Method for evaluating risk of acute cerebral vascular disease using soluble CLEC2

Abstract

Provided is a risk evaluation method for transient ischemic attack (TIA) diagnosis, the method using an easy, rapid, and objective blood test capable of complementing medical interviews regarding clinical symptoms. Also provided is a risk evaluation method for differential diagnosis of cardiac and non-cardiac causes of acute cerebral vascular disorders such as brain infarction and TIA, the method using images, electrocardiogram, and an easy, rapid and objective blood test capable of complementing conventional blood tests. The methods include measuring the concentration of soluble CLEC2 present in the blood collected from a patient.

Description

Method for assessing risk of acute cerebrovascular disease using soluble CLEC2

[0001] The present invention relates to a method for assessing the risk of acute cerebrovascular disease using soluble CLEC2.

[0002] Stroke is a group of diseases that includes ischemic stroke, which occurs when blood vessels in the brain become blocked (i.e., cerebral infarction), and hemorrhagic stroke, which occurs when blood vessels in the brain rupture (i.e., cerebral hemorrhage and subarachnoid hemorrhage), and is the second leading cause of death worldwide after ischemic heart disease. Stroke not only accounts for a large number of deaths, but even if patients survive, they often leave behind severe after-effects, which is a major factor in the need for nursing care and places a heavy burden on both patients and society. Therefore, a system is needed that allows for prevention, early diagnosis, rapid response after diagnosis, and care and risk management by a wide range of medical professionals.

[0003] Among ischemic strokes, cerebral infarction occurs when blood vessels in the brain narrow or when a blood clot formed elsewhere blocks blood vessels in the brain, preventing oxygen and nutrients from reaching the brain. As a result, nerve cells in the brain die, causing various disorders.

[0004] The thrombi that cause cerebral infarction can be broadly divided into cardiogenic cerebral embolism, in which a thrombus formed in the heart travels through blood vessels and blocks an artery in the brain, and non-cardiogenic cerebral infarction, in which a thrombus formed elsewhere than the heart is caused. In the case of cardiogenic embolism, the thrombus is believed to be primarily fibrin, and anticoagulants (warfarin or DOAC) that inhibit fibrin formation are used for treatment and secondary prevention. On the other hand, in the case of non-cardiogenic cerebral infarction, the thrombus is believed to be primarily platelet-derived, and antiplatelet drugs (aspirin, clopidogrel, etc.) that inhibit platelet activation are used for treatment and secondary prevention. In other words, because the treatments for cardiogenic cerebral ischemic stroke and non-cardiogenic stroke are clearly different, it is important to correctly diagnose whether the stroke is cardiogenic or non-cardiogenic.

[0005] Cardiogenic cerebral infarction occurs when a clot formed due to stasis of blood flow in the atria caused by atrial fibrillation travels through the bloodstream and reaches the brain, causing a relatively large infarction. Diagnosis is based on the observation of a large infarction on MRI images and information on past medical conditions such as atrial fibrillation, arrhythmia, and heart disease. On the other hand, non-cardiogenic cerebral infarction is broadly divided into atherosclerotic and lacunar. Atherosclerotic cerebral infarction occurs when a relatively large artery is blocked, while lacunar infarction occurs when a small blood vessel is blocked, so diagnosis is based on information such as the size of the infarction on MRI images, the state of arteriosclerosis, and metabolic abnormalities on blood tests.

[0006] Atrial fibrillation can be detected by electrocardiogram, but there are persistent and sudden types, and sudden types are difficult to detect. In addition, images of atherosclerotic cerebral infarction and cardiogenic cerebral infarction are often difficult to distinguish clearly.

[0007] On the other hand, transient ischemic attack (TIA) is known to present with symptoms similar to those of cerebral infarction. TIA is a condition in which blood vessels narrow or become blocked by a blood clot, just like cerebral infarction, but the event is transient, and symptoms similar to those of cerebral infarction occur transiently.

[0008] TIA was previously defined as a condition in which cerebral ischemic symptoms disappeared within 24 hours. However, advances in imaging technology have led to an increasing number of cases in which cerebral infarction is detected through imaging, even after symptoms have disappeared. Therefore, a 2009 paper by the American Heart Association / Stroke Association (AHA / ASA) defined TIA as "a temporary neurological dysfunction due to focal ischemia of the brain, spinal cord, or retina, without acute infarction," distinguishing infarction from TIA regardless of the duration of symptoms. In other words, a condition in which infarction is visible on imaging even after symptoms have disappeared is considered cerebral infarction, while a TIA is defined as a condition in which symptoms have disappeared and no cerebral infarction is detected on imaging. The World Health Organization's (WHO) International Classification of Diseases (ICD-11, 2018) also stipulates that TIA must not have an infarct, and the global consensus is that diagnosis should be based on imaging.

[0009] In Japan, the Japan Stroke Society defined TIA in 2019 as "a transient episode of neurological dysfunction resulting from focal cerebral or retinal ischemia, without signs of acute infarction. The episode of neurological dysfunction resolves within 24 hours at the longest." Furthermore, although TIA is a type of cerebrovascular disease, it is defined as one that does not present with cerebral infarction, and therefore it is clarified that it is not included in the three types of stroke: cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage.

[0010] Thus, TIA is clearly distinguished from cerebral infarction. Diagnosis of TIA is extremely important, even if the symptoms have disappeared. This is because TIA patients often develop cerebral infarction early. Multiple clinical studies have reported that 10-15% of TIA cases develop cerebral infarction within three months, and half of those cases occur within 48 hours, demonstrating that cerebral infarction often occurs much earlier than previously thought. Based on this data, the Japan Stroke Society recommended in its 2009 "Stroke Treatment Guidelines" that "if TIA is suspected, preventive treatment should be initiated immediately." In other words, TIA is said to be a precursor or precursor to the serious condition of cerebral infarction.

[0011] As mentioned above, the presence or absence of a lesion is necessary for the diagnosis of TIA, but the longer the time between the onset of TIA and the performance of an MRI diffusion-weighted image (DWI), the higher the DWI positivity rate (17% within 6 hours, 37% after 24 hours). It is also said that in many cases where no lesion was found on the first DWI, a lesion was found on the second DWI. In other words, if the definition is that cases where a lesion is found on imaging are cerebral infarction, and cases where no lesion is found are TIA, the timing of DWI greatly influences the diagnosis.

