EA037879B1 - Screening test for determining contact coagulation pathway - Google Patents

Abstract

The invention relates to laboratory diagnostics, particularly to methods of determining a contact coagulation pathway for human blood plasma. A screening test for determining a contact coagulation pathway includes mixing citrated blood plasma with calcium chloride and then photometrically registering coagulation, wherein 96-well flat-bottom plates are used as an activator for the contact coagulation pathway, a reaction is started by adding 25 l of a 0.25 M calcium chloride solution to 75 l of citrated plasma that is diluted 1:2 with a tris imidazole buffer, and, after incubation at 37°C for 30 min, a photometric determination of plasma coagulation is conducted according to a change in the turbidity of samples at a wavelength of 450 nm with measurement intervals of 0, 10, 15, 20, 25 and 30 min. The invention provides for increasing the sensitivity of the test when diagnosing hypercoagulation, increasing the productivity of hemostasis research, and quickly adapting a method for routine research in clinical-diagnostic laboratory settings.

Description

The invention relates to laboratory diagnostics, in particular to methods for determining the contact pathway of coagulation (CPC) of human blood plasma, based on recalcification of citrated blood plasma, and can be used in clinical laboratories of medical institutions for screening studies of the activity of the contact pathway of hemostasis activation.

Currently, among the diseases leading to death, according to the World Health Organization, cardiovascular diseases are in the lead. With all the variety of nosological forms in this group, thrombosis is most often the direct cause of death of patients. Thrombosis is the main pathogenetic mechanism of heart attacks, strokes and pulmonary embolism.

In recent years, many different programs have been intensively developed and implemented aimed at improving the treatment process for cardiovascular diseases: high-tech surgical aid (stenting, shunting), the development of early and active rehabilitation measures, the creation of new pharmaceuticals. In the short term, the task is to develop a preventive direction - screening of risk groups in order to identify patients who require early initiation of therapy.

Despite many years of research, many aspects of the work of the hemostasis system still remain completely unexplored. It is known that more than 300 reactions are involved in the operation of the system. All the factors of plasma hemostasis have already been isolated and characterized, reagents have been created that make it possible to measure the concentration / activity of these factors, and many scientific works have been published on how certain factors change their activity in various pathologies. However, this entire gigantic array of accumulated data is very difficult for a practitioner to use.

One of the main reasons that determine the complexity of the interpretation of the results of laboratory coagulation tests is the lack of a clear relationship between the data of most tests and the clinical symptoms recorded by the doctor (with the exception of such easy-to-interpret complications / diseases as massive blood loss or hemophilia types A and B).

Most cases of incorrect interpretation of the results of laboratory studies of the hemostasis system are due to a lack of understanding of the role of the observed changes in the pathological processes of the body. And the point is often not that the doctor is not sufficiently informed about the principles of the blood coagulation system, but that the data obtained as a result of the study cannot be interpreted as certain pathological changes due to the purely technical features of the test. Classic examples are the APTT (partially activated thrombin time) and prothrombin time tests, which are designed to diagnose hypocoagulant states and bleeding tendencies. To accomplish these tasks, as well as to shorten the test time and better reproducibility during the study, an activator is added to the plasma sample at a concentration that is significantly higher than physiological. - This approach does not interfere with the diagnosis of hemorrhagic conditions, but makes these tests almost completely insensitive to hypercoagulant conditions, because against the background of such a strong activation of the plasma sample, subtle procoagulant changes are leveled (Serebriyskiy II Global and local tests of the hemostasis system in the diagnosis of hypercoagulable syndrome // Handbook of the head of the CDL. - 2012, - No. 12; pp. 27-34).

A somewhat special position is occupied by such a test as measuring the level of D-dimer. His interpretation is often not entirely unambiguous.

