Diagnostics of thromboembolic vein diseases by determination of content of d-dimers and soluble fibrin

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

 

The present application relates to a method and test to determine the activation of blood coagulation, in particular, in the case where coagulation is associated with thromboembolic diseases of the veins, using content analysis of D-dimer and soluble fibrin in the activation process of blood clotting.

Fibrinolysis is the process of decomposition of fibrin in the blood. Fibrinolysis is involved in several pathophysiological processes, run it occurs in situations where tissue plasminogen activator and plasminogen bound to fibrin, forming a ternary complex fibrin-plasminogen, in which t-PA has a high affinity for plasminogen, which leads to the formation of plasmin, the enzyme that breaks down fibrin D-dimers. In the absence of fibrin affinity of t-PA for plasminogen low. This explains the fact that the fibrin in the blood is not destroyed.

Fracture (cleavage) of fibrin or fibrinolysis leads to the formation of cleavage products, containing, in particular, fragments, called "D-dimer". These D-dimer are associated with the E-fragment formed during the destruction of the other monomer molecules of fibrin, with the formation of DDE complex, but even in this form they are usually called D-dimer.

Fibrin, undergoing the process of fibrinolysis, is formed by the conversion of fibrinogen by the action of the enzyme coagulation, namely thrombin. During activation of the coagulation of the formed thrombin induces the formation of fibrin, which is a blood clot, and the formation of soluble fibrin. As a result, the thrombin effect on four of the peptide bond fibrinogen, located, respectively, in 2 chains And alpha and 2 chains In beta, which leads to the release of two fibrinopeptides And two chains And is the alpha and the release of two fibrinopeptides In from the chains In beta, which leads to the formation of fibrin monomers, which spontaneously polymerize to form a polymer by shifting hydrogen bonds formed as a result of interaction between sites of polymerization and In that during the release of fibrinopeptides a and b, and site a and site b, which are located at the ends the gamma and beta chains, respectively. Then the polymer fibrin immediately stabilizes factor XIII(a). While conducting experiments in vitro the formation of thrombin is much more intense than in vivo. Thus, upon activation of blood coagulation in vivo formation of fibrin monomers is much slower than the formation in vitro, resulting in a portion of the monomers formed for polymerization, form insoluble fibrin, forming a blood clot, and the other part of these monomers interacts with the fibrinogen containing access the data sites a and b, or degradation products of fibrinogen with the formation of soluble fibrin in which the monomers of fibrin associated with fibrinogen.

The analysis of concentration of soluble fibrin is important to identify the activation of blood coagulation in a patient. Such analysis can be done using a blood sample or plasma obtained from blood samples taken from the patient.

It is shown that the analysis of soluble fibrin is a useful addition to the analysis of products of destruction on the mechanism of fibrinolysis, as soluble fibrin may help identify ongoing activation of coagulation, while the concentration of D-dimers indicates the degree of destruction of the clot even after termination of the activation of coagulation.

In General, the level of D-dimers in the plasma increases with the destruction of fibrin thrombus in vivo. Thus, if a blood clot exists and subject to destruction, the level of D-dimers is high regardless, does collapse or ended. The level of soluble fibrin, in contrast, increased only if the collapse continues.

Compared with the level of D-dimer measurement is exactly the level of soluble fibrin in plasma allows to detect clotting occurring in a patient in the moment of taking his samples for analysis, simultaneously with the determination of the balance of coagulation.

Thus, the level of D-dimer in the sample, called the basic level, is an indicator of the destruction of a blood clot occurring in vivo, and the level of D-dimers defined after exogenous addition of fibrin specific thrombolytic agent, represents the sum of the baseline D-dimer and D-dimer, formed as a result of the destruction of soluble fibrin, which is also called circulating fibrin.

The application WO-A-02/18628 describes the method of analysis of soluble fibrin in the blood sample, which includes the implementation of contact of plasma plasminogen activator with high affinity to the soluble fibrin (PA-sFb), followed by determination of the level of cleavage products of fibrin (D-dimer): the difference between the concentration of D-dimer in the sample treated PA-sFb, and a base concentration of D-dimers determined in plasma, not treated PA-sFb, is D-dimers associated with the destruction of soluble fibrin. The authors of the present invention believe that the method proposed in the prototype International publication WO-A-02/18628, can be improved for use in the context of the diagnosis of thromboembolic venous disease, as well as diagnostics and monitoring of disseminated intravascular coagulation (DIC). It also allows you to determine the effectiveness of anticoagulation.

T is amoebalike venous disease usually include thrombosis of the veins of the limbs and pulmonary embolism, which is the result of these complications of thrombosis. Thromboses veins, other than thrombosis of the extremities, also included because the thrombosis may occur in the veins of any area. In particular, among the sites pathologies you can specify the veins of the kidneys and veins of the mesentery. Thromboembolic disorders, including deep vein thrombosis (DVT) and/or pulmonary embolism (PE), are life-threatening diseases, which account for a large proportion of disability and death in industrialized countries, and diagnosis of these diseases is necessary to Supplement research methods image visualization, in particular, ultrasonography for the diagnosis of venous thrombosis and scintography or angiography for the diagnosis of pulmonary embolism. These research methods are complex to implement, and it is not always possible to realize quite quickly.

Thus, there is a continuing need in the way of analysis, which will allow quick diagnosis of thromboembolic disease in a patient, with the above diagnosis of the disease should eliminate the need for additional research.

In the diagnosis of diseases associated with blood clotting, it is known that the normal level of D-dimer is negative indicator of thrombosis, PQS is LCU according to the generally recognized principle of the formation of a blood clot leads to either activation of coagulation, and fibrinolysis.

However, the known methods of determining the level of D-dimers do not have sufficient specificity and do not allow to draw definite conclusions about the formation of intravascular thrombus, because D-dimer present in circulating blood, can be formed in the result of the collapse of extravascular fibrin accumulation. D-dimers, formed in situ, can then enter the system circulating blood, resulting in increased levels of D-dimers in the circulating blood. In the context of the present invention, the authors assessed the accuracy of diagnosis based on the analysis of the level of D-dimer in combination with a rapid analysis of soluble fibrin, which is an indicator of the activation of intravascular coagulation. This combination of analyses were preferred for the diagnosis of deep vein thrombosis (DVT) and/or pulmonary embolism (PE), as well as for the diagnosis of disseminated intravascular coagulation (RVS). Analysis of soluble destructible fibrin. to determine the level of soluble fibrin on the content of the products formed in the decomposition of tissue plasminogen activator or other thrombolytic agent, in particular, a plasminogen activator, specific fibrin)that exogenous injected into the sample.

