Method for assessing risk of hemorrhagic stroke using soluble CLEC2

Abstract

Provided is a means of easily supplementing diagnosis using a blood test in medical care for a hemorrhagic stroke. This method comprises a step for measuring (or determining) the concentration of soluble CLEC2 (or a value calculated by dividing soluble CLEC-2 concentration by platelet count) present in blood sampled from a patient who is suspected of having a hemorrhagic stroke or a patient who is diagnosed as having a hemorrhagic stroke.

Description

Method for assessing the risk of hemorrhagic stroke using soluble CLEC2

[0001] The present invention relates to a method for diagnosing and monitoring hemorrhagic stroke using soluble CLEC2.

[0002] Stroke is a group of diseases that includes ischemic stroke, or cerebral infarction, which occurs when blood vessels in the brain become blocked, and hemorrhagic stroke, or cerebral hemorrhage and subarachnoid hemorrhage, which occurs when blood vessels in the brain rupture, 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 those who survive often leave behind severe after-effects, which is a major factor in the need for nursing care. Because this places a heavy burden on both patients and society, a system is needed that allows for prevention, early diagnosis, and rapid response after diagnosis, as well as care and risk management by a wide range of medical professionals.

[0003] Hemorrhagic strokes include subarachnoid hemorrhage, which occurs when an aneurysm in an artery on the surface of the brain ruptures, and cerebral hemorrhage, which occurs when a small artery inside the brain ruptures.

[0004] Subarachnoid hemorrhage is bleeding from blood vessels in the space between the brain and the membrane on the surface of the brain called the arachnoid membrane. The symptom of subarachnoid hemorrhage is almost always a severe headache, often described as "a headache like being hit with a bat" or "a headache like you've never experienced before." The mortality rate for subarachnoid hemorrhage is as high as 30%, making it an extremely serious acute illness that requires immediate treatment, and it is important to diagnose it as soon as possible.

[0005] Subarachnoid hemorrhage can be diagnosed relatively easily with immediate CT scan (hereafter referred to as CT) imaging in patients exhibiting typical clinical symptoms. The sensitivity (positive rate in patients) of CT imaging is said to be 98-100% within 12 hours of onset, making it highly diagnostic and enabling a diagnosis on its own. However, if a CT scanner is not available, patients must be transported to a facility with one. Depending on the region, there may be few hospitals with CT scanners or only CT scanners with low sensitivity. Furthermore, 10-15% of subarachnoid hemorrhage cases are mild cases that patients present to the hospital on foot. In these cases, bleeding may not be detected on CT images due to the time that has passed since the onset of symptoms or the mild bleeding. In such cases, a needle is inserted into the lumbar spine to obtain cerebrospinal fluid and the presence or absence of blood is confirmed. This test has a nearly 100% sensitivity (positive rate in patients) and is highly effective in compensating for negative CT scans. However, the insertion of a needle into the lumbar spine is a highly invasive procedure that places a significant burden on the patient.

[0006] In the past, blood tests such as D-dimer measurement have been proposed as an attempt to diagnose subarachnoid hemorrhage. However, to complement cerebrospinal fluid measurement, the sensitivity (positive rate in patients) must be nearly 100%, but no conventional blood test, not just cerebrospinal fluid test, has achieved such sensitivity, and none are currently in practical use.

[0007] Even if a patient with subarachnoid hemorrhage can survive in the early stages after the onset of the disease, complications such as cerebral vasospasm can be a major problem in treatment. Cerebral vasospasm often occurs between about four days and two to three weeks after the onset of the disease, so monitoring the patient's progress is extremely important. When treating subarachnoid hemorrhage, it is important not only to diagnose whether or not the patient has a subarachnoid hemorrhage, but also to predict whether or not serious complications such as cerebral vasospasm will occur after the onset of the disease.

[0008] Many biomarkers that use cerebrospinal fluid as a sample are known for predicting complications of subarachnoid hemorrhage. It has been reported that many biomarkers in cerebrospinal fluid, such as endothelin, TNF-α, IL-8, IL-6, thrombin-antithrombin complex, and HMGB1, are associated with complications in patients with subarachnoid hemorrhage. However, these require collection of cerebrospinal fluid from the lumbar spine, and have not yet been put to practical use. Even if this were possible, it would be highly invasive and would place a great burden on the patient, so samples could only be collected once, making it impractical to use them to observe changes over time.