The reason for this is the polyetiology of the origin of this marker. D-dimer is a product of the simultaneous operation of three systems: procoagulant and anticoagulant in the formation of a fibrin clot - on the one hand, and fibrinolytic in the destruction of this clot - on the other. An increased level of D-dimer testifies after the fact that the deposition of fibrin filaments has already occurred and at the moment the process of their fibrinolysis is underway, but this test cannot say anything about an increased predisposition to thrombus formation at the time of the analysis.

The tests of the hemostasis system that are currently used in the laboratory can be divided into two types. The first one is local tests, the results of which make it possible to characterize the state of individual factors or links of a cascade reaction. These include routine tests such as APTT, prothrombin time (PT), prothrombin index (PTI), international normalized ratio (MHO), fibrinogen, D-dimer, antithrombin III, protein C, factor VIII, concentration and activity of some others. factors. The second is global coagulation tests, the results of which allow assessing the hemostasis system as a whole - thromboelastography / metry, thrombin generation test and thrombodynamics.

Local tests record changes in the activity / concentration of individual coagulation factors, but at the same time they cannot characterize how these local changes affected (or did not) affect the general ability of the patient's plasma to form a clot. Global tests can give the doctor an integral picture of the cumulative changes that have occurred in the patient's blood coagulation system, but they are not able to characterize the individual factors of the coagulation cascade.

The thrombin generation test is one of the global tests developed in 2001 (Hemker HC, Giesen P., Al Dieri R., Regnault V., de Smedt E., Wagenvoord R., Lecompte T., Begum S. Calibrated au- 1 037879 tomated thrombin generation measurement in clotting plasma // Pathophysiol. Haemost. Thromb. - 2003, V. 33; P. 4-15). The principle of the method is to use a fluorogenic substrate specific to thrombin. After preliminary incubation of platelet-rich blood plasma, a buffer containing ionized calcium and a fluorogenic substrate is added to it. The resulting thrombin breaks down the substrate, resulting in the release of a fluorophore molecule, the emission of which is automatically recorded by a fluorometer at regular intervals. The luminescence intensity is proportional to the concentration of the formed thrombin. Based on the measurements with the help of software, a thrombin generation curve is built. The study evaluates the delay in thrombin formation (lag period), the maximum rate of thrombin formation (peak), the time to reach the maximum rate (peak time), the amount of thrombin formed (area under the curve, endogenous thrombin potential) and some other parameters.

The disadvantage of this test is the use of an excessive amount of tissue factor as an activator of hemostasis, as well as expensive scientific equipment and reagents (fluorimeter and fluorogenic substrate). This test also does not reflect the concentration and activity of the natural substrate of thrombin - fibrinogen. Given these shortcomings, the thrombin generation test has not yet found widespread use in routine practice.

The final hypercoagulable state of hemostasis is a consequence of processes simultaneously occurring in two subsystems: procoagulant changes of any etiology in the work of the coagulation system and unsatisfactory performance of anticoagulant systems (antithrombin III, protein C, TFPI) in an attempt to neutralize the former. Due to the need to simultaneously assess the amount of the contribution of all participants in this process, local tests are poorly applicable to solving this problem. In contrast, global coagulation tests, for the same reason, cannot cope with this issue. That is why the combined use of global and local tests seems to be optimal. Global tests allow you to fix the phenomenon of hypercoagulation itself, and local tests to understand some of the components of its etiology.

Each of the methods for the study of hemostasis has its own pronounced specific features due to various reasons, including the technical features of the implementation of a particular test, the purity and specific properties of the reagents and, of course, not less important, the blood coagulation model, on the basis of which this or another test. This list is far from complete, but it gives an idea of a significant number of reasons that determine the unique sensitivity and specificity of each of the tests in the diagnosis of a particular disorder in the work of the blood coagulation system.

Each of the tests, both global and local, have their own advantages and disadvantages, the knowledge of which allows the doctor to correctly interpret data about the patient, more accurately establish a diagnosis and choose the most adequate treatment.

