or protein C activators are added during the method for analyzing the blood sample. Such embodiments allow for example to study the co-factor properties of protein S for the TFPI functionality in the extrinsic pathway.

[0058] In some embodiments, one or more activators of the coagulation cascade factors are added prior to step d. If the hemostatic regulator with respect to which the blood sample is analyzed is TFPI, the addition of such activators is not necessary or can even be excluded. Such activators are generally configured to activate a factor of the coagulation cascade. These activators are advantageous in cases in which the hemostatic regulator of interest or another coagulation component, e.g. a coagulation component of the extrinsic pathway, requires an activation by such an activator. Particular examples of such activators are FXIa, thrombomodulin, an activator of protein C, in particular a snake venom derived or recombinant activator of protein C, and activated protein C. An example of such a protein C activator are glycoproteins being derived from the copperhead snake Agkistrodon contortrix. Activators of protein C may for example be serine proteinases, the complex of thrombin with thrombomodulin, and / or are preferably configured to convert protein C into activated protein C. In some embodiments, one or more control proteins of the coagulation cascade are added prior to step d.

[0059] In some embodiments, the coagulation trigger reagent comprises an intrinsic pathway inhibitor being configured to inhibit the intrinsic pathway, such as a serine protease inhibitor. Typically, the intrinsic pathway inhibitor may be selective for inhibiting the intrinsic pathway and may particularly not inhibit the extrinsic pathway. In such embodiments, the influence of the hemostatic regulator on the extrinsic pathway, in particular on only the extrinsic pathway is assessed or analyzed.

[0060] In some embodiments, the coagulation trigger reagent comprises a serine protease inhibitor which preferably does not interfere with the extrinsic pathway. Example of suitable serine protease inhibitors may be selected from Corn Trypsin Inhibitor, aprotinin or other inhibitors of the contact phase pathway.

[0061] In some embodiments, the coagulation trigger reagent comprises aprotinin in a concentration of 0.1 to 10 TIU / rnL (trypsin inhibitor unit per mL), in particular 1 to 2 TIU / mL.

[0062] In some embodiments, the coagulation trigger reagent comprises antimicrobial and / or preservative agents such as for example 5-chloro-2-methyl-4-isothiazolin-3-one and / or 2- methyl-4-isothiazloin-3-one, or other antibiotic agents.

[0063] In some embodiments, hexadimethrine bromide is added prior to step d. and may optionally already be present in the coagulation trigger reagent. In particular hexadimethrine bromide may be added in a concentration of 0.01 mg / mL to 0.1 mg / mL. Hexadimethrine bromide can reverse heparin anticoagulation and may thus be used as an antidote for heparin. Alternatively, protamine sulfate (CAS: 9009-65-8) can also be used. In some embodiments, the hemostatic regulator with respect to which the blood sample is analyzed is a factor or protein of the extrinsic pathway. In some embodiments, the hemostatic regulator with respect to which the blood sample is analyzed is selected from tissue factor pathway inhibitor (TFPI), protein Z-related protease inhibitor (ZPI), protein Z (PZ), protein S, protein C, antithrombin III (AT-III), FVa, FVIIIa and heparin co factor II (HCI I) , preferably tissue factor pathway inhibitor (TFPI). In certain embodiments, the hemostatic regulator with respect to which the blood sample is analyzed is selected from tissue factor pathway inhibitor (TFPI), protein Z-related protease inhibitor (ZPI), protein Z (PZ), protein S, protein C, antithrombin III (AT-III) and heparin co factor II (HCI I). In some embodiments, the hemostatic regulator is one of the four major anticoagulant systems, i.e. AT-III, ZPI and / or PZ, protein C respectively activated protein C, or TFPI.

[0064] In certain embodiments in which particularly the hemostatic regulator with respect to which the blood sample is analyzed is AT-III, the coagulation trigger reagent may comprise heparin, or heparin derivatives or heparin like synthetic molecules such as fondaparinux, in particular unfractionated heparin (UFH). For example, heparin or heparin-like drugs may be present in the coagulation trigger reagent in a concentration of 0.1 to 0.5 U / rnL.

[0065] In some embodiments, the method analyzes a blood sample only with respect to one, i.e. a single, hemostatic regulator.

