RU2660706C1 - Screening-test of determining coagulation contact way (stokpk) - Google Patents

Abstract

FIELD: laboratory diagnostics.

SUBSTANCE: invention relates to laboratory diagnostics, in particular to methods for determining a contact pathway for coagulation of human blood plasma. Screening test for determining the coagulation contact path involves mixing citrated blood plasma with calcium chloride and subsequent photometric registration of coagulation. 96-well flat-bottomed plates are used as the activator of the coagulation contact path, start the reaction by adding 25 mcl of 0.25 M calcium chloride solution to 75 mcl of citrated plasma diluted 1:2 with tris-imidazole buffer, and after incubation at 37 °C for 30 minutes, a photometric determination of plasma coagulation is carried out by changing the turbidity of the samples at a wavelength of 450 nm with measurement intervals 0, 10, 15, 20, 25 and 30 min.

EFFECT: invention provides an increase in the sensitivity of the test in the diagnosis of hypercoagulation, increase the productivity of hemostasis research and rapid adaptation of the method for routine studies in clinical diagnostic laboratories.

1 cl, 6 tbl, 3 ex

Description

The invention relates to laboratory diagnostics, in particular to methods for determining the coagulation contact pathway (CCP) 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 leading. With all the variety of nosological forms in this group, the direct cause of death of patients is most often thrombosis. Thrombosis is the main pathogenetic mechanism of heart attacks, strokes and pulmonary thromboembolism.

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

Despite many years of research, many aspects of the hemostasis system are still not fully understood. It is known that more than 300 reactions are involved in the system. All factors of plasma hemostasis have already been identified and characterized, reagents have been created to measure the concentration / activity of these factors, and many scientific papers have been published on how various factors change their activity in various pathologies. However, this whole gigantic array of accumulated data is very difficult to use for a practical doctor.

One of the main reasons for the difficulty in interpreting the results of laboratory coagulological tests is the lack of a clear relationship between the data of most tests and the clinical symptoms registered by the doctor (with the exception of complications / diseases such as massive blood loss or hemophilia type A and B that are easy to interpret).

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 pathological changes due to the purely technical features of the test. A classic example is the APTT (partially activated thrombin time) and prothrombin time tests, which are designed to diagnose hypocoagulant conditions and bleeding tendencies. To perform 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 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 a plasma sample, subtle procoagulant changes are leveled [Serebriisky II "Global" and "local tests of the hemostasis system in the diagnosis of hypercoagulable syndrome // Directory of the head of the CDL. - 2012. - No. 12. - S. 27-34].

A somewhat special position is taken by such a test as measuring the level of D-dimer. His interpretation is often not entirely unambiguous. The reason for this is the polyetiological origin of this marker. D-dimer is the product of the simultaneous operation of three systems: procoagulant and anticoagulant during the formation of a fibrin clot, on the one hand, and fibrinolytic during 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 their fibrinolysis is currently underway, but this test cannot say anything about an increased predisposition to thrombosis at the time of analysis.

The hemostatic test system, which is currently used in the laboratory, can be divided into two types. The first is “local” tests, the results of which allow us to characterize the state of individual factors or units of the cascade reaction. These include routine tests such as APTT, prothrombin time (PV), prothrombin index (PTI), international normalized ratio (INR), fibrinogen, D-dimer, antithrombin III, protein C, factor VIII, concentration and activity of several others factors. The second is “global” coagulological tests, the results of which allow us to evaluate the hemostatic system as a whole - thromboelastography / metry, thrombin generation test and thrombodynamics.

“Local” tests record changes in the activity / concentration of individual coagulation factors, but 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 with the coagulation system of the patient’s blood, but are not able to characterize 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., Béguin S. Calibrated automated thrombin generation measurement in clotting plasma // Pathophysiol. Haemost. Thromb. - 2003.-V.33.-P.4-15.] The principle of the method is the use of a thrombin-specific fluorogenic substrate. After preliminary incubation of the platelet-rich blood plasma, a buffer containing ionized calcium and fluorogenic substrate is introduced into it. The resulting thrombin cleaves the substrate, as a result, a fluorophore molecule is released, the radiation of which is automatically detected by the fluorimeter at regular intervals. The glow intensity is proportional to the concentration of thrombin formed. Based on measurements using software, a thrombin generation curve is built. In the course of the study, the following are estimated: the delay time of the formation of thrombin (lag period), the maximum rate of thrombin formation (peak), the time to reach the maximum speed (peak time), the amount of thrombin formed (the area under the curve, endogenous thrombin potential) and some other parameters.