In donor plasma in vitro after recalcification, even in the absence of activators, the clotting process begins spontaneously in 10-20 minutes in separate centers. Further, the spontaneous clots increase in size and gradually fill the entire volume of the plasma. It has also been shown that the number of spontaneous centers decreases with a decrease in the number of platelets in plasma. After ultracentrifugation of the plasma to remove all platelets and large phospholipid vesicles, spontaneous centers are practically not formed. A contact phase inhibitor (an inhibitor obtained from corn grains) significantly reduces the number of spontaneous clots, but does not completely reduce them (Karotina N.G., Ovanesov M.V., Pliushch O.P., Kopylov K.G., Lopatina E. G., Saenko E.L., Butylin A.A., Ataullakhanov F.I. Research of spontaneous clots in normal plasma and plasma of patients with hemophilia // Hematol and transfusiol. - 2002; - vol. 47; p. 26-30) ...

Known laboratory methods for the study of blood coagulation in conditions of low-contact activation, based on the principle of recalcification of citrated blood [Investigation of the hemostasis system in the clinic. Ed. Barkagana 3.S, Barnaul, 1975, p. 21, 72-73; Ivanov E.P. Diagnostics of hemostasis disorders. Minsk, 1983, p. 120-124; Laboratory methods for studying the hemostasis system. Ed. Baluda V.P. et al. Tomsk, 1980, p. 55-56]. These methods are considered one of the main ones in hemostasiology. However, the accumulated experience and expanding knowledge in the field of blood coagulation mechanisms have shown that these methods in their sensitivity ceased to meet the ever-increasing requirements for the detection of hypercoagulation [Rohrer M.J. et al. Annls of Surgery. - 1988. - V. 208, No. 5. - P. 554557].

The closest technical solution is the thrombodynamics test. Thrombodynamics as a method for studying plasma hemostasis was proposed in 1994 by a group of researchers led by F.I. Ataullakhanov. (Ataullakhanov F.I., Guria G.T., Safroshkina A.Yu. Spatial aspects of the dynamics of blood coagulation. II. Phenomenological model // Biophysics. - 1994; - vol. 39, No. 1; p. 97-106). The method is based on a model of clot propagation, starting from damage to the vascular wall deep into the vessel. The imitation of the damaged vascular wall is achieved by applying a thin (30-50 nm) layer of tissue factor to the surface of the activator insert, which triggers the process of clot formation in the plasma. In the course of the study, the parameters of the spatial growth of the clot (lag-period, Tlag), the initial clot velocity (Vo), the stationary clot velocity (Vst), the clot size (CS), and the clot optical density (CD) are estimated. Thus, the doctor is able to characterize the processes of activation of coagulation with the participation of tissue factor. Also, this method allows you to register the phenomenon of spontaneous clot formation.

The disadvantage of the thrombodynamics method is its low productivity (2 analyzes within 45 min), the need for additional preparation of the sample (plasma) by high-speed centrifugation, the need for a special device for recording thrombodynamics (or several to increase the test performance), a set of reagents and consumables, and, accordingly, a high cost. one analysis for routine research. Also, the disadvantage of the thrombodynamics method is the use of an inhibitor of the contact pathway of coagulation activation, an inhibitor of trypsin from maize, which reduces the information content of the test and assesses only the activation of coagulation through tissue factor.

An object of the present invention is to develop a test for evaluating the contact pathway of plasma hemostasis activation. This will significantly increase the performance of the test, reduce its cost due to the use of an enzyme-linked immunosorbent assay (ELISA) photometer available for clinical diagnostic laboratories, widely available 96-well flat-bottom plates as micro cuvettes and the only reagent for starting the contact pathway of plasma hemostasis activation, calcium chloride solution ...

The technical result of the proposed method is to increase the sensitivity of the test in the diagnosis of hypercoagulation by starting the contact pathway for activating plasma coagulation, a 48-fold increase in the performance of the study of hemostasis in comparison with the prototype, as well as a quick adaptation of the method for routine research in the conditions of clinical diagnostic laboratories.