[0066] In some embodiments, the blood sample and / or the dilution plasma reagent and / or the coagulation trigger reagent are prior to step d. incubated together. Preferably, the blood sample and / or the dilution plasma reagent and / or the coagulation trigger reagent are prior to step d. incubated together for an incubation time of 0 min to 10 min, in particular 0.05 min to 10 min, more particular 0.05 min to 5 min, even more particular 0 to 2 min, even more particular 0.05 min to 2 min.

[0067] In some embodiments, the blood sample and / or the dilution plasma reagent and / or the coagulation trigger reagent are prior to step d. incubated together at 25 to 38 °C, in particular 35 °C to 38 °C, more particular 37 °C. In some embodiments, the tissue factor of the coagulation trigger reagent may be recombinant or tissue derived human, rabbit or bovine tissue factor.

[0068] In some embodiments, the coagulation trigger reagent is added and configured such that the effect of the hemostatic regulator with respect to which the sample shall be analyzed on the coagulation cascade, in particular on the clotting timeAt, becomes visible. In some embodiments, step c. is performed such that the TF concentration after step c. (i.e. in the mixture of the blood sample, dilution plasma reagent and coagulation trigger reagent), in particular after step d., is selected such that the effect of the hemostatic regulator with respect to which the sample shall be analyzed on the coagulation cascade, in particular on the clotting timeAt, becomes visible. An effect becoming visible may in particular mean that a difference between the activity of the hemostatic regulator with respect to which the blood sample shall be analyzed in healthy subjects (being for example set to 100%) and the activity of the hemostatic regulator with respect to which the blood sample shall be analyzed in the subject to be analyzed, becomes visible, respectively determinable. For example, if the activity of the hemostatic regulator with respect to which the blood sample shall be analyzed is higher in the subject to be analyzed, this higher activity can be determined. As another example, if the activity of the hemostatic regulator with respect to which the blood sample shall be analyzed is lower (e.g. between 100% activity for healthy subjects and 0% activity for a sample being devoid of the hemostatic regulator) this lower activity can be determined. In particular, the effect of this difference becomes directly visible, respectively determinable, in a different clotting timeAt.

[0069] In particular embodiments, the coagulation trigger reagent is added and configured such that an activity difference of 50% of the hemostatic regulator with respect to which the sample shall be analyzed as compared to the activity obtained for one or more healthy subjects (being for example set to 100%) on the clotting timeAt may result in a clotting time difference of at least 2 s, in particular at least 5 s, more particular at least 10 s, even more particular at least 15 s. In particular embodiments, step c. is performed such that the TF concentration after step c. (i.e. in the mixture of the blood sample, dilution plasma reagent and coagulation trigger reagent), in particular after step d., is selected such that an activity difference of 50% of the hemostatic regulator with respect to which the sample shall be analyzed as compared to the activity obtained for one or more healthy subjects (being for example set to 100%) on the clotting timeAt may result in a clotting time difference of at least 2 s, in particular at least 5 s, more particular at least 10 s, even more particular at least 15 s.

[0070] In particular embodiments, the coagulation trigger reagent is added and configured such that an activity difference of 50% of the hemostatic regulator with respect to which the sample shall be analyzed as compared to the activity obtained for one or more healthy subjects (being for example set to 100%) on the clotting timeAt may result in a clotting time difference of 5 s to 120 s, in particular of 5 s to 100 s, more particular or 10 s to 80 s, even more particular or 15 s to 30s. In particular embodiments, step c. is performed such that the TF concentration after step c. (i.e. in the mixture of the blood sample, dilution plasma reagent and coagulation trigger reagent), in particular after step d., is selected such that the activity obtained for one or more healthy subjects (being for example set to 100%) on the clotting timeAt may result in a clotting time difference of 5 s to 120 s, in particular of 5 s to 100 s, more particular or 10 s to 80 s, even more particular or 15 s to 30s.

[0071] In some embodiments, the coagulation trigger reagent is added and configured such that tissue factor is present after step c. (i.e. in the mixture of blood sample, dilution plasma reagent and coagulation trigger reagent) or after step d. (i.e. in the mixture of blood sample, dilution plasma reagent, coagulation trigger reagent and calcium ions) in a concentration of less than 100 pM, in particular 0.1 to 100 pM, in particular 1 to 100 pM, more particular 1 to 10 pM. In particular embodiments, the coagulation trigger reagent comprises tissue factor, particularly as the only coagulation component, e.g. coagulation trigger component.

