EA026282B1 - Method for determining the functional status of the hemostasis system - Google Patents

Abstract

The invention relates to medicine, in particular to clinical laboratory research and concerns the diagnosis of disorders of the coagulative blood system, including screening, making a diagnosis, monitoring, planning and evaluating the effectiveness of therapy. The problem addressed by the claimed invention consists in the timely prognosis and prevention of the occurrence of thromboses, hemorrhages and other pathologies of the hemostasis system; the discovery of the tendency of the progression of illnesses associated with a disorder in the functioning of the coagulative blood system and evaluation of the effectiveness of a therapy conducted on a patient. This problem is solved by providing a method for determining the functional status of the hemostasis system, which method comprises the following steps: a) preparing a sample of blood or plasma thereof; b) determining the spatial dynamics, in vitro, of at least one of the processes of coagulation or lysis of a clot in a sample being investigated, comprising mixing the sample with a reagent to inhibit contact activation of coagulation of the sample, adding a reagent for recalcification, providing local activation of coagulation in the sample under investigation by a physiological coagulation activator analogue surface in the unstirred thin layer of the sample, determining the intensity of the light scattering in the clot, the delay time for the formation of the clot, the rate of growth of the clot, the area under the curve representing the dependence of the light scattering on the distance, the rate of propagation of the thrombin curve, the thrombin peak, the dimensions of the clot at a set moment in time, the time of onset of fibrinolysis, the change in the front of the fibrinolysis in time, the change in the front of the thrombin formation in time, the change in the dimensions of the clot in time; c) comparing the results obtained in step b) with a standard and determining the trend and the degree of intensity of deviations from the standard in the patient.

Description

The invention relates to medicine, in particular to clinical laboratory research, and for the diagnosis of disorders of the blood coagulation system, including screening, diagnosis, monitoring, planning and evaluating the effectiveness of therapy.

State of the art

The prior art method for determining the state of the blood coagulation system in a patient, application for invention I8 2009/0130645, publ. 05/21/2012. The indicated method is based on determining the clot formation rate, clot density depending on time, maximum clot density and degree of fibrinolysis. These characteristics are determined in a known manner - viscoelastometry, and then compared with reference values.

The prior art method for determining the formation and lysis of a fibrin clot in a patient, disclosed in application I8 2008/268483 publ. 10/30/2008 This method allows predicting thrombosis and bleeding by determining the delay time (lagtime, CD, maximum amplitude (density of the test sample), time to reach maximum density, and time to drop the density of the test sample.

A known method for the diagnosis of pathologies of the hemostatic system, patent KI 2449281, class. Ο01Ν 33/48, publ. 04/27/2012, The specified method is based on the determination of coagulation time, thromboplastin time, thrombin time and a number of other characteristics by which a preliminary diagnosis or prediction of the outcome of diseases is carried out.

As the closest analogue of the proposed solution can be called a method for determining coagulological characteristics, disclosed in patent I8 6432657, publ. 08/13/2002, Where the degradation of fibrinogen with thrombin occurs in the test sample without clot formation and the dependence of light scattering intensity on time in the clot forming in the sample is measured.

The disadvantages of the mentioned methods include the fact that they are not close to the physiological process of clot formation, i.e., the coagulation process is activated in the entire sample volume, and the amount of activator itself exceeds the physiological need by several times, and thus cannot be sensitive to all pathologies and at the same time reflect all violations of the state of the hemostatic system.

Therefore, for the application of these methods it is necessary to carry out a large number of determinations, which requires additional time and makes these methods inconvenient and not accurate, leading to errors arising in the process of working with them.

Thus, there is a need for a diagnostic system that allows one to determine and evaluate both the initial state of the blood coagulation system and its ability to form a clot, monitor the deterioration of this condition with the progression of the disease, and evaluate the effectiveness of treatment during therapy, and which would overcome the above disadvantages.

SUMMARY OF THE INVENTION

The task to which the invention is directed is the timely prediction and prevention of the occurrence of thrombosis, bleeding and other pathologies of the hemostasis system; revealing the trend in the course of diseases associated with impaired functioning of the blood coagulation system, and evaluating the effectiveness of the patient's therapy.