[0009] On the other hand, cerebral hemorrhage is an acute disease in which a blood vessel in the brain ruptures for some reason, causing bleeding within the brain. Blood clots caused by the bleeding press on the brain, causing swelling, and impairing brain function. Symptoms vary depending on the location and amount of bleeding, but they can range from sensory symptoms such as headache, nausea, and vomiting to motor symptoms such as paralysis and numbness in the hands and feet and difficulty walking. Since some symptoms are similar to those of cerebral infarction, it can be difficult to distinguish. Furthermore, if the amount of bleeding is large, it can be life-threatening. Like subarachnoid hemorrhage, it is often diagnosed using a CT scan.

[0010] After diagnosis, cerebral hemorrhage can be stopped by treatment such as lowering blood pressure, but if it does not stop, the hematoma in the brain will grow and symptoms will worsen. Hematoma growth can be confirmed by taking multiple CT images, but as mentioned above, it is often difficult to perform frequent CT imaging using a CT scanner with a certain level of accuracy. Furthermore, while S100β and GFAP are known at the research level as biomarkers for monitoring the pathology of cerebral hemorrhage, their clinical usefulness is low and none are used in actual clinical settings, so there are no tests that can be easily monitored using blood tests, etc.

[0011] 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 on platelets and megakaryocytes in humans, making it a platelet-specific molecule. It has been reported that sCLEC2 is released into the blood as soluble CLEC2 (hereinafter abbreviated as sCLEC2) upon platelet activation (Patent Document 1, Non-Patent Document 1).

[0012] Patent No. 6078845 Patent Application No. 2020-032797 Patent Application No. 2021-003671

[0013] F. Kazama et al., Platelets 2015; 26(8):711-719Y. Yamashita et al., Thrombosis Research 178 (2019) 54-58

[0014] Hemorrhagic stroke is a serious acute disease with a high mortality rate, and even if death is avoided, it can leave residual damage. Therefore, it is extremely important to diagnose and begin treatment as early as possible. If a patient presents with typical symptoms and visits a medical institution shortly after the onset of symptoms and undergoes imaging tests such as CT or MRI, which confirm intracerebral hemorrhage, a diagnosis can be made. However, in cases where the amount of bleeding is small or the patient is only slightly affected and arrives at a specialized medical institution after some time has passed, bleeding may not be detected by CT or other imaging. Furthermore, depending on the country or region, medical institutions with highly sensitive CT or MRI equipment may be limited. In such cases, patients with subarachnoid hemorrhage undergo a highly invasive test, a lumbar puncture to test the cerebrospinal fluid. Although lumbar puncture is not performed for cerebral hemorrhage, the lack of confirmation by CT or other imaging can make an accurate diagnosis difficult.

[0015] Even if subarachnoid hemorrhage can be prevented from dying by early treatment, it can have a very dangerous complication called cerebral vasospasm, so patients need to be carefully monitored by a doctor for a while after initial treatment. There are biomarkers in the literature that can predict cerebral vasospasm, but there are no biomarkers in actual clinical use that can be easily monitored by blood tests.

[0016] Initial treatment for cerebral hemorrhage involves diagnosis using CT scans, MRI scans, etc., followed by treatment such as lowering blood pressure, but there are no biomarkers that can be used to monitor whether the treatment is effective and whether bleeding has stopped.

[0017] Therefore, an object of the present invention is to provide a means for easily supplementing the diagnosis with a blood test in the treatment of hemorrhagic stroke. Furthermore, an object of the present invention is to provide a method for easily monitoring the condition of a patient undergoing treatment for subarachnoid hemorrhage by a blood test. Furthermore, an object of the present invention is to provide a means for easily assessing the effectiveness of treatment and monitoring the pathology of cerebral hemorrhage by a blood test.