[0012] However, there is a need for a method that allows TIA to be diagnosed not only at specialized hospitals but also by general practitioners who are not able to perform imaging tests, and there is also a need to respond to situations where doctors who are not specialists, such as stroke specialists or neurologists, must make quick decisions in the emergency room of a general hospital.

[0013] Furthermore, at the time of initial consultation, symptoms of TIA often disappear by the time the patient arrives at the hospital, and in such cases, a clear diagnosis is often difficult to make, as the diagnosis of TIA relies on an accurate examination of the patient's condition, including medical history and complications. Furthermore, diseases that must be differentiated from TIA include migraine accompanied by neurological symptoms, seizures, idiopathic transient global amnesia, Meniere's syndrome, hyperventilation syndrome, syncope, hypoglycemia, narcolepsy, and periodic paralysis, and it is often difficult to distinguish between them.

[0014] To improve this situation and increase the accuracy of TIA diagnosis, there is a need for an index that can be used to make objective judgments using biomarkers.

[0015] With recent advances in diagnostic techniques such as MRI imaging and ultrasound, detailed investigation of mechanisms is now possible, and efforts are underway to clarify the mechanisms of TIA, such as cardiogenic, atherothrombotic, lacunar, and other causes, just like with cerebral infarction. However, in the case of TIA, where no infarction images can be seen, it is even more difficult to clearly differentiate and diagnose the cause than with cerebral infarction.

[0016] C-type lectin-like receptor 2 (CLEC2) was identified on platelets as a receptor for the platelet-activating snake venom rhodocytin. CLEC2 is expressed almost exclusively in platelets and megakaryocytes in humans, making it a platelet-specific molecule. It has been reported that CLEC2 is released into the blood as soluble CLEC2 (hereinafter abbreviated as sCLEC2) upon platelet activation (Patent Document 1, Non-Patent Document 1).

[0017] When a blood vessel ruptures and a blood clot forms, platelets are first activated and aggregate to form a primary clot, after which the coagulation system is activated and water-soluble fibrinogen turns into an insoluble fibrin network, which then covers the clot and forms a strong secondary clot. In actual hemostatic clots, the platelet system and the coagulation system activate each other, and a hemostatic clot formed by platelets, fibrin, and red blood cells caught in it is observed.

[0018] Since the blood sCLEC2 concentration increases with platelet activation, it has been shown that the blood sCLEC2 concentration is associated with the formation of thrombosis, and a high level of blood sCLEC2 can be said to suggest the formation of a blood clot somewhere in the body (Patent Documents 1 to 3).

[0019] Patent No. 6078845 Patent Application No. 2020-032797 Patent Application No. 2021-003671 Patent Application No. 2021-060870

[0020] F. Kazama et al., Platelets 2015; 26(8):711-719Y. Yamashita et al., Thrombosis Research 178(2019) 54-58X. Zhang et al., Stroke.2019;50:45-52.

[0021] Cerebral infarction is a serious disease with a high mortality rate, and even if death is avoided, it often leaves behind residual damage. As mentioned above, approximately 15% of TIA cases are known to result in cerebral infarction, making early diagnosis and preventive treatment extremely important. If a TIA is diagnosed, appropriate antithrombotic therapy can significantly reduce the chance of subsequent cerebral infarction. Furthermore, by keeping patients diagnosed with TIA under medical supervision for, for example, one week after onset, even if a cerebral infarction does occur, immediate treatment with thrombolytic therapy or other methods can significantly improve the aftereffects. The sooner cerebral infarction treatment is initiated, the better, as it is a race against time.

[0022] A TIA is diagnosed through a medical interview about clinical symptoms and a risk assessment of the patient. The main symptoms include paralysis of one side of the limbs (motor impairment), numbness on one side (sensory impairment), difficulty speaking or slurred speech (language impairment), difficulty seeing in one eye (visual impairment), and difficulty seeing on one side (visual field impairment). These symptoms usually disappear within a few minutes or up to an hour. The only way to diagnose a TIA is through a medical interview about the symptoms, but symptoms can range from mild to severe, and patients may not be able to accurately describe them to their doctor.

[0023] To compensate for the difficulty of such interviews, patient risk assessments are also widely conducted. The ABCD2 score is a typical example, which evaluates age, blood pressure, clinical features, diabetes, and duration of symptoms by assigning a score. This is merely an assessment of the risk of subsequent cerebral infarction, and does not diagnose TIA itself. As described above, there are no objective imaging tests or blood tests that can supplement the interviews about clinical symptoms when diagnosing TIA.

[0024] As mentioned above, in acute cerebrovascular disorders such as cerebral infarction and TIA, it is extremely important to distinguish between cardiogenic and noncardiogenic causes, but this determination is not always easy. Currently, the diagnosis of cardiogenic or noncardiogenic cerebral infarction is determined comprehensively based on the results of imaging tests, electrocardiograms, echocardiograms, and blood tests such as lipid and blood glucose levels and heart failure markers. Biomarkers such as D-dimer and soluble fibrin have been proposed to support thrombological diagnosis, but they are not yet accurate enough to distinguish between cardiogenic and noncardiogenic causes, and have not yet been used in clinical settings. In the case of TIA, the diagnosis of cardiogenic or noncardiogenic causes is even more difficult because imaging tests do not reveal infarction.

[0025] Therefore, an object of the present invention is to provide a simple, rapid, and objective risk assessment method using a blood test that can supplement a medical interview regarding clinical symptoms in the diagnosis of TIA, and to provide a simple, rapid, and objective risk assessment method using a blood test that can supplement images, electrocardiograms, and conventional blood tests in the differential diagnosis of cardiogenic and non-cardiogenic acute cerebrovascular disorders such as cerebral infarction and TIA.

[0026] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they found that the blood sCLEC2 concentration in TIA patients is significantly elevated compared to that in healthy individuals. They also found that high sCLEC2 levels can occur even in a state where there is no infarction and no detectable thrombus, as in TIA, rather than in a state where there is infarction and no detectable thrombus, as in cerebral infarction. This is consistent with the fact that TIA patients have a high probability of developing cerebral infarction in the near future. In other words, high sCLEC2 levels, even in a state where there is no current infarction and no detectable thrombus, predict that platelets are activated and that there is a high probability that a thrombus of a certain size will form in the near future, clogging the blood vessel and causing infarction.

