Whole blood thrombine activity test

FIELD: medicine.

SUBSTANCE: for thrombin production measurement, a layer of said sample contacts with a fluorogenic substratum of thrombin where the thickness of said layer is 0.05 to 5 mm, while the surface area is 10 to 500 mm2. Further, the thrombin production environment in said sample is provided. It is followed by measuring the fluorescence emitted from the layer surface by a fluorescent group released by the fluorescent substratum as a result of an enzymatic action of produced thrombin on said fluorogenic substratum. Besides, the invention ensures a kit for measuring the thrombin activity in the sample.

EFFECT: higher measuring accuracy.

29 cl, 12 dwg, 5 ex

 

[0001] the Present invention relates to a method of determining, in particular, measurements of the dynamics of the activity of thrombin in vitro, i.e. to measure the number of active thrombin in the sample, in particular, how it changes in the sample of whole blood in the formation of a clot. The result of measuring the activity of thrombin can be represented in the form of so-called "curve generation of thrombin is shown in Figure 1.

[0002] the Present invention also relates to a means to measure the amount of active thrombin after its production in a sample in vitro. The present invention is also directed to the use of this method of measurement for the detection or monitoring of a disease in a patient, including the detection or monitoring of a pathological condition associated with bleeding disorders.

[0003] the Present invention also relates to the application of the above method of measurement for the selection of substances, including screening to search for drugs that can affect the coagulation process, in particular, on the activity of thrombin.

[0004] Thrombotic diseases, such as myocardial infarction, stroke, pulmonary embolism, and some others, are the cause of approximately half of all deaths and cases of disability in Western society. In developing countries the number of these Zab the problems grows with the development. Hemophilia, although not as widespread, is also a significant cause of death. Thus, excessive or insufficient function of the hemostatic system is an extremely important mechanism of pathogenesis. It makes even more remarkable the absence of reliable clinical test.

The role of thrombin in the disorder of hemostasis and thrombosis.

[0005] the Thrombin plays a Central role in hemostasis and thrombosis. It has long been known for thrombotic venous disease (1), and was convincingly demonstrated by the fact that the prevention and treatment of venous thrombosis of the best reduces the activity of thrombin, or by direct inhibition (hirudin, melagatran), or by reducing synthesis (vitamin K antagonists), or by increasing decay (heparins). In recent decades, it becomes clearer that in diseases of the arteries thrombin plays the same important role as in diseases of the veins. Clinical trials have shown that vitamin K antagonists (2), and heparin (3) reduce the frequency of repeated heart attacks. The role of thrombin in the bleeding indicates increased bleeding time, which see under such severe violations of formation of thrombin, as caused by a serious overdose of oral anticoagulants (4) or heparin (5). Gemfi the AI are also diseases of the education system of thrombin (6).

In the formation of thrombin involves all elements of blood.

[0006] Modern research has provided an understanding of the formation of thrombin is the result of joint action of the elements forming the blood and plasma. Red blood cells (erythrocytes) are the least active in this respect, although the membrane a small percentage of red blood cells demonstrates procoagulation activity (7). Much more important is the fact that white blood cells have activity of tissue factor. This activity is normal is hidden, but if the damage is manifested in the interaction with platelets in the blood (8, 9). The main participants are, without doubt, platelets and plasma coagulation system. In textbooks still can read that platelets provide primary hemostasis and thrombosis of arteries, while clotting plasma is responsible for sealing hemostatic plug is the mechanism of thrombosis. This view is based on the fact that plasma and platelets was studied separately. In reality, the interaction between platelets and plasma, and other blood cells, is necessary for both primary and secondary hemostasis and thrombosis of arteries and veins. The formation of platelet plugs plays a role in the formation of thrombin, since the cavity in aggregates of platelets form neprinesiem the niche, where can proceed the formation of thrombin, which will not be carried away by the blood stream. Therefore, the measurement of the generation of thrombin in the clotting of whole blood so close to the physiological reality.

[0007] in Addition to education "sponge", which can be formed thrombin, platelets themselves also make a significant contribution to the production of thrombin. They contain factor V and provide Pro-coagulation phospholipid surface, necessary for the conversion of prothrombin and at various stages of the mechanism of coagulation, which leads to the formation of prothrombinase (10). The speed of thrombin generation and the resulting amount of thrombin depends, therefore, on the activity of platelets, as well as involved in the process of plasma proteins. Especially interesting is the role of polymerizing fibrin. The factor a background of Villebranda (vWf) interacts with polymerizing fibrin and undergoes a conformational change, which makes it able to react with platelet receptor GPIb, and through this link to interact with platelets and become procoagulant (11, 12). This shows that the formation of a fibrin clot is not the final act of hemostasis and the key event of the whole process is the formation of thrombin in the tube (the thrombus or clot). In fact, as we will see is who, >95% of the total generated thrombin is formed after the formation of a blood clot, and this thrombin is necessary for the processes of hemostasis and thrombosis (N&T). Perhaps the best evidence of the close link between platelets and plasma coagulation system is the fact that all the "aggregation inhibitors and other antiplatelet agents also inhibit the production of thrombin in platelet-rich plasma (or whole blood). It has been shown to aspirin (13), abziksimaba (14), MC (15) and clopidogrel (16). Conversely, the fact that antiplatelet tools, and in particular aspirin prevent thrombosis (17), is an additional illustration of the close connection between the function of platelets and the coagulation of blood.

[0008] Thus, the amount of thrombin generated in the blood clot is a significant feature of hemostasis and thrombosis, and all the blood elements involved in its formation.

The generation of thrombin (W) as an indicator of the risk of thrombosis or bleeding.

[0009] Increased TUE invariably indicates the risk of thrombosis, regardless of whether it is due to deficiency of anti-thrombin or excess of prothrombin. Generation of thrombin exceeds the norm also in violation of the chain reactions of protein s deficiency protein S and factor VLeiden). It is true SVER is ivania plasma as a whole, but it becomes especially obvious when you activate thrombomodulin chain reaction protein (figure 1). The tendency to thrombosis, which is called oral contraceptives, can be attributed to acquired resistance to activated protein C, which causes a 10% increase in the production of thrombin and becomes more obvious with the addition of thrombomodulin or concentrate of autologous platelets (18, 19).

[0010] a Particularly interesting case of lupus anticoagulant. This type of antibody causes an increase in the delay time of the formation of thrombin, and, accordingly, increased clotting time, but also significant resistance system activity of the protein (20). This explains the paradox LE", i.e. anticoagulate action, which is accompanied by the tendency to thrombosis.

[0011] it Was found that excessive amounts of factors II, VIII and VII correlate with the incidence of myocardial infarction (21-24). Also, increased von Willebrand factor thrombin generation to levels above normal (12) is a risk factor for arterial thrombosis (25, 26).

[0012] it Was shown that in sub-populations of young stroke patients (approximately 30%) and thrombin generation in platelet-rich plasma (PRP), as well as under the influence of von Willebrand factor is above the norm (27). When all hereditary nedostatocnosti factors swertia is of blood thrombin generation is reduced. This is demonstrated for hemophilia types a, b and C (the absence of factor VIII, IX or XI; 28-31), as well as for all rare species of nedostatochnosty (absence of prothrombin, factor V, VII, X, XII; 32). Bleeding is observed at lower WATTS on 20% below normal. In hemophilia type And not just the infusion of factor VIII or introduction desmopressina (I-diamine-8-D-arginine-vasopressin, DDAVP) compensate for the blood's ability to form thrombin, but also concurrent therapy products containing prothrombin and/or factor VII increases the production of thrombin.

[0013] Heavy trombopenia (<50000 ál-1) causes a decrease in the production of thrombin, as well as thrombopathia the-Glanzmann's (Glanzman) and Bernard-Soulier syndrome. When disease von Willebrand's disease, which is known to cause emerging violation adhesion of platelets, there is a substantial violation of thrombin generation in platelet-rich plasma (see above). This defect in platelet-rich plasma significantly stronger expressed than in platelet-poor plasma, which indicates that they cannot be explained by concomitant, usually mild, decreased factor VIII.

