Method for thrombin and fibrinogen activity and functionality test

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

 

The technical field to which the invention relates.

The described method of measuring the activity of thrombin in the presence of fibrinogen or measurement functionality of fibrinogen in the presence of thrombin.

Prior art

Fibrinogen and thrombin are the most important proteins involved in achieving homeostasis after injury of blood vessels and play an essential role in the formation of blood clot. The fibrinogen and thrombin can be combined in the form of a powder or nonaqueous suspension, without triggering the typical reactions of blood coagulation, thus preventing the formation of fibrin clot up until the whites will not gidratirovana in water or other liquid medium, in which soluble proteins. The mixture of these proteins in powder form has a variety of potential biomedical applications, including local hemostasis, tissue regeneration, drug delivery, etc. in Addition, a mixture of these proteins can be loaded on a carrier or substrate or other medical device in the form of a powder to obtain a product that can be used, for example, as a hemostatic device.

Clotting activity of thrombin is usually measured by a combination of thrombin in solution with a known amount of fibrinogen in solution. In appropriate circumstances, scorodoprasum clot after combining these two proteins depends on the activity of thrombin. The rate of formation of a clot sample with an unknown amount of thrombin compared to the rate of formation of a clot standard thrombin or standard thrombin to determine the activity of the sample.

The activity of thrombin is a critical attribute of any product thrombin/fibrinogen and determines its functionality. Although the measurement of free thrombin is a simple method of measuring the activity of thrombin, when the thrombin and fibrinogen are in the unreacted mixture was a problem, because its measurement usually requires a mixture of proteins was gidratirovana and solubilization, and the formation of fibrin clot between solubilizing thrombin and fibrinogen begins immediately after hydration. In addition, because, as you know, thrombin specifically binds and interacts with immediately formed fibrin clot, thrombin is associated with the fibrin clot and no longer is freely soluble in hydrating solution and becomes unavailable for subsequent measurement of the activity of thrombin. Therefore, the final measurement of the activity of thrombin product thrombin/fibrinogen by hydration and the formation of a clot is only partial and, therefore, inaccurate.

In addition, if the CTL are not entered into reaction mixture and loaded on the carrier, substrate or medical device, it may be necessary to remove proteins from the substrate to accurately measure the activity of thrombin, for example, if the carrier, substrate or medical device adversely affects the measurement of the activity or functionality of proteins due to physical, chemical or optical interference detection and measurement. To overcome the interference of the carrier, substrate or medical device removal or extraction of the proteins to be performed without exposure to the mixture of water conditions that led to the formation of a clot, preventing subsequent measurement.

Fibrinogen is often measured by the method originally described by Clauss, which measures the functionality of fibrinogen on the basis of the rate of formation of the clot. In a typical analysis Clauss sample with an unknown amount of soluble fibrinogen is combined with an excess of thrombin. The proportions of the fibrinogen and thrombin are such that fibrinogen is a limiting velocity of the reactant and the rate of clot formation is a function of fibrinogen concentration. Rapid clotting time would be an indicator of high concentrations of fibrinogen. On the contrary, a longer clotting time would indicate a low concentration of functional fibrinogen. The number of functional fibrin is a gene can be determined by comparing the clotting time of the sample with the clotting time series of standards to plot a standard curve. The fibrinogen concentration in the sample can be determined mathematically based on the equation obtained from the values clotting time standards.

Although the measurement of free fibrinogen in solution, for example plasma, it is possible to perform the approved methods, the assessment of the functionality of fibrinogen, when the fibrinogen present in the presence of thrombin, was a problem. Hydration of the mixture will result in indirect thrombin conversion of fibrinogen into an insoluble fibrin clot. After the generation of fibrin any subsequent measurement of fibrinogen is no longer possible, since the release of fibrinopeptides of fibrinogen, leading to the formation of fibrin, is essentially irreversible.

Therefore, there remains a need to accurately measure the activity of thrombin in the presence of fibrinogen and measurement functionality of fibrinogen in the presence of thrombin.

Detailed description of the drawings

Figure 1 shows the effect of pH inactivation on the activity of the extracted thrombin.