[0027] Furthermore, in order to assess the risk of TIA patients using sCLEC2 concentration, an appropriate threshold, i.e., cutoff value, is required. In the present invention, it was found that the cutoff value for TIA in terms of blood sCLEC2 concentration is preferably 66 pg / mL or more and 148 pg / mL or less.

[0028] In TIA patients, infarction images cannot be used to make a diagnosis, so blood tests are extremely useful in helping to determine whether the cause is cardiogenic or non-cardiogenic.

[0029] Furthermore, when sCLEC2 was measured in the blood of patients with cerebral infarction, the researchers found that sCLEC2 levels were significantly higher in non-cardiogenic patients than in cardiogenic patients. Based on this finding, they simultaneously measured D-dimer, a biomarker indicating the presence of fibrin clots, and calculated the ratio of sCLEC2 to D-dimer (sCLEC2 / D-dimer ratio). They found that the sCLEC2 / D-dimer ratio in non-cardiogenic patients was significantly higher than that in cardiogenic patients. In other words, since sCLEC2 indicates platelet activity and D-dimer indicates the presence of fibrin clots, using the ratio of sCLEC2 to D-dimer to distinguish between non-cardiogenic, which is platelet-dominant and requires treatment with antiplatelet drugs, and cardiogenic, which is fibrin-dominant and requires treatment with anticoagulants, can provide a more accurate diagnostic aid in addition to conventional methods.

[0030] Based on these findings, the present invention has been completed as a method for assessing the risk of acute cerebrovascular disease using soluble CLEC2.

[0031] That is, the present invention provides the following: [1] A method for risk assessment of acute cerebrovascular disease, comprising the step of measuring the concentration of soluble CLEC2 present in blood collected from a patient suspected of having or diagnosed with acute cerebrovascular disease. [2] A method for risk assessment of a patient suspected of having or diagnosed with acute cerebrovascular disease, comprising the steps of: (1) providing a blood sample from the patient, (2) determining the concentration of soluble CLEC2 in the sample, and (3) correlating the concentration of soluble CLEC2 with the presence or absence, likelihood of outcome, or whether the acute cerebrovascular disease is cardiac or non-cardiogenic in the patient. [3] The method of [2], wherein the step of correlating the soluble CLEC2 concentration with the presence or absence, likelihood of outcome, or whether the acute cerebrovascular disorder is cardiogenic or non-cardiogenic in the patient comprises assessing whether the patient is at risk based on changes in the soluble CLEC2 concentration. [4] The method of [2], wherein the acute cerebrovascular disorder is a transient ischemic attack (TIA), and the step of correlating the soluble CLEC2 concentration with the TIA cutoff value of the soluble sCLEC2 concentration is 66 to 148 pg / mL. [5] A method for risk assessment of a patient suspected of suffering from or diagnosed with acute cerebrovascular disease, comprising the steps of: (1) providing a blood sample from the patient; (2) determining the concentration of soluble CLEC-2 in the sample; (3) measuring the number of platelets in the sample; (4) dividing the concentration of soluble CLEC-2 by the number of platelets; and (5) correlating the value obtained by dividing the concentration of soluble CLEC-2 by the number of platelets with the presence or absence of acute cerebrovascular disease in the patient, the likelihood of outcome, or whether the disease is cardiac or non-cardiogenic.[6] The method of [5], which is a method for assessing the risk of acute cerebrovascular disease in a patient suspected of having or diagnosed with acute cerebrovascular disease, wherein the acute cerebrovascular disease is a transient ischemic attack, and in the step of correlating the value obtained by dividing the soluble CLEC2 concentration by the platelet count with the transient ischemic attack, the cutoff value of the value obtained by dividing the soluble sCLEC2 concentration by the platelet count is 0.55 to 0.7. [7] Any of the methods of [1] to [4], which is a method for assessing the risk of acute cerebrovascular disease in a patient suspected of having or diagnosed with acute cerebrovascular disease, wherein the method provides a material for determining whether the acute cerebrovascular disease is cardiogenic or non-cardiogenic. [8] The method of [1], wherein the risk of acute cerebrovascular disease is assessed in a patient suspected of having or diagnosed with acute cerebrovascular disease, the method comprising the steps of: (1) providing a blood sample from the patient; (2) determining the concentration of soluble CLEC-2 in the sample; (3) measuring the platelet count in the sample; (4) dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker; and (5) correlating the value obtained by dividing the concentration of soluble CLEC-2 by the coagulation / fibrinolysis marker with the presence or absence of acute cerebrovascular disease, the likelihood of outcome, or whether the disease is cardiogenic or non-cardiogenic in the patient. [9] The method of [8], wherein the risk of acute cerebrovascular disease is assessed in a patient suspected of having or diagnosed with acute cerebrovascular disease, the method comprising the steps of: (1) providing a blood sample from the patient; (2) determining the concentration of soluble CLEC-2 in the sample; (3) measuring the platelet count in the sample; (4) dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker; and (5) correlating the value obtained by dividing the concentration of soluble CLEC-2 by the coagulation / fibrinolysis marker with the presence or absence of acute cerebrovascular disease, the likelihood of outcome, or whether the disease is cardiogenic or non-cardiogenic in the patient.

[10] A method for assessing the risk of acute cerebrovascular disease in a patient suspected of having or diagnosed with acute cerebrovascular disease, the method according to any one of [1] to [9], wherein the patient-derived sample is collected from the patient within 48 hours after the onset of symptoms of acute cerebrovascular disease.

[11] Any one of the methods according to [1] to

[10] , wherein the step of determining the concentration of soluble CLEC2 is performed by a highly sensitive immunoassay, such as a chemiluminescence immunoassay, an electrochemiluminescence immunoassay, or a fluorescent immunoassay.

[0032] The present invention also provides: a method for assessing the risk of acute cerebrovascular disorders, which involves measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the platelet count, or the value obtained by dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker); a method for assisting in the risk assessment of acute cerebrovascular disorders, which involves measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the platelet count, or the value obtained by dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker); a method for measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the platelet count, or the value obtained by dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker) for the risk assessment of acute cerebrovascular disorders; The present invention includes an in vitro method for risk assessment of acute cerebrovascular disease, characterized by measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the platelet count, or the value obtained by dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker); use of an antibody capable of detecting the concentration of soluble CLEC-2 in the manufacture of a risk assessment kit for acute cerebrovascular disease; and a method for measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the platelet count, or the value obtained by dividing the concentration of soluble CLEC-2 by a coagulation / fibrinolysis marker) in order to provide information necessary for risk assessment of acute cerebrovascular disease.