Thrombogram

[0014] In consideration of the problem to be solved according to the present invention, it is necessary to take into account the following observations.

[0015] Even a simplified diagram of the mechanism of formation of thrombin (Fig) shows it is a very difficult and intense positive and negative reactions to the feedback process. In fact, it is so complex that it is a complex system, i.e. there is no simple relationship between the concentrations of the reactants and the result (output)and the threshold effect may actually cause unpredictable system behavior. Accordingly, the response of the entire system at a specific impulse cannot be predicted based on knowledge of the individual concentrations of the respective reagents (which may even be unknown), and only test, which measure the function of the entire system contained in the blood of a patient, can indicate the hemostatic/tromboliticescoe condition of the individual patient.

[0016] the Result of the entire process for generating thrombin is a temporary appearance and disappearance of the activity of thrombin. For the curve of the activity of thrombin in a time-dependent or Thrombogram (Thrombogram™, TG) is characterized by the initiation phase or time delay (lag), for which only small amounts of thrombin; followed by a burst of activity, known as the growth phase (figure 1). A clot of blood formed at the beginning of the burst, and almost all of thrombin is formed after the formation of a clot. All the formed thrombin subsequently inactivated by antithrombin blood. This is proteins during slow reactions associated stoichiometric amount of thrombin. The rate of inactivation is proportional to the concentration of thrombin and anti-thrombin. While the rate of conversion of prothrombin higher than the rate of inactivation of thrombin, the level of thrombin increases. With increasing levels of thrombin, the rate of inactivation increases. At the peak of both velocities are equal, and then begins to dominate the decay. The obtained curve activity of thrombin shows the different phases and, in particular, the peak production of thrombin, the time to reach the peak and the potential endogenous thrombin (PET).

Prior art

[0017] In the last century, the need to measure the function of hemostasis and thrombosis not escaped the attention of physicians. Solutions to this problem have not changed substantially until the 1990s and suggested tools that were either practical, but inadequate or adequate, but not practical.

[0018] Practical solutions related to the measurement of clotting time and bleeding time. The clotting time is a measure of the length of the phase of initiation of the generation of thrombin and, therefore, reflects only part of the function (33, see also above). The mere fact that in clinical laboratory practice using many varieties of this test, each of which is suitable only for a specific situation, already shows that the clotting time is not reflected in the AET mechanism of coagulation in whole. Regarding bleeding time you can say that this is a very inaccurate method with a coefficient of variation of about 40%, which greatly limits its practical use (34).

[0019] since the 1950s began to emerge in the understanding that the time dimension of the action of thrombin in a collapsible blood is the best way of evaluating the function of hemostasis and thrombosis (35-37). Before 1992, the only way to measure WATTS was sampling from collapsing blood or plasma and the determination of thrombin in the sample. Build one curve is about one man-hour, and therefore suitable for research purposes, but not for use in modern clinic or epidemiology.

[0020] In 1990, Hemker, Béguin et al. (EP-B1-0 420 332) put forward the idea of adding to a collapsible blood chromogenic (give color) substrate with high specificity for thrombin, but a low rate of conversion (low-Kcat) and low affinity binding of thrombin (high Km). This substrate will be present throughout the process W, which allows to measure the total (i.e. integral) the activity of thrombin in time by the total number of the formed product. Ideally, this allows you to measure the potential of endogenous thrombin (PET), i.e. the area under the curve generation of thrombin (AUC).

[0021] Later emker et al. additionally developed this method to get the curve W as a whole (38). This method is based on the principle that under suitable constants of the kinetics of substrate, the reaction rate can, with good approximation, be proportional to the concentration of thrombin during coagulation process, and therefore the first derivative of the concentration of the product gives a curve that is proportional to the activity of thrombin. This method is described in WO 03/093831 A1 represents an addition and improvement procedure that was previously described in EP-B2-0420332, which measured only the final product level, and, accordingly, received the area under the curve W, i.e. PET.

[0022] the Substances used in the specified way, give the yellow product, the measurement of a quantity which requires the measurement of optical density and, therefore, optically transparent reaction medium. Therefore, you should avoid turbidity caused by the formation of a clot from fibrinogen, and you must either remove fibrinogen, or to prevent its polymerization by adding a polymerization inhibitors. However, the removal of fibrinogen has the disadvantage that it represents the destruction of an important reagent and it cannot be performed without removal of cellular elements, such as an important blood platelets. In addition, d is the addition of polymerization inhibitors in high concentrations, necessary to prevent the formation of fibrin, inhibiting prothrombin converting enzyme and biochemical reactions leading to its formation.

[0023] the Fluorescence, in contrast to optical density can be measured in turbid environment. Thus, unlike chromogenic substrates, substrates that yield fluorescent product (fluorogenic substrates)can be applied in the plasma, is not free from fibrin and hence also in the platelet-rich plasma (PRP) (33,39-43). Application fluorogenic substrate, however, has two important drawbacks: 1) the fluorescence intensity is not proportional to the concentration of the fluorophore due to the so-called effect of the inner filter; 2) with the available substrates, the rate of formation of product is not necessarily proportional to the concentration of the enzyme. The last drawback can be circumvented by the use of substrates with low expenses, as in the method using a chromogenic substrate. At the moment, such substrates are not available. Currently in practice both problems can be solved together by continuous comparison of the experimental signal with a constant signal combinatorial activity under conditions identical to the conditions of the analyzed sample (WO 03/093831 A1).

[0024] a Chromogenic method, obviously, Nelly is cannot be applied to whole blood, since blood is not transparent. It was reported that fluorogenic substrates can be used to measure WATTS in whole blood (44). In the practical implementation described in the literature methods in most cases gives the wrong signals that do not reflect the dynamics of thrombin generation, known from a well-developed method of partial samples (figure 2). In addition, the quantitative ratio between the received signal and the number present thrombin varies from experiment to experiment. Briefly, the described method does not give reproducible quantitative results.

[0025] Another possibility is a strong dilution of blood (10 times or more), with the aim to reduce the impact of red blood cells (RBCs) (45). This gives curves that are best received for minimally diluted blood. However, for two reasons it is impossible to imagine that this approach is adequate physiological situation. First, after the formation of a clot (i.e. during the most important phase of thrombin generation), the formation of thrombin is limited by diffusion, since they are insoluble surfaces (the surface of platelets and other cells, immobilized in a fibrin network). This makes them more sensitive to dilution than reactions in free solution, such as reaction inactivation of thrombin. So about the time, the balance between the formation of thrombin and reactions inactivation of thrombin in the diluted blood is not typical for the situation in vivo. This is even more important when the blood contains pathological inhibitors (for example, when not treated hemophilia or inhibitor lupus erythematosus). In clinical practice it is well known that inhibitors of coagulation lose their effect after dilution in vitro.

[0026] therefore, there remains the problem of how to get the signal that it is possible to determine the concentration of thrombin in the sample collapsing blood, which is diluted less than ten times. The present invention is to provide a solution to this problem that will allow you to overcome at least some disadvantages of the current level of technology.

[0027] the Authors present invention have found that reproducible and erroneous results obtained to the present invention has at least two reasons: (a) sedimentation (deposition)occurring before the formation of blood clots and (b) retraction (seal) clot after it happened formation (see figure 4). It follows that the volume from which fluorescence during the reaction changes as the geometric shape of the surface, causing improper focusing and reflection of light, which imageoutputstream signal. The authors of the present invention unexpectedly found that these effects can occur when working with a thin layer of blood, and especially with a thin layer of lattice and/or microspheres. The geometric form of a thin layer together with the specified grid and/or microspheres, and in fact prevents the deposition and compaction of the sample. However, the reaction volume is unknown, and therefore it should be determined during the measurement. Do this by adding in the beginning of the experiment known concentration of fluorescent molecules. The signal is weak, and therefore the measurement on a large surface using any known in the field device effectively increases the ratio of signal to noise. In addition, since a high ratio of volume to surface see a tendency to evaporation of the sample, it is preferable to take appropriate measures to prevent this.

