Thrombosis monitor and method of thrombosis monitoring

FIELD: medicine.

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

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

11 cl, 15 dwg, 23 ex

 

The technical field

The present invention relates to a method of monitoring the effectiveness of antithrombotic drugs, injected into a patient or similar individual, and, in particular, to a device and method overall assessment of blood coagulation and formation of platelet thrombus in the environment, equivalent to the flow, with whole blood or plasma, which contains platelets.

Prior art

For example, aerodromes (such as in myocardial infarction) is a serious blood clot, so atheromatous plaque is destroyed at the site of atherosclerosis, platelets linked to a collagen containing tissue factors, in contact with the bloodstream. Then there are the complex processes of platelet aggregation, activation of the blood coagulation system and the like, leading to the formation of a blood clot, causing a serious obstruction of the vessel. Heart disease, such as myocardial infarction, is a serious disease, and it is the second leading cause of overall mortality in Japan.

However, a blood clot occurs only in the atherosclerotic region, leading to myocardial infarction, and a strong tendency to the formation of blood clots throughout the body is not observed. In vitro studies are not suitable to assess the tendency to tro the BOV and monitoring antithrombotic effect in antithrombotic therapy. Thus, it is important to make an overall assessment of blood coagulation and state of platelets (adhesion and agglutination in the presence of blood flow.

To date, the clotting activity was assessed by determining the activated partial thromboplastin time (ARTT), thromboplastin time (RT) using plasma. ART mainly reflects endogenous coagulation, and RT mainly reflects exogenous coagulation. The study of blood platelets is carried out by use of platelet-rich plasma and adding platelet-activating agents, such as ADP (adenosine diphosphate or collagen, for evaluation by the coagulation of platelets as a result of changes in their velocity penetration or similar phenomena. In addition, the clotting time of whole blood can be determined by the time of the formation of a clot of whole blood, the time of formation of a clot of whole blood after re-addition of calcium, and the like.

Further, in the devices of the survey, using the whole blood is applied thromboelastogram, which controls the activation of clotting factors, the agglutination of platelets and similar parameters.

However, the thrombus grows in the blood in vivo. On the contrary, the above method of research or produces similar definition in vitro, a in the state, closed to flow. Thus, it is impossible to observe the growth of thrombus in vivo.

As suggestions for solving the above problems in the patent document JP 2004-251630 and non-patent documents Blood 1990; 75, str-398; Blood, 1999; August 1:94(3), str-75, disclosed a process comprising passing the blood was put into it antithrombotic drug through collagen and monitoring using a confocal microscope adhesion or agglutination of platelets labeled with a fluorescent label.

However, in the method described in this patent document, the observation is conducted in the presence of anticoagulation drugs. Thus, by monitoring morphological changes in platelets is estimated the fact that the blood clotting system no formation of a blood clot caused by adhesion or agglutination of platelets, or the ability to generate reduced thrombus. Therefore, this assessment does not reflect the relationship of activated platelets with blood clotting system. Therefore this invention are suitable to assess the effectiveness of antiplatelet medications, but it is not able to control the blood clot and the whole process of clot formation. In addition, fluorescent microscope is expensive, so it hardly can be used for the bdih research.

In addition, in patent document JP 2006-145345 And blood flow after injection of anticoagulant is determined by the transmission of the blood through the silicon camera, such small comb. Similarly, this method also uses the blood after administration of the anticoagulant, and the influence of the coagulation system can be determined. In addition, the viscosity in this way has a large individual variability, so it is difficult to assess the impact of drug therapy, using the specified system.

The platelet-activated clotting system, while the clotting system is stimulated by activated platelets. In other words, the introduction of anticoagulants also inhibits platelet activation, so it is impossible to observe the effectiveness of antithrombotic drugs. In addition, if not carried out any anticoagulation therapy, the blood quickly collapsed, so that it cannot be used in the study.

A brief statement of the substance of the invention

The present invention is a device and method overall assessment of blood clots due to blood clotting and formation of platelet thrombus in environmental conditions equivalent to the flow, when the flow of whole blood or plasma containing platelets (in the description of the present and the gain they may together be cited as the "blood"), when monitoring the effectiveness of antithrombotic drugs, injected into a patient or similar individual.

To solve the above problem according to the present invention, an apparatus for monitoring the formation of thrombus, which controls the formation of thrombus by passing the blood after injection of anticoagulant through the channel, which simulates a blood vessel, at the same time doing anticoagulation treatment, or promoting the clotting of blood, and the device comprises: a camera the formation of a blood clot, at least in part hosts causing a blood clot material; an inlet tube which is connected to the camera the formation of thrombus and through which the blood flows into the chamber of clotting; and a tube for medicines, which is connected with the inlet tube and through which served a drug that stimulates the clotting of blood (hereinafter referred to as "stimulator collapse"), or drug, which performs the anticoagulation effect. In the present invention, the term "monitoring" means not only a visual estimate of the formation of a blood clot, visualization, but also the assessment of the degree of clotting in the translation to digital feature by defining a pressure or of such index.

Also, according to the present invention, an apparatus for monitoring the formation of thrombus, which contains the camera, the formation of a blood clot, at least in part hosts causing a blood clot material, an inlet tube which is connected to the camera the formation of thrombus and through which the blood flows into the chamber of clotting, and an inlet tube for inhibitor formation of a blood clot, which is connected with a chamber for formation of a blood clot, and where the mixing inhibitor of clotting of blood after passing through the chamber the formation of thrombus.

In this case, the device monitoring the formation of thrombus preferably placed on the substrate.

Device for monitoring of clotting according to the present invention preferably includes a pump to create excess pressure in the intake pipe and/or tube for a medicinal product or a pump for aspirating the contents of the discharge tube which is connected to the camera the formation of a blood clot and is designed to remove blood from the camera to the formation of a blood clot.

Device monitoring of clotting according to the present invention preferably contains a unit of pressure measurement and a camera for acquiring images, and also the camera of clotting.

In addition, causing a blood clot mother is l contains tissue factor (tissue thromboplastin).

Further, according to the present invention, a method of monitoring the formation of thrombus containing the following steps: direction of blood after injection of anticoagulant into the chamber of clotting, at least in part hosts causing a blood clot material, and simultaneously providing anticoagulation treatment or stimulation of blood coagulation, to control, by means of this, the formation of thrombus. In the present invention the characteristic direction of blood" and "ensuring simultaneously anticoagulation treatment or stimulation of blood coagulation can be a condition in which the channel is a reaction, providing anticoagulation, or reaction, stimulating coagulation, and includes the condition that ensures that the flow of the drug releasing means performing anticoagulation action, or an agent stimulating coagulation, mixing with the blood in the channel, or condition, which is due to the rapid flow of the drug releasing means performing anticoagulation action, or an agent stimulating coagulation, after mixing with blood.

In the method of monitoring of clotting according to the present invention preferably anticoagulation treatment PR is dstable a processing substances, forming chelates of calcium, such as citric acid, and anticoagulation treatment was carried out by the donor of free calcium.

In the method of monitoring of clotting according to the present invention preferably anticoagulation treatment was the treatment with thrombin aptamer, and anticoagulation treatment was performed antisense DNA aptamer, thrombin.

In the method of monitoring of clotting according to the present invention, it is preferable to control the formation of thrombus feed treated with anticoagulant blood in the chamber of clotting, at the same time stimulating the blood clotting without the implementation of anticoagulation treatment. In this case, the means for stimulation of blood coagulation preferably represents adding tissue thromboplastin.

In addition, in the method of monitoring of clotting according to the present invention, the blood clotting which was reduced by one or more anticoagulant means, preferably no longer be subjected to anticoagulation treatment, at least one means of carrying out anticoagulation processing, which corresponds to the used tool anticoagulation treatment. In this case, it is preferable that the means of anticoagula the ion treatment was a factor, inhibitory phase of contact, and the substance forming the chelate calcium, and means engaged anticoagulation treatment, was a donor of free calcium. Further, it is also preferable that the means of anticoagulation treatment was a factor inhibiting phase contact, and heparin, and means engaged anticoagulation treatment, represented heparinase. Also preferably, the means anticoagulation treatment was a factor inhibiting phase contact, such as a factor in blood clotting XII, kallickrein or similar, and the thrombin aptamer, and means engaged anticoagulation treatment, represented the antisense DNA of the thrombin aptamer. Factor inhibitor of blood coagulation XII represents preferably trypsin inhibitor obtained from maize.

In the method of monitoring of clotting according to the present invention, it is preferable to determine the pressure in the inflow and/or outflow of blood from the camera to the formation of a blood clot.

In the method of monitoring of clotting according to the present invention causing a blood clot material preferably includes collagen and tissue factor.

Device monitoring of clotting according to claim 1 of the formula of the present invention contains the Kama is the formation of thrombus, at least part of which is placed causing a blood clot material, an inlet tube which is connected to the camera the formation of thrombus and through which the blood flows into the chamber the formation of a blood clot, and a pipe for medicines, which is connected with the inlet tube and through which is fed the drug, carrying out anticoagulation treatment or drug that stimulates the clotting of blood. Therefore, the blood, which is treated with an anticoagulant to prevent clotting of blood taken from the patient after introducing him anticoagulant, in the canal in the camera the formation of a blood clot, can be controlled by the deliberate formation of a blood clot in the chamber of clotting. Thus, the effectiveness of antithrombotic agents can specifically regulate in an environment similar to the internal environment of the body. In addition, the tool anticoagulation treatment can be applied during the sampling of blood. Therefore, the advantage is that the samples after taking blood samples can be stored for a certain period of time, and the time of the study, you can choose randomly.

In accordance with paragraph 2, the device monitoring of clotting according to the present invention includes a camera of clotting, less is th least in the part where causing a blood clot material, an inlet tube which is connected to the camera the formation of thrombus and through which the blood flows into the chamber of clotting, and the inlet for inhibitor formation of a blood clot, which is connected to the camera the formation of thrombus and mixes inhibitor of clotting of blood after passing through the chamber the formation of thrombus. Therefore, it is possible to monitor the clot as described above. In addition, clotting does not occur downstream in the chamber of clotting, and therefore it is possible to prevent influence on the determination of pressure and more finely control the pressure changes.

A small amount of blood can be controlled when the device for monitoring of clotting according to the present invention formed on the substrate. In addition, the device is equipped with a pump to create excess pressure in the intake pipe and/or tube medicines, or pump for aspiration (suction) of the contents of the discharge tube which is connected to the camera the formation of a blood clot and is designed to remove blood from the camera to the formation of thrombus. Therefore, the blood and the drug to stimulate blood clotting can stably flow in a given period of time at a given pressure or a given speed flux is A.

If the device for monitoring of clotting according to the present invention includes a device for measuring pressure, the degree of formation of thrombus can be translated into numbers, so that it is possible to perform a quantitative assessment.

The device can be easily installed, when the material, causing a blood clot, contains collagen. A blood clot can effectively be called when calling a blood clot material further comprises tissue factor, such as tissue thromboplastin, together with collagen.

If the device for monitoring of clotting according to the present invention includes a camera for obtaining images of clotting, the appearance of the thrombus can be seen in the form of an image, and then the image can be stored.

Further, in accordance with claim 9 of the formula of the present invention a method of monitoring the formation of thrombus contains stages: the direction of the blood after injection of anticoagulant into the chamber of clotting, at least in part hosts causing a blood clot material, while anticoagulation treatment or promoting the clotting of blood. Thus, the formation of thrombus on calling a blood clot material can be monitored during stimulation of the coagulation of blood by passing the blood, which the traveler was received anticoagulation treatment blood taken after administration to the patient of antithrombotic drugs to prevent clotting. Therefore, the effectiveness of antithrombotic drugs can be accurately controlled in an environment similar to the internal environment of the body. In addition, anticoagulation tool can be used for sampling blood. Therefore, there is the advantage that the samples after sampling the blood can be stored for a certain period of time, and time study, you can choose randomly. If anticoagulation treatment is a treatment substance, forming a chelate of calcium, such as citric acid, and anticoagulation treatment is carried out by the donor of free calcium, it is easy to obtain the reagent, and thus it is preferable. If anticoagulation treatment is a treatment with thrombin aptamer, and anticoagulation treatment is antisense DNA aptamer, thrombin, it is possible to conduct the study at the same time reflecting the physiological concentration of calcium ions in the blood.