[0033] The method of the present invention, which involves measuring the sCLEC2 concentration in the blood of TIA patients or using the sCLEC2 / D-dimer ratio, enables simple, rapid, and objective risk assessment of acute cerebrovascular disease. It is also expected to contribute to improving the accuracy of determining the effectiveness of treatment in monitoring preventive treatment for cerebral infarction or TIA after a diagnosis of TIA.

[0034] 1 is a standard curve created using human sCLEC2 protein as a standard. This is a graph comparing the plasma concentrations of sCLEC2 between TIA patients and healthy individuals. This is an ROC curve (Receiver Operating Characteristic Curve) when TIA is diagnosed by measuring blood sCLEC2 concentrations. This is a graph measuring sCLEC2 concentrations in healthy individuals and cerebral infarction patients, and comparing sCLEC2 concentrations in three groups: healthy individuals, patients with cardiogenic cerebral infarction, and patients with non-cardiogenic cerebral infarction. This is an ROC curve when the sCLEC concentration in patients with cerebral infarction is used to make a differential diagnosis between cardiogenic cerebral infarction and non-cardiogenic cerebral infarction. This is an ROC curve when the D-dimer concentration in patients with cerebral infarction is used to make a differential diagnosis between cardiogenic cerebral infarction and non-cardiogenic cerebral infarction. 1 shows an ROC curve for the differential diagnosis between cardiogenic cerebral infarction and non-cardiogenic cerebral infarction using the sCLEC2 / D-dimer ratio in patients with cerebral infarction.

[0035] Although the embodiment of the present invention will be described in detail below, the mode of use is not limited to this.

[0036] Furthermore, as used herein, "CLEC2" refers to a platelet-activating receptor belonging to the C-type lectin family, which is normally present on the platelet membrane and is released into the blood upon platelet activation. As used herein, the term "soluble CLEC2 (sCLEC2)" refers to such CLEC2 or a molecule derived from CLEC2 that is released from platelets and detected in the blood (or in the buffer when incubated in a buffer).

[0037] sCLEC2 is believed to include proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Non-Patent Document 1). The proteins with molecular weights of approximately 40 kDa and approximately 32 kDa are present on the platelet membrane surface and are presumed to be released in a state contained in microparticles produced upon platelet activation. These proteins are thought to have glycosylation attached. On the other hand, the protein with molecular weight of approximately 25 kDa is thought to be cleaved by protease upon platelet activation and released from the platelet. In the present invention, the amount of sCLEC2 as described above is measured. Regarding sCLEC2, the proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa may be detected together, or only the protein with molecular weight of approximately 25 kDa may be detected.

[0038] The sCLEC2 concentration used in the present invention may be the sCLEC2 concentration alone or in combination with other biomarkers. Preferably, for example, in the diagnosis of TIA, the value obtained by dividing the sCLEC2 concentration by the platelet count (hereinafter, sometimes referred to as the C2PAC index) may be used. Unless otherwise specified, the term "sCLEC2 concentration" used herein is interpreted as including both the sCLEC2 concentration and the sCLEC2 concentration divided by the platelet count. Furthermore, in the differential diagnosis of cardiogenic cerebral infarction and non-cardiogenic cerebral infarction, not only the sCLEC2 concentration but also the sCLEC2 / D-dimer ratio, obtained by dividing the sCLEC concentration by D-dimer, can be used.

[0039] Although the sample used for the measurement is preferably derived from a human, samples derived from animals other than humans may be used to understand the pathology of experimental animals, etc. Experimental animals are not particularly limited, and examples thereof include guinea pigs, rats, mice, and dogs.

[0040] The method for detecting the presence of sCLEC2 is not particularly limited, but an immunological method using an antibody that recognizes sCLEC2 (hereinafter, this may be referred to as an "anti-sCLEC2 antibody") is preferred. Immunological protein detection methods include, for example, immunoassays using labeled antibodies such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, and immunochromatography, as well as any commonly known method commonly used in the art, such as Western blotting, latex agglutination, and immunoturbidimetry. However, simple immunoassays may not be sensitive enough to measure sCLEC2 concentrations in healthy individuals, and therefore a method sensitive enough to measure sCLEC2 in the blood of healthy individuals is desirable. For this purpose, a highly sensitive assay is desirable, and chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, and the like are particularly preferred.

[0041] A sample is collected from a subject (especially a patient) using, for example, a blood collection tube for collecting plasma. Typically, a citrate-containing blood collection tube, which has a low residual platelet count, is suitable, but heparin or EDTA-containing tubes are also acceptable. An EDTA-containing blood collection tube is used for measuring blood platelet count, but separate blood collection tubes may be used if blood is collected simultaneously. Plasma sCLEC2 concentration is measured using plasma centrifuged at 2000 g for approximately 20 minutes, for example. However, the centrifugation conditions are not limited to these, and whole blood may also be used. The following explanation uses, but is not limited to, the measurement of plasma sCLEC2 concentration. It is preferable to collect blood from a patient within 48 hours of the onset of symptoms, but blood samples collected after 48 hours can also be used in the present invention. More preferably, blood is collected within 24 hours, even more preferably within 18 hours, and most preferably within 12 hours.

[0042] As used herein, acute cerebrovascular disorders include, but are not limited to, ischemic strokes such as cerebral infarction and TIA. TIA refers to a condition in which cerebral infarction-like symptoms are transiently present, but infarction cannot be confirmed by imaging tests. Examples of cerebral infarction-like symptoms include, but are not limited to, unilateral paralysis of the limbs (motor impairment), unilateral numbness (sensory impairment), speech impair- ment or slurred speech (language impairment), impaired vision in one eye (visual impairment), and impaired vision in one eye (visual field impairment). These symptoms disappear within a few minutes to a maximum of about an hour, so the only way to diagnose them is through questioning. Symptoms range from severe to mild, regardless of severity. A cerebral infarction patient is one in whom, in addition to the above clinical symptoms, MRI, CT, or other scans confirm an infarction in the brain.