Thus, the present izaberete the s refers to the way used for in vitro diagnostics activation of coagulation in the blood sample of the patient, with the specified method includes:

(a) quantitative analysis of the products of destruction of fibrin contained in the test sample, comprising determining the quantity of D-dimers present in the sample and the components of a basic level of D-dimers;

b) processing the sample by incubation with plasminogen activator with high affinity to fibrin (PA-sFb), in conditions that ensure the destruction of soluble fibrin contained in the sample, with the formation of products of destruction, without destruction of fibrinogen, and quantitative determination of D-dimers contained in the treated sample;

C) calculating the difference between the concentration of D-dimers, particular after activation of the activator PA-sFb at the stage b), and the content of D-dimers before the specified activation defined in stage a), with the resulting difference is a measure of the degree of decomposition of soluble fibrin.;

g) comparing the content of D-dimers measured in stage a), with the boundary value of a norm defined for the specified products of decomposition, and the comparison level. calculated on the stage), with the boundary value of a norm defined for..

If the analysis in this way perform, starting with the sample, you can determine the risk of thromboembo the systematic disease: this risk exists, if at least one of the calculated levels of these degradation products of fibrin or. (D-dimers) exceeds the normal value, however, this risk is eliminated if the estimated level of these degradation products of fibrin and level. lower than the corresponding boundary values of the norm.

The diagnostic method according to the present invention used for the diagnosis of blood clotting regardless of whether the clotting process localized (in particular when deep vein thrombosis) or generalized (as in the case of RVS).

In appropriate cases, prior to the addition of plasminogen activator in stage b), described above, in the part of the sample that is used to determine the products of destruction of soluble fibrin, add a mixture of citric acid and sodium citrate.

The reagent used for the analysis of products of destruction, is chosen so that it provided a definition of this group of products of destruction. So, for example, using antibodies installed specificity with respect to a certain type of decomposition products of fibrin.

If the concentration value of the underlying D-dimers measured in stage (a), exceeds the threshold value of 500 ng/ml [nanograms/milliliter], the level of D-dimers is increased. If the value of the concentration of D-dimers, which are appropriate to esthet destruction of soluble fibrin and which rely on stage), which exceeds a limit value of 300 ng/ml, determined in healthy subjects, it is considered to be elevated.

These boundary values is determined by the reagent, which is an antibody from the test set "Lia-test" manufactured by Diagnostica Stago or from the test set VIDAS Bio-Merieux. For other reagents threshold value should be determined by comparison with the results obtained for these reagents.

Measurement of D-dimers on the one hand and soluble destructible fibrin on the other allow to conclude that for the patient, the blood sample which was analyzed, there is a risk of thromboembolic disease if the level of D-dimers, resulting from the decomposition of the underlying fibrin, 500 ng/ml or more, or if the level of soluble fibrin, defined by the difference between the level of D-dimers present in plasma treated with specific plasminogen activator, fibrin, and a basic level of D-dimers, exceeds a limit value, for example, 300 ng/ml.

The biological sample is preferably a biological fluid such as plasma or blood sample, or fluid obtained by puncture, provided that the level of plasminogen in this fluid is identical to the level of plasminogen in which the lazma. In the case of a fluid obtained by puncture, containing a small amount of plasminogen, you must add Glu-plasminogen to ensure that the concentration of plasminogen, is close to its concentration in plasma.

Plasminogen activator with high affinity for fibrin (i.e. the one that activates only the plasminogen to fibrin)used in the method of analysis of soluble fibrin by the formation of specific degradation products, you can choose from a variety of compounds are known that they are the activators of plasminogen. However, some of them, in particular, streptokinase and urokinase, destroy both fibrinogen and fibrin. These compounds are not suitable for use in the method according to the present invention, since the decomposition of fibrin they form also the degradation products of fibrinogen, which interfere with the determination of degradation products of fibrin.

Another group of plasminogen activators comprise the compounds that are described as having high specificity for the cleavage of fibrin compared with fibrinogen. To implement the method according to the present invention, it is preferable to apply this group of compounds, for example:

tissue plasminogen activator (t-PA) or its derivatives, in particular, TNK-tPA, which is a mutant t-PA, has a very high what pecifically against fibrin (S.R. Cannon et al. TNK tissue plasminogen activator compared with front loaded altephase in acute myocardial infarction results of the TIMI 10B trial, Thrombolysis in Myocardial Infarction (TIM1) 10B Investigators, Circulation 98 (25), 2805-14, 1998);

activator isolated from the body Desmodus rotundus (vampire ordinary) (bat-tpa or vPA=plasminogen activator saliva of the vampire), and its derivatives: DSPAs=PA saliva Desmondus rotundus, FEKP=(DSPA alpha 1 and alpha 2, ECR=(DSPA beta, CR=gamma (DSPA (Bringmann et al. Structural features mediating fibrin selectivity values of vampire bat plasminogen activators, J. Biol. Chem. 270, 25596-603, 1995), staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus (Collen D. Staphylokinase: a potent, uniquely fibrin-selective thrombolytic agent, Nat Med, 4-279-84, 1998; Sakharov, D. V. et al. Fibrin specificity of a plasminogen activator affects the efficiency of fibrinolysis and responsiveness to ultrasound: comparison of nine plasminogen activators in vitro. Thromb Haemos, 81, 605-12, 1999) or one of its mutants (Collen D. et al. Recombinant staphylokinase variants with altered immunoreactivity. I: Construction and characterization. Circulation 94, 197-206, 1996).

For the implementation of the diagnostic method described above, use of antibodies to D-dimers, which perform two analyses (the contents of the underlying D-dimer and D-dimer after exposure to specific plasminogen activator, fibrin) in accordance with the present invention. These antibodies are described in the literature and are available commercially, for example, Diagnostica Stago under the brand name "Lia-test or by the company Bio-Merieux under the trademark "Vidas".

For correct comparison of the results for analysis on the operations a) and b) should use the same antibodies to D-dimer.

D-dimers, the cat is which are formed as a result of the destruction of soluble fibrin in the presence of PA-sFb, can be analyzed by any known conventional method, in particular, methods ELISA (enzyme-linked immunosorbent assay), sensitive methods latexes agglutination (for example, used in Lia-test)methods immunostaining, etc. as examples of different commercially available kits for analysis of D-dimers, you can specify ASSERACHROM D-Di or STA LIATEST D-Di, which supplies the company Diagnostic Stago. However, in the context of the present invention, the modes of analysis by the ELISA method using ASSERACHROM D-Di has been successfully modified, allowing us to reduce the analysis time (incubation for 15 minutes with immobilized antibody and within 15 minutes with the antibody, labeled with peroxidase).