In addition, in accordance with section 12 of the formulas of the present invention can also control the formation of a blood clot passing blood anticoagulation after processing in the camera of clotting without anticoagulate the authorized processing, at the same time promoting the clotting of blood. Therefore, a blood clot can be observed when a small amount of blood and can reduce the discomfort of the patient. In addition, in this case, it is not always required tube for medicines, so that the device is monitoring the formation of thrombus can be simplified. If tissue thromboplastin is used as a means of stimulating blood clotting, blood clotting can be stimulated by activation of the coagulation system through an alternate path, which avoids the activation of factor XII and activation of kallikrein to control the formation of thrombus in the chamber of clotting.

In addition, a blood clot can be controlled by a simple operation effects on blood subjected to anticoagulation treatment using one or more types of anticoagulation means, at least one kind of exercising anticoagulation processing means corresponding to the used tool anticoagulation treatment. In this case, when anticoagulant treatment is conducted by the factor inhibitor of the contact phase and an agent to form a chelate of calcium, and anticoagulation treatment is carried out by the donor of free calcium, or when anticoagulation treatment is conducted by the inhibitor factor is the contact phase and heparin, and anticoagulation treatment is conducted by heparinases, anticoagulation processing, rendering the effect in the monitor clot formation, is anticoagulation processing inhibitor factor of the contact phase, therefore, the monitoring of clotting can be done in more physiological conditions, in particular reflecting the divalent metal ion associated with a blood clot, such as calcium or magnesium. In this case, when anticoagulant treatment is conducted by the factor inhibitor of the contact phase, such as factor XII coagulation or kallickrein and the thrombin aptamer, and then anticoagulation treatment antisense DNA aptamer inhibition of thrombin, blood can be stored for a long period. In addition, anticoagulation treatment can be effectively performed when as an inhibitor of factor XII coagulation is used trypsin inhibitor obtained from maize.

If the method of monitoring of clotting according to the present invention is the measurement of pressure during the intake and/or release of blood from the camera to the formation of thrombus, the degree of formation of thrombus can be translated into numbers, so you can easily perform a quantitative assessment using a very simple device.

In addition, if the material is anywaysi a blood clot, contains collagen and tissue factor, the device can be easily installed and can effectively cause a blood clot.

Brief description of drawings

The invention is further explained in the description of the preferred variants of the embodiment with reference to the accompanying drawings, in which:

figure 1 depicts the diagram of the monitoring device of clotting according to the first variant embodiment of the invention;

figure 2(a) diagram of the installation causing a blood clot material 15 in examples 3, 5 and 6 according to the invention;

figure 2(b) diagram of the formation of a blood clot in examples 3, 5 and 6 according to the invention;

figure 3(a) diagram of the installation causing a blood clot material 15 in example 4 according to the invention;

figure 3(b) diagram of the formation of a blood clot in example 4 according to the invention;

4 is a diagram of the main portion of the device monitoring the formation of a blood clot (the main body of the microchip according to the second variant embodiment of the invention;

5 is a diagram of the main portion of the device monitoring the formation of a blood clot (cover microchip) according to the second variant embodiment of the invention;

6 is a diagram of the formation of a blood clot in example 7 according to the second variant embodiment of the invention;

Fig.7. diagram of the main portion of the device monitoring the formation of a blood clot (the main body of the microchip) the agreement is but the second variant embodiment of the invention;

Fig diagram of the main portion of the device monitoring the formation of a blood clot (cover microchip) according to the second variant embodiment of the invention;

figure 9 - diagram of the main parts of the device monitoring of clotting according to the third variant embodiment of the invention; figure 9(a) to the whole scheme, Fig.9(B) - the main body of the microchip, and Fig.9(C) - cover of the microchip according to the invention;

figure 10 - diagram of the system of monitoring the formation of thrombus containing the device for monitoring of clotting according to the invention;

11(a) to form waves thromboelastogram clotting adding 10 μm of each of the aptamers to associte I and associte II in the blood after exposure at room temperature for 15 min and the addition of 40 μm of each of the antisense DNA of both aptamers according to the invention;

11(B) - form of waves thromboelastogram blood immediately after blood collection according to the invention;

Fig diagram of the device according to the fourth variant of implementation of the present invention; Fig(a) to complete the drawing, Fig(In) - cover of the microchip, and Fig(S) - substrate of the microchip according to the invention;

Fig diagram measuring pressure of example 17 (control), example 18 (heparin) and example 19 (Reopro) according to the invention;

Fig diagram of measuring pressure adding heparin in quantities is 0 (control), 0.2, 0.5 and 1 U/ml of heparin according to the invention;

Fig diagram of measuring pressure with the addition of heparin at 0 (control), 2, 5 and 10 µg/ml according to the invention.

Description of the preferred embodiments of the invention

Figure 1 shows the diagram of the first variant of implementation of the monitoring device of clotting according to the present invention.

Device monitoring of clotting according to the present variant implementation contains the camera 10 of clotting, the inlet pipe 11 which is connected to the camera the formation of thrombus and through which the blood flows into the chamber of clotting, and the tube for the medicinal product 12, which is connected with the exhaust pipe and through which is fed the drug, carrying out anticoagulation treatment or promote the clotting of blood.

The camera 10 of clotting is essentially cylindrical in shape and contains material which causes the formation of a blood clot in a part of its inner cavity. The camera can be made of transparent glass, thermoplastic resin or similar material. Examples of the material which causes the formation of a blood clot include collagen, vWF (factor a background of Villebranda)previously received a blood clot and materials fibrous silk, cotton or similar danieto materials can be used singly or in combination of two or more of them. Collagen is particularly preferred because it can be easy to get, easy to manipulate, and it can be provided in the form of the model is similar to the blood vessel. Collagen can include tissue factor. The material, causing a blood clot, collagen or vWF preferably presented in the form of a coating on the internal surface of the chamber formation of a blood clot 10 to prevent leakage causing a blood clot material together with blood stream. The coating can be applied, for example as described in document JP 05-260950 A or document Blood 1995 April 1; 85(7): 1826-35, dissolution of collagen in acid solution and immersing him in the substrate having a hydrophilic nature, such as glass or polystyrene, followed by washing and drying to cover the surface of the material.

Further, it is preferable that the material, causing a blood clot, in the form of a fibrous material or previously received a blood clot could be in a condition suitable for fixing inside the chamber the formation of a blood clot 10. Further, by impregnation collagen hygroscopic fine fibrous material such as cotton, non-woven fabric or knitted fabric, and drying can be obtained causing a blood clot material with a higher ability to induce formation of a blood clot. In addition, the substrate may be preparing Atisa in a solution of collagen, containing tissue thromboplastin, and then dried to further strengthen its ability to cause a blood clot.

Causing a blood clot material can be chosen depending on the inner diameter of the chamber formation of a blood clot 10 and monitoring. If the model presents atheromatous causing myocardial infarction, it is preferable that contained one collagen, or contained and collagen, and tissue thromboplastin. In addition, it is preferable that the channel to form a narrowed portion in the chamber of clotting for the effects of shear stress on the camera the formation of thrombus. Further, in the case study the formation of thrombus in acute myocardial infarction and cerebral infarction cardiac origin or similar condition in which a blood clot may be transferred from another part of the body through the bloodstream and attach, causing occlusion of a blood vessel in another part of the body, it is preferable that previously occurred grip small thrombus with camera of clotting 10, presented in the form of the material, causing a blood clot, with subsequent monitoring of the growth of thrombus formed on it. In the case study of thrombosis of the blood capillary, the inner lumen of the channel in the chamber of clotting can be divided into many to the channels, each of which has a diameter of from 10 to 30 μm. If the camera is the formation of a blood clot 10 has a narrowed portion with a diameter of 100 μm or less, the channel may be subjected to occlusion of small blood clot formed in such a constricted part. So there is no need to use additional material, causing a blood clot, so that a blood clot can be controlled by means of stimulating coagulation, or an anticoagulant. Therefore, the present invention provides this narrowed part in the quality of the material, causing a blood clot.

The material, causing a blood clot, can be coated with collagen or vWF. The camera 10 of the formation of a blood clot in the part of the coating may preferably be narrowed and presents for part 14, so that it is possible to control platelet aggregation induced by shear stress. In the case of coating the collagen in the quality of the material, causing the formation of a blood clot, it is preferable, in order to obtain a high capacity friction with the surface, at least part of the substrate was made of glass or plastic. In addition, the part on which it is necessary to place the camera 10 of the formation of a blood clot, or a part of the chamber 10 the formation of thrombus, where the material, causing a blood clot, can be made in the form of removable cassettes is. This option is preferred, because the resulting thrombus can be easily washed or watch, or the material, causing a blood clot, can be easily replaced with new material. In this case, the cartridge may form a sealed fluid connection ring gasket of silicone rubber or similar material. Preferably, the end of the tape opposite to its end connected to the intake pipe 11 of the camera 10, the formation of a blood clot, can be connected with the exhaust pipe 13, which provides the possibility of manufacturing the blood. Preferably, the discharge tube 13 may be divided cylindrical coil, and its tip can be fitted with a pressure gauge 41, for example a pressure gauge diaphragm type. On the other hand, the tip of the discharge tube 13 is preferably connected to a storage container (not shown).

The inlet pipe 11, connected to the camera 10 of the formation of a blood clot, can be fabricated using transparent glass, thermoplastic resin or similar material. The end of the intake tube 11 opposite to its other end that connects to the camera 10 of clotting, is connected to the syringe 20, which pump the blood. The syringe 20 is connected to the pump 30 and compressing means (not shown), so that the plunger of the syringe 20 nadavlivaya at a given pressure. The pump may be submitted is ü a conventional pump, available on the market. Alternatively, the pump may be a syringe pump, manufactured by extrusion syringe air at constant pressure, or by turning the syringe, so that the piston is at the top, and acting on the piston to its weight.

The inlet tube 11 may be preferably divided cylindrical coil, and its end is preferably provided with a pressure gauge 40, such as a pressure gauge diaphragm type of inlet tube 11 near the camera 10 of clotting.

Blood in the syringe 20 is subjected to anticoagulation treatment. Examples of tools anticoagulation treatment include sodium citrate or potassium citrate, sodium oxalate or potassium oxalate, citrate-dextrose solution (ACD) and ethylenediaminetetraacetate (EDTA). This means anticoagulation treatment can be used in the form of a powder, a lyophilized product or solution such as an aqueous solution. Among these anticoagulation means generally preferred to 3.2% sodium citrate, because it is available. In this case, one volume of funds anticoagulation treatment is preferably mixed with 9 volumes of blood.

Whole blood or plasma without tools anticoagulation treatment is minimized within a few minutes. Coagulation can be reduced or eliminated by adding a substance that forms the hell is calcium, such as citrate. In particular, it was reported that citrate may inhibit agglutination and functions prothrombinase and exogenous and endogenous tenaz.

Processed citrate blood can be stored in liquid form for a specified period of time (e.g., from several hours to several days) and to process blood products, such as product cytopheresis, plasma enriched with platelets, and plasma with a low platelet. Containing citrate plasma can be stored at about -70°C or lower for a long period of time (from several months to several years). In the present invention can also be used whole blood and plasma and, in this case, it is preferable to re-add calcium or other similar substances.

However, whole blood or plasma with newly added calcium spontaneously collapses due to contact activation in any of the majority of containers for storage. In this case, the contact activation may occur within about 2-4 minutes With this in mind, the present invention anticoagulation agent, such as calcium, can be added directly after monitoring the formation of thrombus in the newly prepared by treatment with citrate blood after taking blood samples and freezing for storage and subsequent thawing, or containing blood is the ITA's plasma.

Other anticoagulation means may include heparin, hirudin, hirulog (peptide C-terminal region of hirudin), Aprotinin, antithrombin antibody, aptamer, thrombin, trypsin inhibitor obtained from maize (1977, J. Biol. Chem 252, 8105). These materials inhibit blood clotting by inhibiting the coagulation cascade as a result of inhibition of factor in blood clotting and therefore, sometimes in the description of the present invention will be referred to as "inhibitors of coagulation factors".

Blood samples for monitoring can be rented in any way, such as the way in which the inhibitor of coagulation factors previously placed in a syringe or vacuum vessel for blood collection, and then typed in the blood, or the way in which the inhibitor of coagulation factors quickly into the blood immediately after taking blood samples to get through this blood subjected to anticoagulation treatment.