This result is achieved by the fact that recalcification of citrated blood plasma is carried out by adding a solution of calcium chloride to the wells of 96-well flat-bottomed plates for enzyme-linked immunosorbent assay as cuvettes, samples are incubated for 30 min at 37 ° C, and the determination of plasma coagulation is carried out photometrically by the change in turbidity samples at a wavelength of 450 nm with measurement intervals of 0, 10, 15, 20, 25 and 30 min. Plasma coagulation for 10 minutes is characterized as hypercoagulation, 15 minutes - increased coagulation, 20 minutes - as normal blood plasma coagulation and 25 or more minutes as hypocoagulation.

The method is carried out as follows: a standard blood sampling is carried out into a solution of 3.8% sodium citrate in a ratio of 9: 1, platelet-depleted plasma is prepared by centrifugation at 3000 rpm for 10 minutes. Then, to start the contact pathway of plasma hemostasis activation, the optimal concentration of calcium chloride solution is added to citrate plasma diluted 1: 2 with tris-imidase buffer, pH 7.4. Thoroughly mix the samples prepared in flat-bottom 96-well plates on a shaker and measure the absorbance in the samples at 450 nm on a photometer for enzyme immunoassay (1 measurement - 0 min), the samples are incubated for 30 min at 37 ° C. Measurement of the absorption of samples to control coagulation is carried out after 10 minutes (2nd measurement) and then every 5 minutes (the last measurement at the 30th minute of incubation).

Determination of the optimal concentration of calcium chloride to trigger the contact coagulation pathway

For this purpose, 50 μl of 0.25 M calcium chloride solution was progressively titrated in 96-well flat-bottomed plates, starting from the second well to the seventh well. After that, 25 μl of tris-imidazole buffer, pH 7.4, and pooled citrate plasma were added from relatively healthy donors. They were thoroughly mixed and the absorbance in the samples was measured at 450 nm on a photometer for ELISA analysis (0 min), put on a 30-minute incubation at 37 ° C. The absorption of samples to control coagulation was measured every 10 min (10, 20, and 30 min). The data obtained are presented in table. one.

As can be seen from the data presented in table. 1, the maximum coagulation of citrated plasma is observed when 50 μl of 0.125 M CaCl ₂ is added to 50% of the pooled citrated blood plasma of healthy donors. Thereafter, 25% plasma is recalcified with 0.25 M calcium chloride solution in 96-well flat-bottomed ELISA plates. Then incubation is carried out at 37 ° C for 30 minutes. Plasma coagulation is determined photometrically by the change in the turbidity of the samples at a wavelength of 450 nm with measurement intervals of 0, 10, 15, 20, 25 and 30 minutes.

To confirm the selective activation of the contact pathway of plasma hemostasis, soybean trypsin inhibitor (SIT) was used as an inhibitor of the contact pathway of coagulation. The optimal concentration of SIT is preliminarily determined.

Determination of the optimal concentration of soybean trypsin inhibitor (SIT) to inhibit the contact pathway of hemostasis activation

In a 96-well flat-bottom plate, 25 μl of soybean trypsin inhibitor (1 mg / ml) is progressively diluted, then 25 μl of buffer and 25 μl of pooled citrated blood plasma from healthy donors are added to the wells. The activation of the coagulation contact pathway is initiated by the addition of 25 μL

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0.25 M calcium chloride, mix thoroughly, measure the absorbance of samples at 450 nm on a photometer for enzyme-linked immunosorbent assay. Incubate for 20 min at 37 ° C, measure the change in turbidity of the samples every 10 min to monitor plasma coagulation. Plasma control (without SIT) and plasma blank control (without calcium chloride and SIT) are used as controls. The results are presented in table. 2.

As can be seen from the data presented in table. 2, SIT dose-dependently inhibits the contact pathway of plasma hemostasis activation. At a concentration of 0.25 mg / ml, SIT completely suppresses the contact pathway of plasma hemostasis activation.

