In atherothrombosis such as myocardial infarction, atherosclerotic plaque collapses at the site of arteriosclerosis, and platelets adhere to collagen containing tissue factor which was exposed to the blood flow, followed by complex occurrence of platelet aggregation and activation of the blood coagulation system, leading to formation of a serious occlusive thrombus. Heart diseases such as myocardial infarction are serious diseases which represent the second commonest cause of death among all of the causes of death in Japan.
However, in myocardial infarction and the like, formation of a thrombus proceeds only in areas of arteriosclerosis, and the thrombotic tendency does not extremely proceed throughout the body. In vitro tests are not suited for evaluation of the thrombotic tendency in such thrombosis or for monitoring of the antithrombotic effect in antithrombotic therapy, so that a comprehensive evaluation of coagulation and platelets (adhesion and aggregation) under blood flow is important.
Conventionally, blood coagulation capacity has been evaluated by monitoring activated partial thromboplastin time (APTT) and thromboplastin time (PT) using blood plasma. Mainly, APTT reflects intrinsic coagulation and PT reflects extrinsic coagulation. As monitoring of platelets, by using platelet-rich plasma and by adding a platelet-activating substance such as adenosine diphosphate (ADP) or collagen, the aggregation capacity of the platelets can also be evaluated based on a change in transmittancy or the like. Further, coagulation time of whole blood is also measured based on whole blood coagulation time, whole blood coagulation time on recalcification or the like.
Further, as a monitoring system using whole blood, thromboelastogram is used to monitor activation of coagulation components, aggregation of platelets and the like.
However, a thrombus grows under blood flow in vivo, and since the above-described monitoring method and the like are those carried out in a closed in vitro system, it is impossible to observe a state wherein a thrombus grows in vivo.
As proposals to solve this problem, Patent Literature 1 and Non-patent Literatures 1 and 2 disclose methods for monitoring adhesion and aggregation of platelets by a confocal microscope, the method comprising allowing blood to pass through on a collagen cell, to which blood an antithrombotic agent to be evaluated was added, and fluorescently labeling the platelets.
However, since, in the inventions described in these literatures, monitoring is carried out in the presence of an anticoagulant, absence of formation of a thrombus by adhesion or aggregation of platelets induced by the coagulation system, or decreased ability of thrombus formation is evaluated by monitoring of changes in the morphology of platelets, so that activation of platelets coupled with the coagulation system is not reflected. Therefore, they are inventions preferred for evaluation of pharmacological effects of antiplatelet drugs, but incapable of monitoring a thrombus itself or the entire process of thrombus formation.
Further, in Patent Literature 2, disclosed is a thrombus monitoring device for monitoring thrombus formation by allowing anticoagulated blood to flow into a channel mimicking a blood vessel while releasing the anticoagulation treatment or promoting blood coagulation, the device comprising: a thrombus formation chamber which has been provided with, in at least a part of the inside thereof, a thrombus-inducing material that induces formation of a thrombus; an inlet tube connected to the thrombus formation chamber, for allowing blood to inflow into the thrombus formation chamber; and an agent tube connected to the inlet tube, for feeding into the inlet tube an agent for releasing the anticoagulation treatment or an agent for promoting blood coagulation. By this device, monitoring of blood can be carried out by using a small amount of a blood sample, but there were cases where the blood and the agent for releasing the anticoagulation treatment or the agent for promoting blood coagulation could not be mixed well, so that there was a room for improvement.
Further, in cases where platelets were stained with quinacrine by the method according to Patent Literature 1 or Non-patent Literature 1 or 2, leukocytes were also stained at the same time, so that selective evaluation of only the function of platelets was impossible. Further, there were many points unsuitable for the monitoring, such as color deterioration of the fluorescent dye and expensiveness of the device.
Further, in a conventional evaluation of fibrinolytic capacity, only lysis of fibrin is measured, and there has been no method capable of measuring the lytic effect on a white thrombus formed in an actual artery.
Microfluidic chips formed by engraving a groove to provide a desired channel on a substrate and laminating the substrate with a cover plate are used in a wide variety of uses such as capillary electrophoresis and PCR as well as various monitoring. Since a liquid in a microchip forms a laminar flow due to a very large Reynolds number, it is difficult to mix the liquid in the microchip naturally. To solve this problem, various approaches have been taken.
For example, a stirring bar consisting of a columnar projection is proposed in Patent Literature 3; a stirring bar which vibrates by the piezoelectric effect is proposed in Patent Literature 4; and a stirring bar which rotates by light pressure is proposed in Patent Literature 5. However, in some cases, when a liquid containing whole blood, platelet-rich plasma or a drug-treated liquid thereof is stirred in a microchip using the proposed stirring bars, activation occurs leading to blood coagulation or the like, so that, for example, in cases where blood coagulation, the function of platelets or the like is monitored, accurate monitoring is very difficult. Further, the liquid in a microchip could be made to be naturally mixed by extending the channel, but in this case, the microchip becomes large, which is problematic. And, if the channel is made to wind a plurality of times in switchback style, activation occurs at regions where the liquid remains and the like, leading to blood coagulation or the like, so that the monitoring cannot be carried out accurately.    Patent Literature 1: JP 2004-251630 A    Patent Literature 2: WO 2007/046450    Patent Literature 3: JP 2007-24522 A    Patent Literature 4: JP 2006-205080 A    Patent Literature 5: JP 2001-252897 A    Non-patent Literature 1: Blood. 1990; 75:390-398    Non-patent Literature 2: Blood. 1999 Aug. 1; 94 (3):968-75
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