[0028] According to the present invention are described in vitro method for determining the dynamics of the activity of the thrombin time in the blood sample, whereby the measurement of thrombin generation includes the following stages:

- cast layer of the specified sample in contact with fluorophenyl a thrombin substrate, where the thickness of the specified layer is from 0.05 to 5 mm, and the surface area is from 10 to 500 mm2;

- providing conditions for the development of the Tr is Mina in a specified pattern;

- measurement of the fluorescence emitted from the surface of the specified layer specified fluorescent group, which releases a fluorescent substrate in the enzymatic effects of generated thrombin to the specified fluorogenic substrate.

[0029] the Method for determining the activity of thrombin allows to measure changes in the concentration of thrombin, which, according to the scheme of coagulation depends on his education and subsequent inactivation.

[0030] the Method according to the present invention can be performed on a blood sample, such as a sample of whole blood or a sample of platelet-rich plasma.

[0031] Typically, after contact of the specified sample with components suitable for the initiation of thrombin generation, provide conditions for the generation of thrombin in a specified sample. These components may include coagulation factors, such as tissue factor, and possibly calcium ions.

[0032] While thrombin is produced and present in the reaction mixture (active thrombin), it reacts with fluorogenic substrate, causing the substrate releases the fluorescent group.

[0033] the Reaction can arrange so that fluorogenic substrate was present throughout the reaction in quantities high enough to ensure the measurement of the activity of thrombin. Nab is emer, the concentration of the substrate is approximately equal to or above the Km of the substrate against thrombin, and thanks to the expenditure of the substrate has no significant effect on the reaction rate. The conversion of the substrate by thrombin leads to increased fluorescence emitted from the sample surface.

[0034] the Method defined in the present invention enables the production of thrombin in the blood sample, in particular, in the sample of whole blood, which is generally comparable to the generation of thrombin, which takes place in the body. Therefore, the present invention provides a reliable method of measurement for use in studies of hemostasis and thrombosis.

[0035] measuring the increase of fluorescence emitted from the sample surface could result in the release of the fluorescent group of the substrate under the influence of thrombin, preferably carried out by means of optical devices, which simultaneously allows you to cover large surfaces such as the surface area of from 10 to 500 mm2allows to detect light emitted by the surface. The wavelength chosen for measuring fluorescence, is determined by the selected fluorescent group. One wavelength is determined for the exciting light, which illuminate the sample for measurement. Friend the wavelength to determine the emitted light, generated when the release of the fluorescent group fluorogenic substrate of thrombin. To measure the fluorescence suitable optical devices, such as fluorescent tablet reading device, well known to specialists in this field (e.g., tablet reader Ascent Fluorescent plate reader, Thermolabsystems).

[0036] According to one implementation variant of the present invention, the sample to be analyzed, in particular, a sample of whole blood is placed in one or more containers, which allows simultaneous analysis of several samples. The design of such containers can be placed in them samples, subject to certain conditions and thickness of the surface layer of the sample to be analyzed. For example, flat-bottomed microplates containing wells, or container, or a substrate having other suitable geometrical design, well-known experts in the field, which used similar methods of analysis.

[0037] as the substrate for samples you can use other devices, if they meet the requirement to give samples for the analysis of the shape of the layer corresponding to the definitions of the present invention. Therefore, a description of the ways and means of implementation of the present invention, is made by reference to the wells (microplate), operasie samples, can be applied to other devices (container or substrate)containing samples.

[0038] According to the methods in accordance with the present invention, the concentration of thrombin during analysis is a function of the measured fluorescence of the fluorescent group released fluorogenic substrate of thrombin. The concentration of thrombin, in particular, proportional to the increase in the intensity of appearance of fluorescence.

[0039] Accordingly, the method according to the present invention allows to measure the concentration of thrombin during the whole period of activity of thrombin in the sample, provided the relevant quantities fluorogenic substrate.

[0040] Therefore, the dynamics of the activity of thrombin, before and after the formation of a blood clot, up to the maximum concentrations of thrombin and also for reducing the concentration of thrombin after reaching peak values in the inactivation of thrombin submitted by measuring the curve of the concentration of thrombin from time to time. During the various phases of activity of thrombin fluorogenic substrate reacts to the presence of thrombin, resulting in the hydrolysis of the substrate and the release fluorogenic group.

[0041] a Particular advantage of the present invention is what is the method allows to measure the production of thrombin in the sample, in particular, the sample of whole blood, which is not diluted or diluted in a minimal degree, not more than 10 times, in particular, 4 times or less than 4 times.

[0042] In the specific implementation described method, the layer thickness of the sample for analysis, in particular of a sample of whole blood is about 1 to 3 mm, in particular approximately 2 mm or less.

[0043] In a particular embodiment of the method, the surface of the sample, particularly a sample of whole blood, when it is placed in the wells of a microtiter plate or any other suitable container or substrate, is greater than 20 mm2for example, from 30 mm2up to 200 mm2in particular, more than 100 mm2in particular, in the range from 150 to 200 mm2.

[0044] as an example, the sample is transferred into the wells of the microplate, each of which has a diameter of 15 mm, when the thickness of the layer of the sample is less than 2 mm, which allows the analysis on the surface of approximately 175 mm2.

[0045] the Measurement of the activity of thrombin perform in a way that provides multiple points of reading fluorescence from the surface for each sample.

[0046] According to one implementation variant of the present invention, in that the method uses a sample, particularly a sample of whole blood, which is transferred into wells, which also contain a special grid with razmara the holes 50 microns.

[0047] as an alternative or Supplement to such a grid in one implementation of the present invention the sample, particularly a sample of whole blood is transferred into a well of a tablet or other container or substrate that contains microspheres.

[0048] the Presence of the specified grid and/or microgranules useful for the distribution of blood through the hole and, in particular, to prevent compaction of the bunch when minimizing blood. In other words, the presence of the grid or microgranules can prevent the violation of the surface of the roll of blood that will lead to a weakening of the signal measured during the analysis activity of thrombin. This lattice and/or microspheres can improve the suppression effects of the reduction, which is achieved by using large surface measurements. You can also use other means, which gives this effect.

[0049] In a particular implementation of the present invention wells containing sample, particularly a sample of whole blood, cover to avoid drying blood for measurement of the activity of thrombin as a result of evaporation. Cover the wells can be conventional materials, such as various types of thin plastic film, provided that they do not interfere with the fluorescence measurement.

[0050] the Measurement of the activity of thrombin in the blood sample is performed in the beginning of the Naya since when the sample is transferred into a pit (or any other suitable device, such as a groove) and to the specified sample type components necessary to initiate the generation of thrombin, including tissue factor, and until the disappearance of thrombin in the coagulation process.

[0051] In a particular embodiment of the method according to the present invention, the number fluorogenic substrate of thrombin, which add to the sample is in the range of 50-1000 μm. According to the described method, to find the concentration of active thrombin in absolute terms (i.e. in nm/l), it is necessary to know the volume of the blood sample, in which the measurement of fluorescence. For this to fluorophenol substrate of thrombin, you can add a fluorophore in a known concentration, and the corresponding ratio of the content of the substrate of thrombin to fluorophore lie in the range from 1% to 10% of the number of fluorescent molecules associated with the substrate of thrombin, in particular in the range from 1% to 5%.

[0052] In a particular implementation of the present invention specified fluorophore has the same nature as the fluorescent molecule, which under the action of thrombin releases fluorogenic substrate.

[0053] In another variant of realization of the specified fluorophore belongs to a different species of molecules, than the fluorescent molecule fluorogenic the substrate. In this case, when the measurement of the fluorescence account the presence of this new species fluorophore; in particular, measure the fluorescence specified fluorophore.