The method of in vitro diagnosis according to the present invention preferably also includes the processing of positive control sample, in particular, a positive control plasma.

For the positive control plasma first plasma incubated with a small amount of thrombin within a certain period of time, resulting in the formation of soluble fibrin without fibrin clot. The process of coagulation, the induction of which when this occurs, block by adding thrombin inhibitor, to prevent the continuation of the reaction. As the inhibitor can be used, for example, g is Rudin or heparin.

Incubation time and plasma concentration of thrombin and inhibitor to block preferably chosen in such a way as to ensure the activation of coagulation, which leads to the formation of soluble fibrin without fibrin clot.

Incubation in the presence of an activator of coagulation (thrombin) is preferably carried out for 2 minutes at ambient temperature. Then add the inhibitor in a large excess to securely block the clotting of blood.

In the case of hirudin its final concentration is preferably 100 μg/ml for a final concentration of thrombin 0.18 units/ml.

In the case of the use of heparin his final concentration of 500 units/ml, if the final concentration used thrombin is 0.18 units/ml.

In another preferred embodiment of the invention a method of in vitro diagnosis according to the present invention includes additional processing negative control sample, in particular a positive control plasma. Obtaining these samples, additional details are described in the following examples.

For the determination of soluble fibrin in accordance with the present invention, first apply the splitting of soluble fibrin by the action of RA-sFb, and then set the ut content specific cleavage products under the action of PA-sFb.

For the method according to the present invention it is essential to get these results as quickly as possible, as they are an adequate indicator of the content of soluble fibrin present in the sample. Accordingly, conditions of use PA-sFb must be defined so that the destruction of soluble fibrin occurred quickly and were not accompanied by "polluting" the destruction of fibrinogen plasma circulating blood, which leads to the increase in the content of products of destruction and prevents the determination of soluble fibrin.

The applied dose of PA-sFb and the period of incubation with plasma is chosen in such a way as to increase the level of decay products of fibrin, which is the maximum in the positive control samples and has almost zero increase in negative control samples (i.e. those samples that were not subjected to treatment by coagulation activator).

In the context of the present invention can use various activators of fibrinolysis, providing specific decomposition of soluble fibrin. PA-sFb preferably chosen from the group comprising these activators, namely, t-RA or its derivatives, VPA or its derivatives and staphylokinase or one of its mutants. Preferably use t-PA or staphylokinase, more preferably t-PA

In conditions when the samples incubated for 15 minutes at 37°C. the final concentration of the investigated staphylokinase is in the range from 1 to 12 mcg/ml Final concentration is preferably 10 μg/ml of the incubation Period can vary, and its change is defined as a function of the nature and concentration of the used PA-sFb.

t-PA is preferably used at a final concentration ranging from 1 to 2.5 μg/ml t-PA is preferably used at a concentration of 2 μg/ml in the case of incubation for 15 minutes at 37°C.

In one embodiment of the invention the destruction of soluble fibrin-plasminogen activator without destruction of fibrinogen can be blocked after the destruction of soluble fibrin by adding plasmin inhibitor, such as Aprotinin. Specific conditions for the use of Aprotinin or other equivalent plasmin inhibitor indicated in the examples. The amount used Aprotinin may be, for example, is equivalent to the amount used plasminogen activator. For example, the inhibitor of plasmin added after incubation for 15 minutes at 37°C With plasminogen activator.

In one embodiment of the invention prior to the addition of plasminogen activator in the analyzed sample and a control sample, you can add an anticoagulant in castnet is, the solution containing citric acid and sodium citrate. The amount and mode of addition of citric acid and sodium citrate is indicated in the examples.

In the context of the present invention described above, the diagnostic method used to determine the formation of a venous thrombus.

In one embodiment, the implementation of the method according to the present invention is used for diagnostic exclusion of deep vein thrombosis.

In one embodiment of the invention the method according to the present invention is used for diagnostic exception embolism of the lung.

In one embodiment of the invention the method is carried out on the blood sample taken from the patient before treatment with anticoagulants.

In principle, the analysis of soluble fibrin for diagnostic exclusion of venous thrombosis should be done before any treatment with anticoagulants. If the patient is undergoing treatment with anticoagulants, the concentration of soluble fibrin decreases very rapidly and reaches normal values. In the case of patients undergoing treatment, the concentration of soluble fibrin in plasma allows to determine only the effectiveness of the anticoagulant.

In one embodiment of the method according to the present invention the analysis of soluble fibrin is carried out using t-PA as an asset is ora plasminogen.

Other features of the invention will become apparent from the following examples and figures.

In figures 1 and 2, respectively, shows the comparison of D-dimers and. in patients with suspected pulmonary embolism or with suspected deep vein thrombosis. In all cases, the black circles correspond to patients, and white circles - normal patients. The line shows the upper limit of normal (edge rate).

EXAMPLES

Example 1

The choice of the concentration of thrombin used for the positive control plasma containing soluble fibrin:

Positive control plasma was obtained using the following Protocol:

Normal plasma200 ál
The human thrombin (Stago, No. 00896), 0.5-1 unit/ml (depending on the plasma)20 ál

Incubation for 2 minutes at ambient temperature.

Hirudin (Knoll)100 μg/ml (final concentration)

or

heparin (Choay) 5000 inhibition/ml (final concentration) 20 ál

Established that:

in vitro there is no formation of clot and

- commercially available reagents for analysis of soluble fibrin are positive samples (for example, test FS production Stago Laboratories).

Positive control sampleA negative control sample
Plasma200 ál200 ál
Thrombin 1 unit/ml or 0.5 units/ml20 ál
Physiological serum-20 ál
2 minutes (±10 seconds) when ambient temperature
Heparin, 5000 units of inhibition/ml20 ál
Physiological serum-20 ál

Example 2

The determination of the number of PA-sFb required for use in certain conditions the x incubation

To implement the method according to the present invention, the amount of activator that you want to add in the analyzed sample should be such as to ensure the formation of D-dimers in the positive control plasma as described in example No. 1, with negligibly small formation of D-dimers in the negative control plasma (i.e. a control sample not treated with thrombin).

Conducted incubation of samples negative control plasma and positive control plasma (n=21) with different doses of PA-sFb for 15 minutes at 37°C. At the end of the incubation period the level of D-dimers was determined using the test system Lia-test or rapid assays, ELISA (D-Di Stago) (incubation for 15 minutes at 37°C With immobilized antibody and 15 minutes at 37°C with free antibody).