A brief description of the invention

This document describes the method for determining the activity or functionality, or the first reactive component, or the second reactive component in the mixture of the first reactive component and the second reactive component, which includes stages (and reversible inhibition of the first reactive component to obtain a mixture, containing inactivated first reactive component and the second reactive component; (b) adding to the mixture an excess of the second reactive component in assessing the activity of the first reactive component or an excess of the first reactive component when assessing the activity of the second reactive component; (C) reversible activation of the first reactive component; (d) allowing the first reactive component to interact with the second reactive component in a mixture with an excess of the second reactive component or to provide access to the first reactive component to interact with the second reactive component in a mixture with an excess of the first reactive component; and (e) determining the activity or functionality of the first or second reactive component originally present in the dry mixture.

A detailed description of the invention

As discussed above, to determine the activity of thrombin unreacted mixture of thrombin and fibrinogen, for example, in the form of a powder or nonaqueous suspension, such as a suspension in ethanol, re-hydrated proteins and solubilisate thrombin and fibrinogen in Hydra medium for obtaining an accurate measurement of the activity of thrombin. After the entry of the mixture into contact with a hydrating environment any solubilis the integration of thrombin and fibrinogen will react to the immediate formation of a clot, and any existing thrombin contact with the clot and will not be freely available for its measurement.

In one embodiment, the activity of fibrinogen unreacted mixture temporarily inhibited or reversibly inhibited, thereby preventing the formation of fibrin clot until complete solubilization of thrombin and fibrinogen. By inhibiting the activity of thrombin is possible to avoid the formation of a clot, thrombin is able to dissolve in water hydrating environment and is available for measurement.

Temporary or reversible inhibition activity of thrombin can be achieved, for example, regulation alkaline environment of thrombin. For example, this can be accomplished by dissolving or hydration unreacted mixture of thrombin and fibrinogen in inhibiting or inactivating solution, i.e. an alkaline solution having a pH in the range of about from 8.5 to 11.5, preferably about 9.5 to 10.5, and preferably, about 10 to form a first solution. The maximum extracted the activity of thrombin was observed when the alkalinity of the inactivating solution was at pH 10. Within the pH range of 9.5 to 10.5 was reached at least 80% of the maximum extracted activity of thrombin. At pH levels less than 9.5 and more than 10,5 maximum extracted active is here thrombin was decreased as as the pH is further deviated from 10. When pH levels, 9,25 and below the proof of the formation of a clot was observed during hydration and may explain the reduced maximum extracted the activity of thrombin, which is observed at lower values of pH approaching neutral conditions. In acidic conditions, e.g. pH 4 and 5, the maximum extracted the activity of thrombin was significantly lower than the activity observed under alkaline conditions, which can be an indicator of irreversible inactivation of thrombin.

Inhibiting or inactivating solution may be an alkaline solution or a buffered alkaline solution, including, without limitation, the carbonate solution, TRIS base, borate, glycine, phosphate, methylamine, 2-(cyclohexylamino)econsultancy acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).

After the thrombin and fibrinogen are fully solubilizing in inhibiting or inactivating solution, the first solution or part of it can be combined with a known amount of fibrinogen in the second solution, preferably having an excessive amount of fibrinogen to form a third solution, at the same time maintaining the pH between approximately ranging from 8.5 to 11.5, preferred is entrusted, about 9.5 to 10.5, and preferably about 10. Use the excess fibrinogen so that the amount of thrombin in the mixture consisted of limiting the speed of the reagent in the formation of the fibrin clot, to ensure that the activity of thrombin is correlated with the speed of the formation of a clot. If fibrinogen was present in excess, the rate of coagulation dependent and thrombin, and fibrinogen.