[0054] Adding known concentrations of such fluorophore provides an internal standard in the test specimen and allows you to estimate the amount of sample that is actually happening measurement of fluorescence.

[0055] For implementing the method of determining the generation of thrombin in the sample, and, in particular, in the sample of whole blood, successfully using synthetic substrate of thrombin, which consists of organic reagent, coupled with a fluorescent molecule.

[0056] the Synthetic fluorogenic the substrate may be an Oligopeptide, the sequence of which consists of 2-30 amino acid residues, coupled with a fluorescent molecule.

[0057] it May be especially useful to fluorophenyl group was associated with the terminal residue of lysine or arginine substrate, since thrombin predominantly cleaves group associated with the remnants of these amino acids.

[0058] According to a particular implementation used fluorescent molecule is an AMC (7-amino-4 methylcoumarin) or p-nitroanilide. Synthetic substrates described in Rijkers, D.T., Nsamt, et al. (1996), Int J Pept Protein Res 48(2): 182-93; Rijkers, D.T., S.J.Wielders, et al. (1995), Thromb Res 79(5-6): 491-9;Wielders, S.M.Mukherjee, et al. (1997), Thromb Haemost 77(4): 629-36.

[0059] Specific synthetic fluorogenic substrate of thrombin, suitable for implementing the present invention is Z-Gly-Gly-Arg-AMC (can be purchased at VASHEM).

[0060] In a specific implementation of the present invention wells (or any other suitable device)containing the sample may optionally contain a gel may gel containing calcium ions, and specified the gel is prepared in such a way that it does not cause dilution of the specified sample of whole blood. You can apply these gels, as Sefadex or agarose gels, provided that they are not dried to such an extent to allow fluids or plasma to penetrate into the gel.

[0061] In the case of application of the gel can be placed in the wells to the sample of whole blood or with him.

[0062] Compounds that can be added to the sample to ensure the production of thrombin include tissue factor and calcium ions, which are added in amounts that provide the beginning of coagulation.

[0063] These amounts can be in the range from 0.05 picomol/l to 15 nanomoles/l tissue factor and be approximately 10 mm CA ion++in the case of the use of citrate blood. It is also obvious that the present invention also covers the case of the application of native blood without anticoagulants, in which you add the better the CA ++. Alternatively, in some applications it is useful not to add tissue factor, in order to investigate the ability of blood to spontaneous coagulation. Tissue factor, if needed, add just before the measurement.

[0064] calcium Ions and/or fluorogenic substrate can be added either directly from a blood sample, in particular, in the case of the use of calcium and fluorogenic substrate in the form of a solution. Tissue factor can also be added separately from a blood sample.

[0065] the Measurement is carried out immediately after placing in the hole of the blood sample and different connections.

[0066] In the specific example of implementation of the method according to the present invention analyzed whole blood is a citrate blood.

[0067] the Method of measuring the activity of thrombin in whole blood can be usefully applied for the detection or monitoring of hemostatic disease or thrombotic disease or to detect or monitor the occurrence of such a disease in a patient.

[0068] This method also provides for the detection or monitoring of the impact of a specific substance (s) on the activity of thrombin in a sample of whole blood, and specified substance (substances) or add in a sample to be analyzed, or in the process of developing a centre of the ina.

[0069] Substances that can be tested by the method according to the present invention, are, for example, pharmaceuticals or other compounds that provide clotting effect on the blood, such as coagulation factors or drugs-coagulants, or factors or drugs-anticoagulants. In particular, the method according to the present invention, it is possible to test inhibitors of thrombin.

[0070] In another aspect of the method according to the present invention can be applied for screening substances to determine their ability to influence the activity of thrombin.

[0071] According to the present invention, the method, which was described above and which will be illustrated by means of examples, in particular, be used to measure potential endogenous thrombin (PET) sample of whole blood.

[0072] this method can also be used to measure the time to maximum concentration of thrombin or for measuring clotting time.

[0073] this method is also useful for measuring the maximum level of thrombin generated during analysis.

[0074] the Method according to the present invention allows to measure the so-called curve of thrombin, which is a first derivative of the measured fluorescence, which is the result of the reaction between trombi the om and fluorogenic substrate.

[0075] In a particular embodiment of the method according to the present invention perform the phase calibration as the calibration described in the patent application WO 03/093831.

[0076] the Method according to the present invention has been described with respect to the biological sample, which represents a sample of whole blood. It can also be applied for analysis of a sample which is a platelet-rich plasma (PRP) or platelet-poor plasma (PPP).

[0077] the Present invention relates also to a kit for implementing the above described method and examples, with the specified set contains

- fluorogenic substrate of thrombin,

tissue factor and calcium ions to ensure the production of thrombin,

perhaps the grill or microspheres, which prevents the seal of blood clot and promote the distribution of whole blood,

perhaps gel, possibly containing calcium ions.

[0078] the Kit may also contain instructions for use, to provide specific guidance on the implementation of the method according to the present invention. Other properties of the present invention and its advantages will be described in the following examples and drawings.

Description of figures

Figure 1: Thrombogram obtained in the experiment in partial samples. Main features: BP the two delays (=the time of the formation of a clot), the peak height and area under the curve (=the Potential of endogenous thrombin, PET).

Figure 2: Example curves generation of thrombin obtained in platelet-poor plasma by automated terminography with calibration.

AVK: processing that removes the vitamin K; TM: thrombomodulin.

Figure 3: Simplified diagram of the formation of thrombin. Shows positive and negative feedback.

Figure 4: Schematic representation of the influence of deposition and compaction of the bunch, the fluorescent signal folding of whole blood.

Description: Large ovals: Red blood cells

Star mugs: Excited fluorescent molecules

Squares: non-excited fluorescent molecules

The upper horizontal line (curved in some of the figures): the liquid surface

The lower horizontal line: the glass bottom of the measuring wells

Stage A: Liquid blood directly after placing it in the hole. (Figure 1, t=0)

Stage: Liquid blood directly before the formation of a clot. (Figure 1, t=B)

Stage C: the Blood immediately after the formation of a clot. (Figure 1, t=S)

Stage D: the blood after the beginning of the seal. (Figure 1, t=D)

Figure 5: the Huygens Eyepiece and the condenser.

6: a Thin layer (3 mm) of blood in the holes of a conventional microtiter tablet. Seven identical experiments.

Fig.7: a Thin layer of blood in lung the x microtiter tablet with a large surface. Identical experiments with 32 read points on the hole.

Fig: a Thin layer of blood in the wells of microtiter plate with a large surface with grid and lid. One point of reading through the light-harvesting device (lens Huygens) (1) experimental curve, (2) after correction for α2-thrombin.

Fig.9: From conventional units to thrombin.

Figa: the cartridge with the sample. Shows the possible measuring tape with two departments. One thin strip of material, preferably rigid, for example, glass) (called "slide"), possibly covered with a biocompatible coating that is shared by a suitable partition on the branch. A thin partition may also perform the function of an electrode for registration of application of the sample on the falling resistance and/or increase of the electric current. Each branch can cover such substances as the calibration standard and/or the substrate (or any other substance, such as inhibitors and tissue factor). The second slide is attached on top of the first slide. The distance between the two slides is 5-1000 μm, preferably 50 μm.

On the tape with one or two sides to coat the function dichroism mirror, amplifying the light. This allows vozbujdayuschego light to penetrate into the cartridge, while respecive reflection of the light emitted inside the cassette (see Figv), making it concentrates on the uncoated side of the cassette.

11: Alternative cartridge. Shows a piece of a solid substrate, for example, a porous polymer or cellulose, placed between two slides. This framework may include a substrate and/or calcium (or any other substance, such as tissue factor inhibitor). These substances may be present together with the solvent in the dry or liofilizovannyh form.