The results presented in table II, obtained using ELISA method.

Almost similar results were obtained when using the Lia-test (n=5).

Table II
Decomposition of soluble fibrin by increasing the number of t-PA and SAK
D-dimers (ng/ml)Soluble fibrin* (ng/ml)D-dimers (ng/ml)Soluble is ibrin* (ng/ml)
A negative control samplePositive control sample
Without adding Pa-Fb375375
After staphylokinase (µg/ml)
10400<5017501375
2390<5016151240
1.5375<5017001325
1350<5016571282
0.5410<501125750
After t-PA (µg/ml)
2350<5017901415
1360<5014201045
0.5360<501210835
* Soluble fibrin = level D-dimers after addition of t-PA or staphylokinase - basic level of D-dimers before addition of t-PA or staphylokinase.

The dose of PA-sFb is chosen in such a way as to provide:

- increase <300 µg/ml in untreated control plasma (negative control sample);

the maximum increase in positive control plasma.

From these results it follows that it is preferable to use a final concentration of PA-sFb, is equal to:

- 2 µg/ml for t-PA: under these conditions, the dose of t-PA, which can neutralize inhibitors of plasminogen activator (RO)is negligible;

10 ug/ml for SAK (smaller doses SAK led to the labs destruction of soluble fibrin in some patients or in some positive control samples, most likely it was caused by the presence in the sample of antistaticity, which may occur after infection with staphylococci).

Example 3: analysis of the content of D-dimer and soluble fibrin***

In conducting the study, the level of D-dimer and soluble fibrin was determined in 87 consecutive admissions of patients who were advised in paragraphs emergency with suspected deep vein thrombosis and/or embolism in the lungs, and which did not receive any treatment. These patients were subjected to ultrasound for the diagnosis of deep vein thrombosis, and scintigraphy or pulmonary angiography for the diagnosis of pulmonary embolism. Before beginning treatment with anticoagulants determined the levels of D-dimer and soluble destructible fibrin.). It was shown that the sensitivity analysis for soluble destructible fibrin similar sensitivity analysis for D-dimer (96% for D-dimer and soluble destructible fibrin). Interestingly, the "false negative" levels of D-dimer and soluble destructible fibrin was observed in different patients. Thus, the combination of the two analyses will allow to increase the sensitivity of diagnosis of thrombosis (100%). In addition, the specificity of soluble destructible fibrin in the diagnosis of thromboembolic Zab the diseases of the veins above (86% and 87% for pulmonary embolism and deep vein thrombosis, respectively)than the specificity of D-dimer (36% and 42% for pulmonary embolism and deep vein thrombosis, respectively).

Rapid normalization of the level of soluble destructible fibrin was observed in patients who received treatment in the form of a therapeutic dose of anticoagulant. After treatment with anticoagulant level of soluble fibrin decreased. Thus, soluble destructible fibrin cannot be used as a diagnostic test in patients who received treatment with anticoagulants. However, soluble degradable fibrin may be useful for monitoring anticoagulation. In conclusion it should be stated that the level of soluble destructible fibrin in combination with the level of D-dimers is a useful clinical tool to predict or eliminate the risk of pulmonary embolism and/or deep vein thrombosis.

Soluble fibrin is present during activation of blood coagulation. The increase observed in the early stages of activation.

There are several tests for determining the level of soluble fibrin in patients suffering from thrombosis, however, due to the variability of the results of these tests the importance of determining the level of soluble fibrin in the diagnosis exceptions thromboembolic venous disease to present the second time was underestimated (1-21).

The purpose of this study is to evaluate the possibility of applying the new test, which is based on determining the level of soluble destructible fibrin and which is simple, rapid, sensitive and highly specific with respect to soluble fibrin polymers plasma.

This test is based on determining the level of D-dimers, which are formed after incubation of plasma with t-PA in conditions that encourage the destruction of soluble fibrin, but without inducing the destruction of fibrinogen plasma. Therefore, such a test is called a test for soluble destructible fibrin.). In fact, despite the small content of monomers of fibrin into soluble fibrin monomers, fibrin related to each other transverse relationship, because activation of factor XIII coincides with the release of fibrinopeptide And, and, furthermore, activation of factor XIII by thrombin is accelerated in the presence of fibrin (22). For the formation of thrombin in vivo of markers selected soluble degradable fibrin, namely fibrinopeptide A (FPA, the half-life of 3 minutes (23) or the complex of thrombin-antithrombin (TAT, the half-life of 15 minutes (24)), because its definition may be more sensitive due to the fact that it is less sensitive to anomalies.

The purpose of this study was to determine the effectiveness of the diagnostician is ical test based on the combination of levels of D-dimer and soluble destructible fibrin in untreated patients, consecutively admitted in points emergency in 3 centers with clinical suspicion of pulmonary embolism (n=38) or deep vein thrombosis (n=49). In cases of suspected deep vein thrombosis diagnosis was confirmed by the method of compression ultrasonography, and in the case of pulmonary embolism - method scintigraphy or pulmonary angiography. Boundary value content of soluble destructible fibrin for the test, which was considered to be positive, was 300 ng/ml.

This study aimed to analyze the impact of anticoagulants defined profiles changes in soluble destructible and fibrin D-dimers in patients suffering from pulmonary embolism and/or deep vein thrombosis, after starting treatment with anticoagulants, which allowed to test the effectiveness of therapy for thromboembolic disease.

Materials and methods

Plasma samples: blood samples were collected in 0.13 M citrate (1 part citrate to 9 parts blood). After centrifugation at 2500g for 15 minutes was collected and plasma was frozen at -20°C until use.

However, at a very high level of soluble destructible fibrin, as in the case of intravascular coagulation (RVS), soluble destructible fibrin may OBR is its insoluble complex on the stages of freezing and thawing, therefore, such analysis is recommended with selected fresh plasma.

Blood samples were collected from healthy volunteers or patients in sub-acute care. Patients who received treatment with the anticoagulant, were included in the study only for monitoring purposes. The patient group consisted of consecutive admissions of patients with signs of pulmonary embolism or deep vein thrombosis. To confirm the diagnosis, the patients were examined by ultrasonic analysis of compression of the proximal veins of the lower extremities, scintigraphy or angiography lungs.

Biological tests

Determination of the level of D-dimer

The level of D-dimers was determined by the method of agglutination of the particles of the latex, monoclonal antibodies to D-dimers, with the use of Lia-test (Diagnostica Stago) on the instrument the STA or the ELISA method using VIDAS (Bio-Merieux).