[0018] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they discovered that the blood sCLEC2 concentration in almost all patients with hemorrhagic stroke is elevated compared to healthy subjects, leading to the completion of the present invention. That is, they discovered a method for diagnosing subarachnoid hemorrhage and cerebral hemorrhage by detecting hemorrhagic stroke patients simply and easily using a blood test with excellent sensitivity (i.e., the positive rate of patients).

[0019] Furthermore, the researchers found that by measuring sCLEC2 concentrations over time and observing fluctuations in these concentrations, it is possible to monitor the condition of patients with subarachnoid hemorrhage and cerebral hemorrhage, which may be useful for predicting serious complications such as cerebral vasospasm in patients with subarachnoid hemorrhage.

[0020] To distinguish between patients with hemorrhagic stroke and those without hemorrhagic stroke using sCLEC2, an appropriate threshold is required. In the present invention, it was found that the threshold for blood sCLEC2 concentration for hemorrhagic stroke is preferably 137 pg / mL or less. Based on these findings, the present invention has been completed as a method for assessing the risk of hemorrhagic stroke using sCLEC2.

[0021] That is, the present invention provides the following: [1] A method for assessing the risk of hemorrhagic stroke, comprising the step of measuring the concentration of soluble CLEC2 present in blood collected from a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke. [2] A method for assessing the risk of a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke, 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 of hemorrhagic stroke or the likelihood of outcome in the patient. [3] The method of [2] for risk assessment of a patient suspected of having a hemorrhagic stroke or diagnosed with a hemorrhagic stroke, wherein the step of correlating the soluble CLEC2 concentration with the presence or absence of hemorrhagic stroke or the likelihood of outcome in the patient comprises assessing whether the patient is at risk based on changes in the soluble CLEC2 concentration. [4] The method of [2] or [3] for risk assessment of a patient suspected of having a hemorrhagic stroke or diagnosed with a hemorrhagic stroke, wherein the threshold value in the step of correlating the soluble CLEC2 concentration with hemorrhagic stroke is 137 pg / mL. [5] The method of [1], which performs risk assessment for a patient suspected of having a hemorrhagic stroke or diagnosed with a hemorrhagic stroke, 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 hemorrhagic stroke in the patient. [6] The method of [5], which performs risk assessment for a patient suspected of having a hemorrhagic stroke or diagnosed with a hemorrhagic stroke, wherein the value obtained by dividing the concentration of soluble CLEC-2 by the number of platelets is correlated with hemorrhagic stroke, and the threshold value of the value is 0.7 to 1.0. [7] The method according to any one of [1] to [6], wherein the hemorrhagic stroke is either subarachnoid hemorrhage or cerebral hemorrhage.[8] The method according to any one of [1] to [7], wherein the step of determining the concentration of soluble CLEC2 is performed using a highly sensitive immunoassay, such as a chemiluminescence immunoassay, an electrochemiluminescence immunoassay, or a fluorescent immunoassay.

[0022] The present invention also provides: a method for assessing the risk of hemorrhagic stroke, which comprises 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 number of platelets); a method for assisting in the assessment of the risk of hemorrhagic stroke, which comprises 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 number of platelets); 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 number of platelets) for the assessment of the risk of hemorrhagic stroke; an in vitro method for assessing the risk of hemorrhagic stroke, which comprises 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 number of platelets); use of an antibody capable of detecting the concentration of soluble CLEC-2 in the manufacture of a kit for assessing the risk of hemorrhagic stroke; The present invention includes a method for measuring (or determining) the concentration of soluble CLEC-2 (or the concentration of soluble CLEC-2 divided by the platelet count) in a sample to provide information necessary for assessing the risk of hemorrhagic stroke.

[0023] The method of the present invention, which measures the sCLEC2 concentration in the blood of patients suspected of having hemorrhagic stroke, enables rapid and simple diagnosis of hemorrhagic stroke. Furthermore, it is expected that the accuracy of assessing treatment efficacy and predicting complications will be improved in monitoring the progress of treatment after a diagnosis of subarachnoid hemorrhage. For patients with cerebral hemorrhage, measuring the sCLEC2 concentration in the patient's blood allows for simple assessment of treatment efficacy.