Fig: Shows the curve W, measured in a thin layer of blood in the cell of the filter paper. The concentration of thrombin is translated in nm using a calibration standard - staphylocoagulase.

EXAMPLES

THE FIRST DIMENSION W IN WHOLE BLOOD: THE OBSERVED PHENOMENA AND THE TECHNICAL DIFFICULTIES OF MEASURING WATTS.

[0079] In the case of the use fluorogenic substrate, the fluorescence can be retrieved and measured only at this distance, where the light beam intersects with the red blood cells, i.e. in the space plasma available for exciting light, which is also the space in which we can observe the emitted light. For simplicity, we assume that red blood cells are completely impervious to both types of light. However, even if the red blood cells to some extent permeable, there is no need for fundamental change in logical reasoning.

[0080] the whole Bunch in the blood is formed in the end of the initiation phase, at the beginning of the rapid growth in the production of thrombin. Figure 4 shows the four stages of this process under and above the surface of the blood, the formation of a clot in the hole of the tablet of fluorimetry in the presence fluorogenic substrate and when illuminated from above or, alternatively, below.

[0081] On the stage And the mixing of blood just ended and the red blood cells are evenly distributed in the liquid plasma (Figa). During the delay time before the formation of a clot (usually 3-12 minutes) is settling (sedimentation) red blood cells; at the top for light becomes available to a larger amount of plasma, and bottom - less (Pigv). As soon as the blood rolled (Figs), the appearance of a fibrin clot leads to fixation of the status quo that emerged on the stage of the Century Due to the effect of thrombin on platelets blood, the seal starts as soon as it clots. The deposition and compaction of the bunch - the phenomena occurring at the scale of mm per hour, which can be seen with the naked eye. In the scale of micrometers they occur over minutes, i.e. within the time that is occupied by experiments on TUES. Compaction leads to uneven distribution of red blood cells, and also changes the surface so that it ceases to be flat and becomes uneven. This irregularity leads to neravnomernomu redistribution of plasma and the formation of a clot on the surface and causes unpredictable optical effects. Surface irregularities have dimensions of the order of a millimeter and can be seen with the naked eye. Thus, they have the same order of magnitude as the spot size of the exciting light in the normal fluorimetry.

[0082] the Deposition and compaction cause a change in volume of the liquid in which the stimulating light can reach the fluorescent molecules. Due to these changes the actual volume in which the dimension is variable and unknown. Therefore, reproducible measurement of the amount of thrombin on received signal strength in such conditions it is impossible, even if it were possible to neglect the influence of the deposition and compaction.

[0083] the Experiments conducted with the use of holes of a conventional microtiter tablet and volume filling these holes to normal height (6 mm) made it possible to obtain satisfactory results only in a small percentage of holes. This is explained by the fact that in normal fluorimetry sufficient signal gain only if the illuminated spot on the surface (about 4 mm2) coincides with the area in which it is possible to obtain a sufficient signal thanks to a sufficient number of available plasma, which is not above the dense clot, and in the "depression" of the surface (see Fig.4D). Positive data from the literature about the measurement, in the o f are the result of a careful selection of the received data. In those cases, when receiving a signal of the correct form, quantitative measurement of thrombin is impossible due to the fact that the volume in which the conduct of the measurement is unknown and changing. The measurement from the bottom through the transparent foil would solve the problem of unevenness of the surface, but due to the sedimentation of erythrocytes signal becomes so low that it falls below the noise level.

[0084] it has Been observed that under normal filling holes 96-hole tablet of the modern type, you can get amenable to the interpretation of the signal in 1 or 2 of the 10 holes. If the wells to fill up to a height of less than 2 mm, i.e. below the most commonly used height, the signal obtained when almost all dimensions. However, the signal obtained in this way on a regular fluorimetry very variable and weak, i.e. corresponds to 1-5% of the signal obtained for plasma, and shows a large signal-to-noise ratio (Fig.6). We came to the conclusion that there is no practical way to measure WATTS in whole blood using an optical system based on the normal fluorimetry.

II SCHEME of the METHOD ACCORDING to the PRESENT INVENTION

1. The principle of the invention

[0085] it has been Unexpectedly found that (a) the effect of deposition and compaction gradually decreases with layer thickness of collapsing the blood and, (b), u is the fluorescence from the surface, greater than approximately 10 mm2that leads to the leveling signal from the remaining surface irregularities caused by the seal. Also unexpected was that the effects caused by the deposition and compaction can be further reduced by placing the blood in a complex surface or voids, such as lattice filter (with holes 50-500 μm) or compact spheres (diameter of 50-500 μm). Accordingly, the authors of the present invention is to provide the conditions for obtaining undistorted signal fluorescence from the product activity of thrombin by providing the possibility of carrying out measurements in a thin layer (in particular, less than 2 mm), distributed over the surface, greater than approximately 10 mm2.

[0086] in Order to solve the problem of the unknown volume, which measure the reaction, the authors present invention additionally decided to use not clean the substrate, and a substrate that already contains a fixed low, but well-known and easily measurable concentration of the fluorescent product.

2. An optical device to measure

[0087] the measurement on the surface than usual, requires the use of optical devices that allow you to cover large surfaces and to collect the emitted light from surfaces. One such device is reminiscent of the Huygens eyepiece, and the other on the coolant in the condenser of the microscope (Figure 5). To enhance the fluorescence signal, the blood can be distributed on the reflecting surface, and this surface may be an integral part of the device, described below.

3. The device containing the sample of blood

[0088] the Use of the device in the cavity which contains the blood, allows to simulate the situation when the blood fills the wound, additionally you can make the device contained tissue factor, thrombomodulin and/or other elements known that they are present in the wall normal vessel and influence the process W (e.g., collagen). For comparison, the material from which the device can be selected from inert materials such as, for example, nylon or polypropylene.

[0089] in Order to prevent surface drying, a thin layer of blood can be covered with a thin film of solid or liquid material. Alternatively, blood can be placed in a groove in the transparent material, for example, under the action of capillary forces.

4. Measurement

[0090] the Advantage of measuring in a thin layer is that the fluorescent signal is proportional to the concentration of fluorescent molecules, in other words, there is no effect of the internal filter. However, the expenditure of the substrate still has an impact. It can be compensated in three ways: (a) As in Gromov nom method (38), i.e. by providing substrates with such constants kinetics, in which the influence of consumption of the substrate can be neglected; (b) by carrying out a mathematical correction to the consumption of the substrate, i.e. the application of integral equation speed; and (C) by applying a calibration standard as described in patent WO 03/093831.

[0091] the Dynamics of the concentration of thrombin in the folding of whole blood is determined by the enzymatic action of thrombin on fluorogenic substrate is added to the blood. Stable and sufficient size signal obtained by measuring in a thin layer (layer thickness 2 mm), distributed over a large surface (represented by a surface area of more than 10 mm2). To measure the actual volume in which the reaction, the substrate was added a small amount of fluorophore. To illuminate the entire surface requires special optical device, and another device is required to detect light emitted from the entire surface.

[0092] it is Possible to stabilize a thin layer and prevent it from drying out using a number of mechanical means, including inert lattice and the material is selected so that it imitated certain properties of vessel walls. Alternatively, you can apply a small groove.

[0093] This scheme can be applied is to measure thrombin generation in platelet-poor and platelet-rich plasma.

5. The reaction mixture is

Reagents

[0094] Recombinant relitivaly tissue factor (rTF), not containing polybrene and CA++received from Dade Behring (Marburg, Germany (Marburg, Germany)). Fluorogenic substrate, Z-Gly-Gly-Arg-AMC was obtained from Bachem (Switzerland). After cleavage by thrombin he releases the fluorescent 7-amino-4-methylcoumarin (AMS), which is measured using a set of filters 390 nm for excitation and 460 nm for emission.