Determination of soluble destructible fibrin: conducted in 3 stages

1 - the Destruction of fibrin: 20 μl of t-PA, 20 µg/ml (treated plasma) or 20 μl of physiological serum (raw plasma) was added to 200 ál of plasma. After incubation for 15 minutes at 37°C. the formed plasmin was blocked by the addition of 20 μl of Aprotinin (Pentapharm), 12.5% inhibition of thrombin/ml

2 - Then determined the concentration of D-dimers, with the use of the Lia-test for D-dimer production company Diagnostica Stago.

3 - Calculate the level of soluble destructible fibrin as the difference between the concentration of D-dimers in the treated and untreated plasma.

If the level of D-dimer in plasma exceeded 4000 ng/ml, the sample stage after the destruction diluted.

Soluble fibrin, used as a positive control sample, which was obtained by incubation of normal plasma with small doses of thrombin in a short period of time, after which thrombin was blocked by heparin.

The plasma from the sample, and positive and negative control sample, for determining the content of soluble fibrin, were analyzed, as indicated in table III.

TABLE III
Basic D-dimer. + Basic D-dimer
Plasma200 ál200 ál
Clean AC20 ál20 ál
Tra-20 ál
Physiological serum -
15 minutes at +37°C
Aprotinin-20 ál

Adding Aprotinin blocked plasmin for some time, which led to what happened destruction exclusively fibrin but not fibrinogen.

Positive and negative control sample was prepared with ACd, i.e. speakers with a dilution of 1:5.

Receiving as:

Citric acid, H2AboutMolecular weight = 210.14 0.16 g
Trinitrate sodium, 2 H2OMolecular weight = 294.10 0.44 g
H2About20 ml

Results

The specificity of the measurements

- In normal healthy volunteers (n=180) the level of soluble fibrin was very low - 300 ng/ml or less.

The correlation between the concentration of soluble fibrin in plasma and the concentration of D-dimers was absent, because after the introduction patients with heparin concentration of fibrin decreased sharply, while the concentration of D-dimers decreased much more slowly, reflecting the destruction of the clot, which remains the donkey blocking activation of blood coagulation.

Levels of D-dimer and soluble fibrin in patients with suspected thromboembolic disease venous or pulmonary embolism

In normal healthy volunteers (n=180).

The average value during the examination 180 volunteers was 80±106 ng/ml, with 140 of them, the level of soluble destructible fibrin has not been determined. The boundary is taken as positive samples was 300 ng/ml.

Patients with suspected pulmonary embolism or deep vein thrombosis

Of the 38 patients with suspected pulmonary embolism 23 had a positive performance, according to the visual analysis, and from 49 patients with suspected deep vein thrombosis 25 had positive indicators according to the observation of anomalous compression of the proximal veins of the lower limb ultrasound.

Two groups of patients suffering from pulmonary embolism (n=23) or deep vein thrombosis (25), 2 had levels of soluble fibrin, which were false negatives, one out of a group of patients with pulmonary embolism (confirmed by angiography), and the other from a group of patients with deep vein thrombosis, confirmed by ultrasound). However, these 2 patients had levels of D-dimers, which exceeded the limit value of 500 ng/ml Levels of D-dimers were normal (<500 ng/ml) in 2 patients (one from group embolism light is, and the other group with deep vein thrombosis), while the levels of soluble destructible fibrin was they >300 ng/ml of These false-negative levels of D-dimers was observed in cases when the level of D-dimers was determined by means of tests Lia-test® or Vidas®.

When used as a biological parameters of the combination of the levels of D-dimer and soluble fibrin false-negative results are not received.

Calculated indicators of significance (sensitivity, specificity, predictive value positive and negative results) for D-dimer and soluble destructible fibrin.

TABLE III
Sensitivity, specificity, predictive value positive and negative results for D-dimer and soluble destructible fibrin
.+D-dimer + soluble fibrin
EDVTEDVT
PZPR92%88%71% 60%
PCOR92%95%83%90%
Sensitivity96%96%96%96%
specificity86%87%36%37%
PCPR = predictive value of a positive result
PCOR = predictive value of a negative result
E = pulmonary embolism
DVT = deep vein thrombosis
. = Soluble destructible fibrin

Changes in the levels of D-dimer and soluble destructible fibrin in patients during treatment with anticoagulants

In patients under the action of therapeutic doses nefrackzionirovannam heparin or heparin with low molecular weight was observed rapid normalization of the level of soluble destructible fibrin. After 1 day level of soluble destructible fibrin was normal Il is at the upper limit of the normal range. Daily analysis showed that during treatment with heparin level of soluble destructible fibrin remained within normal limits. In the treatment of heparin level of D-dimers, in contrast, declined slowly and did not reach normal limits. One patient with therapeutic effect of the level of soluble fibrin additionally increased, which pointed to insufficient therapeutic effect.

Discussion

There is a need for non-invasive diagnostic tool that is suitable for the diagnosis of pulmonary embolism and/or deep vein thrombosis, which will in most cases immediately to decide on the necessary treatment.

The objective of this study was to determine the possibility of using combinations of levels of D-dimer and soluble destructible fibrin for the diagnosis of pulmonary embolism and/or thrombosis of deep veins and to evaluate the effectiveness of anticoagulation for inhibiting thrombus formation.

Regarding the sensitivity of the measurements with pulmonary embolism and deep vein thrombosis: 2 patients who had false-negative results associated with the level of D-dimers (one of a group of patients with pulmonary embolism and the other from a group of patients with deep vein thrombosis), the levels of soluble destructible fibrin exceeded upper gr the border value. It could be caused by abnormal structure of fibrin clots (congenital or acquired), these clots are abnormally resistant to fibrinolysis. This anomaly can contribute directly to the increased risk of thrombosis due to violation of thrombolite.

This reduced fibrinolysis may explain 3-5% of cases of "false negative" result for D-dimers in patients with stable thrombosis. When determining the level of soluble destructible fibrin, the latter are easily exposed to fibrinolytic enzymes. The fibrin clot being formed of a thin and very dense filename, in contrast, are less affected by the fibrinolytic enzymes, which explains 3-5% false-negative results determine the level of D-dimers observed in the brain. Negative results determine the level of soluble destructible fibrin found in 2 patients (one of a group of patients with pulmonary embolism and the other from a group of patients with deep vein thrombosis), the level of D-dimers in these 2 patients was higher than 500 ng/ml based On this, we can assume that the formation of blood clots is revolutiony process because the activity of thrombin is temporary.