The disadvantage of this test is the use of excess 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 still not found wide application 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 coagulation system and the unsatisfactory operation of anticoagulant systems (antithrombin III, protein C, TFPI) in an attempt to level the former. Due to the need to simultaneously assess the contribution of all participants in this process, “local” tests are poorly applicable to solve this problem. In contrast, “global” coagulological tests cannot cope with this issue for the same reason. That is why the combined use of "global" and local "tests seems optimal. “Global” tests allow us to fix the phenomenon of hypercoagulation, and “local” - to understand some of the components of its etiology.

Each of the methods for studying hemostasis has its own specific features for various reasons, including the technical features of the implementation of a particular test, the purity and specific properties of the reagents, and, of course, no less important, the model of blood coagulation, 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 test in the diagnosis of a violation in the functioning of the blood coagulation system.

Each of the tests, which belong to both the “global” class and the “local” class, has its own advantages and disadvantages, the knowledge of which allows the doctor to correctly interpret the patient data, more accurately establish the diagnosis and select the most appropriate treatment.

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

Known laboratory methods for the study of blood coagulation in conditions of low-contact activation, based on the principle of recalcification of citrate blood [Study of the hemostatic system in the clinic. Ed. Barkagan Z.S., Barnaul, 1975, p. 21, 72-73; Ivanov E.P. Diagnosis of hemostatic disorders. Minsk, 1983, p. 120-124; Laboratory methods for studying the hemostasis system. Ed. Baludy V.P. et al. Tomsk, 1980, p. 55-56]. These methods are considered one of the main in hemostasiology. However, the accumulated experience and the expansion of knowledge in the field of blood coagulation mechanisms have shown that these methods, by their sensitivity, have ceased to satisfy the ever-increasing requirements for the detection of hypercoagulation [Rohrer M.J. et al. Annls of Surgery.-1988.-V. 208, N 5.-P. 554-557].

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 Ataullakhanov F.I. [Ataullakhanov F.I., Guria G.T., Safroshkina A.Yu. Spatial aspects of the dynamics of blood coagulation. II. Phenomenological model // Biophysics. - 1994. - T. 39, No. 1. - S. 97-106]. The method is based on a model of the propagation of a clot starting from damage to the vascular wall deep into the vessel. Simulation of the damaged vascular wall is achieved by applying a thin (30-50 nm) layer of tissue factor on the surface of the activator insert, which starts the process of clot formation in the plasma. The study estimates the spatial growth parameters of the bunch (lag period, Tlag), the initial velocity of the bunch (Vo), the stationary velocity of the bunch (Vst), the size of the bunch (CS), and the optical density of the bunch (CD). Thus, the doctor gets the opportunity to characterize the processes of coagulation activation 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 for 45 min), the need for additional sample preparation (plasma) by high-speed centrifugation, the need for a special device for recording thrombin dynamics (or several for increasing the test performance), a set of reagents and consumables, and, accordingly High cost of one analysis for routine research. Another drawback of the thrombodynamic method is the use of a coagulation activation contact pathway inhibitor, a trypsin inhibitor from corn, which reduces the information content of the test and only evaluates coagulation activation through tissue factor.

An object of the present invention is to provide a test for evaluating the contact pathway of plasma hemostasis activation. This will greatly increase the productivity of the test, reduce its cost by using a photometer for immunoassay (ELISA) available for clinical diagnostic laboratories, widely available 96-well flat-bottomed plates as microcuvettes and the only reagent for triggering the contact pathway for plasma hemostasis, chloride solution calcium.

The technical result of the proposed method is to increase the sensitivity of the test in the diagnosis of hypercoagulation due to the launch of a contact pathway for activating plasma coagulation, a 48-fold increase in the productivity of hemostasis studies compared to the prototype, as well as the quick adaptation of the method for routine studies in clinical diagnostic laboratories.

This result is achieved by recalcification of citrated blood plasma by adding calcium chloride solution 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 plasma coagulation is determined photometrically by change turbidity of samples at a wavelength of 450 nm with measurement intervals of 0, 10, 15, 20, 25 and 30 minutes. Plasma coagulation for 10 min is characterized as hypercoagulation, 15 min - increased coagulation, 20 min - as normal coagulation of blood plasma and 25 or more minutes as hypocoagulation.