[0024] 1 is a standard curve created using human sCLEC2 protein as a standard. 2 is a diagram comparing the plasma concentration of sCLEC2 between subarachnoid hemorrhage patients and healthy subjects. 3 is a diagram comparing the plasma concentration of sCLEC2 between cerebral hemorrhage patients and healthy subjects. 4 is an example of time-course monitoring of a subarachnoid hemorrhage case. 5 is an example of time-course monitoring of a cerebral hemorrhage case.

[0025] Although the embodiment of the present invention will be described in detail below, the mode of use is not limited to this.

[0026] 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.

[0027] 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 presumed to be present on the platelet membrane surface and released in a state contained in microparticles produced upon platelet activation. These proteins are thought to have glycosylation. On the other hand, the protein with molecular weight of approximately 25 kDa is thought to be cleaved by proteases upon platelet activation and released from the platelets. 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.

[0028] The sCLEC2 concentration used in the present invention may be the sCLEC2 concentration alone, or preferably, the value obtained by dividing the sCLEC2 concentration by the platelet count (hereinafter, sometimes referred to as the C2PAC index).Unless otherwise specified, the sCLEC2 concentration used herein is interpreted as including both the sCLEC2 concentration and the sCLEC2 concentration divided by the platelet count.

[0029] 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.

[0030] 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. ３ Any unit can be used, but a consistent unit should be used for comparison. Although the sCLEC2 concentration / platelet count can take various values ​​by using various units, they are essentially the same concept.

[0031] It is expected that the ratio will often be calculated using the measured values ​​from a clinical testing device measuring sCLEC2 concentration and the measured values ​​from a hemocytometer measuring 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 linking the data from the two measuring devices may also be constructed, or a machine capable of simultaneously measuring sCLEC2 concentration and platelet count may also be constructed. Manual calculations may also be performed using both data. Furthermore, correlations between the plasma sCLEC2 concentration or the sCLEC2 concentration / platelet count and the degree of platelet activation or various diseases may be used, for example, as a threshold value for determination, or as original data or statistically processed data for calculating the threshold value. Dividing the plasma sCLEC2 concentration by the blood platelet count to calculate the amount of sCLEC2 released per platelet and using this as an index for diagnosing thrombotic diseases are preferred, as this allows for evaluation of the degree of platelet activation independent of the 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.

[0032] 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.

[0033] The method for detecting the presence of sCLEC2 is not particularly limited, but an immunological method using an antibody that recognizes sCLEC2 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 commonly known methods such as Western blotting, latex agglutination, and immunoturbidimetry. Among these, immunoassays using labeled antibodies are preferred in terms of ease of operation and measurement accuracy. For diagnosis in the emergency field, rapid results are desired, and therefore chemiluminescent immunoassays, immunochromatography, and the like are particularly preferred.

[0034] 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 for the measurement. The following description will be given using, but not limited to, the measurement of plasma sCLEC2 concentration.

[0035] In the present invention, the hemorrhagic stroke that is the subject of risk assessment refers to cerebral hemorrhage that occurs inside the brain (sometimes referred to as intracerebral hemorrhage) and subarachnoid hemorrhage, which is bleeding that occurs between the pia mater, the inner layer of tissue that covers the brain, and the arachnoid mater, the outer layer, and does not include cerebral hemorrhage caused by head trauma. Known causes of cerebral hemorrhage include hypertension, ruptured cerebral aneurysm, ruptured cerebral arteriovenous malformation, venous sinus occlusion, dural arteriovenous fistula, amyloid deposition, moyamoya disease, etc., but the cerebral hemorrhage that is the subject of the present invention is not limited to those caused by a specific cause.

[0036] As a method for carrying out risk assessment, for example, if the blood sCLEC2 concentration of a patient suspected of having hemorrhagic stroke (subarachnoid hemorrhage or cerebral hemorrhage) is higher than that of healthy individuals or a group of neurological disorders without intracerebral hemorrhage or infarction, it can be said that there is a high possibility of hemorrhagic stroke, and based on such a comparison, the sCLEC2 concentration can be used to diagnose hemorrhagic stroke (subarachnoid hemorrhage or cerebral hemorrhage).

