RU2432577C2 - Thrombosis monitor and method of thrombosis monitoring - Google Patents

Abstract

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

Description

Technical field

The present invention relates to a method for monitoring the effectiveness of an antithrombotic drug administered to a patient or a similar individual, and in particular, to a device and method for the general assessment of blood coagulation and platelet formation in a medium equivalent to blood flow with whole blood or plasma containing platelets.

State of the art

For example, atherothrombosis (such as with myocardial infarction) causes a serious blood clot, so that the atheromatous plaque is destroyed in the area of arteriosclerosis, platelets adhere to collagen, including tissue factors in contact with the bloodstream. Further, complex processes of platelet aggregation, activation of the blood coagulation system and the like occur, leading to the formation of a blood clot, which causes severe obstruction of the vessel. Heart disease, such as myocardial infarction, is a serious illness, and it is the second leading cause of general mortality in Japan.

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

To date, blood coagulation has been evaluated by determining the partial activated thromboplastin time (ARTT), thromboplastin time (PT) using plasma. ARTT mainly reflects endogenous coagulation, and PT mainly reflects exogenous coagulation. A study of blood platelets is carried out by using platelet-rich plasma and adding a platelet activating substance, such as ADP (adenosine diphosphate) or collagen, to thereby evaluate platelet coagulation as a result of a change in their penetration rate or similar phenomena. In addition, the clotting time of whole blood can be determined by the time of formation of a clot of whole blood, the time of formation of a clot of whole blood after repeated addition of calcium and the like.

Further, in examination devices using whole blood, a thromboclastogram is used that controls the activation of coagulation factors, platelet agglutination, and the like.

However, a blood clot grows in blood in vivo. On the contrary, the above research method or the like makes an in vitro determination, i.e. in a condition closed to the bloodstream. Thus, in vivo thrombus growth cannot be observed.

As suggestions for solving the above problems in the patent document JP 2004-251630 A and non-patent documents Blood 1990; 75, pp. 390-398; Blood, 1999; August, 1:94 (3), pp. 968-75, a method is disclosed comprising passing blood with an antithrombotic drug introduced into it through collagen and monitoring with a confocal microscope of adhesion or platelet agglutination labeled with a fluorescent label.

However, in the method described in said patent document, the observation is carried out in the presence of an anticoagulant drug. Thus, monitoring the morphological changes in platelets evaluates the fact that there is no blood clot formation in the blood coagulation system caused by platelet adhesion or agglutination, or the thrombus generation ability is reduced. Therefore, this assessment does not reflect the association of platelet activation with the blood coagulation system. Therefore, this invention is acceptable for evaluating the effectiveness of an antiplatelet drug, but it is not able to control the thrombus itself and the entire process of thrombus formation. In addition, a fluorescence microscope is expensive, so it can hardly be used for general research.

In addition, in JP 2006-145345 A, blood flow after administration of an anticoagulant is determined by passing blood through a silicone chamber similar to a fine comb. Similarly, this method also uses blood after administration of an anticoagulant, so that the effect of the coagulation system cannot be determined. In addition, the viscosity in this method has a large individual variability, so it is difficult to assess the impact of drug therapy using the specified system.

The platelet is activated by the coagulation system, while the coagulation system is stimulated by activated platelets. In other words, the administration of anticoagulants also inhibits platelet activation, so that the effectiveness of an antithrombotic drug cannot be observed. In addition, if any anticoagulation therapy is not performed, then the blood coagulates rapidly, so that it can not be used in the study.

Summary of the invention

The objective of the present invention is to provide a device and method for the overall assessment of blood clots due to blood coagulation and the formation of a platelet thrombus in an environment equivalent to blood flow, with the flow of whole blood or plasma containing platelets (in the description of the present invention they can collectively be referred to as "blood") when monitoring the effectiveness of an antithrombotic drug administered to a patient or similar individual.

To solve the above problem, the present invention provides a device for monitoring the formation of a blood clot, which controls the formation of a blood clot by passing blood after administering an anticoagulant through a channel that simulates a blood vessel, while performing anticoagulation treatment, or stimulating blood coagulation, the device comprising: a thrombus formation chamber, at least in a part of which a thrombus causing material is placed; an inlet tube that is connected to the thrombus formation chamber and through which blood flows into the thrombus formation chamber; and a medicament tube that is connected to the inlet tube and through which a medicament that stimulates blood coagulation (hereinafter referred to as the “coagulation stimulant”) or a medicament that has an anticoagulant effect is supplied. In the present invention, the term “monitoring” means not only a visual assessment of the formation of a blood clot, visualization, but also an assessment of the degree of formation of a blood clot in terms of a digital characteristic by determining pressure or a similar indicator.

In addition, according to the present invention, there is provided a device for monitoring thrombus formation, which comprises a thrombus formation chamber, at least in part of which a thrombus-producing material is placed, an inlet tube that is connected to the thrombus formation chamber and through which blood flows into the thrombus formation chamber and an inlet tube for a blood clot inhibitor that is connected to a blood clot chamber and where the blood clot inhibitor is mixed after passing through of thrombus formation chamber.

In this case, the thrombus formation monitoring device is preferably located on the substrate.

The thrombus formation monitoring device according to the present invention preferably comprises a pump for generating overpressure in the inlet tube and / or drug tube or an aspiration pump for the contents of the outlet tube, which is connected to the thrombus formation chamber and is designed to remove blood from the thrombus formation chamber.

The thrombus formation monitoring device according to the present invention preferably comprises a pressure measuring device and an imaging camera, as well as a thrombus formation chamber.

In addition, the thrombus-causing material contains tissue factor (tissue thromboplastin).

Further, according to the present invention, there is provided a method for monitoring the formation of a blood clot, comprising the steps of: directing blood after introducing an anticoagulant into a thrombus formation chamber, at least in a part of which a material causing a blood clot is formed, and which simultaneously provides anticoagulation treatment or stimulation of blood coagulation, for monitoring , through this, the formation of a blood clot. In the present invention, the sign “blood flow” and “simultaneously providing anticoagulation treatment or stimulation of blood coagulation” can be a condition in which a reaction that provides anticoagulation, or a reaction that stimulates coagulation, occurs in the channel and includes a condition in which the drug flows, releasing an anticoagulant or coagulation promoting agent when mixed with blood in a channel, or a condition in which rum provides a quick course of the drug, releasing an anticoagulant, or coagulation promoting agent, after mixing with blood.

In the method for monitoring the formation of a blood clot according to the present invention, it is preferable that the anticoagulation treatment is a treatment with substances that form calcium chelates, such as citric acid, and the anticoagulation treatment is carried out by a free calcium donor.

In the method for monitoring the formation of a thrombus according to the present invention, it is preferable that the anticoagulation treatment is a thrombin aptamer treatment and the anticoagulation treatment is carried out with the antisense DNA of the thrombin aptamer.

In the method for monitoring the formation of a blood clot according to the present invention, it is preferable to control the formation of a blood clot by supplying anticoagulant-treated blood to the thrombus formation chamber while at the same time stimulating blood coagulation without anticoagulation treatment. In this case, the agent for stimulating blood coagulation is preferably the addition of tissue thromboplastin.

In addition, in the method for monitoring the formation of a blood clot according to the present invention, blood whose coagulability has been reduced by one or more anticoagulation agents, preferably is no longer subjected to anticoagulation treatment with at least one type of anticoagulation treatment that corresponds to the anticoagulation treatment used. In this case, it is preferable that the anticoagulant treatment agent is a contact phase inhibiting factor and the calcium chelate forming substance, and the anticoagulation treatment agent is a free calcium donor. Further, it is also preferred that the anticoagulant treatment agent is a contact phase inhibiting factor and heparin, and the anticoagulation treatment agent is heparinase. It is also preferable that the anticoagulant treatment means be a contact phase inhibiting factor such as blood coagulation factor XII, kallikrein or the like, and the thrombin aptamer, and the anticoagulant treatment agent is the antisense thrombin aptamer DNA. The clotting factor XII inhibitor is preferably a trypsin inhibitor derived from maize.

In the method for monitoring the formation of a blood clot according to the present invention, it is preferable to determine the pressure during the influx and / or outflow of blood from the thrombus formation chamber.

In the method for monitoring thrombus formation according to the present invention, the thrombus-producing material preferably includes collagen and tissue factor.

The thrombus formation monitoring device according to claim 1 of the present invention comprises a thrombus formation chamber, at least in part of which a thrombus-producing material is placed, an inlet tube that is connected to the thrombus formation chamber and through which blood flows into the thrombus formation chamber, and a tube for a drug that is connected to the inlet tube and through which a drug that performs anticoagulation treatment, or a drug that stimulates blood clotting. Therefore, blood that has been treated with an anticoagulant to prevent coagulation of the blood taken from the patient after the anticoagulant has been administered to him, in the channel passing into the thrombus formation chamber can be controlled by deliberate thrombus formation in the thrombus formation chamber. Thus, the effectiveness of an antithrombotic agent can be specifically controlled in an environment similar to the internal environment of the human body. In addition, the anticoagulation treatment can be used during blood sampling. Therefore, the advantage is that the samples after taking blood samples can be stored for a certain period of time, and the study time can be selected randomly.

According to claim 2, a thrombus formation monitoring device according to the present invention comprises a thrombus formation chamber, at least in part of which a thrombus-producing material is placed, an inlet tube that is connected to the thrombus formation chamber and through which blood flows into the thrombus formation chamber, and an inlet for a thrombus formation inhibitor that is connected to the thrombus formation chamber and mixes the thrombus formation inhibitor with blood after passing through the thrombus formation chamber. Therefore, you can monitor the thrombus as described above. In addition, blood coagulation does not occur downstream in the thrombus formation chamber, and therefore it is possible to prevent the influence on the determination of pressure and to more finely control pressure changes.