The technical result obtained from the use of the proposed solution consists in the information content and complexity of assessing the state of hemostasis, i.e., the ability to evaluate the integral state of the coagulation system and its links in a single test, as well as maximally approximating the conditions ίη νίνο, which makes it easier to interpret processes.

The claimed technical solution consists in determining the optimal number of characteristics of the spatial dynamics of the processes of clot formation in the test sample, subsequent comparison of the obtained data with the established norm, and when these characteristics deviate from the norm, the decision is made on whether the patient has hypercoagulation or hypocoagulation, that is, a tendency to the main clinical manifestations pathology of hemostasis, thrombosis or bleeding, respectively. Based on the data obtained on the change in the state of the coagulation system, the doctor decides on the appointment and correction of patient therapy.

This problem is solved by creating a method for determining the functional state of the hemostatic system, which includes the following stages: a) preparation of a blood sample or its plasma, b) determination of the spatial dynamics ίη νίίτο; at least one of the processes of coagulation or lysis of a clot in the test sample, including mixing the sample with a reagent to inhibit contact activation of coagulation of the sample, adding a reagent for re-calcification, providing local activation of coagulation in the test sample with a surface with an analogue of a physiological coagulation activator immobilized on it in a non-stirred thin the layer of the specified sample, determining the intensity of light scattering in the bunch, the delay time of the formation of the bunch, SK GROWTH clot area under the curve of light scattering on the distance the wave propagation velocity of thrombin; thrombin peak, clot size at a given point in time, time

- 1,026,282 of the onset of fibrinolysis, changes in the fibrinolysis front in time, changes in the thrombosis front in time, changes in the size of the clot in time; c) comparing the results obtained in step b) with the norm, and determining the direction and severity of the deviations in the patient.

And also by the fact that at step b) the following processes are additionally recorded in the test sample: clot growth from an activator, spontaneous clot growth, fibrinolysis, or a combination thereof.

The authors found that it is necessary and sufficient to determine at least one of the following processes in the test sample:

1. The growth of a clot from the activator.

Main characteristics:

light scattering intensity in the bunch, characterizes the quality of the bunch;

clot formation delay time (( _{a &} ) characterizes the initial coagulation reactions;

clot growth rate (V beg, V st), initial, stationary, etc. - an integral indicator of the patient's plasma ability to form a clot.

Characteristics in the case of a thrombin wave: velocity of propagation of a thrombin wave; thrombin peak.

Additional characteristics:

the area under the curve of light scattering versus distance (8 g) characterizes both size and quality.

The size of the bunch at a given point in time (1ι cg), an integral indicator.

2. The growth of spontaneous clots.

Main characteristics:

the formation time of the first spontaneous clot (ΐ spont);

time of half-maximum overgrowing with clots of the test sample;

light scattering intensity far from the activator at a given point in time.

Additional characteristics:

time of complete overgrowth of the test sample; rate of overgrowth with spontaneous clots.

3. Fibrinolysis.

Key Features: fibrinolysis delay time; average clot lysis rate.

Additional characteristics: change in clot size over time.

Registration of these processes in the test sample and determination of these characteristics is carried out by means of an effective and time-saving (no more than 30-45 min) diagnostic system for measuring the formation or lysis of a clot in the sample, which allows reproducible results.

The spatial dynamics of at least one of the processes in the test sample is determined by means of a specially developed test by studying the dynamics of growth and lysis of a bunch in a spatially inhomogeneous medium, which was carried out as follows.

Blood is taken from the patient and a test sample is prepared. Next, the sample is mixed with a reagent to inhibit the contact activation of the coagulation of the sample, and a recalcification reagent is added.

A sample (in the form of whole blood or its products (plasma, etc.)) may be freshly prepared or it may be in a frozen state, for example, in the case of samples that do not contain cells. The sample may also contain mixtures of purified natural, synthetic or recombinant proteins and / or other preparations / reagents with hemostatic, fibrinolytic or antifibrinolytic activity. The sample can be obtained from healthy subjects or from subjects that may or may not suffer from a blood clotting disorder.

The sample prepared by this method is placed in a cuvette, and the test sample is brought into contact with an analogue of the physiological coagulation activator. Where as the mentioned activator can be selected tissue factor (TP). Further, by means of a device developed for this test (created by the applicant LLC GemaKor, Russia), clot growth in the space of the test sample is recorded as a function of time.