[0072] In some embodiments, the coagulation trigger reagent is added and configured such that tissue factor is present after step c. (i.e. in the mixture of blood sample, dilution plasma reagent and coagulation trigger reagent) in such a concentration that the clotting timeAt for a blood sample (e.g. a blood sample after calcium ions and the dilution plasma reagent have been added) being devoid of the hemostatic regulator with regard to which the blood sample is analyzed, in particular of TFPI, is 80 to 250 s, in particular 80 to 120 s, more particular 90 to 110 s, even more particular 95 to 105 s. These clotting times may for example be obtained by a KC4 coagulometer (Amelung / Lemgo / Germany) or by the naked eye.

[0073] In some embodiments, the coagulation trigger reagent being added in step c. is lyophilized and preferably dissolved, e.g. in water, prior to step c.. That is, after its preparation, the coagulation trigger reagent is lyophilized. In some embodiments, the coagulation trigger reagent is liquid or solid (e.g. frozen).

[0074] Another aspect of the present disclosure relates to a method of determining the activity or concentration of a TFPI inhibitory therapeutic agent in a blood sample. Such a method may comprise the method for analyzing a blood sample with respect to a hemostatic regulator according to any of the embodiments described herein. The hemostatic regulator may for example be TFPI. Furthermore, the measured clotting time *t is compared to reference data. The reference date may for example be or comprise a calibration curve. In some embodiments, the reference data may in particular associate different clotting times with different activities or different concentrations of the TFPI inhibitory therapeutic agent.

[0075] Another aspect of the present disclosure relates to method for determining the risk of developing a hemostatic disorder. The method may comprise the method for analyzing a blood sample with respect to a hemostatic regulator according any of the embodiments described herein. In the method, the activity or concentration of the hemostatic regulator in the blood sample may be determined and compared to a database or calibration curve associating the hemostatic regulator activity or concentration to a risk for developing the hemostatic disorder. Thereby, a risk for the patient from which the sample may originate may be determined. The database may for example comprise, or consist of a single threshold activity or threshold concentration. If this threshold activity or threshold concentration is exceeded, there may be a risk for developing the hemostatic disorder. It may also be possible that the database comprises multiple activities or concentrations being associated to different risk percentages. The hemostatic disorder may be selected from preeclampsia, thrombosis and hemophilia, respectively the bleeding risk.

[0076] Another aspect of the present disclosure relates to a method for determining a risk of developing preeclampsia. This method may be an in-vitro method. The method may comprise the method for analyzing a blood sample with respect to a hemostatic regulator according to any of the embodiments described herein. Since TFPI serves as a predictor for preeclampsia, the hemostatic regulator in this method may be TFPI. The method may comprise determining the activity or concentration of TFPI in the blood sample and comparing the determined activity or concentration to a database or calibration curve, thereby optionally associating the TFPI activity or concentration to a risk for developing preeclampsia. Thereby, a risk for the patient from which the sample may originate may be determined. The database may for example comprise, or consist of, a single threshold activity or threshold concentration. If this threshold activity or threshold concentration is exceeded, there may be a risk for developing preeclampsia. It may also be possible that the database comprises multiple activities or concentrations being associated to different risk percentages.

[0077] Brief description of the figures

[0078] Fig. 1 shows a schematic representation of the coagulation cascade. The bold T-shaped lines indicate selected inhibition actions. For example, TFPI inactivates FVIIa and FXa. Moreover, in combination with protein S, TFPI regulates the activation of FV by FXa.