The authors of the present invention showed tocombine two tests (determination of the content of soluble destructible and fibrin D-dimers) can be used for differential diagnosis of pulmonary embolism and deep vein thrombosis in patients.

In most cases, D-dimer plasma satisfy the criteria required for the diagnosis of pulmonary embolism and deep vein thrombosis, because they are sensitive markers for thrombosis, however, lack specificity (26-35).

Now clearly established that D-dimer cannot be used for unambiguous diagnosis of thromboembolic venous disease, because this test is not specific enough. However, if the level of D-dimer is normal, the diagnosis of thromboembolic disease can be produced in 95% of patients.

In this study it is shown that the specificity of soluble fibrin is much higher than D-dimers. Low specificity determination of D-dimers in the diagnosis of thromboembolic venous disease may be due to local failure of fibrinolytic enzymes of fibrin present in the tissues. Locally formed in the tissues of the products of decomposition of soluble fibrin is diffused into the blood, due to their relatively low molecular weight. This is confirmed by the observation that the level of D-dimers is often high in patients suffering from inflammatory diseases.

In this study it is also shown that the level of soluble destructible fibrin increases in localremote diseases, however, when therapy with anticoagulants soluble destructible fibrin within a few hours is reduced to normal values. This suggests that anti-clotting medicines (anticoagulants) are able to effectively block the process of thrombus formation. Preservation of the increased level of D-dimers caused by the decomposition of the blood clot that has formed before initiation of therapy with anticoagulants. Therefore, the authors suggest that measuring the level of soluble destructible fibrin is important to determine the effectiveness of therapy with anticoagulants or test the effectiveness of new antithrombotic drugs.

Finally, the results of this study indicate that the determination of the level of soluble destructible fibrin in combination with the level of D-dimers may be a useful clinical tool for the diagnosis of deep vein thrombosis and pulmonary embolism, and other thrombotic diseases. In addition, it can be assumed that the level of soluble destructible fibrin can be a useful indicator for monitoring the effectiveness of anticoagulation therapy.

LITERATURE

1. Arkel YS, Ku DH, Le P, Can - AM. Comparison of a test for soluble fibrin polymer (TpP) with a standard quantitative ELISA for D-dimer in patients without current thrombosis, who have cancer or renal disease. Thromb Haemost. 2001; 86: 1127-8.

2. Arkel YS, Paidas MJ, Ku DH The use of coagulation activation markers (soluble fibrin polymer, TpP, prothrombin fragment of 1.2, thrombin-antithrombin and D-dimer) in the assessment ofhypercoagulability in patients with inherited and acquired prothrombotic disorders. Blood Coagul Fibrinolysis. 2002; 13:199-205.

3. Brimble KS, Ginsberg JS. Evaluation of the combination of a bedside D-dimer assay and enzyme-linked immunosorbent soluble fibrin assay in patients with suspected venous thromboembolism. Thromb Res. 1997; 88:291-7.

4. Dempfle CE, Dollman M, Lill H, Puzzovio D, Dessauer A, Heene DL. Binding of a new monoclonal antibody against N-terminal heptapeptide of fibrin alpha-chain to fibrin polymerization site 'A': effects offibrinogen and fibrinogen derivatives, and pre-treatment of samples withNaSCN. Fibrinolysis. 1993; 4:79-86.

5. Dempfle CE, Pfitzner SA, Dollman M, Huck K, Stehle G, Heene DL. Comparison of immunological and functional assays for measurement of soluble fibrin. Thromb Haemost. 1995; 74:673-9.

6. Dempfle CE, Zips S, Ergul H, Heene DL; FACT study group.The fibrin assay comparison trial (FACT): correlation of soluble fibrin assays with D-dimer. Thromb Haemost. 2001; 86:1204-9.

7. Dempfle CE, Wurst M, Smolinski M, Lorenz S, Osika A, Olenik D, Fiedler F, Borggrefe M. Use of soluble fibrin antigen instead of D-dimer as fibrin-related marker may enhance the prognostic power of the ISTH overt DIC score. Thromb Haemost. 2004; 91:812-8.

8. Derhaschnig U, Laggner AN, Lombardia M, Hirschi MM, Kapiotis S, Marsik C, Jilma B. Evaluation of coagulation markers for early diagnosis of acute coronary syndromes in the emergency room. Clin Chem. 2002; 48:1924-30.

9. Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Stevens P, Brill-Edwards P, Panju A, Patel A. Evaluation of a soluble fibrin assay in patients with suspected deep venous thrombosis. Thromb Haemost. 1995; 74:833-6.

10. Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Donovan D, McGinnis J, Brill-Edwards P, Panju A, Patel A, Weitz JI. Evaluation of a soluble fibrin assay in patients with suspected pulmonary embolism. Thromb Haemost. 1996; 75:551-4.

11. Hetland O, Knudsen A, Dickstein K, Nilsen D.W. Characteristics and prognostic impact of plasma fibrin monomer (soluble fibrin) in patients with coronary artery disease. Blood Coagul Fibrinolysis. 2002; 13:301-8.

12. Koga S. A novel molecular maker for thrombus formation and life prognosis clinical usefulness of measurement of soluble fibrin monomer-fibrinogen complex (SF) Rinsho Byori. 2004; 52:355-61.

13. LaCapra's, Arkel YS, Ku DH, Gibson D, Lake C, Lam X. The use of thrombus precursor protein, D-dimer, prothrombin fragment 1.2 and thrombin antithrombin in the exclusion ofproximal deep venous thrombosis and pulmonary embolism. Blood Coagul Fibrinolysis. 2000; 11:371-7.

14. Mac Gillavry MR, Sanson BJ, de Monye W, Lijmer JG, Huisman MV, Buller HR, Nieuwenhuizen W, Brandjes DP. Use of a new monoclonal antibody-based enzyme immunoassay for soluble fibrin to exclude pulmonary embolism. ANTELOPE-Study Group.Thromb Haemost. 2000; 84:474-7.

15. Nakahara K, Kazahaya Y, Shintani Y, Yamazumi K, Eguchi Y, Koga S, Wada H, Matsuda M. Measurement of soluble fibrin monomer-fibrinogen complex in plasmas derived from patients with various underlying clinical situations. Thromb Haemost. 2003; 89:832-6.

16. Nieuwenhuizen W, Hoegee-De Nobel E, R. Laterveer A rapid monoclonal antibody-based enzyme immunoassay (EIA) for the quantitative determination of soluble fibrin in plasma. Thromb Haemost. 1992; 68:273-7.