A feature of the test is the local activation of coagulation by a surface with an activator immobilized on it, and the registration of a growing clot in an immiscible thin layer of the test sample by light scattering by the dark field method. Coagulation occurs at a set temperature. The term immobilized implies both simple immobilization on a carrier by lyophilization, and immobilization by interaction with a carrier or by attachment to a carrier, such as covalent attachment to a carrier directly or via a linker molecule.

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As a result of the test, graphical and visual (photo) information is obtained, which is processed using the developed software. In particular, the duration of the test can be 45 minutes, preferably 30 minutes, and a sample is taken every 15 seconds.

When processing light scattering data, all the obtained photographs were assumed to be symmetrical with respect to the perpendicular to the activator. This allows us to go from two-dimensional intensity distributions to one-dimensional in segments stretched perpendicular to the activator along the direction of clot growth. For this, a narrow band was chosen in this direction along which for all time instants the intensity curves of the detected light scattering signal were calculated — the graphs of the dependence of the average light scattering intensity (obtained from the pixel intensities) on the distance from the activator. For each curve of the dependence of light scattering intensity on time, the bunch size was calculated as the coordinate at which the light scattering intensity was 50% of the maximum intensity. Based on the dependence of the clot size on time, the following characteristics of the clot growth in space were calculated: the delay time of clot formation (ΐι ₃₆ ), (the coagulation initiation time is the moment at which the clot size becomes nonzero); initial clot growth rate (the slope of the straight line approximating the least squares method is the plot of the curve of the size dependence of the clot near the activator); stationary bunch growth rate (the slope of the straight line approximating the plot of the dependence of the size of the bunch far from the activator).

The inventive method can be illustrated by the following figures of drawings, where in FIG. Figure 1 (a-c) shows the dependence of light scattering on the distance by which the bunch has grown on the surface of the activator.

in FIG. 2 shows the dependence of the clot size on time;

FIG. 3 illustrates clot growth at different times from the start of the experiment;

FIG. 4 illustrates examples of spontaneous thrombosis (clot formation away from the activator);

in FIG. 5 presents examples of clot lysis;

in FIG. Figure 6 shows the dependence of light scattering on the distance by which the clot grows on the boundary of the activator surface for the case of thrombus lysis;

in FIG. Figure 7 shows the dependences of the propagation of a thrombin wave in time and space for different concentrations of the active XI factor.

One of the main characteristics of the first process in the studied sample of Clot growth from the activator is the light scattering intensity in the clot, which is determined based on visual information, an example of which is shown in FIG. 3. It illustrates the growth of a fibrin clot at different times from the start of the test. According to the data obtained in the photographs of FIG. 3, the graphs illustrated in FIG. 1 and 2.

In FIG. Figure 1 shows the dependence of the light scattering intensity in a bunch on the size of the bunch (the distance by which it grew from the boundary of the activator surface) during the test, which is typical of the norm, hypercoagulation state and hypocoagulation state of the coagulation system. The data displayed in FIG. 1, they simultaneously show the size and quality of the bunch, its density, which characterizes the content of fibrinogen, which is another main characteristic of the first process in the test sample — the area under the curve of light scattering versus distance (8 cg).

The characteristic of clot growth — the delay time of clot formation (ΐι ₃₆ ) describes the initial coagulation reaction, which is determined by the graphs presented in FIG. 2. The delay time for the formation of a bunch (ΐι _{3 &} ) is calculated as the time before the formation of a bunch at the boundary of the activator-insert. The delay time for clot formation (ΐι ₃₈ ) reflects the time required to start the growth of the visible fibrin clot, so if the delay time for clot growth (ΐι ₃₈ ) is less than the normal value, we can conclude that the ability of the plasma to form a clot is increased compared to the normal state . The lengthening of the delay time can be a consequence of the reduced ability of the plasma to form a clot compared to the normal state of the plasma, which can be associated with both anticoagulant therapy and other disruption of the blood coagulation system and cause the patient to bleed.

The reduced ability to form a fibrin clot and, accordingly, the risk of bleeding are also indicated by shortened clot growth rates.

Here, the integral indicator is determined - the size of the bunch at a given point in time (1ι cg), from which the main characteristics of the bunch growing from the activator are calculated: the cluster growth rate (V beginning, V article) - initial, stationary, etc., depending on specific tasks .