Determination of activations of plasma hemostasis through tissue factor and contact pathway in the presence of soybean trypsin inhibitor (SIT) and without SIT

To study the effect of SIT on contact coagulation of individual citrate plasmas, comparative studies were carried out:

1) Determined the coagulation by the proposed method (STOKPK) in 14 samples of citrated plasma, as described above;

2) The STOKPK test was carried out in the same samples in the presence of 0.25 mg / ml SIT;

3) To assess the effect of SIT on the activation of coagulation using tissue factor, the APTT test was preliminarily adapted for ELISA plates with the same amount of plasma and calcium chloride. This APTT test is accepted by us as a modified APTT test;

4) APTT test (m) in the presence of 0.25 mg / ml SIT. The results are presented in table. 3.

As can be seen from the data presented in table. 3, the screening test for determining the contact pathway of coagulation (STOKPK) turned out to be more sensitive to both hyper- and hypocoagulation compared to the modified APTT test (APTT (m). The essence of the modification lies in the limitation of the activator in the APTT test at the same concentrations calcium chloride and citrated plasma, which is in the STOKPK test.As can be seen from the data of the APTT (m) test, when the contact coagulation pathway was inhibited by the soybean trypsin inhibitor (SIT), hypercoagulation in 5 samples was due to the high activity of the contact coagulation pathway. how SIT completely suppressed the contact pathway of coagulation.

Thus, experiments with the use of an inhibitor of the contact pathway of plasma coagulation suggest that under these conditions in the proposed test there is a specific activation of the contact phase of coagulation through factor XII.

Example 1.

Study of plasma hemostasis using the proposed test (screening test for determining the contact pathway of coagulation (STOKPK), prototype, thrombodynamics test, and routine methods for assessing hemostasis (APTT, prothrombin time, MHO and fibrinogen). We preliminarily took samples of citrated plasma with hypercoagulation (10 and 15 min coagulation in the proposed test STOKPK. The same samples were studied in the thrombodynamics test and in routine tests for assessing hemostasis (APTT, prothrombin time, MHO and fibrinogen). The results are presented in Table 4. As can be seen from the data presented in Table 4, blood plasma (n = 14) with hypercoagulation in the STOKPK test in 93% of cases coincided with the hypercoagulation detected in the thrombodynamics test, and only in one case out of 14 thrombodynamic parameters were within normal values Hypercoagulation in thrombodynamics test in 93% of cases due to the high rate of fibrin clot generation on the surface of the activator plate, which indicates a high rate of thrombin generation. Only 57% of the samples showed the formation of spontaneous clots, and in 42% of cases of hypercoagulation in the thrombodynamics test were caused by a large size of the fibrin clot (CS> 1200 μm).

Interesting data were obtained in the study of hemostasis by routine methods. So in 3 cases there is a decrease in the APTT index, which indicates hypercoagulation (21%), in 4 cases, an increase in PT (hypocoagulation, 29%). The MHO values in all samples were within the normal range, and only in 3 cases an increased level of fibrinogen was determined.

Thus, the data of STOKPK and thrombodynamics in 93% unidirectionally reveal hypercoagulation, while the data of routine methods of studying hemostasis in 29% determine hypocoagulation and only in 21% - hypercoagulation.

Example 2.

Study of plasma hemostasis using the proposed test STOKPK, prototype, thrombodynamics test and routine methods for assessing hemostasis (APTT, prothrombin time, MHO and fibrinogen). Were pre-selected samples of citrated plasma with hypocoagulation (25 and 30 min coagulation in the proposed test STOKPK). The same samples were investigated in the thrombodynamics test and in the routine tests for assessing hemostasis (APTT, prothrombin time, MHO and fibrinogen). The results are presented in table. 4. As can be seen from the data presented in table. 5, the data of all tests carried out coincided in 100%. Thus, the sensitivity of the STOKPK tests, thrombodynamics and routine methods of hemostasis research turned out to be the same for assessing the state of blood plasma hypocoagulation.