17. Nieuwenhuizen W. Soluble fibrin as a molecular marker for a pre-thrombotic state: a mini - review. Blood Coagul Fibrinolysis. 1993; 4:93-6.

18. Okajima K, Uchiba M, Murakami K, Okabe H, Takatsuki K. Determination of plasma soluble fibrin using a new ELISA method in patients with disseminated intravascular coagulation. Am J Hematol. 1996; 51:186-91.

19. Reber G, Bounameaux H, Perrier A, de Moerloose P. Performances of the fibrin monomer test for the exclusion of pulmonary embolism in symptomatic outpatients. Thromb Haemost. 1999; 81:221-3.

20. Scarano L, Prandoni P, Gavasso S, Gomiero W, Carraro G, Girolami A.Blood Coagul Failure of soluble fibrin polymers in the diagnosis of clinically suspected deep venous thrombosis. Fibrinolysis. 1999; 10:245-50.

21. Wada H, Sase T, Matsumoto T, Kushiya F, Sakakura M, Mori Y, Nishikawa M, Ohnishi K, Nakatani K, Gabazza EC, Shiku H, Nobori T. Increased soluble fibrin in plasma of patients with disseminated intravascular coagulation. Clin Appi Thromb Hemost. 2003; 9:233-40.

22. Brummel KE, Butenas S, and Mann KG. FXIII activation associated with a primary cleavage in the A-subunit at Arg-37 releasing an NH2-terminal activation peptide (54), is coincident with FPA release. J Biol Chem, 1999; 274:22862-22870.

23. Nosse HL, Yudelman I, Canfield RE, Butler VP Jr, Spanondis K. Wilner GD, Qureshi GD. Measurement offibrinopeptide A in human blood. J Clin Invest. 1974; 54:43-53.

24. Shifman MA, Pizzo SV. The in vivo metabolism of antithrombin III and antithrombin III complexes. J Biol Chem. 1982; 257:3243-8.

25. Lee L. V., G. Ewald, A., McKenzie, C. R, Eisenberg P. R. The Relationship of Soluble Fibrin and Cross-linked Fibrin Degradation Products to the Clinical Course of Myocardial Infarction. Arteriosclerosis, Thrombosis, and Vascular Biology. 1997, 17: 628-633.

26. Bounameaux H, Schneider PA, Siosman D, De Moerloose P, Reber g Value of the plasma measurement ofD-dimers in the diagnosis of venous thromboembolism. J Mal Vasco 1991; 16:133-6.

27. Kelly J, Rudd A, Lewis RR, Hunt BJ. Plasma D-dimers in the diagnosis of venous thromboembolism. Arch Intern Med. 2002; 162:747-56.

28. Kevorkian JP, Halimi C, Segrestaa JM, Drouet L, Soria C. Monitoring of patients with deep-vein thrombosis during and after anticoagulation with D-dimer. Lancet. 1998; 351:571-2.

29. Kuruvilla J, Wells PS, Morrow B, MacKinnon K, Keeney M, Kovacs MJ. Prospective assessment of the natural history of positive D-dimer results in persons with acute venous thromboembolism (DVT or PE). Thromb Haemost. 2003; 89:284-7.

30. Nunes JP. D-dimers in the diagnosis of pulmonary embolism. Lancet. 2002 Aug 10; 360(9331):489.

31. van Beek EJ, BE Schenk, Michel MO, van den Ende B, Brandjes DP, van der Heide YT, Bossuyt PM, Buller H R. The role of plasma D-dimers concentration in the exclusion of pulmonary embolism. Br J Haematol. 1996; 92:725-32. Erratum in: Br J Haematol 1996; 95:218.

32. Perrier A, Roy PM, Aujesky D, Chagnon I, Howarth N, Gourdier AL, Leftheriotis G, Barghouth G, Comuz J, Hayoz D, Bounameaux H. Diagnosing pulmonary embolism in outpatients with clinical assessment, D-dimer measurement, venous ultrasound, and helical computed tomography: a multicenter management study. Am J Med. 2004; 116:291-9.

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34. Wells PS, Anderson DR, Rodger M, Forgie M, Kearon C, Dreyer J, Kovacs G, Mitchell M, Lewandowski B, Kovacs MJ. Evaluation of D-dimer in the diagnosis of suspected deep-ein thrombosis. N Engi J Med. 2003; 349:1227-35.

35. Wells PS, Anderson DR, Rodger M, Stielll, Dreyer JF, Bames D, Forgie M, Kovacs G, Ward J, Kovacs MJ. Excluded pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to the emergency department by using a simple clinical model and D-dimer. Ann Intern Med. 2001; 135:98-107.

1. The method of in vitro diagnosis of activation of blood coagulation associated with the risk of thromboembolic disease in a sample of blood taken from the patient, including:
(a) quantitative analysis of the products of destruction of fibrin contained in the test sample, comprising the determination of D-dimers present in the sample and the components of a basic level of D-dimers;
b) processing the sample by incubation with plasminogen activator with high affinity to fibrin (PA-sFb), in conditions that ensure the destruction of soluble fibrin contained in the sample, with the formation of products of destruction, without destruction of fibrinogen, and the determination of the number of D-dimers contained in the treated sample;
C) calculating the difference between the quantity of D-dimers, particular after activation of the activator PA-sFb at the stage b), and the content of D-dimers before the specified activation defined in stage a), with the resulting difference is an indicator of the degree of destruction soluble destructible fibrin.);
g) comparing the level of D-dimers measured in stage a), with the standard threshold value, ODA is defined for the specified products of destruction, and the comparison level. calculated on the stage), with the boundary value of a norm defined for.,
thus they conclude that the risk of thromboembolic disease exists if at least one of the levels: the level of D-dimers measured in stage (a), or current level., determined in step b), exceeds the corresponding specific normal value, and this risk is excluded, if the level of D-dimers defined in stage a), and level., determined at the stage), lower than the corresponding boundary values of the norm.

2. The method according to claim 1, used to determine thromboembolic venous disease.

3. The method according to claim 1 for use in the study of venous thrombus.

4. The method according to claim 1, used for the diagnosis of deep vein thrombosis.

5. The method according to claim 1, used for the diagnosis of lung embolism.

6. The method according to claim 1, characterized in that the blood sample is a plasma sample.

7. The method according to any one of claims 1 to 6, characterized in that the blood sample is collected from a patient undergoing treatment by the method of anticoagulation therapy.

8. The method according to claim 7, characterized in that the plasminogen activator PA-sFb represents t-PA.

9. The method according to any one of claims 1 to 6 or 8, characterized in that the destruction of fibrinogen block by adding an inhibitor of plasmin.