[0095] a Fresh mixture fluorogenic substrate and CaCl2(FluCa) for each experiment was prepared as follows: to 875 μl buffer (Hepes 20 mm, pH 7.35)containing 60 g/l BSA (Sigma A-7030) was added 100 μl of 1 M CaCl2. At 37°C in a thin stream poured in 25 μl of 100 mm solution fluorogenic substrate in DMSO and immediately vigorously stirred. The resulting clear solution, which was identified as FluCa, represents, therefore, a 2.5 mm solution fluorogenic substrate and 100 mm solution of CaCl2.

The buffer contains 20 mm Hepes, 140 mm NaCl, 5 mg/ml BSA, pH=7.35. The buffer contains 20 mm Hepes, 140 mm NaCl, 60 mg/ml, pH=7.35.

Blood and plasma

[0096] the Blood produced by venipuncture (1 volume of tri-sodium citrate 0.13 M to 9 volumes of blood). You should use the method of free flow or minimum aspiration should be avoided vacuum container.

[0097] the Measurement is carried out on a tablet fluorimeter (Ascent reader, Thermolabystems OY, Helsinki, Finland (Helsinki, Finland)equipped with a filter set 390/460 (excitation/emission). Instead of the usual 96-hole tablet, use the tablet with 24 round holes with a diameter of 15 mm and, respectively, the surface area of 175 mm2. Wells were prepared by several washes with buffer a, and drying.

[0098] Then add a mixture of blood and the substrate, which induces the production of thrombin. This mixture contains 80 μl of citrate blood on every hole to be filled.

[0099] 20 μl of Innovin® diluted in a ratio of 1:1000 buffer And 20 ál FluCa or, as the case may be, a multiple number of these volumes.

[00100] Immediately after adding FluCa the mixture is stirred on the Vortex mixer and added to the wells. The tablet is inserted into fluorimeter, shaken for 10 seconds at 1200 rpm and then carry out measurements every 2 minutes for one hour at a wavelength of 390/460 on at 37°C.

[00101] In each well add 120 ál of the mixture.

Example 1

Multiple read points adding mesh and cover.

[00102] the Production of thrombin was launched, as described above, in the three holes. In each hole one by one covered and measured 24 spots. The results are shown in Fig.7. It is seen that the addition of the lattice, in this case nylon filter hole size 600 microns, with 51% open area and thickness of 445 μm (Spectrum Laboratories Inc. Rancho Dominguez, California, USA (To lifornia, USA)), strengthens and stabilizes the signal. However, there is an apparent increase in signal due to evaporation and concentration of the upper layer. Prevent it by covering with a plastic film (Thermosprint clean optical sealing tape for QPCR (Bilatec AG, Mannheim, Germany).

Example 2.

Multiple read points, adding gel and cover.

[00103] the Production of thrombin was launched, as described above, in the three holes. In each hole one by one covered and measured 24 spots. Folded signals from 24 points each reading. Two wells were added to 700 μl of 50% (vol./about.) Spehadex-25 150 mm NaCl and within 5 minutes to allow the powder to settle. Pipette remove supernatant (300-400 µl) of the holes.

[00104] Then add 120 μl of the mixture collapsing the blood. On top of one of these two holes was applied plastic film (Thermosprint optically transparent self-adhesive tape for quantitative PCR (Bilatec AG, Mannheim, Germany (Mannheim, Germany)). The results comparable to the one shown in Fig.7.

Example 3

The light collection optical device

[00105] For this experiment, the dimension in the same hole with a grate and cover as described in example 1 was performed using fluorimetry Fluostar optima (BMG Labtech, Offenburg, Germany (Offenburg, Germany)). The light from the sample was collected in the Huygens eyepiece (10x magnification) and recorded after passing through the optical device. Results n the cauldrons on Fig.

Example 4

From relative units to thrombin

[00106] This experiment was performed almost in the same way as described in example 1 with grid and lid, but with the addition of a known quantity (10 nm) AMC to the substrate Z-Gly-Gly-Arg-AMC. As a result of erythrocyte sedimentation is an increase in volume, which carry out the measurement, so the signal from present AMC growing. At the time of coagulation is "fixing" the situation and the settlement is terminated. At this point, which is determined by the unexpected increase in the signal, we measure the amount of fluorescence generated after addition of 10 nm AMC. This allowed to transfer units of fluorescence (F) in the concentration of AMC. Thus, the measured dF/dt can be translated into d[AMC]/dt. From independent experiment know what d[AMC]/dt corresponds to a concentration. Thus, the rate of change in fluorescence (Figure 9, black line) can be translated into a concentration of thrombin in the sample.

Example 5

The device containing the sample of blood

A cell from the filter paper containing the substrate and ions of CA2+was prepared by adding 50 μl of 100 mm solution fluorogenic substrate in DMSO and 100 μl of 1 M solution of CaCl2to 5850 μl of ethanol. 11 μl of this solution are spread on a strip of hard substrate (1 mm paper chromatography Whatman 1MM) of size 7×9 mm and dried in the atmosphere is e nitrogen. Then it is put on a piece of plastic and cover with another piece of plastic (optically transparent self-adhesive film for quantitative PCR Thermosprint, Bilatec AG, Mannheim, Germany (Mannheim, Germany)), as shown in figure 11.

A similar procedure was used to prepare the cells from the filter paper, containing only the substrate. In this case, 50 μl of 100 mm solution fluorogenic substrate in DMSO was added to 5950 μl of ethanol, placed 11 ál of this solution on a piece of solid substrate and was dried in nitrogen atmosphere.

Multiple read points, the use of the cells from the filter paper.

Shortly before the experiment was prepared by two cells of the filter paper: one containing fluorogenic substrate and calcium ions (a) and one containing only the substrate (b)as described above.

Preparing a mixture of 4:1:1 citrate blood, buffer and Innovin® (diluted 1:1000 in buffer A). As the calibration standard by mixing blood and inhibitor of polymerization prepared mixture of 4:1:1 citrate blood, polymerization inhibitor (H-Gly-Pro-Arg-Pro-OH·AcOH) (Bachem feinchemikalien AG, Bubendorf, Switserland (Bubendorf, Switzerland)) in buffer (1.0 mM) and 20 μm staphylocoagulase. Immediately before the experiment was added staphylocoagulase and well-mixed sample.

Immediately after adding staphylocoagulase began the experiment by the adding the 11 μl of the sample TV in cell a and 11 ál calibration standard in cell C. For this pipette put drops near the solid substrate so that the drops touched the substrate (see 11) and absorbed inside under the action of capillary forces. In each cell were alternately illuminated and measured 4 spots.

The signal from the calibration standard is a straight line, the slope of which is used to translate the magnitude of the signal from the cell with the sample in nanomole thrombin. This standard method of calibration of the signal are known in this area as "not continuous calibration", according to the patent application PCT/EP 03/04705. The results are shown 12.

1. The in vitro method for determining the activity of thrombin in the sample, and the sample is a blood sample, and measuring generation of thrombin includes the following stages:
- cast layer of the specified sample in contact with fluorogenic substrate of thrombin, the thickness of the specified layer is from 0.05 to 5 mm, and the surface area is from 10 to 500 mm2;
- providing conditions for the generation of thrombin in the specified form;
- measurement of the fluorescence emitted from the surface of the specified layer fluorescent group, which releases fluorogenic substrate in the enzymatic effects of verbatime is on thrombin to the specified fluorogenic substrate.

2. The method according to claim 1, characterized in that the concentration of thrombin generated during the analysis, determine as a function of the measured fluorescence released from the fluorescent group.

3. The method according to claim 1 or 2, characterized in that the sample of whole blood is diluted up to a maximum of 10 times.

4. The method according to claim 3, characterized in that the thickness of the layer of the specified sample is approximately 2 mm or less.

5. The method according to claim 1, characterized in that the bringing of the blood sample into contact with fluorogenic substrate of thrombin and/or measurement of fluorescence is carried out in the hole of the tablet, which contains the grid hole size from 50 to 500 microns.