[0043] Cerebral infarction is a condition in which a cerebral artery is blocked (infarction), and in this case, the presence of a thrombus is evident. Therefore, it is reasonable that the level of sCLEC2, a platelet activation marker, would be elevated. Furthermore, disseminated intravascular coagulation (DIC), thrombotic microangiopathy (TMA), and deep vein thrombosis are pathological conditions in which thrombi are clearly observed, so it is natural that sCLEC2 levels would also be elevated. However, in conditions in which infarction cannot be confirmed, such as TIA, the presence of thrombus cannot be confirmed, and thrombus-related markers (such as well-known D-dimer) were thought to be not significantly elevated. However, it was surprising that the concentration of sCLEC2, which has been suggested as a marker for thrombosis, was elevated and could be used as a blood biomarker for risk assessment of TIA.

[0044] In a method for risk assessment, if the sCLEC2 concentration in a patient suspected of having a TIA is higher than that of a healthy person or a group of people with non-thrombotic diseases, it can be determined that the patient is likely to have a TIA. Based on this comparison, the sCLEC2 concentration in a TIA patient can be compared before and after preventive treatment for cerebral infarction, and the result can be used to predict the risk of developing cerebral infarction.

[0045] Regarding the correlation between the measured sCLEC2 concentration in a patient-derived sample and the possibility of TIA, a threshold can be appropriately set by comparing the sCLEC2 concentration in the patient-derived sample with the sCLEC2 concentration in a healthy subject. Furthermore, for predicting the risk of cerebral infarction, TIA can be assessed when a significant change in sCLEC2 concentration is detected from a time-course record of sCLEC2 concentration measured in the same patient before the onset of the disease.

[0046] To cite a specific example, if a patient presenting with symptoms suggestive of TIA is tested for sCLEC2 blood concentration, and if the value is higher than that of healthy individuals, it can be determined that the patient is likely to have TIA, and further testing can be performed to assess the risk, leading to the prophylactic administration of antiplatelet drugs such as aspirin or anticoagulants such as warfarin. Furthermore, if the sCLEC2 concentration is measured after the patient is diagnosed with TIA and begins taking antiplatelet drugs or anticoagulants, and if the value is high, it can be used to determine the treatment plan, such as switching to a different type of drug or administering additional drugs.

[0047] Original data or statistically processed data for calculating the determination threshold (cutoff value) may be used, which indicates the correlation between the sCLEC2 concentration in plasma and the degree of platelet activation or various diseases. For example, a method for calculating the cutoff value may involve performing an analysis to create an ROC curve (Receiver Operating Characteristic Curve) from the measured sCLEC2 levels in plasma, and using the concentration at which both the diagnostic sensitivity and specificity are 80% or higher as the cutoff value. In the methods of the present invention, the cutoff value for sCLEC2-induced TIA is preferably selected from any value between 66 pg / mL and 148 pg / mL. In a more preferred embodiment, a similar analysis can be performed to calculate the cutoff value using the C2PAC index, which is the value obtained by dividing the sCLEC2 concentration by the platelet count. When the C2PAC index is used, the cutoff value can be set to 0.7.

[0048] sCLEC2 is released into the blood upon platelet activation. Existing platelet activation markers, such as platelet factor 4 (PF4) and β-thromboglobulin (βTG), have the problem of nonspecific release due to physical stimulation from blood collection. However, sCLEC2 has a signaling-dependent release mechanism that induces platelet activation, making it a potential marker that more accurately reflects in vivo platelet activation. Furthermore, because CLEC2 expression is almost exclusively limited to the platelet / megakaryocyte lineage in humans, it is a platelet-specific marker with few false positives. Therefore, measuring sCLEC2 enables early diagnosis of platelet activation status and can be used in the diagnosis of TIA.

[0049] Alternatively, the measured sCLEC2 concentration may be divided by the platelet count and used for risk assessment. If the sCLEC2 concentration decreases with treatment, platelet activation is likely to be suppressed. However, if the sCLEC2 concentration remains high or increases, consideration may be given to reviewing, adding, or increasing the dose of antithrombotic drugs. In this specification, the sCLEC2 concentration divided by the platelet count is referred to as the C2PAC index. In the diagnosis of TIA, even if the term "sCLEC2 concentration" is not explicitly stated, the sCLEC2 concentration can be replaced with the C2PAC index.

[0050] When the sCLEC2 concentration is divided by the platelet count to diagnose TIA, the platelet count is usually measured using an automated hemocytometer, but it can also be measured using a hemocytometer and a microscope.

[0051] The sCLEC2 concentration in plasma is expressed in pg / mL, for example, and the platelet count in blood is expressed in 1,000 cells / mm ３ It is preferable to use the C2PAC index, which is expressed as sCLEC2 concentration / platelet count, and calculates the sCLEC2 concentration / platelet count. However, the sCLEC2 concentration used here may be expressed in any unit such as ng / mL or ng / L, and the platelet count is expressed as 10,000 / mm3. ３Any unit such as sCLEC2 concentration / index can be used, but a consistent unit should be used for comparison. Although the sCLEC2 concentration / index can take on various values ​​by using various units, they are essentially the same concept.

[0052] It is expected that the ratio will often be calculated using the measured values ​​from a clinical testing device that measures sCLEC2 concentration and the measured values ​​from a hemocytometer that measures platelet count. In routine clinical practice, this calculation is preferably performed automatically on a hospital testing system, hospital system, or electronic medical record system connected to both measuring devices. However, a system that connects the data from the two measuring devices may also be constructed, or a machine that can simultaneously measure sCLEC2 concentration and platelet count may also be constructed. Manual calculations may also be performed using both data. Furthermore, correlations between plasma sCLEC2 concentration and the degree of platelet activation or various diseases may be used, for example, as a threshold value, or as original data or statistically processed data for calculating a threshold value. Dividing plasma sCLEC2 concentration by blood platelet count to calculate the amount of sCLEC2 released per platelet and using this as an index for diagnosing thrombotic diseases is preferable, as it allows for evaluation of the degree of platelet activation independent of blood platelet count. Specifically, for example, the plasma sCLEC2 concentration is expressed in pg / mL (A), and the blood platelet count is expressed in 1,000 / mm ３ The number obtained by dividing A by B can be used as an index of platelet activation.