[0037] Regarding the correlation between the measured sCLEC2 concentration in a patient-derived sample and the possibility of hemorrhagic stroke, a threshold value may be appropriately set by comparing the sCLEC2 concentration in the patient-derived sample with the sCLEC2 concentration in a sample from a non-hemorrhagic stroke patient, or evaluation may be performed when a significant fluctuation in sCLEC2 concentration is detected from a time-course record of sCLEC2 concentration in the same patient before the onset of the disease.

[0038] Regarding the correlation between the measured sCLEC2 concentration in a patient-derived sample and the likelihood of hemorrhagic stroke, a threshold can be appropriately set and used by comparing the sCLEC2 concentration in the patient-derived sample with the sCLEC2 concentration in a sample derived from a healthy individual. For example, a threshold can be calculated by performing an analysis to create an ROC curve (Receiver Operating Characteristic Curve) from the measured plasma sCLEC2 values, and the concentration at which both the diagnostic sensitivity and specificity are 80% or higher can be used as the threshold. In the methods of the present invention, the threshold for sCLEC2-associated hemorrhagic stroke is preferably selected at an arbitrary value of 137 pg / mL or less. In a more preferred embodiment, a similar analysis can be performed using the C2PAC index, which is the value obtained by dividing the sCLEC2 concentration by the platelet count, to calculate the threshold. When the C2PAC index is used, the threshold can be set arbitrarily between 0.7 and 1.0. When risk assessment is performed by setting such a threshold, for example, if a patient with headache suspected of having hemorrhagic stroke has a blood sCLEC2 concentration below a certain threshold, hemorrhagic stroke can be ruled out.

[0039] Because blood sCLEC2 concentrations increase with platelet activation, it has been suggested that blood sCLEC2 concentrations may be associated with thrombus formation, and it has been reported that they can be used to diagnose cerebral infarction and myocardial infarction. However, their association with hemorrhagic stroke is unknown, and it was surprising that measuring sCLEC2 concentrations is useful for risk assessment. Conventionally, when subarachnoid hemorrhage is suspected and hemorrhagic stroke cannot be confirmed by diagnostic imaging, lumbar puncture and cerebrospinal fluid testing are performed, in which a syringe is inserted into the lumbar spine to collect cerebrospinal fluid. However, this is a highly invasive test and cannot be easily performed anywhere by any medical professional. Therefore, if blood tests could be used as an alternative, it would be very convenient and useful. If a diagnosis of subarachnoid hemorrhage is made, it is recommended that patients remain at rest and avoid invasive tests and procedures immediately after the onset to prevent rebleeding. Therefore, it is preferable that the practice of the present invention can serve as an alternative to highly invasive tests.

[0040] Treatments for a diagnosis of subarachnoid hemorrhage typically include the following: In the early stages, treatments to prevent rebleeding immediately after onset include rest (sedation), painkillers, anti-nausea medications, and analgesics to minimize pain (analgesia), antihypertensive medications to lower blood pressure (antihypertensive) to reduce the risk of rebleeding, and diuretics such as mannitol and glyceol to reduce cerebrospinal fluid volume to prevent brain damage due to increased intracranial pressure. However, in severe cases, careful consideration is often required, as lowering blood pressure can reduce cerebral perfusion pressure and increase cerebral ischemia. Recommended treatments for a diagnosis of cerebral hemorrhage include early reduction of systolic blood pressure and maintaining it for seven days. Calcium channel blockers and nitrates are used as antihypertensives during the acute phase of cerebral hemorrhage. Furthermore, if possible, it is considered appropriate to switch to oral treatment using a calcium channel blocker, angiotensin-converting enzyme (ACE) inhibitor, angiotensin II receptor blocker (ARB), or diuretic at an early stage depending on the patient's condition, etc. Whether it is subarachnoid hemorrhage or cerebral hemorrhage, treatment requires a high level of experience and knowledge, and it is expected that the implementation of the present invention will serve as an indicator for determining a treatment policy.