10. The method according to claim 9, characterized in that the plasmin inhibitor is Aprotinin, which is added after incubating the samples for 15 min at 37°C With plasminogen activator Pa-Fb.

11. The method according to any one of claims 1 to 6, 8 or 10, wherein prior to incubating the sample with plasminogen activator Pa-Fb in the sample add an anticoagulant, in particular, a composition containing citric acid and trinitrate sodium.

12. The method according to any one of claims 1 to 6, 8 or 10, characterized in that it additionally includes the determination of the content of products of destruction soluble destructible fibrin in the negative control sample and a positive control sample.

13. The method according to any one of claims 1 to 6 or 8, characterized in that the content of D-dimers determined using monoclonal antibodies to D-dimer.

14. The diagnostic method according to any one of claims 1 to 6, or 8, characterized in that it additionally includes the determination. that is present in the positive control sample, which represents a sample of normal plasma, with the specified method includes the following stages:
a) incubation of plasma samples with an activator of coagulation, which leads to the formation of soluble fibrin without the formation of a fibrin clot, the trigger is, for example, a small amount of thrombin, methods for the Noah to cause the formation of soluble fibrin;
b) incubating the treated sample with a coagulation inhibitor in an amount sufficient to block coagulation, while the inhibitor is, for example, heparin or hirudin;
C) realization of a contact received samples containing monomers soluble fibrin, plasminogen activator with high affinity for fibrin (PA-sFb), in particular t-PA, in conditions that ensure the destruction of soluble fibrin contained in the sample, with the formation of the products of its destruction, in particular, D-dimers, without destruction of fibrinogen.

15. The diagnostic method according to claims 1-6, 8 or 10, characterized in that the boundary value standards for levels of D-dimers is 500 ng/ml, and a limit value of the content standards. 300 ng/ml, with this method of diagnosis excludes the presence of thromboembolic disease, if measurements of these parameters in the analyzed sample is less than two corresponding boundary values.

16. The kit for the diagnosis of activation of blood coagulation associated with the risk of thromboembolic disease, the method according to any one of claims 1 to 11, containing:
monoclonal antibodies to D-dimers;
plasminogen activator with a high specificity for fibrin (PA-sFb);
the plasmin inhibitor; and
positive control sample of soluble fibrin and, in appropriate cases is, a negative control sample.



 

Same patents:

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: physics.

SUBSTANCE: analyser has a revolving cuvette with a sample in which there is a control ferromagnetic ball, a magnet which can interact with said ball, a coagulation sensor which transmits signals from the ball and having a Hall sensor and a magnet, a signal processing device in form of a power supply unit, a Hall sensor, a microprocessor and a display device included in a common measuring circuit. The analyser is multi-channelled by fitting at least one additional revolving cuvette to form several channels. All cuvettes lie in a temperature-controlled unit included in the common measuring circuit. The longitudinal axis of each cuvette is inclined at an acute angle to the vertical. In the coagulation sensor, the magnet lies opposite the Hall sensor on the opposite side of the cuvette. The magnet is in form of a flat cylinder mounted with possibility of displacement along the cuvette. The analyser is also fitted with a unit for controlling rotation of the cuvettes and, included in the common measuring a circuit, a measurement parameter adjustment unit having rewritable read-only memory, and a timer configured to automatically switch on when a reactant is fed into the cuvette.

EFFECT: use of the invention increases reliability and broadens functional capabilities of the analyser owing to use of a multichannel measuring circuit, and simplifies the measuring procedure by automating the process.

4 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: blood plasma is examined in 4 minutes after the beginning of spontaneous red blood cell aggregation for free red blood cell count and cell count in aggregates. A percentage of non-aggregated red blood cells (PNA RBC) by formula PNA RBC=FRBSC×100/(TRBCA+FRBSC) wherein FRBSC is the free red blood cell count, TRBCA are total red blood cells in aggregates. If the PNA RBC is 56 to 30%, I degree of severity is stated, 30% to 4% - II degree of severity, less than 4% - III degree of severity.

EFFECT: use of the invention enables objectifying and increasing precision of evaluation of red blood cell aggregation, evaluating an intensity of patient's microcirculation disorders in a relatively short time, and thereby ensuring well-timed adequate complex of therapeutic measures or corrected therapy.

3 ex

FIELD: medicine.

SUBSTANCE: quantity of fibrin-monomers, dissolved in 0.5 N sodium hydroxide, is determined spectrophotometrically with application of ethanol test. Claimed method of quantitative determination of fibrin-monomers in blood makes it possible to reveal pathological process in organism with 95% reliability.

EFFECT: increase of determination accuracy.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: thrombosis monitor comprises: a thrombosis chamber, at least in a part of which there is a thrombogenic material; an inlet tube connected to the thrombosis chamber through which blood flows into the thrombosis chamber; a blood supply container connected to the inlet tube; a feed pump for the container; a pressure sensor for measuring pressure applied to the container. A method of thrombosis monitoring consists in the fact that after introduction of an anticoagulant, blood is supplied from the container to the thrombosis chamber by pressing on a fluid placed on a blood layer and having density less than that of the blood layer; it is combined with anticoagulation blood processing or blood coagulation stimulation, and measurement of pressure applied to the container; the thrombogenic material is placed at least in a part of the thrombosis chamber.

EFFECT: group of inventions provides overall assessment of blood coagulation and platelet-cell thrombosis in a medium equivalent to blood flow for evaluation of efficacy of an antithrombotic drug.

11 cl, 15 dwg, 23 ex

FIELD: medicine.

SUBSTANCE: for determination of functional state of hemostasis system record of blood coagulation process is performed, current amplitude of blood resistance in first time moment is registered and second resistance of blood at multiple time moment from initial time value is measured. Two resistances and time moments are used to determine maximum blood resistance and time constant, by which blood resistance at the beginning and end of coagulation process is calculated. Obtained parameters are used to determine indices of beginning and end of blood coagulation process. Obtained indices are compared with of the same name indices of blood coagulation process in norm and in case of differently directed deviations disturbances of functional state of hemostasis system are diagnosed.

EFFECT: invention makes it possible to increase measurement accuracy and reduce examination time.

1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: method is based on a method of observing turbidimetric fibrin clot formation with optical transmission of an incubation medium recorded by ultraviolet radiation band 230 to 320 nm by means of UV-spectrophotometre as a fibrin-polymer detector.

EFFECT: invention enables higher accuracy and sensitivity of the method.

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: 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: 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: 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

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