6. The method according to claim 1, characterized in that the bringing of the blood sample into contact with fluorogenic substrate of thrombin and/or measurement of fluorescence is carried out in the hole of the tablet, which contains micro-granules.

7. The method according to claim 6, characterized in that when determining the activity of thrombin hole containing a blood sample, cover the top.

8. The method according to claim 7, characterized in that fluorophenol substrate of thrombin add the fluorophore, and the relative share of added fluorophore is from 1 to 10% of the number of fluorescent molecules associated with the substrate of thrombin.

9. The method according to claim 8, characterized in that the substrate tro is Bina, added to the sample, ranges from 50 to 1000 microns.

10. The method according to claim 8, characterized in that fluorogenic the substrate is a synthetic substrate of thrombin, coupled with a fluorescent molecule.

11. The method according to claim 10, characterized in that the substrate of thrombin selectively hydrolyzed by thrombin and has moderate affinity binding to thrombin and low kinetic constant.

12. The method according to claim 11, characterized in that fluorogenic substrate is an Oligopeptide, the sequence of which contains from 2 to 30 amino acid residues, and which is connected to a fluorescent molecule.

13. The method according to item 12, characterized in that the Oligopeptide at its end contains lysine or arginine for connection with a fluorescent molecule.

14. The method according to item 13, characterized in that the fluorescent molecule is an AMC (7-amino-4-methylcoumarin).

15. Method according to any one of claims 1 to 2, 4-14, characterized in that the said hole further comprises a gel may contain calcium ions, and the specified gel does not cause dilution of the blood sample.

16. The method according to item 15, wherein the tissue factor and calcium ions added to the blood sample in quantities that provide the possibility of thrombin.

17. The method according to item 15, wherein specify the initial blood sample is a sample of whole blood.

18. The method according to item 15, characterized in that the blood sample is a plasma sample, in particular, platelet-rich plasma (PRP).

19. The method according to 17, characterized in that said sample is whole blood represents the blood treated with citrate.

20. The method according to any one of claims 1 to 2, 4-14, 16-19, according to which the defined activity of thrombin in the sample is judged on the presence or monitor homeostatic or thrombotic disorders.

21. The method according to claim 20, according to which add in the analyzed sample or in the sample on the stage of development of thrombin some substance (substances), with defined activity of thrombin is judged on the presence of impact or monitor the impact of the specified substance (s) the activity of thrombin in a sample of whole blood.

22. The method according to claim 20, whereby a defined activity of thrombin in the sample monitor the impact of coagulation factors or drugs-coagulants.

23. The method according to claim 20, whereby on the basis of the determined activity of thrombin in the sample conduct screening substances to determine their ability to affect the production of thrombin.

24. The method according to claim 20, which is used for measuring the capacity of the Endogenous Thrombin (PET) in a sample of whole blood.

25. With the ESP on item 21, which is used to measure the time to achieve peak concentrations of thrombin.

26. The method according to item 22, which is used to measure the time of formation of the blood clot.

27. The method according to item 23, which is used to measure peak concentrations of generated thrombin.

28. The method according to paragraph 24, which includes a step of calibration.

29. Set for implementing the method according to any one of claims 1 to 28, which contains:
- fluorogenic substrate of thrombin,
tissue factor and calcium ions to ensure the production of thrombin,
perhaps the grill or microspheres, which prevents the seal of blood clot and promote disintegration of whole blood
perhaps gel, possibly containing calcium ions.



 

Same patents:

FIELD: medicine.

SUBSTANCE: blood is examined. A hematocrit level (H), erythrocyte count (E), thrombocyte count (T) are determined. Said parametres are evaluated. In the event if they keep within the determined limits for the patients with acute coronary syndrome (ACS), then adenosine phosphate induced (ADP-induced) clotting time test samples are prepared. Citrated blood sample 0.4 ml is prepared of whole blood and divided on two samples 0.2 ml. Each of these samples is introduced in a measuring cell, recalcified at temperature 37°C for 2 minutes. Then a magnetic ball mixer is placed in each cell. The measurement is activated, and in three seconds the ADP solution 0.1 ml is introduced. After a clotting reaction, a process time duration is recorded separately for each sample. An arithmetical mean of the derived values is calculated (A). The derived values of each of said parameters are scored. Total score Σ=A+H+E+P shows the risk of recurrent thrombotic events. If Σ=4 points, the low risk is observed; the value Σ=5-6 points shows the medium risk, while Σ=7-10 points - the high risk.

EFFECT: method provides more objective risk evaluation of recurrent thrombotic events in the patients with ACS with its simplicity and low cost.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: blood sample is placed in capillary, in whose walls installed are electrodes connected to frequency generator and registering unit, blood electric conductivity is measured at the moment of passing through it of alternating current with frequency 200 Hz, electric coagulogram is registered and used to determine chronometric and amplitude characteristics: A - amplitude of functional curve decline, mV; N - time of functional curve decline to minimal value in minutes. If value of A/T index decreases or increases with respect to normal, conclusion about hemostatic disorders is made. If value of A/T index equals 3-5 - hemostasis state is evaluated as normal, if A/T value is lower than 3, hypocoagulation is determined, and if A/T value is higher than 5 - hypercoagulation.

EFFECT: application of the method makes it possible to obtain data about hemostasis system state in real time mode, without injuring form blood elements in investigated microvolumes of blood, thus making it possible to increase accuracy, self-descriptiveness and efficiency of hemostasis state evaluation and to carry out correction of performed therapy without delay.

4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: in patients with IHD before therapy with acetylsalisylic acid (ASA) ADP-induced and ASA-dependent platelet aggregation are examined and by their difference value of coefficient of aggregation inhibition (CAI) is calculated. CAI value <24% testifies to resistance to ASA, if CAI ≥24% - about sensitivity to ASA.

EFFECT: method ensures high prediction accuracy and makes it possible to prevent development of undesirable coronary events in IHD patients.

1 tbl, 4 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology, and orthopaedics. The intravascular thrombocyte activity correction in children with scoliosis requires the prescription for at least 6 months of a therapeutic complex which involves daily therapeutic physical training, two courses of massage and daily swimming for at least 20 minutes a day.

EFFECT: method allows to normalise intravascular thrombocyte activity in children aged 8-12 with scoliosis, to improve tissue microcirculation considerably, to optimise growth and development of skeleton and internals.

7 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably to haematology and orthopaedics. For thromboplastin synthesis correction in children 8-12 years old with scoliosis is prescribed complex of treatment, including daily remedial gymnastics, two courses of massage and daily swimming for at least 20 minutes a day, during 6 months.

EFFECT: method enables thromboplastin synthesis normalisation in children with scoliosis, significantly sanitate children with scoliosis due to improvement of tissues' microcirculation.

7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to aggressive medical therapy, resuscitation science, critical care medicine, laboratory diagnostics and can be used by resuscitators, intensivists, laboratory doctors for well-timed diagnosis and consequently, for individualised aggressive medical therapy of acute disseminated intravascular coagulation. The integrated assessment of links of haemostatic system and the clinical appraisal of organ dysfunction are applied in a measurer, and when observing structural hypercoagulation characterised by fibrinogen level increase, thrombocyte activity increase, growth of soluble fibrin complex (SFC) level, and also when observing chronometric hypercoagulation characterised by time tests, palette-derived factor 4 activity (P4) with manifested petechial haemorrhage and organ dysfunction, and coagulation cascade activation with underlying depression of antithrombin III and protein C, a hypercoagulation stage of acute DIC is diagnosed. Chronometric hypercoagulation by Activated partial thromboplastin time (APTT), INR, fibrinogen and P4 with manifested signs of structural hypocoagulation by thrombin time prolongation and D-dimer activity increase with underlying further intensification of anticoagulant system deficiency, progression of target organs dysfunction and mixed haemorrhage show a transitive stage of acute DIC. If observing said hypocoagulation changes and disturbed fibrinolytic activity with prevailing either decompensated organ and tissue dysfunction, or hemorrhagic syndrome up to system haemorrhages, or their combination with hemorrhagic syndrome characterised by polymorphism of clinical picture and localisation: petechial-haematoma haemorrhage at the stress-induced stomach ulcers, hematuria, a coagulopathy stage of acute DIC is diagnosed that is characterised either by depression of fibrinolysis, or preserved fibrinolytic activity, or by activation of secondary fibrinolysis, or by acute primary fibrinolysis.