A small amount of blood can be controlled when the blood clot monitoring device of the present invention is formed on a substrate. In addition, the device is equipped with a pump to create excess pressure in the inlet tube and / or drug tube, or a pump for aspiration (suction) of the contents of the outlet tube, which is connected to the thrombus formation chamber and is designed to remove blood from the thrombus formation chamber. Therefore, blood and a blood coagulation stimulation drug can flow stably for a predetermined period of time at a given pressure or predetermined flow rate.

If the device for monitoring the formation of a blood clot according to the present invention contains a device for measuring pressure, the degree of formation of a blood clot can be converted into numbers, so that a quantitative assessment can be performed.

The device can be easily installed when the material causing the formation of a blood clot contains collagen. Thrombus formation can be effectively triggered when the thrombus-forming material further comprises a tissue factor, such as tissue thromboplastin, together with collagen.

If the thrombus formation monitoring apparatus of the present invention comprises a camera for acquiring an image of a thrombus formation chamber, then the appearance of a thrombus can be observed as an image, and then the image can be stored.

Further, in accordance with paragraph 9 of the claims of the present invention, a method for monitoring blood clot formation comprises the steps of: directing blood after administration of an anticoagulant to a thrombus formation chamber, at least in a part of which a material causing a blood clot is placed, while performing anticoagulation treatment or stimulating blood coagulation . Thus, the formation of a blood clot on the material causing the formation of a blood clot can be controlled by stimulating blood coagulation by passing blood, which was obtained by anticoagulation treatment of blood taken after administration of an antithrombotic drug to a patient to prevent coagulation. Therefore, the effectiveness of the antithrombotic drug can be precisely controlled in an environment similar to the internal environment of the human body. In addition, an anticoagulant can be used to take blood samples. Therefore, there is an advantage in that the samples after taking blood samples can be stored for a certain period of time, and the study time can be randomly selected. If the anticoagulation treatment is a treatment with a calcium chelate forming substance, such as citric acid, and the anticoagulation treatment is carried out with a free calcium donor, a reagent can easily be obtained, and thus it is preferred. If the anticoagulation treatment is a thrombin aptamer treatment, and the anticoagulation treatment is carried out by the antisense DNA of the thrombin aptamer, then a study can be carried out while reflecting the physiological concentration of calcium ions in the blood.

In addition, in accordance with paragraph 12 of the claims of the present invention, it is also possible to control the formation of a blood clot by transmitting blood after anticoagulation treatment into a thrombus formation chamber without performing anticoagulation treatment, while stimulating blood coagulation. Therefore, a blood clot can be observed with a small amount of blood and the patient's discomfort can be reduced. In addition, in this case, a tube for the drug is not always required, so that a blood clot monitoring device can be simplified. If tissue thromboplastin is used as an agent that stimulates blood coagulation, blood coagulation can be stimulated by activating the coagulation system in an alternative way, which avoids the activation of factor XII and activation of kallikrein to control the formation of a blood clot in the thrombus formation chamber.

In addition, the formation of a blood clot can be controlled by a simple operation on the blood subjected to anticoagulation treatment using one or more types of anticoagulation agents, at least one type of anticoagulation treatment, corresponding to the used anticoagulation treatment. In this case, when the anticoagulation treatment is carried out by a contact phase factor inhibitor and an agent forming a calcium chelate, and the anticoagulation treatment is carried out by a free calcium donor, or when the anticoagulation treatment is carried out by a contact phase factor inhibitor and heparin, and the anticoagulation treatment is carried out by heparinase, then the anticoagulation treatment that provides effect during monitoring of thrombus formation, is an anticoagulation treatment with a contact factor inhibitor Phase II, however monitoring thrombus formation can be carried out in a more physiological conditions, in particular reflecting the divalent metal ion bound to thrombus, such as calcium or magnesium. In this case, when the anticoagulation treatment is carried out by a contact phase factor inhibitor, such as coagulation factor XII or kallikrein and thrombin aptamer, and then the anticoagulation treatment is carried out by the antisense DNA of the thrombin inhibition aptamer, the blood can be stored for a long period. In addition, anticoagulation treatment can be effectively carried out when a trypsin inhibitor derived from maize is used as an inhibitor of factor XII blood coagulation.

If, in the method for monitoring the formation of a blood clot according to the present invention, pressure is measured during the inflow and / or release of blood from the blood clot chamber, the degree of blood clot formation can be converted into numbers, so that it is easy to quantify using a very simple device.

In addition, if the material causing the formation of a blood clot contains collagen and tissue factor, the device can be easily installed and the formation of a blood clot can be effectively caused.

Brief Description of the Drawings

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

figure 1 depicts a device for monitoring the formation of a blood clot according to the first embodiment of the invention;

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

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

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

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

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

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

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

7 is a diagram of the main part of the device for monitoring the formation of a thrombus (main body of the microchip) according to the second embodiment of the invention;

8 is a diagram of a main part of a blood clot monitoring device (microchip cover) according to a second embodiment of the invention;

Fig.9 is a diagram of the main parts of the device for monitoring the formation of a blood clot according to a third embodiment of the invention; Fig.9 (A) is the whole diagram, Fig.9 (B) is the main body of the microchip, and Fig.9 (C) is the cover of the microchip according to the invention;

figure 10 - diagram of a monitoring system for the formation of a blood clot containing a device for monitoring the formation of a blood clot according to the invention;

11 (A) is the coagulation thromboelastogram waveform when 10 μM of each aptamer is added to exocyte I and exocyte II into the blood after exposure at room temperature for 15 minutes and 40 μM of each of the antisense DNAs of both aptamers according to the invention are added;

11 (B) is a wave form of blood thromboelastogram immediately after taking blood samples according to the invention;

12 is a diagram of an apparatus according to a fourth embodiment of the present invention; Fig. 12 (A) is a complete drawing, Fig. 12 (B) is a microchip cover, and Fig. 12 (C) is a microchip substrate according to the invention;

Fig - diagrams of pressure measurements of example 17 (control), example 18 (heparin) and example 19 (Reopro) according to the invention;

Fig - diagrams of pressure measurements when adding heparin in the amount of 0 (control), 0.2, 0.5 and 1 PIECES / ml of heparin according to the invention;

Fig - diagrams of pressure measurements when adding heparin in the amount of 0 (control), 2, 5 and 10 μg / ml according to the invention.

Description of preferred embodiments of the invention

1 is a diagram of a first embodiment of a thrombus formation monitoring device of the present invention.

The thrombus formation monitoring device according to the present embodiment comprises a thrombus formation chamber 10; the inlet tube 11, which is connected to the thrombus formation chamber and through which blood flows into the thrombus formation chamber, and the drug tube 12, which is connected to the outlet tube and through which the drug is supplied that performs anticoagulation treatment or stimulates blood coagulation.

The thrombus formation chamber 10 is substantially cylindrical in shape and contains material that causes the formation of a thrombus in part of its internal cavity. The camera may be made of clear glass, thermoplastic resin, or the like. Examples of the material that causes the formation of a blood clot include collagen, vWF (von Willebrand factor), the previously obtained blood clot, and fibrous materials from silk, cotton or the like. These materials can be used alone or in combination of two or more of them. Collagen is especially preferred because it can be easily obtained, easily manipulated, and can be provided as a model similar to a blood vessel. Collagen may include tissue factor. The thrombus-forming material from collagen or vWF is preferably presented as a coating on the inner surface of the thrombus-forming chamber 10 to prevent the thrombus-causing material from flowing out along with the blood stream. The coating can be easily applied, for example as described in JP 05-260950 A or in Blood 1995 April 1; 85 (7): 1826-35, dissolving collagen in an acid solution and immersing in it a substrate having hydrophilicity, the same as glass or polystyrene, followed by washing and drying to cover the surface of the material.

Further, it is preferable that the material causing the formation of a thrombus, in the form of a fibrous material or a previously obtained thrombus, can be in a state suitable for fixing inside the thrombus formation chamber 10. Further, by impregnation with collagen of a hygroscopic thin fibrous material such as cotton, non-woven fabric or knitted fabric, and drying them, it is possible to obtain a thrombus-producing material with a higher thrombus-forming ability. In addition, the substrate can be immersed in a collagen solution containing tissue thromboplastin and then dried to further enhance its ability to cause the formation of a thrombus.

The thrombus-producing material can be selected depending on the inner diameter of the thrombus-forming chamber 10 and the monitoring object. If atheromatosis causing myocardial infarction is presented as a model, then it is preferable that it contains only collagen, or both collagen and tissue thromboplastin are contained. In addition, it is preferable that in the channel it was possible to form a narrowed portion in the thrombus formation chamber for the action of shear stress on the thrombus formation chamber. Further, in the case of studying the formation of a blood clot in a myocardial infarction and a cerebral infarction of cardiac origin or a similar condition in which a blood clot can be transferred from another part of the body to the bloodstream and attach, causing occlusion of the blood vessel, in another part of the body, it is preferable that cohesion occurs beforehand a small part of a blood clot with a thrombus formation chamber 10, presented as a material causing a blood clot, followed by monitoring the growth of a blood clot formed on it. In the case of studying thrombosis of a blood capillary, the internal lumen of the channel in the thrombus formation chamber can be divided into many channels, each of which has a diameter of 10 to 30 μm. If the thrombus formation chamber 10 has a constricted portion with a diameter of 100 μm or less, then the canal may be occluded with a small thrombus formed in such a constricted portion. Therefore, there is no need to use additional material that causes the formation of a blood clot, so that the formation of a blood clot can be controlled by an agent that stimulates blood coagulation, or an anticoagulant. Therefore, the present invention contains this constricted portion as a thrombus-forming material.