[0053] Furthermore, the present invention does not exclude its use in the diagnosis of cerebral infarction. Diagnosis of cerebral infarction is made based on symptoms such as unilateral paralysis of the limbs (motor impairment), unilateral numbness (sensory impairment), speech impediment, slurred speech (language impairment), impaired vision in one eye (visual impairment), and impaired vision in one eye (visual field impairment), and a comprehensive assessment is made taking into account whether infarction has been confirmed by imaging tests such as MRI. Measuring the blood sCLEC2 concentration according to the present invention in patients suspected of cerebral infarction is preferable because it can aid in the rapid and simple diagnosis of cerebral infarction. In addition, in clinical settings, it is important not only to diagnose cerebrovascular disorders, including cerebral infarction, but also to be able to objectively determine whether the disorder is cardiac or non-cardiogenic using a rapid and simple method, and the present invention can meet this need.

[0054] To differentially diagnose cardiogenic cerebral infarction or noncardiogenic cerebral infarction, for example, sCLEC2 concentration in the blood of a patient diagnosed with acute cerebrovascular disease is measured. At this time, a significant increase in sCLEC2 concentration can be confirmed. If the sCLEC2 concentration is higher than a predetermined threshold, it can be determined that the probability of noncardiogenic cerebral infarction is high, and if the sCLEC2 concentration is lower, it can be determined that the probability of cardiogenic cerebral infarction is high. Alternatively, sCLEC2 concentration can be combined with other thrombus hemostatic markers and used for differential diagnosis of cardiogenic cerebral infarction or noncardiogenic cerebral infarction. For example, sCLEC2 and D-dimer concentrations in the blood of a patient diagnosed with acute cerebrovascular disease are measured. While measurements are preferably performed using blood samples collected simultaneously, measurements can also be performed using blood samples collected separately. Those skilled in the art can appropriately determine the degree of influence, even if there is a time difference between the blood samples collected for each concentration measurement.

[0055] If the sCLEC2 / D-dimer ratio, calculated by dividing the sCLEC2 concentration by the D-dimer concentration, is higher than a certain threshold, it can be determined that the probability of non-cardiogenic disease is high, whereas if it is lower than a certain threshold, it can be determined that the probability of cardiogenic disease is high. The sCLEC2 / D-dimer ratio is calculated by dividing the sCLEC2 concentration by the D-dimer concentration. Although D-dimer can be expressed in various concentrations, such as μg / mL and ng / L, a unified concentration unit should be used for comparison. The commonly used notation, μg / mL, is preferred, but is not limited to this.

[0056] In addition to D-dimer, other coagulation and fibrinolysis markers that can indicate the presence of fibrin clots and fibrin formation include FDP, a fibrin / fibrinogen degradation product, available fibrin, fibrin monomer, thrombin-antithrombin complex (TAT), and prothrombin fragment F1+2, which are markers of thrombin activation, and can be used to determine whether the condition is cardiogenic or non-cardiogenic by calculating the ratio to sCLEC2.

[0057] A diagnosis of cardiogenic cerebral infarction is easy when a relatively large infarction is observed on MRI or other imaging, and atrial fibrillation or arrhythmia is also present. While typical atherosclerotic cerebral infarction can be easily diagnosed in some cases, such as when the infarction is moderate and atherosclerotic stenosis of the carotid artery or stenosis of an artery adjacent to the infarct is present, diagnosis is often difficult in cases where neither of these conditions are present. However, because the diagnosis significantly influences treatment, in actual clinical practice, a comprehensive diagnosis is made based on imaging tests such as MRI, MRA, CT, and ultrasound, as well as clinical symptoms, medical history, electrocardiogram, and blood tests. However, thrombotic information, such as whether the thrombus causing the infarction is fibrin- or platelet-dominant, is extremely useful for diagnosis. In the case of cerebral infarction, initiating treatment is a matter of urgency, but the time elapsed before the patient is seen in an outpatient clinic, such as an emergency department, is often unknown. If a differential diagnosis between cardiogenic cerebral infarction and non-cardiogenic cerebral infarction can be made by a simple method such as that of the present invention, it will be possible to quickly select an appropriate treatment method, and therefore the use of such a risk assessment method is preferable.

[0058] Anticoagulants and antiplatelet drugs are known as intravenous therapies for acute cerebral infarction. However, even the same anticoagulant may have different indications. For example, between the anticoagulants argatroban and heparin, argatroban is indicated for non-cardiogenic cerebral infarction, while heparin is indicated for cardiogenic cerebral infarction. Furthermore, the appropriate therapeutic agent may differ depending on the time elapsed since the onset of cerebral infarction. For example, the anticoagulant (argatroban) is indicated if the infarction is 1.5 cm or larger within two days of onset, but is not indicated if the infarction is not large or if more than three days have passed. Thus, the implementation of the present invention is preferable in determining a treatment strategy because it not only diagnoses cerebral infarction but also quickly distinguishes between cardiogenic and non-cardiogenic infarction.

[0059] Previously, therapeutic drugs such as edaravone, which has an action mechanism completely different from that of anticoagulants and antiplatelet drugs and is applicable to both cardiogenic and non-cardiogenic strokes, and is thought to reduce the exacerbation of cerebral infarction by removing free radicals, were proposed. However, in recent years, the US treatment guidelines have not mentioned edaravone, and there are cases where the approach to treatment differs from country to country. In light of the above situation, and in order to respond to the ever-evolving decisions on treatment plans, the implementation of the present invention is preferable because it not only enables the diagnosis of cerebral infarction but also enables rapid differentiation between cardiogenic and non-cardiogenic stroke.

[0060] In acute-phase cerebral infarction treatment, an anticoagulant and an antiplatelet drug are often administered in combination, with the effectiveness monitored for approximately one to two weeks before deciding on a subsequent treatment plan. In this case, oral aspirin or warfarin is often the next choice. However, while the choice of drug is straightforward if imaging tests can identify lacunar, atherosclerotic, or cardiogenic cerebral infarction, even specialists often disagree on the issue, making it difficult to determine which drug to use. When imaging tests alone are insufficient, a comprehensive assessment is made based on the results of tests such as 24-hour electrocardiograms, implantable electrocardiograms, carotid artery echocardiograms, transthoracic echocardiograms, and transesophageal echocardiograms. Even after these tests, determining whether the infarction is cardiogenic or noncardiogenic is still often difficult. Therefore, the present invention's ability to easily and accurately determine whether the infarction is cardiogenic or noncardiogenic through biomarker-based testing is extremely valuable in such cases.