The initial clot growth rate — the clot growth rate near the activator is calculated as the slope of the straight section (Fig. 2). The delay time (ΐι ₃₈ ) of clot growth and the initial speed calculated over a certain period of time reflect the external activation pathway of coagulation and may be sensitive to drugs such as Ν0ν08ΕνΕΝ®. While stationary

- 3,026,282 speed reflects the internal pathway of coagulation activation, being sensitive, for example, to hemophilia. The stationary growth rate of a bunch is the growth rate of a bunch away from the activator is calculated as the slope of the straight section (Fig. 2).

Any other necessary clot growth rates are calculated in the selected time range.

For each curve, the clot size was calculated as the coordinate at which the light scattering intensity was 50% of the maximum intensity, which corresponds to the conversion of half of all fibrinogen to fibrin.

The second defined process in the test sample of the proposed method, spontaneous thrombosis characterizes a state of increased tendency to thrombosis (hypercoagulation). Normally, a clot grows only from the activator, however, with hypercoagulation, a phenomenon of activator-independent coagulation is observed - the appearance and growth of fibrin clots far from the activator in the volume of the test sample. This phenomenon will hereinafter be referred to as spontaneous thrombus formation, and such clots as spontaneous clots. The presence of spontaneous clots and their characteristics indicate that the patient’s blood sample contains a large number of active components that trigger coagulation. The risk of blood clots in a patient in such cases is especially high.

In FIG. Figure 4 presents examples of spontaneous thrombosis (clot formation away from the activator). Spontaneous clot formation indicates the presence of coagulation activating components in the test sample (increased content of active factors or, for example, phospholipid surfaces), the indicator does not depend on the action of the activator, and shows a strong tendency to thrombosis. This process reflects the characteristics of clots that form independently of the activator. To assess this process, there are specific characteristics: the time of formation of the first spontaneous clot (онт spont), the time of half-maximum overgrowing with clots of the test sample (percentage of overgrowth of the sample at some point in time), the time of complete overgrowth of the test sample, light scattering far from the activator at a given moment time, the rate of spontaneous clumping.

Spontaneous clots and their characteristics are special indicators of thrombogenicity in patients. It is they that make it possible to assess the presence of a pronounced tendency to pathological thrombosis and / or the initial stages (hypercoagulation phase) of the DIC syndrome in a patient.

The third process to be determined in the studied sample of the proposed method of fibrinolysis is the thrombus lysis process, estimated by the delay time of fibrinolysis, the average clot lysis rate obtained from the fibrinolysis front, the light scattering function and the distance from time χ (ΐ) and y (1). which are shown in FIG. 5 and 6.

With fibrinolysis, reduced clot growth rates can be observed, as well as using additional indicators to assess the severity of fibrinolysis in a patient and decide on the need for treatment correction. In FIG. Figure 5 presents photographs of an example of a thrombus lysis, when after some time the thrombus begins to lysate from top to bottom from the activator (with a certain patient therapy, disease or a specific test ίη νίΐτο for checking medications received by the patient).

The dependence shown in FIG. 6, the light scattering from the distance over which the clot has grown, for the case of clot lysis, allows one to determine indicators for assessing fibrinolysis, based on which graphs of the dependence of the fibrinolysis front on time, the thrombogenesis front on time, and the dependence of the light scattering and clot size on time are plotted. To assess lysis, there are characteristics - fibrinolysis delay time, average clot lysis rate (ΐ l).

A characteristic of clot growth is the propagation of a thrombin activity wave in space and time using the developed video microscopy method using a fluorogenic substrate. Coagulation is also triggered by the activator and spreads from it in the cell space. However, a thrombin-specific protein fluorogenic substrate was added to the test sample. In the process of coagulation in the test sample, the resulting thrombin promotes the production of fluorescent molecules, which can be used to judge the propagation of the thrombin activity wave. In FIG. Figure 7 shows the dependences of the propagation of a thrombin wave in time and space for different concentrations of the active XI factor. The thrombin concentration nM is indicated along the y axis, the distance in mm along the x axis, and the time in minutes along the ζ axis. FIG. 7a and 7b reflects a situation of deficiency of the XI coagulation factor of 0 and 0.2 U / ml. FIG. 7c displays the normal thrombin wave for a normal factor XI content of 1 U / ml. FIG. 7g reflects a thrombin wave for a high content of XI factor 5 U / ml. Using these figures, the propagation velocity of the thrombin wave and the peak of thrombin are calculated.