The method is as follows: conduct a standard blood sampling in a solution of 3.8% sodium citrate in a ratio of 9: 1, prepare platelet-depleted plasma by centrifugation at 3000 rpm for 10 minutes Then, to start the contact pathway for the activation of plasma hemostasis, the optimal concentration of calcium chloride solution is added to the citrate plasma diluted 1: 2 with Tris-imidase buffer, pH 7.4. The samples prepared in flat-bottomed 96-well plates are thoroughly mixed on a shaker and the absorbance in the samples is measured at 450 nm using an enzyme-linked immunosorbent assay (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 (2 measurement) and then every 5 minutes (last measurement at 30 minutes of incubation).

Determining the optimal concentration of calcium chloride to trigger the coagulation contact path. For this purpose, previously in 96-well flat-bottomed plates, 50 μl of a 0.25 M calcium chloride solution was progressively grown from the second well to the seventh well. After that, 25 μl Tris-imidazole buffer, pH 7.4 and pooled citrate plasma were added relative to healthy donors. Thoroughly mixed and absorbed in samples at 450 nm was measured on a photometer for ELISA analysis (0 min), set for 30-minute incubation at 37 ° C. Measurement of sample absorption to control coagulation was carried out every 10 min (10, 20 and 30 min). The data obtained are presented in table 1.

As can be seen from the data presented in table 1, the maximum coagulation of citrate plasma is observed with the addition of 50 μl of 0.125 M CaCl ₂ to 50% pulsed citrated blood plasma of healthy donors. After this, 25% plasma is recalcified with a 0.25 M calcium chloride solution in 96-well flat-bottomed ELISA plates. Then incubate at 37 ° C for 30 minutes. Determination of plasma coagulation is carried out photometrically by changing 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, a soybean trypsin inhibitor (SIT) was used as an inhibitor of the coagulation contact pathway. Preliminary determine the optimal concentration of SIT.

Determination of the optimal concentration of soybean trypsin inhibitor (SIT) for inhibiting the contact pathway of hemostasis activation. A soybean trypsin inhibitor (1 mg / ml) of 25 μl is progressively diluted in a 96-well flat-bottom plate, then 25 μl of buffer and 25 μl of pulsed citrated blood plasma from healthy donors are added to the wells. Activation of the coagulation contact pathway is started by adding 25 μl of 0.25 M calcium chloride, mix thoroughly, absorbance of samples is measured at 450 nm using an enzyme-linked immunosorbent assay. Incubated for 20 min at 37 ° C, measure the change in turbidity of the samples every 10 min to control plasma coagulation. The following controls are used: plasma control (without SIT) and plasma blank control (without calcium chloride and SIT). 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 activation of plasma hemostasis. At a concentration of 0.25 mg / ml, SIT completely inhibits 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 conducted:

1. Coagulation was determined by the proposed method (STOKPK) in 14 citrate plasma samples as described above;

2. 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. We took this APTT test 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 coagulation contact pathway (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 is to limit activator in the APTT test at the same concentrations of calcium chloride and citrate plasma as in the STOKPK test.As can be seen from the APTT test (m), inhibition of the coagulation contact pathway using a soybean trypsin inhibitor (SIT), hypercoagulation in 5 samples was due to ene high activity of the contact coagulation path. While, SIT completely suppressed pin coagulation pathway.

Thus, experiments using 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

A study of plasma hemostasis using the proposed test (screening test for determining the coagulation contact pathway (STOKPK), prototype, thrombodynamics test, and routine methods for evaluating hemostasis (APTT, prothrombin time, INR and fibrinogen). To evaluate the informativeness of STOKPK for the diagnosis of hypercoagulation studies. Pre-samples of citrate plasmas with hypercoagulation were taken (10 and 15 min coagulation in the proposed STOKPK test. The same samples were investigated in the thrombodynamics test and in routine tests to evaluate hemostasis (APTT, prothrombin time, INR and fibrinogen). The results are shown 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 in only one case out of 14, the thrombodynamics indicators were within the normal range. Hypercoagulation in the thrombodynamics test in 93% of cases is due to the high rate of generation of the fibrin clot on the plate-act surface Vator, which indicates a high rate of thrombin generation. Only 57% of the samples revealed the formation of spontaneous clots and in 42% of cases of hypercoagulation in the thrombodynamics test they were due to the 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 indicator, which indicates hypercoagulation (21%), in 4 cases, an increase in PV (hypocoagulation, 29%). INR indices were found to be within the normal range in all samples, and only in 3 cases was an elevated level of fibrinogen determined.