[0061] Although the use in the differential diagnosis of whether cerebral infarction is cardiogenic or noncardiogenic has been described, it can also be used in the differential diagnosis of whether TIA is cardiogenic or noncardiogenic. The differential diagnosis of cardiogenic TIA and noncardiogenic TIA can be performed in the same manner as the differential diagnosis of cardiogenic cerebral infarction or noncardiogenic cerebral infarction. Since infarction images such as MRI are not observed in TIA, such differential diagnosis is possible, which is even more preferable.

[0062] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0063] Example 1: Measurement of sCLEC2 in human plasma In this example, the concentration of sCLEC2 in plasma was measured according to the following procedure. (Preparation of measurement reagent and test sample) Sample diluent: A sample diluent was prepared by combining 0.1 mol / L HEPES buffer (pH 7.5) containing a preservative with 2% sodium octanoate and 0.5% n-octyl-β-D-glucoside (OG). The antibody contained in the reagent was the antibody described in the Examples of Japanese Patent No. 6078845, and was prepared as follows: First antibody solution: A mouse monoclonal antibody (11D5) that recognizes sCLEC2 was bound to magnetic latex particles (JSR Corporation) and dispersed in 0.01 mol / L MES buffer (pH 6.0) containing a preservative. Secondary antibody solution: Another mouse monoclonal antibody (11E6) that recognizes sCLEC2 was labeled with alkaline phosphatase (ALP) using the maleimide method and dispersed in 0.01 mol / L MES buffer (pH 6.5) containing a preservative. Luminescent substrate solution: 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenylphosphate disodium (CDP-Star®: Applied Biosystems) was used. B / F washing solution: A buffer containing 0.1 mol / L citric acid (pH 6.5), 0.15 mol / L NaCl, and 0.1% Triton X-100 was used. Test sample: Recombinant hCLEC-2 protein diluted with buffer (0.025 mol / L HEPES, 0.14 mol / L NaCl, 0.1% sodium octanoate, 0.3% BSA) was used as test sample 1, and recombinant hCLEC-2 protein diluted with citrated plasma was used as test sample 2.

[0064] (Measurement using measurement reagents) A fully automated clinical testing system, STACIA (registered trademark, manufactured by LSI Medience Corporation), was used for the measurement. The prepared specimen dilution solution, first antibody solution (magnetic latex reagent), and second antibody solution (enzyme-labeled antibody reagent) were each filled into a STACIA-specific bottle and placed in the instrument. Measurements were performed according to the operating method of the instrument. Specifically, 40 μL of specimen dilution solution was added to 10 μL of sample, and the mixture was heated at 37°C for several minutes. Then, 25 μL of first antibody solution (magnetic latex reagent) was added and heated at 37°C for several minutes. Next, B / F separation was performed, 50 μL of second antibody solution (enzyme-labeled antibody reagent) was added, and the mixture was heated at 37°C for several minutes. After B / F separation again, 100 μL of luminescent substrate solution was added, and the mixture was incubated at 37°C for several minutes. The signal intensity (counts) was measured. Figure 1 shows a standard curve created using hsCLEC2 protein as a standard.

[0065] Example 2 Measurement of sCLEC2 in Plasma Samples from TIA Patients and Healthy Individuals Using citrated plasma obtained from patients diagnosed with TIA based on a clinical interview and MRI scans revealing no new infarctions, and from healthy individuals, the plasma sCLEC2 concentration, platelet count, and sCLEC2 / platelet ratio (C2PAC index) were measured using the method of Example 1 (Tables 1 and 2).

[0066]

[0067]

[0068]

[0069] Using these measurements, statistical data on sCLEC2 levels in healthy subjects and TIA patients were compiled (Table 3). The mean, standard deviation, and median sCLEC2 values ​​(pg / mL) were 198.3, 94.9, and 166.0 in TIA cases, respectively, and 51.9, 16.7, and 54.6 in healthy subjects measured separately. Therefore, the sCLEC2 values ​​in TIA patients were significantly higher than those in healthy subjects (P<0.0001). The individual measurement values ​​are shown in Figure 2. Furthermore, the upper limit of the C2PAC index measured separately in healthy subjects was 0.7.

[0070] Example 3: Sensitivity and specificity of TIA diagnosis using sCLEC2 When the measured values ​​in Example 2 were analyzed by changing the cutoff value of sCLEC2, an ROC curve (Receiver Operating Characteristic Curve) like that shown in Figure 3 was obtained, with an area under the curve of 0.994, which was very good. Table 4 shows the diagnostic sensitivity and specificity when the cutoff value in this analysis was changed from 60 pg / mL to 150 pg / mL. The cutoff values ​​showing a sensitivity and specificity of 80% or more were 66 pg / mL to 148 pg / mL.

[0071]

[0072] If the cutoff value for sCLEC2 concentration is set at 100 pg / mL, the diagnostic ability of sCLEC2 for TIA is as follows: sensitivity (11 / 12) = 91.6%, specificity (70 / 71) = 98.6%, and accuracy (81 / 83) = 97.6% (Table 5). Furthermore, if the cutoff value for the C2PAC index is set at 0.55, 10 TIA patients had a C2PAC index of 0.7 or higher, resulting in a sensitivity of 83.3%. These findings suggest that sCLEC2 measurement and the sCLEC2 / platelet ratio (C2PAC index) provide simple, accurate, and objective data for the diagnosis of TIA.