All three of the above main processes in the test sample of the proposed method are determined by setting one test described above.

The present invention is further illustrated by the following examples, but is not limited to.

To conduct control tests and determine the range of reference values of the process characteristics, 74 plasma samples of healthy volunteers were used. The age range of volunteers ranged from 20 to 62 years.

Based on an analysis of the processes in the test sample and a comparison of their characteristics with reference values, a conclusion is drawn on the direction and severity of the deviations in the coagulation system of the patient. Based on the definition of a hypercoagulable or hypocogulation state, the doctor makes further diagnostic and therapeutic decisions in relation to a particular patient

Example 1

A patient A with severe type A hemophilia was taken a blood sample. Platelet-free plasma (ρίρ) was prepared from blood taken from a patient. 120 μl of plasma ρίρ was mixed with a reagent to inhibit contact activation of the coagulation of the sample, after which they were incubated for 15 min in an incubator, and then, adding a reagent for recalcification, immediately placed the sample in a cuvette, brought the contents of the cuvette into contact with the activator and started the study on a diagnostic device, as stated earlier.

As a result of the test, the following data were obtained:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 1.1 0.3-1.5 The initial growth rate of the clot, microns / min 25 36-56 Stationary clot growth rate, μm / min 12 20-30 Clot size for 30 min, microns 530 740-1120 The density of the bunch, conventional units 13776 14000-30000

This test allows you to determine not only quantitative characteristics, but also illustrative material in the form of graphs and photographs. So in FIG. 1c shows graphs of clot growth profiles. The concentration of the curves in the range from 0 to 1000 μm indicates the hypocoagulation state of the patient's hemostasis, as well as this condition can be judged by photographs, which clearly show that the growth of the clot is practically absent.

Significantly reduced initial and stationary clot growth rates and a reduced clot size for 30 min of research indicate a patient's hypocoagulation state, he is prone to bleeding and needs some therapy.

Example 2

Patient B, early postoperative period after hip replacement. A blood sample of this patient was prepared and tested as described in Example 1.

As a result of the test, the following data were obtained:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 1.4 0.3-1.5 The initial growth rate of the clot, microns / min 62 36-56 Stationary clot growth rate, μm / min 46 20-30 Clot size for 30 min, microns 1501 740-1120 The density of the bunch, conventional units 14664 14000-30000 Half max thrombosis time sample min 24 Not discovered Complete thrombosis (overgrowing) time sample min 42 Not discovered The formation time of the first spontaneous clot, min nine Not discovered

So in FIG. 1b shows graphs of clot growth profiles. The curves are distributed over almost the entire length of the distance coordinate, which indicates the hypercoagulable state of the patient's hemostasis, and this condition can also be judged from the photographs in FIG. 4, which clearly shows the formation of spontaneous clots and accelerated clot growth, which confirms the state of hypercoagulation.

The patient has an increase in the initial clot growth rate to 62 μm / min and a stationary speed to 46 μm / min. The total overgrowing time was 42 minutes. The first spontaneous clot formed after 9 minutes. All these characteristics confirm the patient’s hypercoagulable state, that is, the patient has a very high risk of thrombosis, and, therefore, he needs anticoagulant therapy.

Example 3

Donor C, a conditionally healthy person. A blood sample of this patient was prepared and tested in the manner indicated in Example 1.

As a result of the test, the following data were obtained:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 0.7 0.3-1.5 The initial growth rate of the clot, microns / min 54 36-56 Stationary clot growth rate, μm / min 27 20-30 Clot size for 30 min, microns 1072 740-1120 The density of the bunch, conventional units 16155 14000-30000

According to the data obtained, we can conclude that all indicators are within the range of normal values, which confirms the lack of data for pathology.

Example 4

Patient Ό with hemophilia A, who is on substitution therapy with a recombinant coagulation factor-agaric USA preparation in a standard dosage. A blood sample of this patient was prepared and tested as described in Example 1.