The material causing the formation of a blood clot can only be a collagen or vWF coating. The thrombus formation chamber 10 in the coating portion may preferably be narrowed and presented for portion 14, so that platelet aggregation caused by shear stress can be controlled. In the case of collagen coating as a thrombus forming material, it is preferable that at least a portion of the substrate be made of glass or plastic to obtain high adhesion to the surface. In addition, the part on which to place the thrombus formation chamber 10, or the part of the thrombus formation chamber 10 where the material causing the formation of the thrombus is located, can be made in the form of a removable cartridge. This option is preferable because the resulting blood clot can be easily washed or observed, or the material causing the blood clot can be easily replaced with new material. In this case, the cassette may form a fluid tight seal with an O-ring of silicone rubber or the like. Preferably, the end of the cassette opposite its end connected to the inlet tube 11 of the thrombus formation chamber 10 may be connected to the outlet tube 13, which allows blood to be released. Preferably, the exhaust pipe 13 may be divided by a cylindrical bobbin, and its tip may be provided with a pressure gauge 41, for example a diaphragm type pressure gauge. 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 thrombus formation chamber 10 may be made using clear glass, thermoplastic resin, or the like. The end of the inlet tube 11, opposite its other end, connecting to the thrombus formation chamber 10, is connected to a syringe 20 that delivers blood. The syringe 20 is connected to the pump 30 and a squeezing means (not shown), so that the piston of the syringe 20 is pressed at a given pressure. The pump may be a commercially available pump. Alternatively, the pump may be a syringe pump made by extruding the syringe with air at constant pressure, or turning the syringe so that the piston is on top and acting on the piston with its weight.

The inlet tube 11 may preferably be separated by a cylindrical bobbin, and its end is preferably provided with a pressure gauge 40, such as a diaphragm type pressure gauge, in the portion of the inlet tube 11 near the thrombus formation chamber 10.

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

Whole blood or plasma without anticoagulation treatment coagulates within a few minutes. Coagulation can be reduced or eliminated by the addition of a calcium chelate forming substance such as citrate. In particular, citrate has been reported to inhibit the agglutination and function of prothrombinase and exogenous and endogenous tenase.

Blood treated with citrate can be stored in liquid form for a predetermined period of time (for example, from several hours to several days) and processed into blood products such as a product of cytopheresis, platelet rich plasma, and low platelet plasma. Plasma containing citrate can be stored at about -70 ° C or lower for a long period of time (from several months to several years). Whole blood and plasma can also be used in the present invention, and in this case, calcium or the like can preferably be added again.

However, whole blood or plasma with newly added calcium coagulates spontaneously due to contact activation in any of most storage containers. In this case, contact activation can occur within about 2-4 minutes. With this in mind, in the present invention, an anticoagulant, such as calcium, can be added immediately after monitoring the formation of a blood clot into a newly prepared citrate-treated blood after taking a blood sample and freezing it for storage and subsequent thawing, or in platelet-containing plasma.

Other anticoagulant agents may include heparin, hirudin, hirulog (peptide of the C-terminal region of hirudin), aprotinin, antithrombin antibody, thrombin aptamer, trypsin inhibitor derived from maize (1977, J. Biol. Chem 252, 8105). These materials inhibit blood coagulation by inhibiting the coagulation cascade as a result of inhibition of coagulation factor, and therefore are sometimes referred to in the description of the present invention as “coagulation factor inhibitors”.

A blood sample for monitoring can be taken in any way, such as a method in which a coagulation factor inhibitor is first placed in a syringe or a vacuum vessel for taking blood, and then blood is collected, or a method in which a coagulation factor inhibitor is quickly added to the blood immediately after sampling blood for receiving through this blood, subjected to anticoagulation treatment.

Next, blood is collected in a vacuum blood collection vessel containing a coagulation factor inhibitor such as heparin, and then heparinase and an anticoagulation agent suitable for monitoring are added to break down the heparin, so that heparin is replaced with an anticoagulation agent suitable for the purpose of monitoring. In addition, blood is collected in a vacuum blood collection vessel containing citric acid, and then calcium chloride and a coagulation factor inhibitor suitable for the monitoring purpose, such as a trypsin inhibitor derived from maize, or a thrombin aptamer are added. Therefore, blood subjected to anticoagulation treatment can be collected depending on the purpose of monitoring.

The drug tube 12 connected to the thrombus formation chamber 10 may be made of clear glass, thermoplastic resin, or similar materials. The end of the drug tube 12, which is opposite to its other side, connected to the thrombus formation chamber 10, is connected to the syringe 21 for supplying the anticoagulant drug or blood coagulation drug. The syringe 21 can be connected to the pump 31 and a squeezing means (not shown), so that the piston of the syringe 21 can be pressed at a given pressure. The pump may be a commercially available pump. Alternatively, the pump may be a syringe pump made by extruding the syringe with air at a given pressure, or by turning the syringe so that the piston is on top and acting on the piston with a load. The drug tube 12 is filled with an anticoagulation treatment agent or a blood coagulation promoting agent, as described below.

To monitor thrombus, the thrombus formation monitoring device according to the present invention, the syringe 20 is filled with whole blood or plasma containing platelets, and subjected to anticoagulation treatment with sodium citrate (solution A). The syringe 21 is filled with a drug that provides anticoagulation treatment, such as a solution of calcium chloride (solution B). Solution A and solution B are fed into the inlet tube 11 by pumps 30 and 31, respectively, so that solution B can reach a concentration of 5 to 20 mmol, at which the coagulation cascade of solution A can be initiated. Subsequently, solution A and solution B are mixed in the inlet tube 11, so that the mixture is directed into the thrombus formation chamber 10. Then, collagen or the like, capable of causing a thrombus, is preliminarily applied to a portion of the inner surface of the thrombus formation chamber 10 to form a thrombus-forming material. The thrombus formation chamber can be made of a transparent plastic tube. The formation of a thrombus can be easily controlled by a blood monitoring device passing through such a thrombus formation chamber 10, which is viewed through transparency.

Monitoring the formation of a blood clot can be carried out by a visual assessment of blood flowing for a predetermined period of time through a chamber (chamber 10 of a blood clot) treated with collagen, and then removing blood from it. When the pump sucks in air for solution A and solution B, solution A and solution B can be supplied under constant pressure. Therefore, the formation of a blood clot on collagen can be controlled by reducing the rate of blood flow flowing from the exhaust tube 13. Alternatively, when a pressure gauge is installed near the thrombus formation chamber 10 on the part of the inlet tube 11 and the outlet tube 13, the formation of a blood clot on the collagen can be controlled by observing a change in internal pressure in thrombus formation chamber 10. Alternatively, the formation of a blood clot on collagen can be observed under a microscope in the manufacture of a blood clot chamber 10 with a thickness of 500 μm or less. In particular, it is easy to observe a blood clot in a platelet-rich plasma because it is clearly visible, and the formation of a blood clot can also be observed with the naked eye. Further, it is also possible to fluorescently label blood platelets and control their fluorescence with a fluorescence microscope.

Examples of a medicament for performing anticoagulation treatment with a chelating agent, such as citric acid, comprise: calcium halides 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 calcium compounds provided as free calcium donors.

The anticoagulation agent may be selected and used depending on the coagulation factor inhibitor when the anticoagulation treatment is carried out by the coagulation factor inhibitor (anticoagulation treatment agent). For example, as an anticoagulant, when the anticoagulation treatment is carried out with heparin, a protamine, heparinase or an anti-heparin antibody can be used. As an agent having an anticoagulant effect, when the anticoagulant treatments are performed with hirudin, hirulog and aprotinin, an anticoagulant agent such as an anti-hirudin antibody, an anti-hirulog antibody and an anti-aprotinin antibody can be used.

Examples of an anticoagulant agent when the anticoagulation treatment is performed using an anti-thrombin antibody as an inhibitor of coagulation factor include: fully inactivated thrombin, such as PPAC-thrombin (D-Phe-Pro-Arg-chloromethylketone) -thrombin; thrombin disruption fragment; and a synthetic polypeptide comprising an antibody-recognizing thrombin epitope.

Antibodies used in anticoagulation treatment or anticoagulation, preferably include antibodies from which the Fc domains are removed by papainase or similar substances to minimize their effect on the complement system, or antibodies such as chicken egg antibodies that are unable to activate the human complement system.

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, is used as a means for anticoagulation treatment, a substance that binds to the thrombin aptamer and inhibits its functions, such as antisense DNA or antisense RNA, can be used as an anticoagulant. When two types of thrombin aptamers, one recognizing exocyte I, and the other recognizing exocyte II, are used in combination, a very high effect of anticoagulation treatment can be obtained compared to the case in which each of them is used separately. The antisense DNA used in this case may be antisense DNA against a portion of the thrombin aptamer, while it substantially inactivates the antithrombin function of the thrombin aptamer.

In addition, the thrombin aptamer and its antisense DNA are effective, respectively, as a means of anticoagulation treatment and a means that performs anticoagulation action, which is described with reference to the following reference examples.

The coagulation system cannot be activated within a few hours when the anticoagulation treatment is carried out with heparin. Thus, blood can be stored for a long period of time while monitoring the formation of a blood clot. However, for example, there is a case when anticoagulation treatment is not suitable for the study of blood taken from a patient who was injected with heparin.

On the other hand, hirudin, hirulog and antithrombin antibody are inhibitors for inhibiting the conversion of fibrinogen to fibrin by inhibiting thrombin, which acts on the final stage of the coagulation system. However, the contact phase factor (such as pre-kallikrein or factor XII) of the coagulation cascade is gradually activated even if thrombin is completely inhibited, so activation upstream of the coagulation cascade can occur. Therefore, there is a case that is not suitable for long-term storage of blood.

Aprotinin also inhibits the activity of kallikrein in the contact phase to delay the endogenous blood coagulation cascade. However, the coagulation cascade is gradually activated even under conditions of inhibition of kallikrein activity, so that blood coagulates after a few hours even in the presence of aprotinin.