Then the activity of thrombin can be eliminated, for example, by bringing the pH of the third solution to a range in which the activity of thrombin is no longer inhibited, i.e. from about 6.0 to less than 8.5, preferably from about 7.0 to less than 8.5, and preferably about 7.5. Alternatively, the inactivating solution containing solubilization protein or portion thereof, that is, the first solution can be combined with a known amount of fibrinogen in the second solution, preferably, an excess amount of fibrinogen to form a third solution, whereby at the same time eliminates the inhibition of the activity of thrombin. Examples of the second solution include, without limitation buffer solutions TRIS-HCl, imidazole, 4-(2-hydroxyethyl)piperazine-1-econsultancy acid (HEPES), phosphate, barbitala, 4-morpholinepropanesulfonic acid (MOPS), 3-morpholine-2-hydroxypropanesulfonic sour is (MOPSO), 1,4-piperazineethanesulfonic acid (PIPES), N,N-bis(2-hydroxyethyl)-2-emiratesunited acid (BES), citrate or carbonate.

Volume and tabularasa the ability of the second solution should be sufficient to cast to obtain a third solution having a pH from about 6.0 to less than 8.5, preferably from about 7.0 to less than 8.5, and preferably, about 7.5, adding to the first solution. For example, the ratio of volumes between the first and second solutions is typically in the range of from about 1:1 to 1:20, and preferably from about 1:4 to 1:10, for example, when both molarity of the second solution is from about 25 mm to 500 mm buffer TRIS-HCl, and preferably, from about 100 mm to 150 mm buffer TRIS-HCl.

The activity of thrombin may be determined by the coagulation analyzer with a mechanical device identifying boundary points for detecting the formation of a clot, such as coagulation analyzer Diagnostica Stago ST4, or a device that measures changes in turbidity due to the formation of the fibrin clot. Solubilization inactivating proteins in solution can be combined with the second solution in one of these devices, and you can measure the time until coagulation, which can then be correlated with the values of the clotting time for known levels of activity of thrombin.

The other pic is BOM, which to measure the activity of thrombin, is the use of chromogenic or fluorogenic peptide substrate for thrombin. In this way the solubilized thrombin is combined with an excess of a chromogenic or fluorogenic substrate. Thrombin will cause cleavage of the substrate, releasing the chromophore or fluorophore, the monitoring of which can be done in a spectrophotometer or fluorimeter. Examples of chromogenic or fluorogenic substrates respectively include β-Ala-Gly-Arg-p-nitroanilide and Z-Gly-Pro-Arg-AMC [Z = benzyloxycarbonyl; AMC = 7-amino-4-methylcoumarin]. The rate of release of the chromophore or fluorophore can be correlated with the activity of thrombin.

In another embodiment, the functionality of fibrinogen in the unreacted mixture of thrombin can be measured by the inhibition of the activity of thrombin regulation alkaline environment of thrombin. For example, this can be accomplished by dissolving or hydration of a mixture of thrombin and fibrinogen in inhibiting or inactivating solution, i.e. an alkaline solution having a pH in the range of about from 8.5 to 11.5, preferably about 9.5 to 10.5, and preferably, about 10 to form a first solution. Inhibiting or inactivating solution may be an alkaline solution or a buffered alkaline solution, including without ogran the treatment solution of the carbonate, TRIS (Tris(hydroxymethyl)aminomethan)base, borate, glycine, phosphate, methylamine, 2-(cyclohexylamino)econsultancy acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO). Additionally and optionally, to the inhibitory or inactivating solution or to the first solution, you can add a thrombin inhibitor, such as bivalirudin (Angiomax) to achieve maximum inhibition activity of thrombin, thus providing the ability to solubilize the most part fibrinogen for subsequent testing. Other thrombin inhibitors include antithrombin, heparin, heparin, low molecular weight analogues of heparin low molecular weight, fondaparinux, argatroban, melagatran, efegatran, inogatran, dabigatran, hirudin and derivatives of hiragana, such as lepirudin and desirudin.

After the activity of thrombin was ingibirovany, the functionality of fibrinogen can be defined by combining the first solution, or part thereof with a known amount of thrombin in the second solution, preferably containing an excess amount of thrombin to form a third solution, at the same time maintaining the pH between approximately ranging from 8.5 to 11.5, preferably from about 9.5 to 10.5 and preferably about 10. Uses the I excess of thrombin with the that the amount of fibrinogen in the mixture consisted of limiting the speed of the reagent in the formation of the fibrin clot to ensure that the fibrinogen concentration was highly correlated with the rate of formation of a clot. If there was an excess of thrombin, the rate of coagulation dependent and thrombin, and fibrinogen.