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

Example 2

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

Example 3

Comparative studies of coagulation contact pathway activity and coagulogram indices (APTT, PV. INR, FG). Investigations of 96 samples by the proposed STOKPK test and coagulogram indices were carried out. The results obtained are presented in table 6. As can be seen from the data presented in table 6, the proposed test in 62.5% of cases reveals hypercoagulation, 25% normal, and 12.5% hypocoagulation. While tests of routine hemostasis (APTT, PV, and INR) do not reveal hypercoagulation in the studied samples of citrate plasma. Routine coagulation methods in total in 51% of cases noted normocoagulation and in 49% - hypocoagulation. In every fifth sample, hypercoagulation is detected in the STOKPK test, while signs of hypocoagulation are recorded according to the coagulogram. The discovered fact may be directly related to the problem of restenosis after stenting of the arteries, when blood clots are observed in patients with anticoagulant therapy.

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

The proposed method is simple, reagents and consumables are available for diagnostic laboratories. To register plasma coagulation, a thermostat is required for 96-well flat-bottomed plates and a photometer for enzyme immunoassay. Using a simple and informative test for determining the activation of the coagulation contact pathway allows screening and identification of people prone to thrombosis both against the background of anti-coagulant therapy and practically healthy people, which will allow early prevention of thrombosis, strokes and heart attacks at the pre-cardiovascular stage disasters.

Table 1

The effect of the concentration of 0.25 M calcium chloride solution on the coagulation of 25% donated citrated plasma donors

0.25 M CaCl ₂ , μl in 100 μl of 25% citrate plasma fifty 25 12.5 6.25 3,125 1,56 0.78 0
BUT ₄₅₀ 0 min 0.269 0.239 0.213 0.244 0.214 0.236 0.211 0.201 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 plasma activation

hemostasis soybean trypsin inhibitor (SIT)

one 2 3 four Controlled plasma K (bl) SIT (1 mg / ml) 25 12.5 6.25 3,125 0 0 0 0
BUT ₄₅₀ 0 min 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

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

Plasma number STOKPK,
min STOCKPK +
CIT, min APTT (m),
min APTT (m) +
CIT, min one 10 No coagulation for 120 minutes or more 10 5 2 10 10 10 3 fifteen 10 10 four fifteen 10 10 5 fifteen 10 10 6 10 5 10 7 twenty 10 10 8 10 5 10 9 5 5 5 10 10 5 10 eleven 10 5 10 12 thirty 10 10 13 25 thirty 10 fourteen fifteen 10 10

Table 4

Comparative hemostasis indicators obtained using the proposed test (STOKPK), thrombodynamics test and coagulogram

No.
samples STOKPK,
min
(Hyperco-
agulation) Thrombodynamics Coagulogram T _lag
min
V
(μm /
min D
(conv.
units) T _sp
(min) CS,
(microns) APTT,
(from) PV
(from) INR FG
(g / l) Norm 20 minutes 0.6-1.5 20-29 15000-
32000 from 800-
1200 24.3-
35.0 9.1-12.1 0.8-1.2 2-4 8 10 N 37.4 N 17 N 23.4 11.0 1.02 3.0 9 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 16 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 / t - not tested

Table 5

Comparative hemostasis indicators obtained using the proposed test (STOKPK), thrombodynamics test and coagulogram

No.
samples STOKPK,
min
(Hypocoa-
walk) Thrombodynamics Coagulogram T _lag
min
V
(μm /
min D
(conv. unit) T _sp
(min) CS,
(microns) APTT,
(from) PV
(from) INR FG
(g / l) Norm 20 minutes 0.6-1.5 20-29 15000-
32000 from 800-
1200 24.3-
35.0 9.1-12.1 0.8-1.2 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 / t - not tested

Table 6

Generalized STOKPK test data and coagulograms

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

Claims (1)

Coagulation contact pathway screening test (STOCC), including mixing citrated blood plasma with calcium chloride followed by photometric coagulation, characterized in that 96-well flat-bottom plates are used as the coagulation contact pathway activator, initiating the reaction by adding 25 μl 0.25 M solution of calcium chloride to 75 μl of citrate plasma diluted with 1: 2 Tris-imidazole buffer, pH 7.4, then incubated for 30 min at 37 ° C, determination of plasma coagulation is carried out photometrically a change in the turbidity of the samples at a wavelength of 450 nm with measurement intervals of 0, 10, 15, 20, 25, and 30 min, plasma coagulation for 10 min is estimated as hypercoagulation, 15 min as increased, 20 min - normal coagulation of blood plasma and 25 min and more like hypocoagulation.