[0073]

[0074] Example 4 sCLEC2, D-dimer, and sCLEC2 / D-dimer ratio in various types of cerebral infarction and TIA Among patients diagnosed with cerebral infarction, blood sCLEC2 levels were measured in patients with cardiogenic cerebral infarction, atherosclerotic cerebral infarction, and lacunar infarction using the method of Example 1 ( FIG. 4 ). When the Mann-Whitney U test was used to confirm the significance of the difference between the sCLEC2 levels in patients with cardiogenic cerebral infarction and those in patients with non-cardiogenic cerebral infarction, the p-value was 0.002720 (<0.05), confirming that sCLEC2 is useful for distinguishing between cardiogenic and non-cardiogenic cerebral infarction. Furthermore, to more accurately differentiate between cardiogenic and noncardiogenic cerebral infarction, D-dimer levels in samples collected from the cerebral infarction patients were measured using an LPIA-Genesis D-Dimer (manufactured by LSI Medience). Measurements were performed using a fully automated clinical laboratory analyzer, STACIA (manufactured by LSI Medience). The differential diagnostic ability of D-dimer, sCLEC2, and the sCLEC2 / D-dimer ratio to differentiate between noncardiogenic cerebral infarction (atherosclerotic cerebral infarction and lacunar infarction) and cardiogenic cerebral infarction was analyzed using receiver operating characteristic curves ( ROC curves) ( Figures 5 to 7 ). The areas under the curves for D-dimer, sCLEC2, and the sCLEC2 / D-dimer ratio were 0.67, 0.73, and 0.79, respectively, with the sCLEC2 / D-dimer ratio demonstrating the best diagnostic ability. We also examined representative cases of TIA patients suspected to be cardiogenic using sCLEC2 and the sCLEC2 / D-dimer ratio, and found that, as with cerebral infarction, the values ​​tended to be lower than those of patients with non-cardiogenic TIA.

[0075] As described above, the measurement of blood sCLEC2 and the sCLEC2 / platelet ratio of the present invention can be used as a clinical test for diagnosing TIA, and the sCLEC2-measuring reagent can serve as a clinical diagnostic reagent for TIA. Furthermore, the measurement of blood sCLEC2 and the sCLEC2 / D-dimer ratio can be used as a clinical test for diagnosing cerebral infarction and cardiogenic / non-cardiogenic TIA, and the sCLEC2-measuring reagent can serve as a clinical diagnostic reagent for cerebral infarction and cardiogenic / non-cardiogenic TIA.

Claims

1. A method for risk assessment of acute cerebrovascular disorder, comprising the step of measuring the concentration of soluble CLEC2 present in blood collected from a patient suspected of having acute cerebrovascular disorder or a patient diagnosed with acute cerebrovascular disorder.

2. A method for risk assessment of a patient suspected of having acute cerebrovascular disorder or a patient diagnosed with acute cerebrovascular disorder, comprising: (1) the step of providing a blood sample derived from the patient; (2) the step of determining the concentration of soluble CLEC2 in the sample; (3) the step of correlating the concentration of soluble CLEC2 with the presence or absence of acute cerebrovascular disorder, the possibility of outcome, or whether it is cardiogenic or non-cardiogenic in the patient. The method according to claim 1.

3. A method for risk assessment of a patient suspected of having acute cerebrovascular disorder or a patient diagnosed with acute cerebrovascular disorder, wherein the step of correlating the concentration of soluble CLEC2 with the presence or absence of acute cerebrovascular disorder, the possibility of outcome, or whether it is cardiogenic or non-cardiogenic in the patient includes evaluating whether the patient has a risk based on the change in the concentration of soluble CLEC2. The method according to claim 2.

4. A method for risk assessment of acute cerebrovascular disorder in a patient suspected of having acute cerebrovascular disorder or a patient diagnosed with acute cerebrovascular disorder, wherein the acute cerebrovascular disorder is a transient ischemic attack, and in the step of correlating the concentration of soluble CLEC2 with the transient ischemic attack, the cut-off value of the soluble sCLEC2 concentration is 66 to 148 pg / mL. The method according to claim 2 or 3.

5. A method for risk assessment of a patient suspected of having acute cerebrovascular disorder or a patient diagnosed with acute cerebrovascular disorder, comprising: (1) the step of providing a blood sample derived from the patient; (2) the step of determining the concentration of soluble CLEC2 in the sample; (3) the step of measuring the platelet count in the sample; (4) the step of dividing the concentration of soluble CLEC-2 by the platelet count; (5) the step of correlating the value obtained by dividing the concentration of soluble CLEC2 by the platelet count with the presence or absence of acute cerebrovascular disorder, the possibility of outcome, or whether it is cardiogenic or non-cardiogenic in the patient. The method according to claim 1.

6. A method for risk assessment of acute cerebrovascular disorder in a patient suspected of having acute cerebrovascular disorder or diagnosed with acute cerebrovascular disorder, wherein the acute cerebrovascular disorder is a transient ischemic attack, and a step of correlating the value obtained by dividing the soluble CLEC2 concentration by the platelet count with the transient ischemic attack, and the cut-off value of the value obtained by dividing the soluble sCLEC2 concentration by the platelet count is 0.55 to 0.7, the method according to claim 5.

7. A method for risk assessment of acute cerebrovascular disorder in a patient suspected of having acute cerebrovascular disorder or diagnosed with acute cerebrovascular disorder, which provides a material for determining whether the acute cerebrovascular disorder is cardiogenic or non-cardiogenic, the method according to any one of claims 1 to 4.

8. A method for risk assessment of acute cerebrovascular disorder in a patient suspected of having acute cerebrovascular disorder or diagnosed with acute cerebrovascular disorder, comprising: (1) a step of providing a blood sample derived from the patient; (2) a step of determining the soluble CLEC2 concentration in the sample; (3) a step of measuring the platelet count in the sample; (4) a step of dividing the soluble CLEC-2 concentration by a coagulation-fibrinolysis marker; (5) a step of correlating the value obtained by dividing the soluble CLEC2 concentration by the coagulation-fibrinolysis marker with the presence or absence of acute cerebrovascular disorder, the possibility of prognosis, or whether it is cardiogenic or non-cardiogenic in the patient, the method according to claim 1.

9. A method for risk assessment of acute cerebrovascular disorder in a patient suspected of having acute cerebrovascular disorder or diagnosed with acute cerebrovascular disorder, wherein the coagulation-fibrinolysis marker is D-dimer, the method according to claim 8.

10. A method for risk assessment of acute cerebrovascular disorder in a patient suspected of having acute cerebrovascular disorder or diagnosed with acute cerebrovascular disorder, wherein the provision of the sample derived from the patient is taken from the patient within 48 hours after the onset of symptoms of acute cerebrovascular disorder, the method according to any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, wherein the step of determining the soluble CLEC2 concentration is a highly sensitive immunoassay method, for example, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay.

Images