Therefore, when monitoring the formation of a blood clot, after approximately several tens of minutes, it is possible to control the formation of a blood clot by anticoagulation treatment with a thrombin inhibitor and / or aprotinin. However, this may not be preferable in the case of the required long time to start monitoring or the required long time for monitoring.

When thrombus formation is monitored after 1 hour or more from the time a blood sample is taken, anticoagulation can be performed using the thrombin aptamer in combination with a trypsin, hirudin, and aprotinin inhibitor derived from maize. Alternatively, a small amount of heparin, for example less than 1 U / ml, which is a level that does not have a large effect on clotting time, can be used in combination with a contact phase factor inhibitor or thrombin inhibitor.

A much more pronounced effect of anticoagulation treatment can be obtained when the thrombin aptamer is used in combination with two types of aptamers for exocyte I and exocyte II. Further, the antisense DNA of the respective aptamers can immediately carry out anticoagulation treatment.

A contact phase inhibitor or thrombin inhibitor, such as an inhibitor obtained from maize (factor XII inhibitor), or aprotinin (a kallikrein inhibitor), and a thrombin inhibitor, such as a thrombin aptamer, are also added to the blood sample in advance, and then the antisense thrombin aptamer DNA is added (DNA inhibitor) or the like. Thus, the function of a single thrombin inhibitor is inhibited to effect a functional inhibition of thrombin, followed by a rapid flow of the sample into the thrombus formation chamber. As a result, for example, an exogenous coagulation cascade can be activated by a tissue factor (tissue thromboplastin) or the like, so that blood coagulation is stimulated, and the physiological formation of a blood clot can be controlled.

Alternatively, anticoagulation treatment can be performed with an anticoagulation agent inhibitor and maize trypsin, which are capable of performing anticoagulation treatment, such as chelating agents (citric acid), heparin or the like; and a free calcium donor such as calcium chloride; or heparinase can then be allowed to carry out appropriate anticoagulation treatment, followed by allowing blood to flow into the thrombus formation chamber to stimulate blood coagulation by activating an exogenous coagulation cascade.

In this case, the thrombus-forming material may preferably contain an appropriate amount of tissue factor (tissue thromboplastin). In particular, it is preferable that the thrombus-forming material can be coated with a mixture obtained by mixing collagen with tissue thromboplastin, followed by drying, because thrombus formation is stimulated only in the thrombus-forming chamber. When a thrombus causing material is used coated with a collagen solution containing tissue factor (tissue thromboplastin) as an atheromatous model, monitoring can be performed that reflects a pathological mechanism.

In addition, the formation of a blood clot can be controlled by the addition of a free calcium donor and a trypsin inhibitor to the blood, subjected to anticoagulation treatment with a calcium chelating compound such as citric acid, and by allowing rapid blood flow to the blood clot chamber, or by adding heparinase and a trypsin inhibitor to the blood, subjected to anticoagulation treatment with heparin, and then providing a rapid flow of blood into the thrombus formation chamber. In the method described above, in which the formation of a blood clot is controlled by mixing the anticoagulated blood obtained using one or more types of anticoagulation treatment with at least one anticoagulation treatment corresponding to the agent used and then ensuring a fast blood flow into the thrombus formation chamber, a thrombus formation monitoring device can be used (Fig. 9).

The above thrombin inhibitors, such as hirudin and thrombin aptamers, synthesized low molecular weight contact phase factor inhibitors, and protein anticoagulation agents are expensive compared to any anticoagulation agent such as citric acid and EDTA that form chelates with calcium and it like that. However, they do not change the concentration of divalent metal ions, such as calcium, magnesium and zinc, so that the formation of a blood clot at the initial concentration of these ions in the patient’s blood can be reflected during monitoring. Therefore, monitoring becomes possible, which more closely reflects the clinical condition of the patient.

Blood coagulation can be suppressed by inhibiting the contact phase factor, such as activated factor XII or kallikrein. However, it was reported that activation of XIIa and kallikrein does not contribute much to the actual physiological formation of a blood clot or hemostasis. In fact, there is generally no evidence of bleeding or the like even in a patient with congenital deficiency of factor XII, pre-kallikrein or the like. In particular, with atherothrombosis, such as myocardial infarction, it is widely known that the coagulation system is activated by a tissue factor due to a plaque caused by arteriosclerosis. Factor XII can be activated mainly by thrombin on an activated platelet. Therefore, when performing anticoagulation treatment, when an inhibitor for inhibiting the activation of factor XII, or prekallikrein, or an inhibitor for inhibiting activated factor XII, or kallikrein is used as an anticoagulant, there is no need to add an anticoagulating agent. By adding to the blood any substance that activates exogenous coagulation, such as tissue factor (e.g. tissue thromboplastin), or a thrombus-producing material instead of an anticoagulation agent, or by adding a substance to the thrombus-forming material, the coagulation system is activated in an alternative way which avoids the activation of factor XII and the activation of kallikrein to stimulate blood coagulation, so that the formation of a thrombus can be controlled in the thrombus formation chamber.

As anticoagulation agents that can be used to monitor blood clot formation, after stimulating blood coagulation by adding a substance that activates exogenous coagulation, such as tissue factor, without adding an anticoagulation agent, the synthesized low molecular weight inhibitors, such as the PKSI-kallikrein inhibitor, are illustrated 527 (Thromb Res 57: 889, 1990) and D-Phe-Arg-CK, which is an activated factor XII inhibitor (Cal Biochem. Co., Ltd.). In addition, protein inhibitors include anti-contact phase protease antibodies in the coagulation cascade, such as an anti-kallikrein antibody, an anti-precallicrein antibody, an anti-coagulation factor XII antibody, an anti-coagulation factor XII antibody, and a trypsin inhibitor derived from maize.

To monitor the formation of a blood clot after stimulating blood coagulation by adding a substance that activates exogenous coagulation, without adding an anticoagulant, in particular in terms of accessibility and anticoagulation ability, it is preferable that the blood be subjected to anticoagulation treatment once with a combination of citric acid and obtained from maize with a trypsin inhibitor or a combination of thrombin aptamer and a maize trypsin inhibitor, and the blood is then stored It familiarizes because it can increase the degree of freedom of the monitoring operation. Further, immediately before monitoring the formation of a blood clot, anticoagulation treatment with a trypsin inhibitor obtained from maize continues, while other anticoagulation treatment is partially carried out by a donor of free calcium or antisense DNA of the thrombin aptamer. In this case, as a model of an atheromatous thrombus, it is preferable to activate exogenous coagulation using a material that causes the formation of a thrombus coated with collagen or collagen-containing tissue thromboplastin to stimulate coagulation.

The containers used to monitor blood clot formation, such as a syringe for blood supply and a vacuum vessel for collecting blood, are preferably coated with heparin or a material with antithrombotic ability and blood compatibility, such as polyvinyl lactoamide (PVLA) or poly-2-methoxyethyl acrylate (PMEA) .

In a thrombus formation monitoring device according to another embodiment of the present invention, the tube and the thrombus formation chamber can be combined with each other on a substrate by a thin channel, such as a microchip.

4 is a plan view of a main part of a blood clot monitoring device in accordance with a second aspect of the present invention. 4, a groove is formed in the substrate 100, and a contour is formed on it. This embodiment is configured as follows. On a small piece of the substrate, a thrombus formation chamber is made as the main part of the thrombus formation monitoring device, an inlet tube which is connected to the thrombus formation chamber, and through which blood flows into the thrombus formation chamber, and a drug tube which is connected to the inlet tube and through which is injected into the thrombus formation chamber a drug that performs anticoagulation treatment, or a drug that stimulates blood coagulation, all the elements are combined in a single whole with each other and are presented in the form of a microchip contour. In this embodiment, the parts, except the main part, are integrated into a single unit on the substrate, similar to the first embodiment.

4, the substrate 100 is the main body of the microchip, and its materials can be any of the group consisting of metal, glass, plastic, silicone, and the like. When observing a blood clot, a transparent material is preferred. A plastic material is preferred to form the contour. Therefore, transparent plastic material is particularly preferred. When selected from a silicone resin such as polydimethylsiloxane (PDMS), its adhesion is very high. Thus, the contour can be formed by contacting with a cap without glue or the like. When a polystyrene substrate is used, the channel can be easily coated with PVLA and subjected to antithrombotic treatment. Further, PMEA can also provide the possibility of simple, effective antithrombotic treatment (see Reference Example 4).

FIG. 5 is a plan view of a substrate that serves as a microchip cover that is overlaid and connected to the substrate 100 (FIG. 4). The substrate 200 (figure 5) is a transparent glass slide or plate or sheet, which are made of plastic or similar material. A cover substrate 200 is overlaid and connected to the substrate 100, so that the outline of the substrate can be formed from a thrombus formation chamber 110, an inlet tube 111, and a drug tube 112.

To provide the inner surface of the thrombus formation chamber 110 with a material causing the development of the thrombus 115, for example, collagen or the like can be applied to a predetermined mounting position of the substrate 200. A device for monitoring the formation of a blood clot can be obtained when the substrate 100 and the substrate 200 are bonded to each other by a connection or by fitting inwardly the collagen-coated surface of the substrate 200. In view of the simple application, collagen can preferably be applied to a flat substrate 200 that does not have grooves. In addition, tissue thromboplastin is preferably applied after mixing with collagen, because it is possible to obtain a thrombus-producing material having a higher thrombus-producing ability. Further, for easy formation of a blood clot by collagen-coated glass, the glass having the collagen-coated portion may undergo further processing, such as replacing the glass with frosted glass or the like, so that the surface area can be increased.

The connecting part 100A, the connecting part 100B, and the connecting part 100C of the substrate 100 are connected to tubes (not shown) that form respectively parts of the inlet tube, drug tube, and outlet tube. Thus, each of the connecting part 100A and the connecting part 100B is connected to pumps (not shown) through tubes. Subjected to anticoagulation treatment, blood is injected from the connecting part 100A, and an anticoagulation agent corresponding to the anticoagulation treatment is injected from the connecting part 100B.