Then the activity of thrombin can be eliminated, for example, by bringing the pH of the third solution to a range in which the activity of thrombin is no longer inhibited, i.e. from about 6.0 to less than 8.5, preferably from about 7.0 to less than 8.5, and preferably about 7.5. Alternatively, the inactivating solution containing solubilization protein or portion thereof, that is, the first solution can be combined with a known amount of thrombin in the second solution, preferably, an excess amount of thrombin to form a third solution, whereby at the same time eliminates the inhibition of the activity of thrombin. Examples of the second solution include, without limitation buffer solutions TRIS-HCl, imidazole, 4-(2-hydroxyethyl)piperazine-1-econsultancy acid (HEPES), phosphate, barbitala, 4-morpholinepropanesulfonic acid (MOPS), 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO), 1,4-piperazineethanesulfonic acid (PIPES), N,N-bis(2-hydroxyethyl)-2-aminotadalafil to the slot (BES), citrate or carbonate at a pH of about 7.5.

The functionality of fibrinogen may be determined using a coagulation analyzer with a mechanical device identifying boundary points for detecting the formation of a clot, such as coagulation analyzer Diagnostica Stago ST4, or a device that measures changes in turbidity due to the formation of the fibrin clot. Solubilization inactivating proteins in solution can be combined with the second solution in one of these devices, and you can measure the time until coagulation, which can then be correlated with the values of the clotting time for known levels of functionality of fibrinogen.

Alternatively, the functionality of fibrinogen may be determined by the inhibition of the activity of thrombin using a thrombin inhibitor, such as bivalirudin (Angiomax). Optionally, the alkaline environment of thrombin can be adjusted in combination with the inhibitor of thrombin. Other examples of inhibitors include thrombin antithrombin, heparin, heparin, low molecular weight analogues of heparin low molecular weight, fondaparinux, argatroban, melagatran, efegatran, inogatran, dabigatran, hirudin and derivatives of hiragana, such as lepirudin and desirudin. After the activity of thrombin was ingibirovany, the functionality of fibrinogen can determine what elite the use of such thrombin enzyme, which is able to act on fibrinogen to form a clot, but not exposed to thrombin inhibitor. Examples of such thrombin enzymes include, without limitation batroxobin (obtained from the venom of South American rattlesnake Bothrops atrox) and ancrod (obtained from the venom of Calloselasma rhodostoma.).

When proteins are in the unreacted mixture and loaded, for example, on a carrier, substrate or medical device, the mixture can be represented in the form of a powder, where proteins are dry or dried, remove proteins before rehydration and solubilization can be carried out by extraction of proteins using non-aqueous fluids, including, without limitation, perforated hydrocarbons, such as HFE-7000, HFE7001, HFE7003, HFE-7300 and PF-5060 (commercially available from the 3M company of Minnesota), and can be used with any other carrier fluid, in which not dissolved proteins, such as alcohols, ethers or other organic liquid. After proteins were extracted using a non-aqueous solvent, the activity of thrombin or functionality of fibrinogen can be measured as described above.

Alternatively, when the proteins loaded on the carrier, substrate or medical device, the activity of thrombin or functionality of fibrinogen can be defined, as described above, be the removal of proteins. For example, proteins can hydrogenate itself location of the carrier, substrate or medical device that has them proteins directly in inhibiting or inactivating solution, samples of which can be taken for testing the activity of thrombin or functionality of fibrinogen as described above.