Next, the blood is collected in the vacuum vessel for blood collection, containing the inhibitor of coagulation factors, such as heparin, and then for the destruction of heparin are added heparinase and means anticoagulation treatment that is appropriate for monitoring, so heparin is replaced by means of anticoagulation treatment, suitable for the purpose of monitoring. In addition, blood is collected in a vacuum is the first vessel for blood collection contains citric acid, and then add the calcium chloride and inhibitor of coagulation factor, suitable for the purpose of monitoring, such as trypsin inhibitor obtained from maize, or the thrombin aptamer. Therefore, the blood is subjected to anticoagulation treatment, can be collected depending on the purpose of monitoring.

Tube for medicines 12, connected to the camera 10 of the formation of a blood clot, can be made of transparent glass, thermoplastic resin or similar materials. The end of the tube for the medicinal product 12, which is opposite to its other side connected to the camera 10 of clotting, is connected to the syringe 21 to supply medicines, exercising anticoagulation treatment, or drugs that stimulate blood clotting. The syringe 21 may be connected to the pump 31 and a compressing means (not shown), so that the plunger of the syringe 21 may nadavlivaya at a given pressure. The pump may be a conventional pump, commercially available. Alternatively, the pump may be a syringe pump, manufactured by extrusion syringe air at a given pressure, or by turning the syringe, so that the piston is at the top, and acting on the piston load. Tube for drugs 12 is filled with the tool, carry out what they anticoagulation treatment, or as a means to stimulate blood clotting, as described below.

For monitoring thrombus device monitoring of clotting according to the present invention, the syringe 20 is filled with whole blood or plasma containing the platelets, and subjected to anticoagulation treatment with sodium citrate (solution A). The syringe 21 will fill medicine that provides anticoagulation treatment, such as calcium chloride (solution B). Solution a and solution served in the intake pipe 11 of the pumps 30 and 31, respectively, so that the solution could reach a concentration of from 5 to 20 mmol, in which you can initiate a cascade of coagulation solution A. In the following, the solution and solution b are mixed in the intake pipe 11, so that the mixture is directed into the chamber 10 of clotting. Then the collagen or a similar substance capable of causing a blood clot, pre-applied to the portion of the inner surface of the chamber 10 the formation of thrombus formation material, causing a blood clot. Luggage the formation of thrombus can be made of a transparent plastic tube. A blood clot can easily control device monitoring blood passing through such viewable due to the transparency of the camera 10 of clotting.

Monitoring the formation of a blood clot is possible to carry out a visual assessment of blood occurring within a specified period of time through the camera (the camera 10 of the formation of a blood clot), processed collagen, and then removing the blood from it. When a pump of solution a and solution sucks In air, then the solution a and the solution can be fed under constant pressure. Therefore, the formation of thrombus on the collagen can be controlled to reduce the speed of blood flow resulting from the discharge tube 13. Alternatively, when the gauge is installed near the camera 10 the formation of thrombus on the part of the inlet tube 11 and the discharge tube 13, a blood clot on the collagen can be monitored by monitoring changes in the internal pressure in the chamber 10 of clotting. Alternatively, the formation of thrombus on the collagen can be observed under the microscope in the manufacture of the camera 10, the formation of thrombus thickness of 500 μm or less. In particular, you can easily observe the blood clot in platelet-rich plasma, because it is highly visible, and a blood clot can also be seen with the naked eye. Next, you can also fluorescente to tag the blood platelets and monitor their fluorescence fluorescence microscope.

Examples of drugs for the implementation of anticoagulation treatment to form a chelating agent such as citric acid, contain: halide calcium, such as calcium chloride,calcium bromide and calcium iodide; inorganic calcium salts such as calcium phosphate, calcium sulfate, calcium nitrate and calcium bicarbonate; and calcium salts of organic acids such as formic acid, acetic acid, propionic acid, butyric acid, alginic acid, lactic acid, gluconic acid, glyceric acid and glycerophosphoric acid, which are compounds of calcium provided in the form of a donor of free calcium.

The tool implementing anticoagulation effect, you can choose to use depending on the inhibitor of coagulation factors, when anticoagulant treatment is conducted by the inhibitor of coagulation factors (tool anticoagulation treatment). For example, as a means of carrying out anticoagulation effect when anticoagulation treatment with heparin, can be used Protamine, heparinase or antibody against heparin. As a means of having anticoagulant effect, when anticoagulant treatment is conducted by hirudin, verulagam and Aprotinin, you can use the tool engaged anticoagulation action, such as, respectively, the antibody against hirudin, an antibody against hirulog and antibody against Aprotinin.

Examples of means of carrying out the anticoagulant action is e, when anticoagulant treatment is carried out using antibodies against thrombin as an inhibitor of coagulation factor include: fully inactivated thrombin, such as RISK-thrombin (D-Phe-Pro-Arg-chloromethylketone)-thrombin; fragment destruction of thrombin and synthetic polypeptide containing the antibody recognizes an epitope of thrombin.

Antibodies used for anticoagulation treatment or the implementation of anticoagulation, preferably include antibody from which the Fc domains removed by papanti or similar substances, to minimize their effect on the complement system, or such antibodies as antibodies to chicken eggs, unable to activate the complement system of the person.

When the thrombin aptamer (Blood. 1993 Jun 15; 81(12): 3271-6 or J Mol Biol. 1997 Oct 10; 272(5): 688-98), which is a single-stranded oligo DNA was used as a tool for anticoagulation treatment, a substance which binds to the aptamer, thrombin and inhibits its function, such as antisense DNA or antisense RNA can be used as a means of carrying out the anticoagulant effect. When two kinds of thrombin aptamers, one that recognizes ecsatic I, and another that recognizes ecsatic II, are used in combination, it is possible to obtain a very high effect of anticoagulation treatment, cf is the ranking with the case, in which each is used separately. Antisense DNA used in this case may be an antisense DNA against the side of the thrombin aptamer, while it is substantially inactivates antithrombin function of the thrombin aptamer.

In addition, thrombin aptamers and antisense DNA respectively effective as a means of anticoagulation treatment and means of carrying out anticoagulation effect as described with reference the following reference examples.

The clotting system cannot be activated within a few hours, when anticoagulation treatment with heparin. Thus, the blood can be stored for a long period of time while monitoring the formation of thrombus. However, for example, is the case when anticoagulant treatment is not suitable for studies of blood taken from the patient who was administered heparin.

On the other hand, hirudin, hirulog and antithrombin antibody are inhibitors to inhibit the conversion of fibrinogen into fibrin by inhibiting thrombin, which acts on the final stage of the coagulation system. However, the factor of the contact phase (such as pre-kallickrein or factor XII coagulation cascade gradually activated even in the case of complete inhibition of thrombin, so can the be higher activation in the coagulation cascade. Therefore, there is a case that is not suitable for long term storage of blood.

Aprotinin also inhibits the activity of kallikrein on the phase of contact for delay endogenous cascade of blood coagulation. However, the coagulation cascade is gradually activated even in terms of inhibiting the activity of kallikrein, so that the blood coagulates after a few hours even in the presence of Aprotinin.

Therefore, when monitoring the formation of a blood clot after about several tens of minutes, you can control the formation of thrombus, anticoagulation treatment with an inhibitor of thrombin and/or Aprotinin. However, this may not be preferred in case required a long time to start monitoring or required a long time for monitoring.

When a blood clot is controlled within 1 hour or more from the time of sampling blood, anticoagulant treatment may be performed using thrombin aptamer in combination with derived from maize trypsin inhibitor, hirudin and Aprotinin. Alternatively, a small amount of heparin, for example less than 1 IU/ml, which is a level that does not have a significant effect on the clotting time can be used in combination with an inhibitor of factor phase contact or inhibitor of thrombin.

Much more in the expressed effect of anticoagulation treatment can be obtained, when the thrombin aptamer is used in combination with two kinds of aptamers for associte I and eksotika II. Further, the antisense DNA of the respective aptamers can immediately implement anticoagulation treatment.

In a blood sample is also preferable to add the inhibitor to factor phase a contact or a thrombin inhibitor such as an inhibitor, obtained from maize (inhibitor of factor XII), or Aprotinin (inhibitor of kallikrein, and thrombin inhibitor, such as an aptamer, thrombin, and then add antisense DNA aptamer, thrombin (inhibitor of DNA) or a similar substance. Thus, the function of one inhibitor of thrombin inhibited by functional inhibition of thrombin followed by rapid flow direction of the sample into the chamber of clotting. As a result, for example, exogenous clotting cascade can be activated by tissue factor (tissue thromboplastin) or similar, so that stimulates clotting of blood, and you can control the physiological formation of thrombus.

Alternative anticoagulation treatment may be performed and inhibitor means anticoagulation treatment, and obtained from maize trypsin, which is capable of anticoagulation treatment, such as substances forming chelates (citric acid), heparin, or the like, and don is R free calcium, such as calcium chloride; or heparinase can then be given the opportunity to perform anticoagulation treatment with subsequent enabling blood to flow into the chamber of clotting to stimulate blood clotting activation of exogenous coagulation cascade.

In this case, causing a blood clot material may preferably contain the appropriate amount of tissue factor (tissue thromboplastin). In particular, preferably, causing a blood clot material would be covered with a mixture obtained by mixing collagen with tissue thromboplastin, followed by drying, because the blood clot is stimulated only in the chamber of clotting. When used, causing a blood clot material, covered with a solution of collagen containing tissue factor (tissue thromboplastin) as atheromatous model, it is possible to conduct monitoring, which reflects the pathological mechanism.

In addition, a blood clot can be controlled by adding a donor of free calcium and trypsin inhibitor to the blood subjected to anticoagulation treatment compound, forming chelates of calcium, such as citric acid, and rapid blood flow into the chamber the formation of a blood clot, or adding heparinase the trypsin inhibitor to the blood, subjected to anticoagulation treatment with heparin, and then securing a rapid flow of blood into the chamber of clotting. In the above-described method, in which a blood clot is controlled by mixing subjected to anticoagulation treatment blood obtained using one or more kinds of funds for anticoagulation treatment, at least one means of implementing anticoagulation treatment that are specific to your vehicle, and then securing a rapid flow of blood into the chamber of the formation of a blood clot, you can use the device monitoring the formation of a blood clot (Fig.9).

Above thrombin inhibitors such as hirudin and thrombin aptamers synthesized small molecule inhibitors of factor phase contact, and means anticoagulation treatment of protein costly compared to any of the funds anticoagulation treatment, such as citric acid and EDTA, which form chelates with calcium and the like. However, they do not alter the concentration of divalent metal ions, such as calcium, magnesium and zinc, so that the formation of a blood clot in the initial concentrations of these ions in the blood of the patient may be reflected in the monitoring. Therefore, it becomes possible monitoring, which reflects the patient's clinical status.

Blood clotting can be suppressed by inhibition of factor phase contact, such as activated factor XII or kallickrein. However, it was reported that activation Ha and kallickrein does not make a large contribution to the actual physiological blood clot or hemostasis. In fact, in General there is no evidence of bleeding or similar phenomenon even in a patient with congenital deficiency of factor XII, pre-kallikrein or the like. In particular, when atherothrombosis, such as myocardial infarction, it is widely known that the coagulation system is activated tissue factor due to the plaque, caused by arteriosclerosis. Factor XII can be activated mainly by thrombin on the activated platelet. So when anticoagulation treatment, when the inhibitor for inhibiting the activation of factor XII, or prekallikrein, or inhibitor for the inhibition of activated factor XII, or kallikrein is used as an anticoagulant solution, no need to add a means of carrying out the anticoagulant effect. By adding in the blood of any substance that activates exogenous coagulation, such as tissue factor (e.g., tissue thromboplastin), or material that causes a blood clot, instead anticoagulation treatment, or the user is receiving the substance in the material, causing a blood clot, clotting system is activated by alternative, which avoids the activation of factor XII and activation of kallikrein to stimulate coagulation of blood, so a blood clot can be controlled in the chamber of clotting.

As a means anticoagulation treatment that can be used for monitoring the formation of a blood clot, after stimulation of blood coagulation by the addition of a substance that activates exogenous coagulation, such as tissue factor, without adding means of carrying out the anticoagulant effect, illustrated synthesized small molecule inhibitors, such as inhibitors of kallikrein PKSI-527 (Thromb Res 57: 889, 1990) and D-Phe-Arg-CK, which is an inhibitor of activated factor XII (Cal Biochem. Co., Ltd.). In addition, protein inhibitors include antibodies against the protease of the contact phase in the coagulation cascade, such as antibody against kallikrein, antibody against prekallikrein, antibody against coagulation factor XII antibody against the activated coagulation factor XII, and trypsin inhibitor obtained from maize.