In accordance with this embodiment, a blood clot can be controlled with an extremely small amount of blood, so that discomfort for the patient can be reduced. In addition, platelets adhered to the substrate 200 from the side of the substrate 200 can be controlled by platelet labeling with a fluorescent reagent such as mepacrine.

Further, the circuit 100D has a sealed end at which air is blocked. Thus, the circuit 100D is provided as a pressure gauge. The end of circuit 100D may preferably be provided with a control valve 100E to relieve pressure or increase sensitivity when circuit 100D is provided as a pressure gauge. The control valve 100E may be closed with a cap or adhesive tape, and its volume may be changed by filling, such as resin, depending on the required sensitivity. The circuit 100D is formed low in order to prevent mixing of blood with air and to prevent air leakage. The thickness of the contour 100D depends on the material of the substrate 100, but is about 0.1 to 0.5 mm. The internal pressure of the inlet tube 111 of the thrombus formation monitoring device is increased by the formation of a thrombus, the air in the circuit 100D is compressed by blood, so that the amount of anticoagulated blood can be introduced into the circuit 100D in such an amount. Internal pressure can be controlled by the movement of blood in the 100D circuit at any time.

The adhesion of platelets to collagen, the activation of the coagulation system on activated platelets and the accumulation of activated platelets by platelet agglutination can be controlled respectively by a thrombus formation monitoring device and / or a method for monitoring blood clot formation according to the present invention.

Further, to reproduce the formation of a thrombus during atherothrombosis, the narrowed portions 14 and 114 are formed respectively on the thrombus formation chambers 10 and 110. Therefore, monitoring can be carried out, which also reflects shear stress, which creates platelet agglutination.

Fig. 9 shows a main part of a blood clot monitoring device in accordance with a third embodiment of the present invention. Fig. 9 shows an inlet tube 322 for a blood coagulation inhibitor and an inlet tube 317 for applying a pressure that mixes the blood coagulation inhibitor with blood downstream of the thrombus formation chamber. In this embodiment, the drug tube is not provided upstream of the thrombus formation chamber.

According to a third embodiment, blood collected after the addition of an anticoagulant treatment agent (e.g., citric acid and a trypsin inhibitor obtained from maize) is further added to an anticoagulation treatment agent (e.g. a free calcium donor) immediately before measurement and then is introduced into the syringe 320. Subsequently, a filling fluid under pressure of blood, such as mineral oil, is pressed into the syringe from the inlet tube 317 to supply pressure connected to Prica 320, thereby forcing the blood into the microchip 300.

An increase in the pressure exerted on the syringe 320 with the sample shows the state of occlusion of the channel by the formed thrombus, so monitoring the formation of a blood clot by changing the pressure is especially suitable for monitoring the model of a durable atheromatous thrombus in which an obstructive thrombus forms. The thrombus-causing material may, without particular limitation, be material derived from collagen and tissue factor. For example, a blood clot-producing material is effective in monitoring blood clot formation when blood coagulation is stimulated by activation of exogenous coagulation.

During the measurement of pressure, to more accurately measure the pressure in the channel during the formation of a blood clot, the blood coagulation inhibitor is introduced through the inlet tube 322 for the blood coagulation inhibitor and mixed with the blood located downstream of the thrombus formation chamber through the channel formed in the microchip 300. Thus, it is possible to prevent the formation of a blood clot in the blood located in the channel following the thrombus formation chamber. Therefore, such a configuration is preferable because it is possible to definitely and accurately measure the change in pressure in the canal, which is caused by the narrowing or closure of the canal by the formation of a blood clot in the thrombus formation chamber.

The coagulation inhibitor may preferably, without particular limitation, be the anticoagulation treatment agent used in the present invention. When considering cost effectiveness and the possibility of manipulation, it is preferable to choose any of those means that prevent blood coagulation by albuminoid deformation, including alkaline or acid solution, alcohol, urea and sodium dodecyl sulfate.

In the described embodiment, the thrombus formation monitoring device of the present invention includes a suction pump on an exhaust pipe 313 instead of a pressure inlet pipe 317 so that the device can determine the degree of thrombus formation based on a change in the suction pressure (negative pressure). Such a configuration can lead to a more efficient measurement, while at the same time preventing the occurrence of a situation where the liquid for supplying pressure of mineral oil or the like is mixed with blood in the contact area, and the mixture is supplied when the quantity is small, in the case of using a pump that produces indirect ejection of blood through a liquid separated as a layer, as described below.

Further, there is no need to use a closed container, such as a syringe, as a container (sample container) for supplying blood to the blood clot monitoring device of the present invention. Thus, the container may not have a lid and may be open to air. As a result, a blood clot monitoring device can be simplified. Further, in the thrombus formation monitoring device of the present invention, the internal pressure in the channel is negative. Thus, when the device for monitoring the formation of a blood clot according to the present invention is made in the form of a microchip, for example, even in the case of incomplete adhesion between the main body of the microchip and the cover of the microchip, there is no blood leak at all. In some cases, it can be used as a blood clot monitoring device by fitting the main body of the microchip and the lid of the microchip together when the fit is accurate.

Figure 10 shows an additionally systematic device A for monitoring the formation of a blood clot.

The thrombus formation monitoring device A fills the supply micropump 330 with a liquid having a density lower than blood. Then, the liquid is pressed into the syringe for sample 320, into which blood enters through the pressure inlet tube 317, and then is layered on the blood, thereby squeezing the blood into the microchip 300. The squeezed blood is mixed with the anticoagulation agent injected from the drug tube 312, followed by entry into the chamber 311 of the formation of a blood clot. The fluid for pumping blood into the delivery micropump 330 may be any of the oils and fats, such as liquid paraffin, mineral oil, and silicone oil, and physiological saline. Thus, it becomes possible to prevent contamination of both the supply micropump 330 and the blood pressure sensor 340 by indirect expulsion of blood.

Such a pump for indirect extrusion of blood with a liquid separated by a layer from the blood can be used in a blood clot monitoring device in accordance with any embodiment of the present invention.

The pressure sensor 340 can determine the pressure applied to the syringe of the sample 320 by the micropump 330 feed.

In this thrombus formation monitoring device A, a thrombus formation state can be recognized in more detail by image analysis. In particular, in the case of labeling platelets and leukocytes with quinacrine and then monitoring their adhesion and agglutination with collagen, the illumination per unit area due to the manifestation of fluorescent color is controlled by image analysis. Thus, monitoring results can be evaluated and stored as data. Image analysis can be performed by processing an image captured by a fluorescence stereoscopic microscope 308, a camera controlled by a computer 307, and displaying the image.

Therefore, in the device A monitoring the formation of a blood clot, it is possible to comprehensively determine the state of the formation of a blood clot from the results of image analysis and the change in pressure supplied to the piston with a sample of 320.

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

12 is a diagram of a blood clot monitoring device of a fourth embodiment of the present invention. The device for monitoring the formation of a blood clot is equipped with a camera for observing the formation of a blood clot.

For example, in a device for monitoring the formation of a blood clot in a blood subjected to anticoagulation treatment with citric acid and a trypsin inhibitor obtained from maize, an anticoagulation treatment agent (for example, a free calcium donor) is added and the syringe 420 is quickly filled with it. Syringe 420 is aspirated by pump 414, connected to the outlet tube 413 to allow blood to pass through the thrombus formation chamber 411, thereby forming a thrombus. The degree of thrombus formation can be determined by changing the suction pressure (negative pressure).

To prevent blood clotting after passing through the thrombus formation chamber with blockage, as a result of the outlet tube 413 or affecting the measurement of pressure, a thrombus formation inhibitor is mixed in the inlet tube for the thrombus formation inhibitor 422 after passing through the thrombus formation chamber.

A camera 430 (for example, a computer-controlled camera) is located under the microchip 400 to obtain an image of a thrombus formation chamber, so that space above the microchip can be effectively used. Along the guides 433, the chamber 430 can move back and forth and around the site of the formation of the thrombus. By using the camera 430, which has the function of preserving its position along the X-axis and Y-axis, it can receive images, while at the same time regularly moving around many specific points. With the above configuration, it is possible to control the process of thrombus formation over time over a wide area of the thrombus formation chamber 411, even if the magnification of the chamber 430 is set to a high level. Further, the optical source 432 (for example, a light emitting diode) is preferably located around the camera 430, since the optical source 432 can simultaneously move, while maintaining its positional relationship with the camera 430. It is preferable that the optical source 432 be able to emit light at a wavelength that can excite a particular fluorescent substance along with white light, depending on the fluorescent material that serves as a visualization target, allowing excitation various fluorescent materials.