Test data before drug administration:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 0.5 0.3-1.5 The initial growth rate of the clot, microns / min 46 36-56 Stationary clot growth rate, μm / min 17 20-30 Clot size for 30 min, microns 755 740-1120 The density of the bunch, conventional units 19456 14000-30000

Test data half an hour after drug administration:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 0.6 0.3-15 The initial growth rate of the clot, microns / min 52 36-56 Stationary clot growth rate, μm / min 27 20-30 Clot size for 30 min, microns 1029 740-1120 The density of the bunch, conventional units 19723 14000-30000

The results of the test clearly showed that before the start of substitution therapy, the patient was in a state of hypocoagulation with a reduced stationary rate of clot growth of 17 μm / min. These characteristics confirm the patient's bleeding tendency. After administration of the drug, the obtained test characteristics show that the condition of hemostasis in this patient was normalized, i.e., the stationary clot growth rate began to be 27 μm / min. We can conclude that the test clearly showed the effect of the drug administered to the patient and made it possible to confirm the correct choice of its dosage.

Therefore, this test allows you to quickly and clearly monitor replacement therapy in patients with blood clotting disorders.

Example 5

Patient E with portal vein thrombosis undergoing anticoagulant therapy. A blood sample of this patient was prepared and tested as described in Example 1.

Test data before anticoagulant administration:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 0.7 0.3-1.5 The initial growth rate of the clot, microns / min 65 36-56 Stationary clot growth rate, μm / min 37 20-30 Clot size for 30 min, microns 1374 740-1120 The density of the bunch, conventional units 17844 14000-30000

Test data 3 hours after administration of the anticoagulant:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 0.7 0.3-1.5 The initial growth rate of the clot, microns / min 56 36-56 Stationary clot growth rate, μm / min fifteen 20-30 Clot size for 30 min, microns 840 740-1120 The density of the bunch, conventional units 17398 14000-30000

The results of the test clearly showed that the patient was in a state of hypercoagulation with increased initial and stationary clot growth rates of 65 and 37 μm / min, respectively, and an increased clot size of 1374 μm. These characteristics confirm propensity

- 6,026,282 patients with pathological thrombosis. After the introduction of an anticoagulant drug, the obtained test characteristics show that the patient's hemostasis was brought into a state of mild hypocoagulation with a stationary clot growth rate of 15 μm / min, which is necessary to prevent the progression of thrombosis. We can conclude that the test clearly showed the effect of the drug administered to the patient and made it possible to determine the correct choice of dosage.

Therefore, this test allows you to quickly and clearly monitor the effectiveness of anticoagulant therapy in patients with blood clotting disorders.

Example 6

Patient P with newly occurred portal vein thrombosis receives anticoagulant therapy and at the same time fibrinolytic therapy. A blood sample of this patient was prepared and tested as described in Example 1.

As a result of the test, the following data were obtained:

Clot growth characteristics testimony patient norm Clot growth inhibition, min 0.8 0.3-1.5 The initial growth rate of the clot, microns / min 14 36-56 Stationary clot growth rate, μm / min 12 20-30 Clot size for 30 min, microns 405 740-1120 The density of the bunch, conventional units 20874 14000-30000 Fibrinolysis Delay Time, min 3Ι Clot lysis rate, μm / min 3,5

As a result of testing a patient’s blood sample, the appearance of a fibrinolysis front (dissolution front of the formed clot) was recorded and its characteristics, fibrinolysis delay time of 31 min and the clot lysis rate of 3.5 μm / min were calculated. Characteristics allow us to judge that in this patient, a blood clot cleavage drug works. The size of the clot and the rate of clot formation are calculated from the distance dependence of the thrombus formation and light scattering front of a given sample. Reduced clot formation characteristics reflect the effects of anticoagulant therapy.

Example 7

Patient O, early postoperative period, pneumonia. I have not received anticoagulation therapy due to thrombocytopenia. On the 2nd day after surgery, the patient developed portal vein thrombosis.

Test data before thrombosis:

Clot growth characteristics testimony patient norm Clot growth inhibition, min one 0.3-1.5 The initial growth rate of the clot, microns / min 59 36-56 Stationary clot growth rate, μm / min 34 20-30 Clot size for 30 min, microns 1227 740-1120 The density of the bunch, conventional units 14602 14000-30000

The blood sample of this patient was tested on the first day after surgery, and the test results showed an increase in the initial and stationary growth rates of the clot (59 and 34 μm / min, respectively), which already confirmed the tendency of this patient to pathological thrombosis. However, the patient was not prescribed the necessary therapy, and thrombosis that occurred on the 2nd day after surgery was a consequence of this pathological condition.