[0079] Examples

[0080] 1. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor (TFPI) - Initial Coagulation Trigger Reagent Formulation

[0081] Determination of TFPI functionality was performed for SSC / ISTH Secondary Coagulation

[0082] Standard Lot #5 Plasma containing different concentrations of TFPI, using a coagulation trigger reagent (a commercially available rabbit brain thromboplastin, such as diluted PTr), comprising a low concentration of tissue factor in a buffer solution as prepared as follows: The coagulation trigger reagent ( Technoplastin HIS, Technoclone, Ref 5003030 / 5003026 I 5003009 / 5003021 , or STA®-Neoplastine® Cl 5, Stago Ref. 00605, or, STA®- Neoplastine® Cl Plus 5, Stago, Reff 00606, or Pacific Hemostasis™ Prothrombin Time reagent, REF 100354TS, BIO-TP, Low ISI; Biolabo, France, REF 13702, or Thromboral S, a human placenta based thromboplastin, Siemens, OLIHP29 / OLIHP49, or Innovin, human recombinant TF, B4212-40 / B4212-50 / B4212-100) is generally prepared by dissolving 1 vial according to manufacturer’s instruction and diluted 1 :4000 using the following buffer: 25 mM HEPES, 150 mM NaCI, 0.5 % Bovine Serum Albumin, 0.05 % ProCiin 300. The coagulation trigger reagent generally prepared (diluted PTr) contains 2-5 pM of Tissue Factor, the pH is 7.2.

[0083] The different TFPI concentrations were established as comparison to the dilution of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma with TFPI-depleted plasma (containing less than 5% TFPI activity as compared to normal pool plasma, e.g. the SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma). The TFPI-depleted plasma (BioMedica, Catalogue Number: 848DP) is commercially available and is manufactured using immunodepletion techniques. Twenty microliters of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma were mixed with thirty microliters of TFPI-depleted plasma to establish the corresponding 100 % TFPI activity. The solution containing fifty microliters of TFPI-depleted plasma establishes the correspondent to 0 % TFPI activity. The 50 % TFPI activity is produced using ten microliters of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma and forty microliters of TFPI-depleted plasma. Mixing thirty microliters of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma and twenty microliters of TFPI-depleted plasma generate a sample corresponding to 150 % TPI activity.

[0084] The samples containing the different TFPI concentrations were used to prepare a calibration curve based on the clotting times (see table la). Fifty microliters of each sample were mixed with fifty microliters of the prepared coagulation trigger reagent (diluted PTr) in a KC4 coagulometer (Amelung / Lemgo / Germany). Coagulometer cuvette and incubated for two minutes at 37°C. Then, fifty microliters of a 25 mM aqueous solution of CaCh were added and the clotting time was recorded. As shown in Table lb, the functionality of TFPI is correlated with the clotting times.

[0085] In general, sensitivity means the relative shortening of the clotting time at 100% TFPI activity versus 0% TFPI. For example, 150 sec / 100 sec = 1.5. The coefficient of variation is in general in the examples described herein less than 5%.

[0086] Table la

[0087] Table lb

[0088] 2. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor (TFPI) - Initial Coagulation Trigger Reagent Formulation containing PL

[0089] Determination of TFPI functionality was performed for SSC / ISTH Secondary Coagulation

[0090] Standard Lot #5 Plasma containing different concentrations of TFPI, using the coagulation trigger reagent (diluted PTr) as described in Example 1 , which further comprised phospholipids (PL). The coagulation trigger reagent (diluted PTr, prepared according to Example 1), was prepared with different concentration of PL (0 - 0.5 mg / mL. The samples containing different concentrations of TFPI, as in Example 1 , were tested in the absence or presence of PL. Again, fifty microliters of each sample were mixed with fifty microliters of the coagulation trigger reagent (diluted PTr) in the absence or presence of PL in a KC4 Coagulometer cuvette and incubated for two minutes at 37 °C. Then, fifty microliters of a 25 mM aqueous solution of CaCh were added and the clotting time (CT) was recorded. As shown in table II, the addition of PL, shortens substantially the clotting times.

[0091] Table II

[0092] 3. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor (TFPI) - Initial Coagulation Trigger Reagent Formulation containing PL and Aprotinin

[0093] Determination of the clotting time was performed for SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma using the coagulation trigger reagent (diluted PTr) as described in Example 2, comprising 0.02 mg / mL of PL and the serine protease inhibitor aprotinin. The aprotinin was tested in the range of 0.1 to 10 TIU. The samples containing different concentrations of TFPI, as in Example 1 , were tested in the absence or presence of aprotinin. Again, fifty microliters of each sample were mixed with fifty microliters of the coagulation trigger reagent (diluted PTr) comprising 0.1 to 10 TIU of aprotinin in a KC4 Coagulometer cuvette and incubated for two minutes at 37°C. Then, fifty microliters of a 25 mM aqueous solution of CaCh were added and the clotting time was recorded. As shown in Table III, the addition of aprotinin increases the sensitivity of the detection of TFPI functionality.