To monitor the formation of a blood clot after stimulation of blood coagulation by the addition of a substance that activates exogenous coagulation, without adding means of carrying out anticoagulation the second action, in particular, from the viewpoint of availability and anticoagulation ability, it is preferable that the blood was once subjected to anticoagulation treatment with a combination of citric acid and derived from maize trypsin inhibitor, or a combination of thrombin and aptamer obtained from maize trypsin inhibitor, and the blood is then stored, because it is possible to increase the degree of freedom of operation monitoring. Further, immediately before monitoring the formation of thrombus, anticoagulation continues processing obtained from maize trypsin inhibitor, while the other anticoagulation treatment partly carried by the donor of free calcium or antisense DNA aptamer, thrombin. In this case, as a model of atheromatous blood clot, it is preferable to activate exogenous coagulation of the material, causing a blood clot, covered with collagen, or containing collagen tissue thromboplastin, to stimulate clotting.

Containers used for monitoring the formation of a blood clot, such as a syringe for blood flow and vacuum vessel for collecting blood, preferably coated with heparin or a material which has antithrombotic properties and compatibility with blood, such as polyvinylacetate (PVLA) or poly-2-methoxyethylamine (RMEA).

In mouth is oiste monitoring of clotting in accordance with another embodiment of the present invention, the tube and the chamber of clotting can be combined with each other on a substrate in a thin channel, such as a microchip.

Figure 4 shows a view in plan of the main part of the device, monitoring the formation of a blood clot in accordance with the second aspect of the present invention. Figure 4 in the substrate 100 is formed of the groove, and it formed a loop. This implementation is configured as follows. On a small piece of substrate made Luggage the formation of thrombus in the main part of the device, monitoring the formation of a blood clot, an inlet tube which is connected to the camera the formation of thrombus, and through which the blood flows into the chamber of clotting, and the tube for medicines, which is connected with the inlet tube and through which chamber the formation of a blood clot is injected drug, carrying out anticoagulation treatment or drug that stimulates the clotting of blood, all elements integrated with each other and presented as the contour of the microchip. In this embodiment, parts other than the main part are integrated on a substrate, similar to the first option implementation.

In figure 4, the substrate 100 is a main body of the microchip, and its materials can be any from the group consisting of metal, glass, plastic, silicone and the like. When observing a blood clot preferred transparent material. D. the I formation outline the preferred plastic material. It is therefore particularly preferred transparent plastic material. When it is selected from a silicone resin such as polydimethylsiloxane (PDMS), its bonding capacity is very high. Thus, the circuit can be formed by the contact binding with cover without adhesive or the like. When used as a substrate made of polystyrene, the channel can easily cover PVLA and expose antithrombotic treatment. Next, RMAA can also provide the possibility of a simple, effective antithrombotic treatment (see reference example 4).

Figure 5 presents a view in plan of the substrate, which serves as a cover of the microchip, which is superimposed over and connected to the substrate 100 (figure 4). The substrate 200 (figure 5) is a transparent glass slide or plate or sheet, which is made of plastic or similar material. The substrate 200 in the form of a cover superimposed over and connected with the substrate 100, so that the circuit substrate can be formed from the chamber 110 of clotting, the inlet tube 111 and tube for medicines 112.

To ensure the inner surface of the chamber 110 of clotting material, causing the development of thrombus 115, for example, collagen or similar material can be applied to the specified installation position of the substrate 200. Device for monitoring the formation of thrombus can be obtained codeology 100 and the substrate 200 are connected to each other by the connection or fitting in the direction of the inside surface coated with collagen substrate 200. Due to the simple application of collagen can preferably be applied to a flat substrate 200 having no grooves. In addition, tissue thromboplastin preferably applied after mixing with collagen, because you get a caller a blood clot material having a higher capacity to cause a blood clot. Further, for easy formation of a blood clot caused by collagen glass having a portion coated with collagen, may be subjected to additional processing, such as replacing glass, frosted glass or similar material, so the surface area can be increased.

The connecting portion 100A, the connecting portion 100B and the connecting portion 100C of the substrate 100 are connected with tubes (not shown), which respectively form part of the inlet tube, tube for medicines and discharge tube. Thus, each of the connecting part 100A and the connecting portion 100B are connected to pumps (not shown) through the tube. Subjected to anticoagulation treatment blood injected from the connecting portion 100A, and anticoagulation tool corresponding to the tool anticoagulation treatment, injected from the connecting portion 100B.

In accordance with this embodiment, the formation of thrombus can be controlled extremely small amounts is the your blood, so you can reduce the discomfort for the patient. In addition, platelets, coupled with the substrate 200 from the substrate 200, it is possible to control the tagging of platelets fluorescent reagent such as mepacrine.

Further, the circuit 100D has a resealable end, which is blocked in the air. Thus, the circuit 100D is provided in the form of a manometer. The end of the path 100D may be preferably provided with a regulating valve 100E to relieve the pressure or increasing the sensitivity when the circuit 100D is provided in the form of a manometer. Control valve 100E may be closed by a cap or masking tape, and its volume can change content, such as resin, depending on the required sensitivity. The circuit 100D is formed low in order to prevent mixing of the blood with air and prevent air leakage. The thickness of the circuit 100D depends on the material of the substrate 100, but is approximately from 0.1 to 0.5 mm, an Internal pressure of the inlet tube 111 monitors the formation of thrombus increases the blood clot, air circuit 100D is compressed by the blood, so exposed to anticoagulant-treated blood can be introduced into the circuit 100D in this number. The internal pressure can be controlled by the movement of blood in the circuit 100D at any time.

Adhesion of platelets with collagen, Akti is the situation of the coagulation system on activated platelets and the accumulation of activated platelets by agglutination of platelets can be controlled respectively by the device monitoring the formation of thrombus and/or method monitoring of clotting according to the present invention.

Further, to reproduce the formation of a blood clot in atherothrombosis, the narrowed part 14 and 114 are formed respectively in the chambers 10 and 110 of clotting. So you can monitor that also displays the shear stress that creates the agglutination of platelets.

Figure 9 shows the main part of the device monitoring the formation of a blood clot in accordance with the third embodiment of the present invention. Figure 9 shows the inlet tube 322 to an inhibitor of blood coagulation and the inlet tube 317 to supply pressure, which mixes the inhibitor of coagulation of blood below the stream from the camera to the formation of thrombus. In this embodiment, tube for medicines not provided upstream from the chamber of clotting.

In accordance with the third embodiment in blood collected after adding means anticoagulation treatment (for example, citric acid and derived from maize trypsin inhibitor), also added a tool which implements anticoagulation treatment (for example, a donor of free calcium), immediately before the measurement and then introduced into the syringe 320. Subsequently, the liquid to fill the blood pressure, such as mine is real butter, pressed into the syringe from the inlet tube 317 for pressure, connected to a syringe 320, squeezing through this blood in the microchip 300.

The increase in pressure exerted on the syringe 320 sample, shows the state of occlusion of the channel formed by the blood clot, so monitoring the formation of thrombus on the pressure change is particularly suitable for monitoring model durable atheromatous blood clot which is formed obstructive thrombus. Causing a blood clot material may in no particular limitations to represent material derived from collagen and tissue factor. For example, causing a blood clot material effective for monitoring the formation of a blood clot when blood clotting is stimulated by exogenous activation of coagulation.

During measurement of the pressure for more accurate measurement of the pressure in the channel when the blood clot, an inhibitor of coagulation of the blood introduced through the inlet tube 322 to an inhibitor of blood coagulation and mixed with blood, which is downstream from the camera to the formation of thrombus through the channel formed in the microchip 300. Thus, it is possible to prevent formation of a blood clot in the blood, which is in the channel next to the camera the formation of thrombus. Therefore, this configuration is preferred because you definitely and accurately measure the changing pressure in the channel, which is caused by a narrowing or closing of the channel by the formation of a blood clot in the chamber of clotting.

Inhibitor of blood coagulation may preferably no particular restrictions to provide a means for anticoagulation treatment used in the present invention. When taking into account cost effectiveness and the potential for manipulation is preferable to choose any of those means, which prevent blood clotting albuminoidal deformation, including alkaline or acid solution, alcohol, urea and sodium dodecyl sulphate.

In the described embodiment, the monitoring unit of clotting according to the present invention includes a suction pump on the outlet pipe 313, instead of the inlet tube 317 feed pressure, in order to determine the degree of formation of thrombus on the basis of the change of the suction pressure (negative pressure). This configuration can lead to more effective measurement, at the same time preventing the situation where the fluid pressure mineral oil or the like is mixed with blood in the contact region, and the mixture is fed, when the number is small, if the use of a pump that produces indirect pushing blood through the liquid separated in the form of a layer, as described below.

<> Further, there is no need to use a closed container such as a syringe, as a container (tank sample) for the supply of blood in the device monitoring of clotting according to the present invention. Thus, the container may have a lid and may be open to air. As a result, it is possible to simplify the device monitoring the formation of thrombus. Further, in the device monitoring of clotting according to the present invention, the internal pressure in the channel is negative. Thus, when the device for monitoring of clotting according to the present invention is made in view of the microchip, for example, even in the case of incomplete coupling between the main body of the microchip and the cover of the microchip, there is no leakage of blood. In some cases, it can be used as a monitoring device of clotting by fitting the main body of the microchip and the cover of the microchip together when the fit is accurate.

Figure 10 shows additional systematic device And monitoring the formation of thrombus.

The device And monitoring the formation of a blood clot fills Micronase 330 feed fluid having a density less than the blood. The liquid is then pressed into the syringe for sample 320, in which the blood flows through the inlet tube 317 feed pressure is, and then accumulates in the blood by squeezing the blood in the microchip 300. Squeezed out the blood mixed with the tool engaged anticoagulation treatment, injected from the tube for medicines 312, with subsequent admission to the chamber 311 of clotting. Fluid for inflating the blood Micronase 330 feed can represent any of the fats and oils such as liquid paraffin, mineral oil and silicone oil, and saline. Thus, it becomes possible to prevent contamination and Micronase 330 supply, and the pressure sensor 340 indirect blood pushing blood.

This pump for indirect extrusion of blood fluid, separated by a layer of blood, can be used to monitor the formation of a blood clot in accordance with any of the embodiments of the present invention.

The sensor 340 pressure can determine the pressure applied to the syringe sample 320 Micronesian 330 submission.

In this device, And monitoring of clotting condition of clotting can be recognized in more detail by analyzing the image. In particular, in the case of labeling of platelets and leukocytes by chinachina and then monitoring their adhesion and agglutination with collagen, the illuminance per unit area due to manifestations of fluorescent color to ntralized image analysis. Thus, the monitoring results can be evaluated and stored in the data view. The image analysis can be carried out by processing the images captured fluorescent stereoscopic microscope 308, camera, computer controlled 307, and view the image on the display.

Therefore, in the device And monitoring the formation of thrombus can fully determine the state of clotting according to the results of image analysis and change in the pressure applied to the piston with the sample 320.

The device monitoring the formation of thrombus can be classified using the device monitoring the formation of thrombus in any of the embodiments according to the present invention.

On Fig shows a diagram of the monitoring device of clotting fourth variant of implementation of the present invention. The device monitoring the formation of a blood clot is equipped with a camera for monitoring the formation of thrombus.

For example, in the device monitoring the formation of a blood clot in the blood subjected to anticoagulation treatment with citric acid and derived from maize trypsin inhibitor is added to the tool engaged anticoagulation treatment (for example, a donor of free calcium), and is quickly filling her syringe 420. The syringe 420 is aspirated by the pump 414, connected to vypuskno the pipe 413, to allow blood to pass through the camera of clotting 411, forming thereby a blood clot. The degree of clotting may be determined by the change of the suction pressure (negative pressure).

To prevent clotting of blood after passing through the chamber the formation of a blood clot obstruction as a result of the discharge tube 413 or impact on the measurement of the pressure in the intake tube of an inhibitor of clotting 422 is mixing inhibitor of clotting of blood after passing through the chamber the formation of thrombus.