EXAMPLES

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

Example 1

A blood clot monitoring device (FIG. 1) is used to fill a syringe with 20 50 ml of citrate-treated blood (solution A) containing 9 parts by volume of blood immediately after mixing a blood sample with 1 part by volume of 3.2% sodium citrate, and to fill syringe 21 10 ml of 0.2 M CaCl ₂ (solution B). Syringes 20 and 21 are connected to a transparent nylon tube (inlet tube 11 and drug tube 12) with an inner diameter of 3 mm. Both tubes are connected together at a T-shaped connection (cylindrical bobbin) and then connected to a polycarbonate thrombus formation chamber 10 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 cm. The thrombus formation chamber 10 itself is designed as a removable cartridge. The connecting part between the cartridge and the nylon tube (inlet tube 11) is made liquid tight by means of an annular seal made of silicone rubber. The glass element is fixed on the inside of the cassette with epoxy-based adhesive, thereby forming a narrowed part 14. The narrowed part 14 is designed so that the most narrowed section of the narrowed part 14 can have an inner diameter (maximum gap between the narrowed part 14 and the inner wall) 1,5 mm In addition, the thrombus formation chamber 10 is connected hermetically to a liquid with a tube, such as an outlet tube 13 having the same diameter and made of the same material, by means of an o-ring made of silicone rubber, and thus a thrombus monitoring device 1 is manufactured (figure 1). It should be noted that the manometers 40 and 41 of the flange type are respectively mounted on the parts of the inlet tube 11 and the outlet tube 13 near the thrombus formation chamber 10 by means of connections (cylindrical bobbins). In addition, the glass material of the constricted portion on the inner surface of the thrombus formation chamber 10 is prepared so that collagen is coated as a thrombus-forming material 15 on the glass constricted portion of the inner surface by immersion in a 0.1N acetic acid solution containing 1% insoluble Type I collagen (manufactured by Wako Pure Chemical Industries, Ltd.) and then dried. The syringes 20 and 21 are turned upside down so that the pistons are on the upper side, and then gravity forces are applied to the pistons to allow solution A and solution B to flow into syringe pumps at a rate of 5 ml / min and a speed of 0.5 ml / min

When solution A and solution B flow for 10 minutes, after a few minutes there is a difference between the readings of the pressure gauges 40 and 41 in the inlet pipe 11 and the outlet pipe 13, and then this difference increases over time. At the same time, it is confirmed that the blood flowing from the discharge tube 13 also gradually decreases. When the flow of all solutions is completed, physiological saline flows to the thrombus formation monitoring device 1 to flush the thrombus formation chamber 10. Thus, a thrombus formation can be detected in the thrombus formation chamber 10 by visual observation.

Example 2

A thrombus formation monitoring device 1 was made and the thrombus formation was then monitored in a manner analogous to Example 1, except that unfractionated heparin (obtained from pig mucous membrane) was added to the solution A of Example 1 at a concentration of 1 mg / ml.

In this case, there is no pressure difference, and there is no decrease in blood flow, and a blood clot cannot be found in the thrombus formation chamber 10 by visual observation after washing with physiological saline.

Example 3

The thrombus formation monitoring device 1 was made in a manner analogous to Example 1, except that a glass tube with a diameter of 3 mm and a length of 5 cm was used as a cassette-type thrombus formation chamber 10, each of the syringes 20 and 21 was connected to syringe pumps driven by electric motors, and instead of the narrowed part 14 and collagen, applied as a coating, as a material causing the formation of a blood clot 15, a piece of silk 1.5 cm long was attached with glue to the narrowed part 14 (figa), immersed collagenized in Example 1 and dried at 4 ° C as a thrombus forming material 15 onto the inner surface of the thrombus forming chamber 10, which is 5 cm in size from the connecting part of the glass tube and the inlet tube 11.

After solution A flowed into syringe 20 at a rate of 3 ml / min and solution B into syringe 21 at 0.3 ml / min for 15 min, the thrombus formation chamber 10 was disconnected and its inner surface was then washed with physiological saline. As a result, visual observation confirmed that the thrombus 16 was about 1 cm long and about 1 mm thick in the shape of a comet (Figure 2b), which appeared to have formed from the downstream side of the silk as a starting point and spread downstream , was attached to the inner surface of the glass tube. It is believed that the influence of blood flow can cause the formation of 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 thrombus formation monitoring device 1 was made by a method similar to Example 3, except that instead of silk (Fig. 3a), 50 μl of blood without anticoagulation was instilled onto a glass tube’s inner surface with a Pasteur pipette and then left to stand at room temperature for 15 minutes, thereby obtaining a blood clot in the form of a disk with a diameter of about 2 mm as the material causing the formation of a blood clot 15.

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

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

Example 5

A thrombus formation monitoring device 1 was made and the thrombus formation was then monitored in a manner analogous to Example 3, except that Argatroban (registered brand manufactured by Daiichi Pharmaceutical Co., Ltd.) was added to solution A as a means of anticoagulation treatment in a concentration of 0.1 mg / ml relative to 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.

Example 6

A thrombus formation monitoring device 1 was made and thrombus formation was then monitored in a manner analogous to Example 3, except that the anticoagulated blood was obtained by adding hirudin at a concentration of 1 μg / ml relative to whole blood and used as solution A, and a polyclonal anti-hirudin antibody (manufactured by COSMO BIO CO., LTD.) dissolved at a concentration of 1 mg / ml in physiological saline, relative to blood, was used as solution B.

In this case, almost the same thrombus formation was confirmed as in Example 3.

Example 7

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

For the manufacture of a blood clot monitoring device, a substrate 100 (FIG. 4) was used, which is made of polydimethylsiloxane 40 mm wide, 70 mm long and 1.5 mm thick, and a substrate 200 shown in FIG. 5, which has a thickness of 1 mm and is made made of glass of the same size.

The contour containing the thrombus formation chamber 110 was formed by cutting a groove 0.5 mm deep on the surface of the substrate 100 (Fig. 4). The depth of the groove portion to be provided in the form of inlet tube 111 is 1 mm. The length of the 100D loop is 30 mm. A through hole with a diameter of 1 mm is formed at the end of the groove and is presented in the form of a control valve 100E, which is closed by an actuated and closed cap. The thrombus formation chamber 110 has a length of 30 mm and a width of 2.5 mm in the wider part and a narrowed part 114 having a width of 0.5 mm. A through hole of 1 mm diameter is then provided 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.

Ribbon-shaped collagen with a width of 10 mm was applied as a blood clot material 115 onto the substrate 200 to a position covering the narrowed portion 114 of the substrate 100 (FIG. 5). The application of collagen is carried out so that a rectangular masking tape is attached corresponding to the narrowed portion 114 of the substrate 200, and then it is coated with a desorption agent based on silicone SRX-211 (Dow Corning Toray Co., Ltd.). Subsequently, the masking tape exfoliates. Then, a solution of collagen type 1 (Wako Pure Chemical Industries, Ltd.) dissolved in 0.1 N acetic acid so that 1% was free of a desorbing agent was instilled, and then left to stand for 1 h at 25 ° C. After that, the collagen on the layer of the desorbing agent was washed with purified water and the collagen was applied only to the rectangular glass region corresponding to the narrowed portion 114, where the desorbing agent was not applied.

Subsequently, the surface of the collagen coated substrate 200 and the surface of the formed contour 100 are joined together face to face.

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

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

Solution A and solution B were allowed to flow respectively from the connecting part 100A and the connecting part 100B for 5 minutes. Physiological saline was injected from the connecting part 100A for washing the blood. As a result, the formation of a thrombus is confirmed mainly on region a and region b (FIG. 6) on the collagen-treated surface of the substrate 200.

Example 8

A thrombus formation monitoring device was made and the thrombus formation was then monitored in a manner analogous to Example 7, with the following exceptions: solution A was prepared so that a polyclonal antibody against factor XII (manufactured by COSMO BIO CO., LTD.) Was added to the blood immediately after taking a blood sample. from which papain was excised Fc domain, at a concentration of 0.3 mg / ml and unfractionated heparin of porcine origin (Wako Pure Chemical Industries, Ltd.) at a dose of 0.3; Solution B was prepared by diluting 50 times with a thromboplastin reagent (manufactured by Daiichi Pure Chemicals Co., Ltd.); collagen deposition was carried out on a pre-treated area of a transparent polystyrene substrate 200 (1 mm thick) with collagen deposited (Fig. 8) by dripping a solution obtained by dissolving potassium permanganate in concentrated sulfuric acid. At a concentration of 2 g / l, reacting at 25 ° C for 10 min followed by a quick wash with purified water, the collagen was applied by dripping a solution in which type 1 collagen (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 a narrowed portion was used for the thrombus formation chamber 110 (Fig. 7). It should be noted that the connecting part 100C of the substrate 100 has a through hole of 1 mm diameter connected to a silicone tube with an inner diameter of 1 mm as an outlet tube.

After the flow of solution A and solution B for 10 min, physiological saline was injected from the connecting part 100A to flush the blood, thereby confirming the adhesion of the red blood clot to the channel portion of the collagen supported region 200.

Example 9

A thrombus formation monitoring device was made and the thrombus formation was then monitored in a manner analogous to Example 7, except that:

solution A was obtained so that blood immediately after taking a 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 quinacrine (Sigma Co., Ltd.);

the connecting part 100B was closed with a cap and only solution A was then injected from the connecting part 100A at a flow rate of 1 ml / min.

When observed with a fluorescence stereoscopic microscope focused on the surface of a slide glass of a collagen coated substrate 200, the manifestation of fluorescence light of green hinkrin was confirmed on a part of the collagen coated surface, and this region spread over time as a spotted area. After 10 minutes, the manifestation of green fluorescent light, which could be caused by platelet adhesion, was confirmed on most of the collagen-coated surface.

Example 10

A thrombus formation monitoring device was made and thrombus formation was then monitored in a manner analogous to Example 9, except that solution A was obtained without the addition of quinaline.

Blood was injected from the connecting part 100A at a flow rate of 1 ml / min. When the tube of the connecting part 100C was compressed to overlap for 2 s during injection, the blood immediately moved 20 mm from the branch point in the circuit 100D, which was accompanied by an increase in internal pressure. Even after opening the connecting part 100C, the blood did not move from the 20 mm point, so that an increase in pressure could be confirmed.

Example 11

A thrombus formation monitoring device was manufactured and thrombus formation was then monitored in a manner analogous to Example 8, except that:

polyclonal anti-thrombin antibody (COSMO BIO CO., LTD.) was added, from which the Fc domain was removed by papain to a concentration of 30 μg / ml instead of the heparin solution A of Example 8;

and then solution B of Example 8 with thrombin PPAC at a concentration of 5 mg / ml was added.