[0041] In another embodiment, blood samples are collected over time from a patient diagnosed with subarachnoid hemorrhage, and the patient's condition can be monitored by measuring sCLEC2. Since sCLEC2 is believed to represent the state of platelet activation, a sustained high level of sCLEC2 can be expected to indicate continued bleeding and continued formation of hemostatic thrombi, while a decreasing level can be expected to indicate a decrease in hemostatic thrombus formation. Since cerebral vasospasm, a serious complication, is said to be caused by blood leakage from blood vessels, it is important and useful to understand the state of bleeding based on blood sCLEC2.

[0042] Furthermore, risk assessment using such monitoring can also be used for risk prediction by comparing sCLEC2 concentrations before and after treatment in hemorrhagic stroke patients. For example, if sCLEC2 concentrations decrease as a result of treatment, platelet activation is likely to be suppressed, but if sCLEC2 concentrations remain high or increase, it is likely that bleeding is continuing. For example, if antihypertensive treatment is not effective, this can provide information for considering the selection of a treatment method, such as further antihypertensive measures. Because fluctuations in blood platelet count may be observed during the monitoring process, monitoring using the C2PAC index is also more suitable for observing the patient's condition.

[0043] In another embodiment, even in the case of a patient diagnosed with cerebral hemorrhage, blood samples can be collected over time and sCLEC2 can be measured to monitor the patient's condition. Since sCLEC2 is believed to represent the state of platelet activation, if sCLEC2 levels remain high, it can be predicted that bleeding is continuing and hemostatic clot formation is continuing, while if sCLEC2 levels decrease, it can be predicted that hemostatic clot formation is decreasing. If bleeding continues, the intracerebral hematoma will grow, compressing the brain and worsening symptoms, so it is important and useful to understand the state of bleeding based on blood sCLEC2.

[0044] Furthermore, risk assessment using such monitoring can be used to predict risk by comparing sCLEC2 levels before and after treatment in patients with hemorrhagic stroke. For example, if sCLEC2 levels decrease with treatment, platelet activation is likely to be suppressed, but if sCLEC2 levels persist or increase at elevated levels, a review of antihypertensive therapy may be considered. Because fluctuations in blood platelet counts may be observed during the monitoring process, monitoring using the C2PAC index is also more suitable for observing the patient's condition.

[0045] Even when risk assessment is performed by monitoring, blood sCLEC2 concentration can be measured for patients who present with symptoms suggestive of hemorrhagic stroke (subarachnoid hemorrhage or cerebral hemorrhage), and if the value is higher than that of healthy individuals, it can be determined that the patient has a high possibility of hemorrhagic stroke. Furthermore, the patient's condition can be monitored by collecting blood samples from patients diagnosed with hemorrhagic stroke over time, measuring the sCLEC2 concentration, and comparing the results over time.

[0046] Even when risk assessment is performed by monitoring, the correlation between the measured sCLEC2 concentration in a patient-derived sample and the possibility of hemorrhagic stroke may be assessed by appropriately setting a threshold value based on a comparison of the sCLEC2 concentration in the patient-derived sample with the sCLEC2 concentration in a sample from a patient who has not had hemorrhagic stroke, or assessment may be performed when a significant fluctuation in the sCLEC2 concentration is detected from a time-course record of the sCLEC2 concentration in the same patient before the onset of symptoms.

[0047] 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 non-specific platelet release due to the physical pressure of blood collection, which stimulates platelets. However, sCLEC2 is a signal-dependent release mechanism that triggers 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 estimation of platelet activation status and can be used for diagnosing hemorrhagic stroke and monitoring pathology.

[0048] 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.

[0049] Example 1: Measurement of sCLEC2 in human plasma The concentration of sCLEC2 in plasma was measured as follows. (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 hCLEC2 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 hCLEC2 protein diluted with citrated plasma was used as test sample 2.

[0050] (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.

[0051] Example 2 Measurement of sCLEC2 in Plasma Samples from Patients with Subarachnoid Hemorrhage, Cerebral Hemorrhage, and Healthy Subjects Using citrated plasma obtained from patients diagnosed with subarachnoid hemorrhage or cerebral hemorrhage during outpatient consultations, plasma sCLEC2 concentrations, platelet counts, and sCLEC2 / platelet ratios (C2PAC index) were measured by the method of Example 1 (Tables 1 and 2). sCLEC concentrations were also measured in healthy subjects (Table 3).