The camera 430 (e.g., camera, computer-controlled) is located under the chip 400 for receiving a camera image formation of a blood clot, so it is possible to effectively use the space on a microchip. The guides 433 Luggage 430 can move forward and back and around the place of formation of thrombus. By using the camera 430, which has the function of keeping its position along the X-axis and Y-axis, it can acquire images at the same time regularly moving around a set of specific points. With the above configuration, you can control the process of clotting time on the wide area chamber of clotting 411, even if the increase in chamber 430 is set to a high level. Further, the optical and the source 432 (e.g., light-emitting diode) is preferably located around the camera 430, because the optical source 432 can move at the same time maintaining their positional relationship with the camera 430. Preferably, the optical source 432 was capable of emitting light at a wavelength that can excite a particular fluorescent substance together with white light, depending on the fluorescent material, which serves as the target visualization, providing the possibility of excitation of different fluorescent materials.

EXAMPLES

Hereinafter the present invention will be explained in detail by specific examples. However, the present invention is not limited to these examples.

Example 1

The device monitoring the formation of thrombus (figure 1) is used to fill the syringe 20 50 ml treated with citrate blood (solution A)containing 9 parts by volume of blood immediately after mixing the blood sample with 1 part by volume of 3.2% sodium citrate, and to fill the syringe 21 10 ml of 0.2 M CaCl2(solution B). Syringes 20 and 21 are connected with transparent nylon tube (inlet tube 11 and the tube 12 for medicines) with an inner diameter of 3 mm, Both tubes are connected together at a T-shaped connection (cylindrical bobbin) and then connected with polycarbonate camera 10 education the Finance thrombus with an inner diameter of 3 mm and a length of 1 cm through a single nylon tube (inlet tube 11) with an inner diameter of 3 mm and a length of 3 see The camera 10 of clotting constructed in the form of a removable cartridge. The connecting portion between the tape and nylon tube (inlet tube 11 is made pressurized liquid through an annular gasket made of silicone rubber. The glass element is fixed on the inner side of the tape adhesive based on epoxy resin, forming through this narrowed part 14. The tapered part 14 is designed so that the narrowed area constricted part 14 may have an inner diameter (the maximum gap between the constricted part 14 and the inner wall) is 1.5 mm in Addition, the camera 10 of clotting connected tightly to the liquid tube, such as a discharge tube 13, having the same diameter and are made of the same material, by means of an annular gasket made of silicone rubber, and thus made the device 1 for monitoring thrombus (figure 1). It should be noted that the gauges 40 and 41 of the flange type mounted respectively on the parts of the inlet tube 11 and the discharge tube 13 near the camera 10 the formation of thrombus through connections (cylindrical bobbins). In addition, the glass material constricted portion on the inner surface of the camera 10 of clotting receive so that collagen is deposited in the form of a coating as a calling education is the W thrombus material 15 on the glass narrowed portion on the inner surface by immersion in 0,1N solution of acetic acid, containing 1% insoluble collagen type I (manufactured Wako Pure Chemical Industries, Ltd.) and then drying. Syringes 20 and 21 of the inverted so that the pistons are on the top side, and then on the pistons influence of gravity in order to give the solution a and the solution To flow into the syringe pumps with a speed of 5 ml/min and at a rate of 0.5 ml/min

When the solution a and the solution To flow for 10 minutes, after a few minutes, there occurs a difference between the readings of the pressure gauges 40 and 41 in the intake pipe 11 and an outlet pipe 13, and then this difference will increase with time. At the same time confirmed that the blood flowing from the discharge tube 13, is also gradually reduced. When the flow of all solutions is completed, the device 1 monitoring of clotting flows physiological saline solution for rinsing chamber 10 of clotting. Thus, a blood clot can be found in the camera 10 of clotting visual observation.

Example 2

The device 1 monitoring of clotting produced and the formation of a blood clot is then controlled in a manner analogous to example 1, except that the solution a of example 1, in addition, was added nefrackzionirovannam heparin (obtained from the mucosa of pigs) in a concentration of 1 mg/ml

In this case there is no pressure difference, which does not reduce blood flow, and thrombus cannot be found in the camera 10 of clotting visual observation after washing with physiological saline solution.

Example 3

The device 1 monitoring of clotting produced in a manner analogous to example 1, except that the glass tube with a diameter of 3 mm and a length of 5 cm was used in the camera 10 of clotting cassette type, each of the syringes 20 and 21 was connected to syringe pumps driven by electric motors, and instead narrowed part 14 and collagen deposited in the form of a coating in the quality of the material, causing a blood clot 15, a narrowed part 14 (figa) were attached with glue a piece of silk with a length of 1.5 cm, immersed in the collagen of example 1 and dried at 4°C, the quality of the material, causing a blood clot 15, on the inner surface of the camera 10, the formation of thrombus, which has a size of 5 cm inward from the fitting of the glass tube and the inlet tube 11.

After the flow of solution into the syringe 20 with a speed of 3 ml/min and the solution In the syringe 21 with a speed of 0.3 ml/min for 15 min, the camera 10 of clotting was disconnected and its inner surface is then washed with physiological saline solution. As a result, visual observation confirmed that the thrombus 16 length of about 1 cm and a thickness of about 1 mm in the form of a comet (fig.2b), the which, seems to be formed with the downstream side of the silk as a starting point and spread down the stream, was attached to the inner surface of the glass tube. Consider that the influence of blood flow can cause a blood clot in the form of a comet.

The widest part of the thrombus had a width of about 3 mm

Example 4

The device 1 monitoring of clotting produced in a manner analogous to example 3, except that instead of silk (figa) 50 ml of blood without anticoagulant treatment was buried on the inner surface of glass tubes Pasteur pipette and then left to stand at room temperature for 15 min, thereby obtaining a blood clot in the form of a disc with a diameter of about 2 mm as causing a blood clot material 15.

In a manner analogous to example 3, after the flow of solution a and solution B, the camera 10 of the formation of a blood clot was removed and then its inner surface was rinsed with physiological saline solution. As a result, visual observation confirmed that the inner surface of the glass tube was attached thrombus 16 length of about 1 cm and a thickness of about 1 mm in the form of a comet (fig.3b), passing in the direction downstream, at the same time inverting the clot as causing education material is 15. Consider that the influence of blood flow can cause a blood clot in the form of a comet.

The widest part of the thrombus had a width of about 3 mm

Example 5

The device 1 monitoring of clotting produced and the formation of a blood clot is then controlled in a manner analogous to example 3, except that in the solution And, in addition, was added Argtroban (registered trademark, manufactured Daiichi Pharmaceutical Co., Ltd.) as a means of anticoagulation treatment in a concentration of 0.1 mg/ml relative to the solution a of example 3.

As a result, visual observation did not confirm the presence of a blood clot in a glass tube after washing with physiological saline solution.

Example 6

The device 1 monitoring of clotting produced and the formation of a blood clot is then controlled in a manner analogous to example 3, except that subject to the anticoagulant-treated blood was obtained by addition of hirudin concentration of 1 μg/ml relative to whole blood and used as solution a, And a polyclonal antibody against hirudin (manufactured by COSMO BIO CO., LTD.), dissolved in a concentration of 1 mg/ml in physiological saline solution, relative to blood, was used as solution C.

In this case it was proved by almost the same blood clot, as in when the ore 3.

Example 7

Received 10 ml of whole blood (solution A), which was subjected to anticoagulation treatment adding to the blood immediately after the taking of the blood sample of 10 mg/ml Aprotinin and 1 μg/ml of recombinant hirudin (manufactured by Wako Pure Chemical Industries, Ltd.); and 1 ml of a solution (solution B), which was obtained by adding polyclonal antibodies against hirudin (manufactured by COSMO BIO CO., LTD.), removed domain Fc immobilized papain resin to 1000-fold diluted with physiological saline solution 1 vial/ml thromboplastin reagent (manufactured by Sysmex Corporation) as a drug to stimulate blood clotting.

For the fabrication of device monitoring the formation of thrombus used substrate 100 (figure 4), which is made of polydimethylsiloxane width 40 mm, length 70 mm and thickness of 1.5 mm, and the substrate 200, is shown in figure 5, which has a thickness of 1 mm and made of glass of the same size.

The path containing the camera of clotting 110, formed by cutting a groove depth of 0.5 mm on the surface of the substrate 100 (figure 4). The deep part of the groove, which shall be provided in the form of an inlet tube 111 is 1 mm, the length of the circuit 100D is 30 mm Through hole diameter of 1 mm formed on the end of the groove and presented in the form of regulating valve 100 is, which is actuated and closed by a cap. The camera 110 of clotting has a length of 30 mm and a width of 2.5 mm in the wider portion and a narrowed portion 114 having a width of 0.5 mm and Then provided with a through hole diameter of 1 mm as each of the connecting parts 100A and 100B. In addition, a through hole with a diameter of 2.5 mm is provided in the form of a connecting part 100C.

The collagen in the form of a tape with a width of 10 mm was applied as causing a blood clot material 115 on the substrate 200 in the position covering the narrowed portion 114 of the substrate 100 (figure 5). Deposition of collagen is carried out so that the attached rectangular masking tape corresponding to the narrowed portion 114 of the substrate 200, and then it is covered by desorbers agent based on silicone SRX-211 (Dow Corning Toray Co., Ltd.). Subsequently, the masking tape is peeled. Then made the instillation of a solution of collagen type 1 (Wako Pure Chemical Industries, Ltd.), dissolved in 0.1 N acetic acid to 1% it was free from Stripping agent, and then left to stand for 1 h at 25°C. After that, the collagen layer Stripping agent was washed with purified water and collagen inflicted only on a rectangular glass pane corresponding to the narrowed portion 114, where deformirujuschij agent was not applied.

Subsequently, the surface at which Oki 200 coated with collagen and the surface of the substrate 100 formed with the circuit connected together face to face.

The connecting portion 100A and 100B are connected with silicone tubing having an inner diameter of 1 mm from the rear surface (free from circuit side) of the substrate 100, and then are connected respectively with 10-ml syringe filled with solution A, and 1-ml syringe, filled with a solution Century. Each of these syringes respectively connected to syringe pumps. It should be noted that the other connecting portion 100C is connected to a silicone tube having an inner diameter of 2.5 mm, which is provided as the discharge tube.

A solution is injected from the connecting portion 100A at a flow rate of 0.3 ml/min Solution injected from the connecting part 100 at a flow rate of 0.03 ml/min

Solution a and solution b were given the opportunity to flow respectively from the connecting part 100A and the connecting part 100 V for 5 min with Physiological saline solution was injectively of the connecting portion 100A to launder blood. As a result, the formation of a blood clot is confirmed mainly on region a and region b (6) on the treated collagen surface of the substrate 200.

Example 8

The device monitoring the formation of a blood clot produced and the formation of a blood clot is then controlled in a manner analogous to example 7, with the following exceptions: a solution was obtained so that the blood of poslusate sample of blood was added polyclonal antibody against factor XII (manufactured by COSMO BIO CO., LTD.), from which papain was isseen Fc domain, at a concentration of 0.3 mg/ml and nefrackzionirovannam heparin porcine origin (Wako Pure Chemical Industries, Ltd.) dose of 0.3; the solution was obtained by dilution of thromboplastin reagent (manufactured by Daiichi Pure Chemicals Co., Ltd.) 50 times; deposition of collagen was performed on the pre-treated area of transparent polystyrene substrate 200 (1 mm thick) coated with collagen (Fig) by dripping a solution obtained by dissolving potassium permanganate in concentrated sulphuric acid. At a concentration of 2 g/l, subjecting interaction at 25°C for 10 min followed by rapid washing with purified water, collagen was applied by dripping the solution in which the collagen type 1 (Wako Pure Chemical Industries, Ltd.), dissolved in 0.1 N acetic acid, was dissolved in a concentration of 1% on the pre-treated area and left to stand at 25°C for 1 h followed by washing with purified water, and the substrate 100 without constricted part used for camera 110 the formation of a blood clot (Fig.7). It should be noted that the connecting portion 100C of the substrate 100 has a through hole with a diameter of 1 mm, is connected to a silicone tube with an inner diameter of 1 mm as a discharge tube.

After the flow of solution a and solution b for 10 min with physiological saline solution was injectively from connecting cha is ti 100A to flush blood, through this, confirming the adhesion of the red blood clot with a channel part of the area of the substrate 200 is coated with collagen.