EFFECT: method allows optimising classification of acute disseminated intravascular coagulation, improving diagnostic significance of the classification and simplifying a diagnostic prospecting which provide a basis to consider the staging of acute disseminated intravascular coagulation.

1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology and orthopaedics. Correction of blood microvesicle level in children aged 8-12 with scoliosis is ensured by the prescription of a therapeutic complex that involves daily therapeutic physical training, two courses of massage and daily swimming for at least 20 minutes a day, for at least 6 months.

EFFECT: method enables correcting blood microvesicle level in children aged 8-12 with scoliosis, optimising microcirculation in tissues, normalising functions of the internal organs, growth and development of a child.

7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology, and orthopaedics. Thrombocyte aggregation normalisation in children aged 8-12 with scoliosis is ensured by the prescription of a therapeutic complex that involves daily therapeutic physical training, two courses of massage and daily swimming for at least 20 minutes a day. The therapeutic complex is performed for at least 6 months.

EFFECT: method improves the effectiveness of thrombocyte aggregation correction in children aged 8-12 with scoliosis.

9 ex

FIELD: medicine.

SUBSTANCE: blood is drawn off, stabilised to recover thrombocyte-enriched plasma (TEP) and applied on a slide where thrombocyte aggregation test with a number of inducers is performed. In this environment, the visual evaluation of thrombocyte aggregation allows creating a real model of blood flow with small volume (0.02 ml.) of plasma and small volume of inducers with using minimum mutually potentiating concentrations of inducers, with necessary application of collagen in the combinations and calculation of "СЗИАК" the value of which is used to observe fine disorders of thrombocyte aggregation.

EFFECT: improved clinical effectiveness.

4 dwg, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention can be used to evaluate thrombocyte aggregation in the intravascular-related conditions to diagnose thrombocyte hypo- and hyperaggregation in physiological and pathological statuses. A blood sample is drawn, stabilised with 3.8% sodium citrate, divided into plasma and erythrocytes by centrifugation. That is followed with the thrombocyte aggregation test with simultaneously addition of at least three aggregation inducers chosen of the group including ADP, adrenaline, collagen, thrombin. The time of aggregate generation while adding the aggregation inducers is recorded with calculating the average aggregation within a damage area (AADA). If the AADA is 20.0-23.75 sec, the thrombocyte aggregation is considered to be normal. The AADA 12.1-19.9 sec indicates the risk of hyperaggregation; while the value 12.0 sec and less indicates high thrombocyte aggregation with potential thromboses in the nearest future. If the AADA is 23.76-29.9, there is a risk of hypoaggregation; the values 30.0 sec and higher show hypoaggregation with developing hemorrhagic diathesis in the nearest future.

EFFECT: application of the method allows diagnosing thrombocyte hypo- and hyperaggregation in small plasma amount.

3 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: specified kit contains a dried complex including tissue factor/phospholipid and a dried mixture containing thrombin substratum and CaCl2. There is disclosed method for preparing the dried complex including tissue factor (TF/phospholipid (PL). There is offered method for preparing said dried mixture containing thrombin substratum and CaCl2. Besides, there is described method for measuring thrombin formation in a sample by measuring the concentration of thrombin.

EFFECT: invention allows detecting kinetic changes in thrombin formations after introduction of agents action of which is not related to inhibitor.

22 cl, 5 dwg, 2 tbl, 6 ex

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

The invention relates to immunology

The invention relates to the diagnosis and treatment of diseases such as atherosclerosis and thrombosis

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: specified kit contains a dried complex including tissue factor/phospholipid and a dried mixture containing thrombin substratum and CaCl2. There is disclosed method for preparing the dried complex including tissue factor (TF/phospholipid (PL). There is offered method for preparing said dried mixture containing thrombin substratum and CaCl2. Besides, there is described method for measuring thrombin formation in a sample by measuring the concentration of thrombin.

EFFECT: invention allows detecting kinetic changes in thrombin formations after introduction of agents action of which is not related to inhibitor.

22 cl, 5 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: for thrombin production measurement, a layer of said sample contacts with a fluorogenic substratum of thrombin where the thickness of said layer is 0.05 to 5 mm, while the surface area is 10 to 500 mm2. Further, the thrombin production environment in said sample is provided. It is followed by measuring the fluorescence emitted from the layer surface by a fluorescent group released by the fluorescent substratum as a result of an enzymatic action of produced thrombin on said fluorogenic substratum. Besides, the invention ensures a kit for measuring the thrombin activity in the sample.

EFFECT: higher measuring accuracy.

29 cl, 12 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: method for thrombin activity test in an initially non-reacted mixture of thrombin and fibrinogen (versions) involving the stages: (a) reversible thrombin inhibition by adding an inhibitory solution having pH varying within the range of 8.5 to 11.5; (b) addition of the known amount of fibrinogen to the mixture (or the known amount of a chromogenic or fluorogenic thrombin substrate), (c) reversible thrombin activation by pH reduction to approximately 6.0 to less than 8.5, (d) enabling thrombin reacting with fibrinogen, (e) thrombin activity test initially found in the dry mixture. The method for fibrinogen functionality test in an initially non-reacted mixture of thrombin and fibrinogen (versions) involving the stages: (a) reversible thrombin inhibition by adding an inhibitory solution having pH varying within the range of 8.5 to 11.5; (b) addition of the known amount of thrombin to the mixture (or a thrombin-like enzyme), (c) reversible thrombin activation by pH reduction to approximately 6.0 to less than 8.5, (d) enabling thrombin reacting with fibrinogen, (e) fibrinogen functionality test initially found in the dry mixture.

EFFECT: group of inventions enables higher accuracy of thrombin and fibrinogen activity test.

32 cl, 1 dwg

FIELD: medicine.

SUBSTANCE: invention includes determination of content of soluble fibrin and D-dimers, formed in the process of fibrinolysis, activated in blood sample. In method, in accordance with the claimed invention, level of D-dimers, corresponding to destruction of soluble fibrin and level of D-dimers in sample with border values of the norm, are compared.

EFFECT: test in accordance with the claimed invention can be applied for determining whether resistance to blood coagulation in patient is sufficient.

4 tbl, 3 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to medical microbiology and a method of determining activation of plasminogen with bacteria. The method involves adding protamine sulphate to a prepared supernatant fluid, incubating the obtained mixture, depositing cells by centrifuging, incubating the supernatant fluid with the protamine sulphate, depositing protein and detecting activation of plasminogen with bacteria from the amount of split arginine, content of which is determined by Sakaguchi method from the red colour of the sample.

EFFECT: invention enables to detect activation of plasminogen with bacteria in vitro using protamine.

4 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention is a method of controlling autoactivation of factor VII or its analogue and a method of prevention of cleavage of activated factor VII or its analogue. The methods comprise measuring the initial concentration of factor VII/VIIa or its analogue. The initial part of activated factor VII or its analogue is measured. The reaction time of activation of factor VII or its analogue is calculated by correlating the values measured at the abovementioned steps, with the value of the required part of activated factor VII or its analogue. The reaction of activation of the factor VII is carried out at pH of from 6.0 to 8.0. The reaction is terminated by reducing pH to the value below about 6.0.

EFFECT: invention enables to determine the optimum reaction time to achieve the desired levels of the modified factor VII or its analogue, to obtain a purer protease product, to reduce the cost of manufacturing base.

15 cl, 5 dwg, 1 tbl, 2 ex

Up!