1. The method for determining the activity or functionality of thrombin or fibrinogen in the original unreacted mixture of thrombin and fibrinogen, which includes stages
(a) reversible inhibition of thrombin to obtain a mixture containing inactivated thrombin and fibrinogen, the addition of inhibitor solution having a pH in the range from 8.5 to 11.5;
(b) adding to the mixture a known amount of fibrinogen in assessing the activity of thrombin or a known amount of thrombin in the assessment activity fibrinogen;
(c) reversible activation of thrombin bringing the pH from approximately 6.0 to less than 8.5;
(d) allowing thrombin to interact with fibrinogen, originally present in the mixture, and with a known amount of fibrinogen or allowing thrombin to interact with fibrinogen, originally present in the mixture and with a known amount of thrombin;
(e) determining the activity or functionality of thrombin or fibrinogen, original prisutstwu vsego in the dry mixture.

2. The method according to claim 1, where thrombin is a recombinant or derived from animal or human source, and fibrinogen is a recombinant or derived from animal or human source.

3. The method according to claim 1, where steps (b) and (C) are performed simultaneously.

4. The method according to claim 3, where the mixture is in the form of powder, and stage reversible inhibition of thrombin involves the dissolution of a mixture of thrombin and fibrinogen in the first solution, having a pH in the range from 8.5 to 11.5.

5. The method according to claim 4, where the first solution has a pH from 9.5 to 10.5.

6. The method according to claim 5, where the first solution has a pH of 10.

7. The method according to claim 4, where the first solution comprises an alkaline solution of at least one component selected from the group consisting of carbonate, TRIS (Tris(hydroxymethyl)aminomethane)Foundation, borate, glycine, phosphate, methylamine, 2-(cyclohexylamino)econsultancy acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO).

8. The method according to claim 7, where a known amount of fibrinogen contained in the second solution, which is capable of lowering the pH of the first solution containing an inactivated thrombin and fibrinogen, up to a level of from about 6.0 to less than 8.5.

9. The method of claim 8, where a known amount of fibrinogen contained in the second solution, which str is Aubin to reduce the pH of the first solution, containing an inactivated thrombin and fibrinogen, up to a level of from about 7.0 to less than 8.5.

10. The method according to claim 9, where a known amount of fibrinogen contained in the second solution, which is capable of lowering the pH of the first solution containing an inactivated thrombin and fibrinogen, to the level of about 7.5.

11. The method of claim 8, where the second solution, which is able to decrease pH of the first solution is a solution of at least one component selected from the group consisting of TRIS-HCl, imidazole, 4-(2-hydroxyethyl)piperazine-1-econsultancy acid (HEPES), phosphate, barbitala, 4-morpholinepropanesulfonic acid (MOPS), 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO), 1,4-piperazineethanesulfonic acid (PIPES), N,N-bis(2-hydroxyethyl)-2-aminotadalafil acid (BES), citrate or carbonate.

12. The method according to claim 11, where the second solution, can reduce the pH of the first solution contains from 25 mm to 500 mm buffer TRIS-HCl.

13. The method according to item 12, where the second solution, can reduce the pH of the first solution is from 100 mm to 150 mm buffer TRIS-HCl.

14. The method of claim 8, where the famous excess fibrinogen is added to the first solution comprising an inactivated thrombin and fibrinogen, in stage (b).

15. The method according to claim 1, where the phase (b) includes adding a second solution comprising a known amount of fibrinogen is a, to the first solution comprising an inactivated thrombin and fibrinogen to form a third solution having a pH in the range from 8.5 to 11.5.

16. The method according to clause 15, where stage (C) reversible activation of thrombin includes bringing the pH of the third solution to a level approximately from 6.0 to 8.5.

17. The method of claim 8, where the activity or functionality of thrombin or fibrinogen, initially present in the mixture, determined by the kinetic analysis of coagulation, which measures the clotting time and clotting time is correlated with a known activity or functionality of thrombin or fibrinogen.

18. The method according to 17, where the activity or functionality of thrombin or fibrinogen is converted to units of activity or function number.

19. The method of claim 8, where the activity or functionality of thrombin or fibrinogen, initially present in the mixture, is determined using turbidimetry, nephelometry, or viscosity, or mechanical way analysis of boundary points.

20. The method according to claim 1, where the mixture of thrombin and fibrinogen in powder form is located on the substrate, and stage (b) is carried out by placing the substrate with a mixture of inactivated thrombin and fibrinogen, the second solution having a known amount of fibrinogen.