Example 9

The device monitoring the formation of a blood clot produced and the formation of a blood clot is then controlled in a manner analogous to example 7, except that:

a solution was obtained so that the blood immediately after the taking of the blood sample was subjected to anticoagulation treatment by adding Aprotinin at a concentration of 0.05 mg/ml and recombinant hirudin (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 1 μg/ml and then added chinacan (Sigma Co., Ltd.);

the connecting portion 100B closed cap and the only solution then was injectively of the connecting part 100A with a flow rate of 1 ml/min

When observing a fluorescent stereoscopic microscope is focused on the surface of the glass substrate 200 is coated with collagen, expression of fluorescent light green hendrina was confirmed on part of the surface coated with collagen, and this area was distributed over time in the form of blotching. After 10 min the expression of green fluorescent light, which could be caused by the adhesion of platelets was confirmed in the greater part of the surface coated with collagen.

Example 10

Device monitoring clicks the education clot produced and the formation of a blood clot then controlled way, same as example 9, except that the solution a was obtained without adding hineline.

The blood was injectively of the connecting part 100A with a flow rate of 1 ml/min When the tube connecting portion 100C clutched to overlap on 2 during the injection, the blood was immediately transferred to 20 mm from branching points in the circuit 100D, which was accompanied by increase of the internal pressure. Even after the opening of the connecting part 100C blood did not move from the 20-mm point, so it was possible to confirm the increase in pressure.

Example 11

The device monitoring the formation of a blood clot produced and the formation of a blood clot is then controlled in a manner analogous to example 8, except that:

added a polyclonal antibody against thrombin (COSMO BIO CO., LTD.), removed domain Fc papain, to a concentration of 30 μg/ml instead of heparin solution And example 8;

and then added to the solution In example 8 with thrombin of RISK at a concentration of 5 mg/ml

After the flow of solution a and solution for 15 min with physiological saline solution was injectively of the connecting portion 100A for washing away the blood. As a result, it was confirmed that the red thrombus was attached to part of the channel region of the substrate 200, processed collagen.

Example 12

The device monitoring the formation of thrombus, Isrotel the Wali and the formation of a blood clot then controlled way, similar to example 7, except that:

a solution was obtained so that 50 ml of blood, which immediately after taking the sample of blood was added to 0.5 U/ml heparin and 10 μg/ml Aprotinin, centrifuged at 800 rpm and platelet-rich plasma (PRP) was obtained from the supernatant, thereby obtaining a solution; and

the solution, in which the thromboplastin reagent at a concentration of 1 vial/ml (Sysmex Corporation) was diluted 30-fold with physiological saline, was used as solution C.

A blood clot in the constricted portion 114 controlled for 15 min, using a stereoscopic microscope. Confirmed the presence of multiple platelet clots, is attached around the constricted portion 114. It was also observed that the number of bunches was increased, and their size is also increased, and platelet clots repeatedly attached and detached.

Example 13

A blood clot was controlled by passing the solution a and the solution In a manner analogous to example 7, with the following exceptions:

a solution was obtained by adding 500 g of red blood cells collected from the centrifuged precipitate in 20 ml of PR solution a of example 12;

the solution, in which the thromboplastin reagent at a concentration of 1 vial/ml (Sysmex Corporation) was diluted 30-fold with physiological saline solution, used in the image quality is as the sample solution;

a glass slide was obtained by coating the entire surface of the substrate 200 desorbers agent SRX-211 (Dow Corning Toray Co., Ltd.) silicone-based; and

approximately 1 μl of whole blood were linked to surface about the end point of expansion of the expanding portion, subsequently spreading to a width of 2.5 mm, which is adjacent to the located upstream from the constricted portion 114 of the camera of clotting 110 on the substrate 100, and then whole blood was left to stand at room temperature for 10 min in order to cause its collapse, and pre-formed thrombus, intended for use as a material causing a blood clot.

The area of attachment of the thrombus controlled stereoscopic microscope for 15 minutes the Formation of new red blood clot, which was located downstream from the pre-clot as a starting point, it was confirmed after about 5 min, it was generally spread downstream in the period up to 15 minutes, and its width was increased to 2.5 mm, it Was confirmed that the blood flowed along the formed thrombus or flowed through the gaps between the formed thrombus.

Example 14

A blood clot was controlled by passing the solution a and the solution In a manner analogous to example 7, with the following exception is:

in 50 ml of blood immediately after the taking of the blood sample was added to the thrombin aptamer of associte I (GGTTGGTGTGGTTGG: SEQ ID No. 1) at a final concentration of 5 μm and obtained from maize trypsin inhibitor (COSMO BIO CO., LTD.) at a final concentration of 30 μg/ml and was centrifuged at 800 rpm for 10 min, and supernatant formed platelet-rich plasma (PRP) was provided as a solution A;

antisense DNA (CCAACCACACCAACC: SEQ ID No. 2) was dissolved in physiological saline solution so that its concentration was 150 μm, and provided in the form of a solution; and

during deposition on a glass slide inhibitor of clotting 115 solution, which was obtained by mixing a solution of collagen of example 7 with a solution obtained by dissolving 1 vial thromboplastin reagent (Sysmex Corporation) in 1 ml of purified water and then deliberately in the ratio of 5:1, Ngapali area without applied silicone and dried by air at 4°C.

A blood clot in the constricted portion 114 controlled within 5 min, using a stereoscopic microscope. It was confirmed the presence of multiple platelet clots, is attached around the constricted portion 114. The monitoring also showed that the number of bunches was increased with a simultaneous increase of their sizes, and platelet clots repeatedly attached and detached.

Note the R 15

The device monitoring the formation of a blood clot produced and the formation of a blood clot controlled in a manner analogous to example 14, with the following exceptions:

after receiving PRP same procedure as in example 14, immediately before the measurement was added to the antisense DNA (CCAACCACACCAACC: SEQ ID No. 2)so that its concentration was 15 μm, thereby realizing antithrombin processing, and the resulting solution was provided as a solution A;

the injection hole of the fitting 100V closed in a manner analogous to example 9;

collagen type I (collagen reagent to cover the Cup for cell culture collagen, the mother liquor of the type I-A, Cellmatrix Co., Ltd.) used instead of the solution of collagen of example 14; and

only solution was passed with a flow rate of 0.2 ml/min for 5 minutes

A blood clot in the constricted portion 114 controlled using a stereoscopic microscope. It was confirmed the presence of multiple platelet clots, is attached around the constricted portion 114. The monitoring also showed that the number of bunches was increased with a simultaneous increase of their sizes, and platelet clots repeatedly attached and detached.

Example 16

The device monitoring the formation of thrombus was produced in a manner analogous to example 7, with the following exceptions:

on the surface of the substrate formed with the groove depth of 0.1 mm cutting path (7), at the same time leaving the dividing wall so that the received concave series, each of length 1 mm and a width of 40 μm, passing across the surface of the lower part around the center of the camera of clotting 110 at intervals of between 40 μm;

the injection hole of the fitting 100V closed in a manner analogous to example 9; and

the substrate 200 was a glass slide without surface treatment. Gap (channel) between the dividing walls simulates blood capillary.

Whole blood immediately after the sample was subjected to anticoagulation treatment by adding derived from maize trypsin inhibitor at a final concentration of 30 μg/ml and associte I) thrombin at a final concentration of 10 μm. Immediately before the monitoring was added to the antisense DNA associte I of thrombin aptamer so that its concentration was 30 μm, followed by injection of 100A at a flow rate of 0.05 ml/min for 5 minutes

A blood clot in the gap between the dividing walls kontrolirowali using a stereoscopic microscope. As a result, the channels sequentially closed during monitoring within 5 minutes the Monitoring revealed that approximately half of the channel was closed educated tro the BOM.

Example 17

Used feeder liquid syringe type (Fig.9). The microchip 300, where the substrate and cover of the microchip (Fig.9(b) and Fig.9(C)) were made from the same materials as the materials of example 7, connected by compression. The microchip 300 includes an inlet tube 322 to an inhibitor of blood coagulation and the discharge tube 313, which were attached to the substrate 100, as in the case of the discharge tube of example 7. However, all of the grooves of the microchip 300 (Fig.9) were generated by a depth of 200 μm. They also made a channel so that the blood could be injected into the channel from a syringe for sample 320, the channel had a width of 1 mm and a length of 40 mm and was connected to the camera of clotting 311, consisting of a narrow channel having a width of 200 μm and a length of 10 mm, and an inlet tube for inhibitor of blood coagulation 322. The solution was prepared so that the contents of 1 vial of reagent RT Sysmex Corporation (tissue thromboplastin) was dissolved in 1 ml of purified water and then deliberately in purified water. Validirovannyj product in purified water was mixed with collagen type I (manufactured by Nitta Gelatin Inc.) in the ratio of 1:1. The solution was applied onto the surface of the material, causing a blood clot 315. After this part of the coated solution was dried at 4°C in the air and the camera 311 of clotting covered and used as a material causing coord the clot 315. Blood was taken after adding means anticoagulation treatment, so that the final concentration of the respective components in the syringe was 25 mcg/ml obtained from maize trypsin inhibitor, 10 µm for associte I of thrombin aptamer and 10 μm for eksotika II of thrombin aptamer (5'-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3': seq id No. 3). Immediately before the monitoring collected in this way the blood was added to the antisense DNA against thrombin aptamers of associte I and II so that each of them has reached a concentration of 40 μm. Then the syringe (syringe 1 ml, manufactured by Terumo Corporation) was filled with blood and was provided as a syringe for sample 320, shown in Fig.9. My Flow (manufactured Arbiotec Co., Ltd.), which is a pump micropolicy capable of controlling the internal pressure of the feed liquid pump, it is connected with a pipe for supplying pressure 317. Mineral oil was pressed into the microchip 300 from the top of the syringe 320, and the blood would come in the microchip 300 a flow rate of 50 ál/min while controlling the pressure exerted on the syringe for sample 320.

As an inhibitor of coagulation from the inlet tube 322 for inhibitor of blood coagulation was applied 1 M Tris-HCl (pH 10) with a flow rate of 50 ál/min Pressure began to grow after 6 min after the start of measurement of pressure.

The pressure surface is raushee it changes in the direction of increase or decrease due to the movement of a blood clot ("control", shown in Fig), rose to 80 kPa.

Example 18

The taking of blood samples was performed with the addition of anticoagulation treatment in a manner analogous to example 17. Then carried out the same procedure as the procedure of example 17, except that nefrackzionirovannam heparin was added at a concentration of 1 U/ml Then there was the pressure measurement. The pressure was increased to approximately 10 kPa ("Heparin", shown in Fig).

Example 19

The taking of blood samples was performed with the addition of anticoagulation treatment in a manner analogous to example 17. Then carried out the same procedure as the procedure of example 17, except that the added ReoPro (LILLY Co., Ltd.) at a concentration of 50 µg/ml Then there was the pressure measurement. When measuring for 18 min, it was impossible to confirm the increase in pressure ("ReoPro"shown in Fig).

Example 20

The taking of blood samples was performed with the addition of 3.2% citric acid so that the ratio between citric acid and blood was 1:9. Then added blood obtained from maize trypsin inhibitor at a concentration of 25 μg/ml for holding anticoagulation treatment. Immediately after the addition of calcium chloride in the blood subjected anticoagulation processed so that its concentration reached 15 mm relative to the blood, lead is bent anticoagulation processing during monitoring of clotting, the blood was added into the syringe (syringe 1 ml, manufactured by Terumo Corporation) and then flow into the blood in the same microchip as the microchip in example 17, at a flow rate of 40 µl/min Then the pressure change was measured using the same device that in example 17. The pressure began to increase after 15 min after the start of measurement of pressure, and it grew to about 60 kPa.

Example 21

The taking of blood samples was performed with the addition of anticoagulation treatment in a manner analogous to example 20. Then carried out the same procedure as the procedure of example 20, except that the heparin was added respectively at concentrations of 0.2 U/ml, with 0.5 U/ml and 1 U/ml Then there was the pressure measurement. The results are shown in Fig.

Example 22

The taking of blood samples was performed with the addition of anticoagulation treatment in a manner analogous to example 20. Then carried out the same procedure as the procedure of example 20, except that the added ReoPro (LILLY Co., Ltd.) at a concentration of 2 µg/ml Then there was the pressure measurement. The pressure began to grow after 20 min after the start of measurement of pressure, and it grew to about 15 kPa after 25 min (Fig).