[0052]

[0053]

[0054]

[0055]

[0056] The sCLEC2 (pg / mL) levels in patients with hemorrhagic stroke (both subarachnoid hemorrhage and cerebral hemorrhage) were significantly higher than those in healthy subjects (P<0.0001) (Table 4). The respective values ​​are shown in Figure 2 (subarachnoid hemorrhage) and Figure 3 (cerebral hemorrhage). Furthermore, the upper limit of the C2PAC index measured separately in healthy subjects was 0.55.

[0057] Example 3: Sensitivity and specificity of sCLEC2 in diagnosing hemorrhagic stroke (subarachnoid hemorrhage and cerebral hemorrhage) When the threshold value was set at 120 pg / mL, the diagnostic ability of sCLEC2 for hemorrhagic stroke was as follows: sensitivity (34 / 34) = 100%, specificity (71 / 71) = 100%, and accuracy (105 / 105) = 100% (Table 5). Thus, measurement of sCLEC2 and the sCLEC2 / platelet ratio (C2PAC index) provide simple and accurate data for diagnosing hemorrhagic stroke.

[0058]

[0059] Example 4 Monitoring of Patients with Subarachnoid Hemorrhage Using sCLEC2 Various test values ​​on days 1, 8, and 10 of illness in one case diagnosed with subarachnoid hemorrhage are shown in Figure 4. sCLEC2 increased slightly from day 1 to day 8 of illness, but because the platelet count also increased significantly, the C2PAC index showed a slight downward trend.

[0060] Example 5 Monitoring of a Patient with Cerebral Hemorrhage Using sCLEC2 Various test values ​​on days 1, 2, 12, and 14 of illness in one patient diagnosed with cerebral hemorrhage are shown in Figure 5. sCLEC2 and C2PAC index increased on day 12 and decreased on day 14, while D-dimer increased from day 12 to day 14.

[0061] As described above, the measured value of blood sCLEC2 and the sCLEC2 / platelet ratio in the present invention are clinical tests that can be used to diagnose subarachnoid hemorrhage and cerebral hemorrhage, which are hemorrhagic strokes, and can also be used to monitor the condition of patients with subarachnoid hemorrhage and cerebral hemorrhage, which are hemorrhagic strokes. Therefore, measuring sCLEC2 is useful as a method for assessing the risk of hemorrhagic stroke patients.

Claims

1. A method for assessing the risk of hemorrhagic stroke, comprising a step of measuring the concentration of soluble CLEC2 in blood collected from a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke.

2. The method of claim 1, wherein the method comprises 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 of hemorrhagic stroke or the likelihood of outcome in the patient.

3. The method of claim 2, wherein the step of correlating the soluble CLEC2 concentration with the presence or absence of hemorrhagic stroke or the likelihood of outcome in the patient comprises assessing whether the patient is at risk based on changes in the soluble CLEC2 concentration.

4. A method for risk assessment of a patient suspected of having hemorrhagic stroke or diagnosed with hemorrhagic stroke, the method described in claim 2 or 3, wherein the threshold value in the step of correlating soluble CLEC2 concentration with hemorrhagic stroke is 137 pg / mL.

5. The method of claim 1, wherein the method comprises 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 hemorrhagic stroke in the patient.

6. A method for assessing the risk of hemorrhagic stroke in a patient suspected of having hemorrhagic stroke or a patient diagnosed with hemorrhagic stroke, wherein in the step of correlating the value obtained by dividing the soluble CLEC2 concentration by the platelet count with hemorrhagic stroke, the threshold value of the value obtained by dividing the soluble sCLEC2 concentration by the platelet count is 0.7 to 1.

0.

7. The method according to any one of claims 1 to 6, wherein the hemorrhagic stroke is either subarachnoid hemorrhage or cerebral hemorrhage.

8. The method according to any one of claims 1 to 7, wherein the step of determining the concentration of soluble CLEC2 is performed using a highly sensitive immunoassay, such as a chemiluminescence immunoassay, an electrochemiluminescence immunoassay, or a fluorescence immunoassay.

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