After the flow of solution A and solution For 15 min, physiological saline solution was injected from the connecting part 100A to flush the blood. As a result, it was confirmed that a red blood clot was attached to a portion of the channel region of the collagen-treated substrate 200.

Example 12

A thrombus formation monitoring device was made and the thrombus formation was then monitored in a manner analogous to Example 7, except that:

Solution A was prepared so that 50 ml of blood, to which immediately 0.5 U / ml heparin and 10 μg / ml aprotinin were added immediately after blood sampling, was centrifuged at 800 rpm and platelet-rich plasma (PRP) was obtained from the supernatant, such thus obtaining a solution A; and

a solution in which a thromboplastin reagent at a concentration of 1 vial / ml (Sysmex Corporation) was diluted 30 times with physiological saline was used as solution B.

The formation of a blood clot in the narrowed portion 114 was monitored for 15 minutes using a stereoscopic microscope. The presence of a plurality of platelet clots attached around the narrowed portion 114 was confirmed. It was also observed that the number of clots increased and their size also increased, and platelet clots were reattached and detached.

Example 13

The formation of a blood clot was controlled by passing solution A and solution B in a manner analogous to example 7, with the following exceptions:

Solution A was prepared by adding 500 μg of red blood cells collected from a centrifuged pellet in 20 ml of PRP solution A of Example 12;

a solution in which a thromboplastin reagent at a concentration of 1 vial / ml (Sysmex Corporation) was diluted 30 times with physiological saline was used as solution B;

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

approximately 1 μl of whole blood adhered to the surface near the extension end point of the expanding part, subsequently spreading to a width of 2.5 mm, which is adjacent to the thrombus formation chamber 110 located upstream of the narrowed part 114 on the substrate 100, then the whole blood was left to settle at room temperature for 10 min in order to cause it to coagulate, and formed a preliminary thrombus, intended for use as a material that causes the formation of a thrombus.

The thrombus attachment area was monitored with a stereoscopic microscope for 15 minutes. The formation of a new red thrombus, which was located downstream of the preliminary thrombus as a starting point, was confirmed after about 5 minutes, it generally spread downstream for up to 15 minutes, and its width increased to 2.5 mm. It was confirmed that blood flowed along the formed blood clot or flowed through the gaps between the formed blood clot.

Example 14

The formation of a blood clot was controlled by passing solution A and solution B in a manner analogous to example 7, with the following exceptions:

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

antisense DNA (CCAACCACACCAACC: SEQ ID No. 2) was dissolved in physiological saline so that its concentration was 150 μM, and provided as solution B; and

during application of a thrombus formation inhibitor 115 onto a glass slide, a solution which was prepared by mixing the collagen solution of Example 7 with a solution obtained by dissolving 1 vial of thromboplastin reagent (Sysmex Corporation) in 1 ml of purified water and then dialyzed in a 5: 1 ratio was applied to a region without applied silicone and air dried at 4 ° C.

The formation of a thrombus in the narrowed portion 114 was monitored for 5 minutes using a stereoscopic microscope. The presence of a plurality of platelet clots attached around the narrowed portion 114 was confirmed. Monitoring also showed that the number of clots increased with a simultaneous increase in their size, with platelet clots reattaching and detaching.

Example 15

A thrombus formation monitoring device was manufactured and thrombus formation was monitored in a manner analogous to Example 14, with the following exceptions:

after obtaining PRP by the same procedure as in Example 14, antisense DNA (CCAACCACACCAACC: SEQ ID No. 2) was added immediately before measurement so that its concentration was 15 μM, thereby performing an antithrombin treatment, and the resulting solution was provided as solution A;

the injection hole of the connecting part 100B was closed in a manner analogous to example 9;

type I collagen (collagen reagent for coating a cell culture dish with collagen, type I-A mother liquor, Cellmatrix Co., Ltd.) was used instead of the collagen solution of Example 14; and

only solution A was passed at a flow rate of 0.2 ml / min for 5 minutes.

The formation of a thrombus in the narrowed portion 114 was monitored using a stereoscopic microscope. The presence of a plurality of platelet clots attached around the narrowed portion 114 was confirmed. Monitoring also showed that the number of clots increased with a simultaneous increase in their size, with platelet clots reattaching and detaching.

Example 16

A thrombus formation monitoring device was made by a method similar to Example 7, with the following exceptions:

a groove of 0.1 mm deep was formed on the surface of the substrate by cutting the contour (Fig. 7), while leaving the dividing walls so that concave rows were obtained, each 1 mm long and 40 μm wide, extending over the entire surface of the lower part around the center thrombus formation chambers 110 with 40 μm intervals between them;

the injection hole of the connecting part 100B was closed in a manner analogous to example 9; and

substrate 200 was a glass slide without surface treatment. The gap (channel) between the separation walls simulates a blood capillary.

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

The formation of a thrombus in the gap between the dividing walls was monitored using a stereoscopic microscope. As a result, the channels were sequentially closed during monitoring for 5 minutes. Monitoring revealed that approximately half of the canals were blocked by an formed thrombus.

Example 17

A syringe type fluid supply device was used (FIG. 9). The microchip 300, where the substrate and the cover of the microchip (Fig. 9 (B) and Fig. 9 (C)) were made of the same materials as the materials of example 7, were connected by compression. The microchip 300 includes an inlet tube 322 for a blood coagulation inhibitor and an outlet tube 313 that were attached to the substrate 100, as in the case of the outlet tube of Example 7. However, all grooves of the microchip 300 (FIG. 9) were formed with a depth of 200 μm. A channel was also formed so that blood could be introduced into the channel from the syringe for samples 320, while the channel had a width of 1 mm and a length of 40 mm and was connected to a thrombus formation chamber 311, consisting of a narrow channel having a width of 200 μm and a length of 10 mm, and an inlet tube for a blood coagulation inhibitor 322. The solution was prepared so that the contents of 1 vial of RT Sysmex Corporation reagent (tissue thromboplastin) were dissolved in 1 ml of purified water and then dialyzed in purified water. The dialyzed product in purified water was mixed with type I collagen (manufactured by Nitta Gelatin Inc.) in a 1: 1 ratio. The solution was applied to the surface of the material causing the formation of thrombus 315. After that, the part with the applied solution was dried at 4 ° C in air and the thrombus formation chamber 311 was covered and used as the material causing the formation of thrombus 315. Blood was taken after the addition of anticoagulation treatment, so that the final concentrations of the respective components in the syringe were 25 μg / ml for the trypsin inhibitor obtained from maize, 10 μM for the thrombin aptamer exocyte I and 10 μM for the thrombin aptamer exocyte II (5'-AGTCCG TGGTAGGGCAGGTTGGGGTGACT-3 ': seq id No. 3). Immediately prior to monitoring, antisense DNA was added to the blood collected in this way with respect to thrombin aptamers of exocyte I and II so that each of them reached a concentration of 40 μM. Then, the syringe (1 ml syringe manufactured by Terumo Corporation) was filled with blood and provided as a syringe for sample 320 shown in FIG. 9. A My Flow device (manufactured by Arbiotec Co., Ltd.), which is a micro-feed pump capable of controlling the internal pressure of a sediment supplying fluid, was connected to a pressure supply tube 317. The mineral oil was pressed into the microchip 300 from the top of the syringe 320, and blood pushed into the microchip 300 with a flow rate of 50 μl / min while monitoring the pressure exerted on the syringe for sample 320.

As a coagulation inhibitor, inlet tube 322 for a blood coagulation inhibitor was supplied with 1 M Tris-HCl (pH 10) at a flow rate of 50 μl / min. The pressure began to increase 6 minutes after the start of the pressure measurement.

The pressure, repeating its changes in the direction of increase and decrease due to the movement of the thrombus ("control", shown in Fig.13), rose to 80 kPa.

Example 18

Blood sampling was carried out by adding an anticoagulation agent in a manner analogous to Example 17. Then, the same procedure as the procedure of Example 17 was carried out, except that unfractionated heparin was added at a concentration of 1 U / ml. Then a pressure measurement was performed. The pressure increased to about 10 kPa (Heparin shown in FIG. 13).

Example 19

A blood sample was taken with the addition of an anticoagulation agent in a manner analogous to Example 17. Then, the same procedure as the procedure of Example 17 was carried out, except that ReoPro (LILLY Co., Ltd.) was added at a concentration of 50 μg / ml. Then a pressure measurement was performed. When measured for 18 minutes, it was not possible to confirm the increase in pressure (“ReoPro” shown in FIG. 13).

Example 20

A blood sample was taken with the addition of 3.2% citric acid, so that the ratio between citric acid and blood was 1: 9. Then, blood was added with a maize-derived trypsin inhibitor at a concentration of 25 μg / ml for anticoagulation treatment. Immediately after adding calcium chloride to the anticoagulant treated blood, so that its concentration reaches 15 mM relative to the anticoagulant treated blood during monitoring of blood clot formation, blood is added to the syringe (1 ml syringe manufactured by Terumo Corporation) followed by blood flowing 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 as in Example 17. The pressure began to increase 15 minutes after the start of the pressure measurement, and it increased to about 60 kPa.

Example 21

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

Example 22

A blood sample was taken with the addition of an anticoagulation agent in a manner analogous to Example 20. Then the procedure was the same as that of Example 20, except that ReoPro (LILLY Co., Ltd.) was added at a concentration of 2 μg / ml. Then a pressure measurement was performed. The pressure began to increase 20 minutes after the start of the pressure measurement, and it grew to about 15 kPa after 25 minutes (Fig. 15).