Example 3.

Comparative studies of the activity of the contact pathway of coagulation and coagulogram indices (APTT, PT. MHO, FG). Studies of 96 samples were carried out with the proposed STOKPK test and coagulogram indices. The results are presented in table. 6. As can be seen from the data presented in table. 6, the proposed test reveals hypercoagulation in 62.5% of cases, normoagulation in 25%, and hypocoagulation in 12.5%. At the same time, tests of routine hemostasis (APTT, PT, and MHO) did not reveal hypercoagulability in the studied citrated plasma samples. Routine coagulation methods in total in 51% of cases stated normocoagulation and in 49% - hypocoagulation. In every fifth sample, hypercoagulation is detected in the STOKPK test, while according to the coagulogram data, signs of hypocoagulation are recorded. The discovered fact may be directly related to the problem of restenosis after arterial stenting, when blood clots are observed in patients against the background of anticoagulant therapy.

Thus, the STOKPK test is a new test for assessing the contact pathway of hemostasis activation. Comparative studies of the proposed test with thrombodynamics for the diagnosis of hypercoagulation showed a high degree of agreement (93%) and 100% - agreement in the diagnosis of hypocoagulation. The new data obtained from comparative studies of the proposed STOKPK test and routine hemostasis tests (APTT, PF, and MHO) may be useful for the prevention of thrombosis during anticoagulant therapy during surgery.

The proposed method is simple, reagents and consumables are available for diagnostic laboratories. Plasma coagulation registration requires a 96-well flat-bottom plate thermostat and an ELISA photometer. The use of a simple and informative test for determining the activation of the contact pathway of coagulation allows screening and identification of people prone to thrombosis both on the background of anticoagulant therapy and in practically healthy people, which will allow starting early prevention of thrombosis, strokes and heart attacks at the stage before cardiovascular catastrophes ...

Table 1. Influence of the concentration of 0.25 M calcium chloride solution on the coagulation of 25% pooled citrate plasma of donors

0.25 M CaSH, μl in 100 μl 25% citrated plasma fifty 0.269 25 0.239 12.5 0.213 6.25 0.244 3.125 0.214 1.56 0.236 0.78 0.211 0 0.201 0 minutes A450 10 min 0.261 0.23 0.201 0.233 0.203 0.225 0.198 0.192 20 minutes 0.26 0.387 0.299 0.239 0.191 0.218 0.21 0.173 30 minutes 0.283 0.391 0.299 0.227 0.19 0.218 0.187 0.167

Table 2. Inhibition of the contact pathway of activation of plasma hemostasis by soybean trypsin inhibitor (SIT)

one 2 3 four Control pooled plasma K (bl) SIT (1 mg / ml) 25 12.5 6.25 3.125 0 0 0 0 A450 0 minutes 0.091 0.085 0.078 0.100 0.1 0.09 0.093 0.093 5 minutes 0.092 0.222 0.306 0.352 0.397 0.383 0.386 0.092 10 min 0.091 0.223 0.307 0.354 0.41 0.395 0.392 0.092 20 minutes 0.092 0.235 0.307 0.367 0.405 0.388 0.387 0.092

Table 3. Data for determining the activation of plasma hemostasis through tissue factor (APTT (m) and without SIT and contact pathway (STOKPK) in the presence of soybean trypsin inhibitor (SIT)

Plasma No. STOKPK, min STOKPK + SIT, min APTT (m), min APTT (m) + SIT, min one 10 Lack of coagulation in for 120 minutes or more 10 five 2 10 10 10 3 fifteen 10 10 four fifteen 10 10 five fifteen 10 10 6 10 five 10 7 twenty 10 10 eight 10 five 10 nine five five five 10 10 five ________10 AND 10 five 10 12 thirty 10 10 13 25 thirty 10 fourteen fifteen 10 10

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Table 4. Comparative data of hemostasis indicators obtained using the proposed test (STOKPK), thrombodynamics test and coagulogram