Example 23

Used feeder liquid syringe of the type shown in Fig. In addition, microchip 400, where the substrate 200 and the cover mi is recipe and the substrate 100 in the case of the microchip, shown in Fig () and Fig(C), were connected by compression. The discharge tube 413, made of Teflon (registered trademark), was filled with mineral oil and one end connected with the chip 400 through the connection tube 412 made of silicone rubber, and its other end connected with a suction pump 414, in which was inserted a pressure sensor. The inlet tube forming the channel of the microchip 400, connected with the syringe 420 through the connecting tube 412 made of silicone rubber. One end of the intake tube for inhibitor of blood coagulation 422 made of Teflon (registered trademark), connected with the pump liquid supply 423 other end of which is connected with the end of the camera the formation of thrombus through the connecting tube 412 made of silicone rubber. Thus, the microchip 400 connected to the exhaust pipe 413, syringe 420 and an inlet tube for inhibitor of blood coagulation 422, received the device In monitoring the formation of thrombus. A computer controlled camera 430, supplied by the optical light source 432 (light emitting diode), which was established and was able to move on the guide 433, established under the camera of clotting.

All of the grooves on the microarray had a depth of 120 μm.

The channel, which was rst is s blood, had a width of 800 μm and a length of 7 mm, Narrowed the channel had a width of 200 μm and a length of 10 mm

The solution was received so that the contents of 1 vial of reagent RT (tissue thromboplastin, Sysmex Corporation) was dissolved in 1 ml of purified water and then deliberately in purified water, validirovannyj product in purified water was mixed with collagen type I (manufactured by Nitta Gelatin Inc.) in the ratio of 1:1. Then the solution was applied to a region of the substrate 200 microchip facing the channel narrowed portion on the surface, subject to connection by squeezing with the substrate 100 of the microchip. After this part of the coated liquid was subjected to vacuum drying at 4°C, and then the substrate 100 and the substrate 200 connected by squeezing with each other, providing microchip 400 having the chamber 411 of clotting.

Sampling of blood was performed using a vacuum vessel for blood collection, which was concluded sodium citrate as anticoagulant funds. Just before the introduction of the syringe 420 in the blood was added calcium chloride at a final concentration of 12.5 mm and obtained from maize trypsin inhibitor at a final concentration of 25 μg/ml

My Flow (manufactured Arbiotec Co., Ltd.), which is a pump micropolicy capable of controlling the internal pressure of the feed liquid pump was connected to the exhaust pipe 413 as n is Sosa 414. Thus, the absorption was performed with a speed of 15 ál/min while controlling the internal pressure in the channel.

As an inhibitor of coagulation 1 M Tris-HCl (pH 10) was passed from the inlet tube to an inhibitor of blood coagulation 422 at a flow rate of 8 μl/min Education mainly white thrombus was observed after 4 min from the start of measurement of pressure, as was confirmed by the decrease of the internal pressure. In addition, it was confirmed that the internal pressure decreased to 30 kPa in 10 min from the beginning.

Further, the formation of thrombus was observed images when the camera is moving around 430 education clot in the channel. The position in which we observed formation of a blood clot, was memorable, and the camera 430 received image with regular movement around a set of specific points. As a result, over time was recorded the growth and destruction of many large blood clots.

Next will be described made reference experiments to demonstrate the effectiveness of thrombin aptamers and antisense DNA as a means respectively anticoagulation treatment and means of implementing anticoagulation effect.

Control experiment 1

Was given the blood immediately after its capture, and blood obtained by adding one is th tenth volume of 300 μg/ml obtained from maize trypsin inhibitor in the first portion of the blood, and blood obtained by adding thrombin aptamer that recognizes ecsatic I, in the second portion of the blood that was obtained concentration of 5 μm. Then, for these three kinds of blood in Eppendorf tubes (made of polypropylene) was measured by the time required for coagulation. The test tube with the sample turned up-side down every minute, and confirmed its fluidity. Blood without additives showed the clotting time of 9 minutes on the Contrary, the blood with the addition of an inhibitor of trypsin showed clotting time 22 minutes Later, the blood after the addition of thrombin aptamer showed clotting time 62 minutes

Further, when 5 μm thrombin aptamer that recognizes ecsatic I, and 5 µm thrombin aptamer that recognizes ecsatic II, used in combination, blood clotting was not confirmed even after 3 h even in the absence of blood inhibitor of trypsin.

The thrombin aptamer that recognizes ecsatic I: 5'-GGTTGGTGTGGTTGG-3' SEQ ID No. 1,

The thrombin aptamer that recognizes ecsatic II: 5'-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' SEQ ID No. 3).

Control experiment 2

Measurement of ART was carried out on 200 µm plasma relative to each of the sample And with the addition of 10 μm physiological salt solution and the sample with the addition of 10 μm to 500 μm thrombin aptamer that recognizes ecsatic I, and the sample with the addition of 10 μl of a solution containing 500 μm up the Amer thrombin, recognize ecsatic I, and 1500 μm antisense DNA thrombin aptamer that recognizes ecsatic I. ART sample And amounted to 44, the sample In 2 min or more and a sample of 45 C.

Control experiment 3

11(A) represents the analysis of the waveform collapse on thromboelastogram during storage of blood with the addition of 10 μl of aptamers to associte I and associte II at room temperature for 15 min, and then adding 40 μm antisense DNA of both aptamers. 11(B) represents the waveform of thromboelastogram blood immediately after the taking of the blood sample.

As is evident by the results presented figure 11(a) and 11(B), it was found that the addition of two kinds of thrombin aptamers provides the ability to store blood, and antisense DNA may exercise its anticoagulation treatment.

According to the results of control experiments 1-3 it is obvious that the combination obtained from maize inhibitor of trypsin and thrombin aptamer can effectively inhibit the coagulation of whole blood, and that the anticoagulant effect of thrombin aptamer can inactivate antisense DNA to the thrombin aptamer.

The control experiment 4

Next will be described the efficiency impacts of pavement, RMEA during storage of blood and the adhesion of platelets to the substrate.

(1) a Solution of methanol containing 1% of RMAA, Paul is obtained in accordance with JP 04-152952 A, put on a 2.5-ml container of acrylic resin (internal dimensions: 1 cm × 1 cm × 4.5 cm) and then dried at 90°C for 10 minutes In the future, 760 μl of blood subjected to anticoagulation treatment 2% citric acid was added to each not covered container and the container is covered with RMAA, followed by aspiration with a pipette every 5 min to confirm the clotting of blood.

Consequently, uncovered container showed an obvious increase in viscosity after 30 min and collapse after 60 minutes In contrast, the container is covered with RMAA, showed an obvious increase in viscosity after 35 min, but the time of onset of coagulation lengthened to 90 minutes

(2) the Specified RMEA flat layer was applied on a transparent acrylic plate. It was used instead of the coated collagen plate of example 9 (Fig), and the experiment adhesion labeled chinachina platelet and white blood cell count was performed in a manner analogous to example 9.

As a result, the apparent adhesion or agglutination of platelets and leukocytes was observed after 10 min from the beginning of blood flow. In contrast, adhesion or agglutination of platelets and leukocytes on the acrylic plate, covered with RMAA, was not observed even after 30 minutes

According to the above results in the device monitoring of clotting according to the present invention by coating RMEA syringes on the I storage of blood, tube, the substrate and such surfaces can suppress the formation of a blood clot at a location other than the camera the formation of thrombus. Therefore, it is assumed that can be achieved in the monitoring of clotting specific to the camera monitoring the formation of thrombus.

In the above description the present invention has been described with reference to preferred options for implementation. However, the present invention is not limited to the examples and the methods of implementation described above, and it is possible to apply various modifications, without departing from the essence of the present invention. For example, you can install the high pressure created by the pump, and install a small diameter discharge tube. In this case, in the inlet tube and outlet tube may create back pressure, so you can monitor the formation of thrombus at the same time controlling the volume flow in the load conditions of the internal pressure corresponding to the pressure of the blood. Further, the velocity of blood flow at this time can be freely controlled by the pressure generated by the pump, and the degree of throttling of the discharge tube.

Industrial applicability

The device monitoring the formation of thrombus and the method of monitoring of clotting according to the present invention can be ospedalesorsola with a comprehensive assessment of coagulation and formation of platelet thrombus in the environment, equivalent to the flow, using whole blood or plasma containing the platelets to assess the effectiveness of antithrombotic drugs, administered to the patient, or the like.

1. The device monitoring the formation of thrombus by passing subjected to anticoagulation treatment of blood through the channel, simulating a blood vessel, and while anticoagulation treatment or stimulation of blood coagulation, containing:
the camera on the formation of a blood clot, at least part of which is placed the material, causing a blood clot,
the inlet tube which is connected to the camera the formation of thrombus and through which the blood flows into the chamber of clotting,
the container for the blood flow connected to the inlet tube,
the supply pump to the container,
the pressure sensor designed to measure the pressure applied to the container.

2. The device according to claim 1, additionally containing an inlet tube for feeding inhibitor of clotting connected with camera of clotting and providing mixing inhibitor of clotting of blood after passing through the chamber the formation of thrombus.

3. The device according to claim 1, additionally containing a tube for the supply of a medicinal product that is connected with the inlet pipe, and through this the felling served drug, providing anticoagulation treatment or drug that stimulates the clotting of blood.

4. The device according to claim 1, in which the specified tube and the chamber of clotting combined with each other on a substrate and represents a diagram of the microchip.

5. Device according to any one of claims 1 to 4, in which the material, causing a blood clot, contains collagen.

6. The device according to claim 5, in which the material, causing a blood clot, contains tissue factor.

7. Device according to any one of claims 1 to 4, which further comprises a camera for obtaining images of clotting.

8. Method of monitoring the formation of thrombus, namely, that sent the blood after injection of anticoagulant from the container into the chamber of clotting by pressure on the liquid, having a density less than that of the layer of blood placed on the layer of blood that you are carrying out anticoagulation treatment of blood or stimulate the clotting of blood,
determine the pressure applied to the container in which the material, causing a blood clot, placed in at least part of the chamber of clotting.

9. The method according to claim 8, in which subjected to anticoagulation treatment blood obtained by use of an inhibitor of factor of the contact phase and the calcium chelator, and found antique is agulation treatment is carried out by the donor of free calcium.

10. The method according to any of PP and 9, in which the quality of the material, causing a blood clot, use collagen and tissue factor.

11. The method according to claim 8, in which monitor formation of thrombus through the use of the device according to any one of claims 1 to 4.



 

Same patents:

FIELD: medicine.

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

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

1 tbl, 4 dwg

FIELD: medicine.

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

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

4 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: for thrombin production measurement, a layer of said sample contacts with a fluorogenic substratum of thrombin where the thickness of said layer is 0.05 to 5 mm, while the surface area is 10 to 500 mm2. Further, the thrombin production environment in said sample is provided. It is followed by measuring the fluorescence emitted from the layer surface by a fluorescent group released by the fluorescent substratum as a result of an enzymatic action of produced thrombin on said fluorogenic substratum. Besides, the invention ensures a kit for measuring the thrombin activity in the sample.

EFFECT: higher measuring accuracy.

29 cl, 12 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: blood is examined. A hematocrit level (H), erythrocyte count (E), thrombocyte count (T) are determined. Said parametres are evaluated. In the event if they keep within the determined limits for the patients with acute coronary syndrome (ACS), then adenosine phosphate induced (ADP-induced) clotting time test samples are prepared. Citrated blood sample 0.4 ml is prepared of whole blood and divided on two samples 0.2 ml. Each of these samples is introduced in a measuring cell, recalcified at temperature 37°C for 2 minutes. Then a magnetic ball mixer is placed in each cell. The measurement is activated, and in three seconds the ADP solution 0.1 ml is introduced. After a clotting reaction, a process time duration is recorded separately for each sample. An arithmetical mean of the derived values is calculated (A). The derived values of each of said parameters are scored. Total score Σ=A+H+E+P shows the risk of recurrent thrombotic events. If Σ=4 points, the low risk is observed; the value Σ=5-6 points shows the medium risk, while Σ=7-10 points - the high risk.

EFFECT: method provides more objective risk evaluation of recurrent thrombotic events in the patients with ACS with its simplicity and low cost.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: blood sample is placed in capillary, in whose walls installed are electrodes connected to frequency generator and registering unit, blood electric conductivity is measured at the moment of passing through it of alternating current with frequency 200 Hz, electric coagulogram is registered and used to determine chronometric and amplitude characteristics: A - amplitude of functional curve decline, mV; N - time of functional curve decline to minimal value in minutes. If value of A/T index decreases or increases with respect to normal, conclusion about hemostatic disorders is made. If value of A/T index equals 3-5 - hemostasis state is evaluated as normal, if A/T value is lower than 3, hypocoagulation is determined, and if A/T value is higher than 5 - hypercoagulation.