21. The method according to claim 1, where the mixture of thrombin and fibrinogen which is a non-aqueous suspension.

22. The method according to item 21, where non-aqueous suspension includes alcohol, thrombin and fibrinogen.

23. The method according to item 22, where the alcohol is an ethanol.

24. The method of determining the functionality of fibrinogen in the original unreacted mixture of thrombin and fibrinogen, which includes stages
(a) reversible inhibition of thrombin to obtain a mixture containing an inactivated thrombin and fibrinogen, by dissolving a mixture of thrombin and fibrinogen in the first solution, having a pH in the range from 8.5 to 11.5;
(b) adding to the mixture a known amount of thrombin in the second solution, which is capable of lowering the pH of the first solution comprising an inactivated thrombin and fibrinogen, from about 6.0 to less than 8.5;
(c) allowing thrombin to interact with fibrinogen; and
(d) determine the functionality of fibrinogen, originally present in the mixture.

25. The method according to paragraph 24, where stage is the inhibition of thrombin, in addition, includes the addition of an inhibitor of thrombin.

26. The method according A.25, where thrombin inhibitor selected from the group consisting of anti-thrombin, heparin, heparin low molecular weight analogues of heparin low molecular weight, fondaparinux, argatroban, melagatran, efegatran, inogatran, dabigatran, bivalirudin, hiragana and derivatives of hiragana, such as lepirudin and desire is in.

27. The method of determining the functionality of fibrinogen in the original unreacted mixture of thrombin and fibrinogen, which includes stages
(a) reversible inhibition of thrombin to obtain a mixture containing an inactivated thrombin and fibrinogen, by dissolving a mixture of thrombin and fibrinogen in the first solution, having a pH in the range from 8.5 to 11.5;
(b) adding to the mixture a known amount of such thrombin enzyme, and reducing the pH of the first solution comprising an inactivated thrombin and fibrinogen, up to a level of from about 6.0 to less than 8.5;
(c) providing opportunities similar to the thrombin enzyme to interact with fibrinogen; and
(d) determine the functionality of fibrinogen, originally present in the mixture.

28. The method according to item 27, the stage where inhibition of thrombin, in addition, includes the addition of an inhibitor of thrombin.

29. The method according to item 27, where the thrombin inhibitor is selected from the group consisting of anti-thrombin, heparin, heparin low molecular weight analogues of heparin low molecular weight, fondaparinux, argatroban, melagatran, efegatran, inogatran, dabigatran, bivalirudin, hiragana and derivatives of hiragana, such as lepirudin and desirudin.

30. The method according to item 27, where similar to thrombin, the enzyme is batroxobin or ancrod.

31. Method definition wide-angle the activity of thrombin in the original unreacted mixture of thrombin and fibrinogen, incorporating the following stages:
(a) reversible inhibition of thrombin to obtain a mixture containing an inactivated thrombin and fibrinogen, by dissolving a mixture of thrombin and fibrinogen in the first solution, having a pH in the range from 8.5 to 11.5;
(b) adding to the mixture a known amount of fibrinogen in the second solution, which is capable of lowering the pH of the first solution is from about 6.0 to less than 8.5;
(c) allowing fibrinogen to interact with thrombin; and
(d) evaluating the formation of a clot,
(e) determining the activity of thrombin initially present in the mixture.

32. The method for determining the activity of thrombin in the original unreacted mixture of thrombin and fibrinogen, which includes stages:
(a) reversible inhibition of thrombin to obtain a mixture containing an inactivated thrombin and fibrinogen addition of inhibitor solution having a pH in the range of from about 8.5 to 11,5;
(b) adding to the mixture a known amount of a chromogenic or fluorogenic thrombin substrate;
(c) reversible activation of thrombin by bringing the pH to from about 6.0 to less than 8.5; and
(d) allowing thrombin to react with chromogenic or fluorogenic thrombin substrate;
(e) determining the activity of thrombin, originally present in the mixture.



 

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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: 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: 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

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