The test described here has the ability to predict and can be used to prescribe therapy for the correction of hemostasis.

The given examples confirm the fact that if the determined characteristics deviate from the established norms, the patient may have the presence of hypercoagulation or hypocoagulation in time, that is, a tendency to the main clinical manifestations of hemostasis pathology, thrombosis or bleeding, respectively. Based on the data obtained, the doctor may decide on the appointment and correction of patient therapy.

This forecasting method is applicable to patients with various pathologies of hemostasis.

Thus, the totality of the characteristics determined as a result of the test, allows you to diagnose a tendency to thrombosis, bleeding and other pathologies of the hemostasis system. It also allows you to monitor changes and conditions of coagulation during the replacement therapy of coagulation factors, anticoagulant, fibrinolytic therapy and therapy with other drugs that affect the blood coagulation system.

Example 8

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A patient H, having a severe form of hemophilia C (coagulation factor deficiency XI less than 1% normal), took a blood sample. Platelet rich plasma was prepared using a standard centrifugation procedure, divided into four samples. Different concentrations of factor X1a were added to these samples: 0 (Fig. 7a) 0.2 U / ml (Fig. 7b), 1 U / ml (Fig. 7c) and 5 U / ml (Fig. 7d), respectively. A concentration of 1 U / ml is considered normal (Fig. 7c). Thus, the first sample corresponds to hemophilia proper, the second to mild deficiency, the third to normal plasma and the fourth to a strong excess of factor (500% of the norm).

Clot growth characteristics 0 units / ml 0.2 PIECES / ml 1 U / ml 5 units / ml The speed of propagation of a thrombin wave, microns / min 14 35 70 90 Thrombin peak, nM. No peak 12 one hundred 120

In the case of normal plasma (1 U / ml), the propagation of thrombin in space with a peak amplitude of 100 nM and a speed of 70 μm / min was observed. At a reduced concentration (0.2 U / ml), the wave formation was weakly expressed, the amplitude sharply decreased (by an order of magnitude), and the velocity fell by half. With the complete absence of factor XI, the rate of thrombin propagation decreased even more, and there was no pronounced peak at all, which demonstrates a qualitative transition. These results illustrate that the thrombin peak can be an important diagnostic indicator, in some situations more sensitive and specific than the clot growth rate: when the speed changes many times, the peak changes by orders of magnitude and even undergoes qualitative changes (disappears).

Example 9

A patient I, a conditionally healthy person, was taken a blood sample. A sample of platelet-poor plasma PPP was prepared, on which the study was conducted.

As a result of the test, the following data were obtained:

Clot growth characteristics

Clot growth inhibition, min

Initial clot growth rate, µm / min. Stationary clot growth rate, µm / min. Clot size for 30 min, µm

The density of the bunch, conventional units

The obtained indicators are within the range of normal values, which confirms the lack of data for pathology.

Claims (2)

CLAIM 1. The method of determining the functional state of the hemostatic system, comprising the following stages: a) preparing a sample of blood or its plasma; b) the definition of spatial dynamics ίη νίίΓο; at least one of the processes of coagulation or lysis of a clot in the test sample, including mixing the sample with a reagent to inhibit contact activation of coagulation of the sample, adding a reagent for re-calcification, providing local activation of coagulation in the test sample with a surface with an analogue of a physiological coagulation activator immobilized on it in a non-stirred thin the layer of the specified sample, determining the intensity of light scattering in the bunch, the delay time of the formation of the bunch, SK GROWTH clot area under the curve of light scattering on the distance the wave propagation velocity of thrombin; thrombin peak, clot size at a given point in time, the onset time of fibrinolysis, changes in the fibrinolysis front in time, changes in the thrombosis front in time, changes in clot size in time; c) comparing the results obtained in step b) with the norm and determining the direction and severity of the deviations in the patient. 2. The method according to claim 1, where in step b) the following processes are additionally recorded in the test sample: growth of a clot from an activator, growth of spontaneous clots, fibrinolysis, or a combination thereof.