Sample no. STOKPK, min (Hypercoagulation) Thrombotic dynamics Coagulogram Flag, MIN V, (μm / min D, (arb. Units) Tsp, (min) CS, (μm) APTT, (s) pv, (s) many FG, (g / l) Norm 20 minutes 0.6- 1.5 20-29 15000- 32000 from 800- 1200 24,335.0 9.1- 12.1 0.8- 1,2 2-4 eight 10 N 37.4 N 17 N 23.4 11.0 1.02 3.0 nine fifteen N 41.6 N 25.3 N 25.5 11.1 1.03 4.2 eleven fifteen N 40.6 N 25.2 N 24.7 11.2 1.04 4.3 13 10 N 46.2 N from 1488 24.1 11.3 1.05 3.3 fifteen 10 N 51.3 N 17.7 1653 26.3 12.0 1.11 3.1 sixteen fifteen N 33.5 N from 1300 n / t n / t n / t n / t 17 fifteen N 33.5 N from 1282 28.8 12.5 1.16 3.3 eighteen fifteen N 40.7 N 21.1 1487 24.5 12.6 1.17 3.5 twenty 10 N 32.9 N from 1245 26.5 11.8 1.09 3.5 21 10 N 52.9 N 12.8 N 30.8 14.2 1.31 5.1 25 10 N 31.7 N from N 23.7 12.0 1.11 3.7 26 10 N 38.8 N 28.9 N 26.4 11.8 1.09 3.3 27 10 N 51.7 N 10.1 N 22.2 11.6 1.07 3.4 28 fifteen N N N N N 41.7 13.7 1.27 3.6

N is the norm;

n / a - not tested

Table 5. Comparative data of hemostasis indicators obtained using _____ the proposed test (STOKPK), thrombodynamics test and coagulogram

Sample no. STOKPK, min, (Hypocoagulation) T rhombo dynamics Coagulogram Flag, MIN V, (μm / min D, (conventional units) Tsp, (min) CS, (μm) Black body in, (s) PV, (s) INR FG, (g / l) Norm 20 minutes 0.61.5 20-29 15000- 32000 from 800- 1200 24,335.0 9.1- 12.1 0.81.2 2-4 one thirty N 18.6 N from N 29.9 11.3 1.05 4.4 3 thirty N 15.2 N from - 25.1 12.6 1.17 5.5 four thirty 2.9 N N from N 23.6 n / t 2.22 2.9 7 thirty 3.7 13.0 N from 550 n / t 44.7 4.26 n / t 12 thirty N N N from N 29.9 13.1 1.22 2.6 22 thirty 1.9 8.4 N from 506 n / t 12.3 1.14 n / t 23 25 N 16.1 N from N 29.5 13.1 1.21 4.4

N is the norm;

ots - absent;

n / a - not tested

Table 6. Generalized data of the STOKPK test and coagulogram

Test Hyper- | Normo- | Hypo- -coagulation STOKPK (n = 96 samples) 60 samples 24 samples 12 samples Coagulogram (n = 96 samples) Not found 49 samples 47 samples

CLAIM

Claims (1)

CLAIM Screening test for determining the contact coagulation pathway (STOKPK), including mixing citrated blood plasma with calcium chloride followed by photometric registration of coagulation, characterized in that 96-well flat-bottomed plates are used as an activator of the contact pathway of coagulation, the reaction is started by adding 25 μl of 0.25 M solution calcium chloride to 75 μl of citrated plasma diluted 1: 2 with tris-imidazole buffer, pH 7.4, then incubated for 30 min at 37 ° C, the determination of plasma coagulation is carried out photometrically by the change in the turbidity of the samples at a wavelength of 450 nm with measurement intervals 0, 10, 15, 20, 25 and 30 minutes, plasma coagulation within 10 minutes is assessed as hypercoagulation, 15 minutes as increased, 20 minutes normal blood plasma coagulation and 25 minutes or more as hypocoagulation.