EFFECT: application of the method makes it possible to obtain data about hemostasis system state in real time mode, without injuring form blood elements in investigated microvolumes of blood, thus making it possible to increase accuracy, self-descriptiveness and efficiency of hemostasis state evaluation and to carry out correction of performed therapy without delay.

4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: in patients with IHD before therapy with acetylsalisylic acid (ASA) ADP-induced and ASA-dependent platelet aggregation are examined and by their difference value of coefficient of aggregation inhibition (CAI) is calculated. CAI value <24% testifies to resistance to ASA, if CAI ≥24% - about sensitivity to ASA.

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1 tbl, 4 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology, and orthopaedics. The intravascular thrombocyte activity correction in children with scoliosis requires the prescription for at least 6 months of a therapeutic complex which involves daily therapeutic physical training, two courses of massage and daily swimming for at least 20 minutes a day.

EFFECT: method allows to normalise intravascular thrombocyte activity in children aged 8-12 with scoliosis, to improve tissue microcirculation considerably, to optimise growth and development of skeleton and internals.

7 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably to haematology and orthopaedics. For thromboplastin synthesis correction in children 8-12 years old with scoliosis is prescribed complex of treatment, including daily remedial gymnastics, two courses of massage and daily swimming for at least 20 minutes a day, during 6 months.

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7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to aggressive medical therapy, resuscitation science, critical care medicine, laboratory diagnostics and can be used by resuscitators, intensivists, laboratory doctors for well-timed diagnosis and consequently, for individualised aggressive medical therapy of acute disseminated intravascular coagulation. The integrated assessment of links of haemostatic system and the clinical appraisal of organ dysfunction are applied in a measurer, and when observing structural hypercoagulation characterised by fibrinogen level increase, thrombocyte activity increase, growth of soluble fibrin complex (SFC) level, and also when observing chronometric hypercoagulation characterised by time tests, palette-derived factor 4 activity (P4) with manifested petechial haemorrhage and organ dysfunction, and coagulation cascade activation with underlying depression of antithrombin III and protein C, a hypercoagulation stage of acute DIC is diagnosed. Chronometric hypercoagulation by Activated partial thromboplastin time (APTT), INR, fibrinogen and P4 with manifested signs of structural hypocoagulation by thrombin time prolongation and D-dimer activity increase with underlying further intensification of anticoagulant system deficiency, progression of target organs dysfunction and mixed haemorrhage show a transitive stage of acute DIC. If observing said hypocoagulation changes and disturbed fibrinolytic activity with prevailing either decompensated organ and tissue dysfunction, or hemorrhagic syndrome up to system haemorrhages, or their combination with hemorrhagic syndrome characterised by polymorphism of clinical picture and localisation: petechial-haematoma haemorrhage at the stress-induced stomach ulcers, hematuria, a coagulopathy stage of acute DIC is diagnosed that is characterised either by depression of fibrinolysis, or preserved fibrinolytic activity, or by activation of secondary fibrinolysis, or by acute primary fibrinolysis.

EFFECT: method allows optimising classification of acute disseminated intravascular coagulation, improving diagnostic significance of the classification and simplifying a diagnostic prospecting which provide a basis to consider the staging of acute disseminated intravascular coagulation.

1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology and orthopaedics. Correction of blood microvesicle level in children aged 8-12 with scoliosis is ensured by the prescription of a therapeutic complex that involves daily therapeutic physical training, two courses of massage and daily swimming for at least 20 minutes a day, for at least 6 months.

EFFECT: method enables correcting blood microvesicle level in children aged 8-12 with scoliosis, optimising microcirculation in tissues, normalising functions of the internal organs, growth and development of a child.

7 ex

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the suggested studying should be carried out on the glass simultaneously with several inductors by applying minimal inter-taking antilogarithms concentrations of aggregation inductors which correspond at double combination of inductors: ADP 5.0 x 10-8 M, adrenaline 3.0 x 10-9, collagen - dissolving the main suspension 1:8, thrombin 0.075 U/ml; at triple combination of inductors: ADP 10-9 M, adrenaline 10-9, collagen - dissolving the main suspension 1:9, thrombin 0.060 U/ml. The development of aggregation means thrombocytic activation in patients with arterial hypertension at metabolic syndrome. The method enables to evaluate the changes of thrombocytic functional state with combination of inductors more probably present in area of vascular lesion by applying minimal necessary concentrations that develops real conditions at hemostatic initiation in human vessels.

EFFECT: higher efficiency of studying.

3 dwg, 3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: method involves checking consciousness, blood coagulation state, peripheral blood leukocytes number, K+ ions, bilirubin, fibrinogen, hemolysis and hemoglobinuria availability, prothrombin index and exotoxic shock development. Each value is calculated in points as follows. Lucidity is evaluated as -2 points; depression - +3 points; coma - +6 points; lack of changes in blood coagulation system - -2 points; coagulation availability without clinical injuries - +2 points; coagulopathy with clinical manifestation signs - +19 points; K+ ions concentration being less than 3.0 mmole/l - +3 points, from 3.1 to 3.5 mmole/l - -5 points, from 3.6 to 5.0 mmole/l - 0 points, greater than 5.0 points - +7 points, failure in determining K+ ions concentration - 0 points; hemolysis availability - +6 points, its lack - -3 points; hemoglobinuria availability - +8 points, its lack - -1 points; leukocytes number being less than 12.0x109/l - -2 points, from 12,1 to 18.0x109/l - 0 points, higher than 18.0x109/l - +8 points; hourly urine output being less than 30 ml/h - +6 points, greater than 30 ml/h - -2 points; bilirubin content being less than 31 mcmole/l - -2 points, from 30.1 to 50.0 mcmole/l - 0 points, greater than 50.0 mcmole/l - +2 points, failure in determining bilirubin content due to hemolysis being available -+6 points; prothrombin index being equal to or less than 60% - +3 points, greater than 60% - 0 points, failure in determining prothrombin index due to hemolysis being available - +12 points; fibrinogen concentration in blood plasma being less than 2.1 g/l - +4 points, from 2.1 to 4.0 g/l - -1 point, from 4.1 to 6.0 g/l - +1 point, failure in determining fibrinogen concentration due to erythrocyte hemolysis being available - +13 points; exotoxic shock development - +9 points, its lack - -1 point. The points are summed up. The value being greater than +13, admission for treatment in resuscitation department is indicated. The value being less than -13, admission for treatment in therapeutics department is indicated. The value being from -13 to +13, resuscitation expert consultation is advised.

EFFECT: high evaluation accuracy.

3 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: one should evaluate the time for clotting of plasma under testing in phospholipid-dependent test, moreover, one should apply high- and low-sensitive thromboplastin reagents to lupus anticoagulant to calculate the ratio of indices of prothrombin time prolongation and at its value being either equal to or above 1.1 one should diagnose APS.

EFFECT: shortened terms of research.

1 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: method involves analyzing symptoms manifesting initial disseminated intravascular blood coagulation syndrome danger like burn area, availability of upper air passages burn, shock with its severity degree taken into consideration, sepsis development; clinical manifestations of disseminated intravascular blood coagulation syndrome like lung, kidney, liver function insufficiency, cerebral dysfunction, local and multiple hemorrhages, thrombosis, infarction; homeostasis system laboratory analysis data, hyper- and hypocoagulation based on chronometry test data, number of blood platelets, fibrin-monomer complexes, D-dimers, activity of antithrombin III, C and S proteins, XIIa-dependent fibrinolysis plasminogen content, availability of injured erythrocytes, combinations of laboratory tests for recognizing disseminated intravascular blood coagulation syndrome. Each sign under consideration receives a number of points corresponding to its diagnostic significance and integral value is calculated DIBCSIV=(X1+X2+…+Xn)/n, where n is the number of signs taken into consideration. DIBCSIV value equal to 1.0-1.5 units shows physiological norm. The value being between 1.6 and 2.5 units, light disseminated intravascular blood coagulation syndrome is diagnosed. The value being between 2.6 and 3.5 units, disseminated intravascular blood coagulation syndrome of medium severity is diagnosed; 3.6-4.5 points to one heavy severity degree; 4.6 and greater indicates highly severe case of disseminated intravascular blood coagulation syndrome.

EFFECT: high accuracy and objectiveness in differentiating syndrome severity degrees.

1 tbl

FIELD: medicine, diagnostics.

SUBSTANCE: one should study blood components to detect anticoagulant-fibrinolytic activity. Moreover, patient's blood should be sampled: in whole blood one should detect the presence of affected erythrocytes and evaluate the quantity of thrombocytes, in plasma it is necessary to study the activity of antithrombin III, XIIa-dependent fibrinolysis, the content of soluble fibrin-monomeric complexes, in blood serum of the sample taken one should detect the concentration of urea, creatinine, sodium, albumin, total cholesterol and the activity of aspartate aminotransferase, moreover, one should calculate integral value of renal-hepatic deficiency, to put corresponding point for the degree of parameters under testing, then one should calculate integral value of disseminated intravascular clotting (IVDIC) and at its value being 6.3 U and more DIC-syndrome should be diagnosed, moreover, at IVDIC value ranged 6.3-10.1 U it is possible to diagnose latent DIC-syndrome, at 10.2-14.6 - subacute DIC-syndrome and at 14.7 and higher - acute DIC-syndrome should be concluded.

EFFECT: higher accuracy and efficiency of diagnostics.

4 ex, 2 tbl

FIELD: medicine, obstetrics.

SUBSTANCE: the present innovation deals with predicting disadaptive processes in women in dynamics of menstrual cycle. During menstrual cycle beginning since the 1st d to the 21st d one should detect the dynamics for alteration in coefficient of activity of syntoxic adaptation programs (CASAP), calculated by the following formula:

where CST - concentration of blood serotonin, AAT-III - activity of antithrombin III, Aaoa - total antioxidizing activity of plasma, CCD8+ - concentration of T-suppressors, Cad - concentration of blood adrenalin, Cα2MG - concentration of α2-macroglobulin, CMDA - concentration of malonic dialdehyde, CCD4+ - concentration of T-helpers. Moreover, normally CASAP value alters two-fold against the first day of the cycle - since 0.70 up to 1.40 on the 21st d of the cycle, at no alterations in CASAP value one should diagnose female disadaptive alterations leading to failed pregnancy. The innovation enables to perform diagnostics of disadaptive processes in women in dynamics of menstrual cycle followed by prognostic conclusion upon future pregnancy.

EFFECT: higher accuracy of diagnostics.

2 ex

FIELD: medicine.

SUBSTANCE: method involves determining spontaneous blood platelets aggregation and one induced by adrenalin and collagen, thrombocytospecific peptides activity of β-thromboglobulin and thrombocytic factor 4 in blood plasma.

EFFECT: high accuracy of diagnosis.

2 tbl

FIELD: medicine.

SUBSTANCE: method involves determining coagulating blood viscosity values like reaction period r, thrombin constant K, maximum amplitude MA, time T for forming fibrin-thrombocytic blood clot, spontaneous blood platelets aggregation intensity Ar, retraction and spontaneous clot lysis total FA. The r being within 5-7 min, Ar from -2 to -6 relative units, K being within 4-6 min, MA within 500-700 relative units, T within 40-60 min and FA equal to 10-20%, low inflammatory process activity is considered to be the case. The r being less than 5 min, Ar equal to -8 to -12 relative units, T less than 40 min and FA less than 10% with no changes in K and MA being observed, inflammatory process activity in chronic glomerulonephritis case is considered to be of high severity degree.

EFFECT: high accuracy of diagnosis; enhanced effectiveness of treatment method selection.

1 dwg

FIELD: medicine, clinical neurology, neurosurgery.

SUBSTANCE: one should study both activation and aggregation of thrombocytes in blood of carotid artery, at the quantity of thrombocytic active forms being above 70% and the number of aggregated thrombocytes being above 9.0% one should predict the development of cerebral ischemic lesion along with stable focal neurological symptomatology, and at the quantity of thrombocytic active forms being below 30% and the number of aggregated thrombocytes being below 8.0% it is possible to predict positive dynamics in the course of the disease mentioned without developing cerebral ischemic lesion.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, clinical neurology, neurosurgery.

SUBSTANCE: one should study the level of von Willebrand's factor in patient's carotid artery blood. At its content being below 105% one should predict the development of repeated AICH. The innovation improved information value of testing due to possibility to obtain reliable prediction in latent period, as well.

EFFECT: higher accuracy of prediction.

2 ex, 1 tbl

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