[0094] Table III 4. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor

[0095] (TFPI) - Coagulation Trigger Reagent prepared using different buffers

[0096] Determination of the clotting time was performed for SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma using the coagulation trigger reagent (diluted PTr) as described in Example 3, testing the effect of different buffer compositions (Table IVa) on the clotting times, lyophilization process and stability. The buffers tested were 25 mM HEPES, 25 mM Imidazol and 25 mM Tris. Each buffer solution contained as well, 150 mM NaCI, 0.5 % Bovine Serum Albumin, 1 TIU / rnL Aprotinin and 0.05 % ProCiin 300, pH adjusted to 7.2. Generally, the addition of any buffer improves the performance, compared to the variant without buffer, however, none of the buffer substances tested had significant advantages or disadvantages, both before and after freeze drying (Table IVb). Nevertheless, imidazole clearly provided the best results in terms of the physical appearance of the freeze-dried cake. Table IVa:

[0097] Table IVb 5. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor

[0098] (TFPI) - Reagent with Imidazole Buffer containing hexadimethrine bromide

[0099] The coagulation trigger reagent with imidazole buffer containing hexadimethrine bromide (i.e. commonly known under the trade name “polybrene”). Determination of the clotting time was performed_for SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma using the coagulation trigger reagent (diluted PTr) as described in Example 4, testing the effect of hexadimethrine bromide to neutralize different heparins. The coagulation trigger reagent (diluted PTr) as described in Example 4, was prepared containing no or 0.02 mg / mL hexadimethrine bromide. The SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma was spiked with Liquemin® (unfractioned heparin)(1 IIJ / mL), Fragmin® (low molecular weight heparin) (1.6 lll / mL) or Arixta® (synthetic heparin like pentasaccharide) (1.5 .g / mL) and clotting times were analyzed. Table V shows the results for the efficacy of 0.02 mg / mL hexadimethrine bromide. The results show that the assay tolerates the presence of hexadimethrine bromide and can neutralize the effect of heparins spiked.

[0100] Table V

[0101] 6. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor (TFPI) - Coagulation Trigger Reagent with Imidazole Buffer and the optimization of NaCI concentration

[0102] Determination of the clotting time was performed for SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma using the coagulation trigger reagent (diluted PTr) and imidazole buffer as described in Example 4, testing the effect of NaCI on the clotting times and lyophilization process. The coagulation trigger reagent (diluted PTr) as described on Example 4, was prepared containing 10 - 175 mM of NaCI and the clotting times and aspect after lyophilization were analyzed. Table Via shows the results for the impact of NaCI concentration on clotting times before lyophilization and Table VI b after lyophilization and storage at 4-8 °C. The results demonstrate that, before lyophilization, the NaCI concentrations of 125, 150 and 175 mM present similar sensitivity. In particular, the concentrations 150 and 175 mM perform slightly better. After the lyophilization, the concentrations of 150 and 175 mM allow a stable lyophilization-cake in contrast to a concentration of 125 mM. The dissolution was not affected. In general, it is possible to observe that the clotting times are reduced after lyophilization. After storage for 72 h at 2- 8° C a similar sensitivity for 150 and 175 mM NaCI was found, and both were similar to the sensitivity obtained before lyophilization. However, the clotting time corresponding to 100% is shortened as compared to the values observed before lyophilization.