Example 23

The syringe type fluid supply device shown in FIG. 12 was used. In addition, the microchip 400, where the substrate 200 and the microchip cover and the substrate 100 in the form of the microchip body shown in Fig. 12 (B) and Fig. 12 (C), were connected by compression. An outlet pipe 413 made of Teflon (registered trademark) was filled with mineral oil and one end thereof was connected to a microchip 400 through a connecting tube 412 made of silicone rubber, and the other end was connected to a suction pump 414 into which a pressure sensor was inserted . The inlet tube forming the channel of the microchip 400 was connected to the syringe 420 through a connecting tube 412 made of silicone rubber. One end of the inlet tube for a coagulation inhibitor 422 made of Teflon (registered trademark) was connected to a fluid supply pump 423, the other end of which was connected to the end of the thrombus formation chamber through a connecting tube 412 made of silicone rubber. Thus, the microchip 400 was connected to an outlet tube 413, a syringe 420 and an inlet tube for a blood clotting inhibitor 422, and a blood clot monitoring device B was obtained. A computer-controlled camera 430, equipped with an optical light source 432 (light emitting diode), which was installed and could move on the guide 433, was installed under the thrombus formation chamber.

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

The channel into which the blood was introduced had a width of 800 μm and a length of 7 mm. The narrowed channel had a width of 200 μm and a length of 10 mm.

The solution was prepared so that the contents of 1 vial of RT reagent (tissue thromboplastin, Sysmex Corporation) were dissolved in 1 ml of purified water and then dialyzed in purified water, the dialyzed product in purified water was mixed with type I collagen (manufactured by Nitta Gelatin Inc.) in a ratio of 1 :one. Then, the solution was applied to the region of the microarray substrate 200 facing the channel of the narrowed part on the surface to be connected by compression with the microarray substrate 100. After that, the liquid-coated part was vacuum dried at 4 ° C, and then the substrate 100 and the substrate 200 were connected by compression with each other, providing a microchip 400 having a thrombus formation chamber 411.

Blood sampling was carried out using a vacuum blood collection vessel in which sodium citrate was enclosed as an anticoagulant. Immediately prior to injection into the syringe 420, calcium chloride was added to the blood at a final concentration of 12.5 mM and a maize derived trypsin inhibitor at a final concentration of 25 μg / ml.

A My Flow device (manufactured by Arbiotec Co., Ltd.), which is a micropump pump capable of controlling the internal pressure of a sediment supplying fluid, was connected to the outlet pipe 413 as a pump 414. Thus, suction was performed at a rate of 15 μl / min at simultaneous control of internal pressure in the channel.

As a coagulation inhibitor, 1 M Tris-HCl (pH 10) was passed from the inlet tube for the blood clotting inhibitor 422 at a flow rate of 8 μl / min. The formation of a predominantly white blood clot was observed 4 minutes after the start of the pressure measurement, while a decrease in internal pressure was confirmed. In addition, it was confirmed that the internal pressure decreased to 30 kPa after 10 minutes from the start.

Further, thrombus formation was observed by imaging as the camera 430 moved around a thrombus formation region in the channel. The position in which the formation of a blood clot was observed was remembered, and the camera 430 received images by regularly moving around a plurality of defined points. As a result, the growth and destruction of many large blood clots were recorded over time.

The following reference experiments will be described to demonstrate the effectiveness of thrombin aptamers and their antisense DNAs as anticoagulation agents and anticoagulation agents, respectively.

Control experiment 1

Blood was provided immediately after it was taken, as well as blood obtained by adding one tenth of a volume of 300 μg / ml of trypsin inhibitor obtained from maize to the first portion of the blood, and blood obtained by adding the thrombin aptamer, which recognizes exocyte I, to the second portion of the blood, so that a concentration of 5 μM was obtained. Then, for these three types of blood, the time required for clotting was measured in Eppendorf tubes (made of polypropylene). The tube with the sample was turned upside down every minute, and its fluidity was confirmed. Blood without additives showed a clotting time of 9 minutes. In contrast, blood supplemented with a trypsin inhibitor showed a clotting time of 22 minutes. Further, the blood after the addition of the thrombin aptamer showed a clotting time of 62 minutes.

Further, when 5 μM thrombin aptamer that recognizes exocyte I and 5 μM thrombin aptamer that recognizes exocyte II were used in combination, blood coagulation was not confirmed even after 3 hours even if there was no trypsin inhibitor in the blood.

Thrombin aptamer recognizing exocyte I: 5'-GGTTGGTGTGGTTGG-3 'SEQ ID No. 1,

Thrombin aptamer recognizing exocyte II: 5'-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3 'SEQ ID No. 3).

Control experiment 2

ARTT was measured on 200 μM plasma relative to each of sample A with the addition of 10 μM physiological saline, sample B with the addition of 10 μM 500 μM thrombin aptamer recognizing exocyte I, and sample C with 10 μl of a solution containing 500 μM thrombin aptamer, recognizing exocyte I, and 1500 μM antisense DNA of the thrombin aptamer recognizing exocyte I. The ARTT of sample A was 44 s, sample B 2 min or more, and sample C 45 s.

Control experiment 3

11 (A) is a thromboelastogram coagulation waveform analysis during blood storage with the addition of 10 μl aptamers to exocyte I and exocyte II at room temperature for 15 min, and then 40 μM antisense DNA of both aptamers. 11 (B) is a blood thromboelastogram waveform immediately after taking a blood sample.

As is evident from the results presented in FIGS. 11 (A) and 11 (B), it was found that the addition of two types of thrombin aptamers provides the ability to store blood, and antisense DNA can carry out its anticoagulation treatment.

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

Control experiment 4

Next, the effectiveness of the effects of the PMEA coating during blood storage and platelet adhesion to the substrate will be described.

(1) A methanol solution containing 1% PMEA obtained in accordance with JP 04-152952 A was applied to a 2.5 ml acrylic resin container (internal dimensions: 1 cm × 1 cm × 4.5 cm) and then dried at 90 ° C for 10 minutes Subsequently, 760 μl of blood subjected to anticoagulation treatment with 2% citric acid was added to each uncoated container and a container coated with PMEA, followed by pipetting every 5 minutes to confirm blood coagulation.

Consequently, the uncoated container showed an obvious increase in viscosity after 30 minutes and coagulation after 60 minutes. In contrast, the PMAA coated container showed an obvious increase in viscosity after 35 minutes, but the coagulation onset time lengthened to 90 minutes.

(2) The indicated PMEA was applied flat on a transparent acrylic plate. It was used instead of the collagen-coated plate of Example 9 (Fig. 8), and an experiment on the adhesion of quinacrine-labeled platelets and leukocytes in the blood was carried out in a manner analogous to Example 9.

As a result, obvious adhesion or agglutination of platelets and leukocytes was observed 10 minutes after the onset of blood flow. In contrast, platelet and white blood cell adhesion or agglutination on an acrylic plate coated with PMAA was not observed even after 30 minutes.

According to the above results, in the thrombus formation monitoring device of the present invention by coating the PMEA syringes for storing blood, a tube, a substrate, and similar surfaces, thrombus formation can be suppressed at a place other than the thrombus formation chamber. Therefore, it is contemplated that thrombus formation monitoring specific to the thrombus formation monitoring chamber can be achieved.

In the above description, the present invention has been described with reference to preferred embodiments. However, the present invention is not limited to the examples and embodiments described above, and various modifications can be applied without departing from the spirit of the present invention. For example, you can set the high pressure generated by the pump and set the small diameter of the exhaust pipe. In this case, backpressure can be created in the inlet tube and in the outlet tube, so that the formation of a blood clot can be monitored while at the same time controlling the volumetric blood flow under load conditions with internal pressure corresponding to the blood pressure. Further, the blood flow rate 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 thrombus formation monitoring device and the thrombus formation monitoring method according to the present invention can be successfully used in a comprehensive assessment of blood coagulation and platelet formation in an environment equivalent to blood flow, using whole blood or plasma containing platelets to evaluate the effectiveness of an antithrombotic drug administered to a patient , or the like.

Claims (11)

1. A device for monitoring the formation of a blood clot by passing subjected to anticoagulation treatment of blood through a channel simulating a blood vessel, and while performing anticoagulation treatment or stimulation of blood coagulation, containing:
a thrombus formation chamber, at least in a part of which a material causing thrombus formation is placed,
an inlet tube that is connected to the thrombus formation chamber and through which blood flows into the thrombus formation chamber,
a blood supply container connected to the inlet tube,
container feed pump
pressure sensor for measuring the pressure applied to the container. 2. The device according to claim 1, additionally containing an inlet tube for supplying a thrombus formation inhibitor connected to a thrombus formation chamber and for mixing the thrombus formation inhibitor with blood after passing through the thrombus formation chamber. 3. The device according to claim 1, additionally containing a tube for supplying a drug connected to the inlet tube, and through the specified tube is supplied a drug that provides anticoagulation treatment, or a drug that stimulates blood coagulation. 4. The device according to claim 1, wherein said tube and thrombus formation chamber are combined with each other on a substrate and are a microchip circuit. 5. The device according to any one of claims 1 to 4, in which the material causing the formation of a blood clot contains collagen. 6. The device according to claim 5, in which the material causing the formation of a blood clot contains a tissue factor. 7. The device according to any one of claims 1 to 4, which further comprises a camera for acquiring an image of a thrombus formation chamber. 8. A method for monitoring the formation of a blood clot, which consists in directing blood after administration of an anticoagulant from the container into the blood clot chamber by pressing on a fluid having a density lower than the blood layer placed on the blood layer, while performing anticoagulation treatment of blood or stimulating coagulation blood
determine the pressure applied to the container, while the material causing the formation of a thrombus, is placed at least in part of the thrombus formation chamber. 9. The method according to claim 8, in which the blood subjected to anticoagulation treatment is obtained by using a contact phase factor inhibitor and a calcium chelator, said anticoagulation treatment being carried out by means of a free calcium donor. 10. The method according to any one of claims 8 and 9, in which collagen and tissue factor are used as the material causing the formation of a blood clot. 11. The method of claim 8, in which monitoring the formation of a blood clot by using the device according to any one of claims 1 to 4.

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