[0103] Table Via

[0104] Table Vlb 7. Clotting assay to evaluate the functionality of Antithrombin III (AT-III)

[0105] Determination of AT-III functionality was performed for SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma containing different concentrations of AT-III, using the dilution plasma reagent (diluted PTr) as in Example 3. The coagulation trigger reagent (in this representative example BIO-TP, Low ISI; Biolabo.REF 13702 was used) is prepared dissolving 1 vial according to manufacturer’s instruction and diluted 1 :4000 using the following buffer: 25 mM Imidazole, 150 mM NaCI, 0.02 mg / mL of Cephalin, 1 TIU / mL of Aprotinin, 1 % Bovine Serum Albumin, 0.05% ProCiin™ 300 (SigmaAldrich 48912-U). (coagulation trigger reagent - diluted PTr, containing 2-5 pM of Tissue Factor and pH adjusted to 7.2). Twenty microliters of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma were mixed with thirty microliters of AT-lll-immunodepleted plasma (Enzyme Research Laboratories, REF. ATI I l-DP) to establish the corresponding 100 % AT-III activity. The solution containing fifty microliters of AT-III -depleted plasma establishes the correspondent to 0 % AT-III activity. The 50 % AT-III activity is produced using ten microliters of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma and forty microliters of AT-lll-depleted plasma. Mixing thirty microliters of SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma and twenty microliters of AT-lll-depleted plasma generate a sample corresponding to 150 % AT-III activity. The samples containing the different AT-III concentrations were used to prepare a calibration curve based on the clotting times. Fifty microliters of each sample were mixed with fifty microliters of the coagulation trigger reagent (diluted PTr) in a KC4 Coagulometer cuvette and incubated for two or five minutes at 37 °C. Then, fifty microliters of a 25 mM aqueous solution of CaCh were added and the clotting time was recorded. As shown in Table VII, the functionality of AT-III is correlated with the clotting times.

[0106] Table VII

[0107] 8. Clotting assay to evaluate the functionality of Antithrombin III (AT-III) - Coagulation Trigger Reagent containing Unfractionated Heparin (UFH) Determination of AT-III functionality was performed for SSC / ISTH Secondary Coagulation Standard Lot #5 Plasma containing different concentrations of AT-III, using the coagulation trigger reagent (diluted PTr) as in the Example 7, testing the potential enhancing effect of UFH. The coagulation trigger reagent (diluted PTr) as described in Example 7, was prepared containing 0.1 to 0.5 lll / mL of Liquemin (UFH) and clotting times were analyzed.

[0108] Table VIII shows the results for the efficacy of 0.1 and 0.15 lU / mL of Liquemin. The results show the AT-III enhancing activity of UFH, and therefore the sensitivity of the assay is increased.

[0109] Table VIII

[0110] 9. Clotting assay to evaluate the functionality of Tissue factor pathway inhibitor (TFPI) - Impact of reagent concentration of tissue factor on sensitivity for TFPI

[0111] TF activator reagents for common comparable applications contain usually 4 - 40 pmol / mL Tissue Factor (TF). To achieve an optimal sensitivity of the assay, the TF concentration is reduced to obtain a concentration of approximately 1 pmol / mL (1:4000).

[0112] During development, the ratio between the CT of 100% TFPI and the CT of 0% TFPI is used to indicate the sensitivity for TFPI.

[0113] Table IX

[0114] The data below shows that sufficient sensitivity for TFPI requires to operate with a very low TF concentrations in the activator reagent.

[0115] Table X

Claims

Claims1. A method, in particular an in vitro method, for analyzing a blood sample with respect to a hemostatic regulator, the method comprising the steps: a. Providing a blood sample to be analyzed, wherein the blood sample comprises a chelating agent being configured to chelate calcium ions; b. Selectively reducing the concentration of the hemostatic regulator in the blood sample with respect to which the blood sample shall be analyzed by the addition of a dilution plasma reagent; c. Adding a coagulation trigger reagent comprising tissue factor; d. Adding calcium ions at starting time to in at least such an amount that saturation of the chelating agent in the blood sample is achieved; e. Measuring the clotting timeAt between starting time to and end time te.

2. The method according to claim 1, wherein the measured clotting timeAt is in a subsequent step compared to reference data, in particular a calibration curve or the clotting time of another subject, in particular a healthy subject.

3. The method according to claim 2, wherein the reference data is a calibration curve associating a clotting time with an activity of the hemostatic regulator or with a concentration of the hemostatic regulator.

4. The method according to any of the previous claims, wherein step b. comprises adding the dilution plasma reagent to the blood sample in excess with respect to the blood sample, wherein the dilution plasma reagent is depleted of the hemostatic regulator to be analyzed.

5. The method according to claim 4, wherein the dilution plasma reagent comprises all other coagulation components which are necessary for coagulation.

6. The method according to any of the previous claims, wherein phospholipids are added during or prior to step d.

7. The method according to any of the previous claims, wherein an antidote for anticoagulant agents, in particular heparin and heparin derived anticoagulant agents, is added during or prior to step d.

8. The method according to any of the previous claims, wherein the dilution plasma reagent is provided in a volumetric excess with respect to the blood sample of at least 2-fold, in particular 2 to 20-fold, more particular 3 to 10-fold.

9. The method according to any of the previous claims, wherein the end time te is reached when thrombin formation or fibrin formation is detected.

10. The method according to claim 9, wherein thrombin formation is detected by a clotting assay or by adding a selective detecting agent which is configured to undergo a reaction with thrombin upon which a detectable signal or detectable entity is generated and by detecting the detectable signal or detectable entity or wherein or fibrin formation is detected by optical methods, in particular measuring the change of transmission or viscoelastic methods.

11. The method according to any of the previous claims, wherein one or more activators of coagulation cascade factors or control proteins of the coagulation cascade are added prior to step d.

12. The method according to claim 11 , wherein the one or more activators is selected from FXIa, thrombomodulin, an activator of protein C, in particular a snake venom derived or recombinant activator of protein C, and activated protein C.

13. The method according to any of the previous claims, wherein the hemostatic regulator is selected from TFPI, ZPI, protein Z, protein S, protein C, AT-III, FVa, FVIIIa and HCII.

14. The method according to any of the previous claims, wherein prior to step d. the blood sample and / or the dilution plasma reagent and / or the coagulation trigger reagent are incubated together, in particular for 0 to 10 min, more particular 0.05 to 5 minutes, more particular 0.05 to 2 min.

15. The method according to any of the previous claims, wherein the coagulation trigger reagent is added and configured such that the effect of the hemostatic regulator with respect to which the sample shall be analyzed on the coagulation cascade, in particular on the clotting timeAt becomes visible.

16. The method according to any of the previous claims, wherein the coagulation trigger reagent is added and configured such that tissue factor is present after step c. in a concentration of 0.1 to 100 pM, in particular 1 to 100 pM, more particular 1 to 10 pM.

17. The method according to any of the previous claims, wherein the coagulation trigger reagent is added and configured such that tissue factor is present after step c. in such a concentration that the clotting timeAt for a blood sample being devoid of the hemostatic regulator, in particular of TFIP, is 80 to 250 s, in particular 80 to 120 s, more particular 90 to 110 s, even more particular 95 to 105 s.

18. The method according to any of the previous claims, wherein the concentration of added calcium ions added in step d. is 5 to 200 mM, in particular 20 to 30 mM.

19. The method according to any of the previous claims, wherein step d. is performed such that the calcium ion concentration after step d. is at least 2.5 mM.

20. A method, in particular an in vitro method, for analyzing a blood sample with respect to a hemostatic regulator, the method comprising the steps:a. Providing a blood sample to be analyzed, wherein the blood sample comprises a chelating agent being configured to chelate calcium ions; b. Adding a dilution plasma reagent to the blood sample in excess with respect to the blood sample, wherein the dilution plasma reagent is depleted of the hemostatic regulator to be analyzed and wherein the dilution plasma reagent comprises all other coagulation components which are necessary for coagulation; c. Adding a coagulation trigger reagent comprising tissue factor; d. Adding calcium ions at starting time to in at least such an amount that saturation of the chelating agent in the blood sample is achieved; e. Measuring the clotting timeAt between starting time to and end time te.

21. A method for determining the concentration or activity of a TFPI inhibitory therapeutic agent in a blood sample, the method comprising the method for analyzing a blood sample with respect to a hemostatic regulator according to any of the previous claims, wherein the hemostatic regulator is TFPI.

22. A method for determining the risk of developing a hemostatic disorder, comprising the method for analyzing a blood sample with respect to a hemostatic regulator according any of claims 1 to 20, wherein the activity or concentration of the hemostatic regulator in the blood sample is determined and compared to a database or calibration curve associating the hemostatic regulator activity or concentration to a risk for developing the hemostatic disorder.

23. The method according to claim 22, wherein the hemostatic disorder is selected from preeclampsia, thrombosis and hemophilia.

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