PERIPHERAL BLOOD MARKER FOR CERABRAL HEMORRHAGE AND APPLICATION THEREOF

Abstract

Disclosed are a peripheral blood marker for cerebral hemorrhage and an application thereof. The peripheral blood marker is a peripheral blood protein belonging to leucine-rich repeat (LRR) protein family. The peripheral blood protein is leucine-rich α2-glycoprotein-1 (LRG1). This disclosure further provides a method for diagnosing the cerebral hemorrhage.

Claims

1. A method for diagnosing cerebral hemorrhage, comprising: determining a content of a peripheral blood protein; and predicting a risk of cerebral hemorrhage in a subject according to the content of the peripheral blood protein; wherein the peripheral blood protein is a member of a leucine-rich repeat (LRR) protein family.

2. The method of claim 1, wherein the peripheral blood protein is leucine-rich α2-glycoprotein-1 (LRG1).

3. The method of claim 1, wherein with respect to an expression level of the peripheral blood protein in a healthy subject or a subject with dizziness, syncope and coma that are not caused by cerebral hemorrhage, an up regulation in an expression level of the peripheral blood protein indicates a risk of cerebral hemorrhage; and the dizziness, syncope and coma that are not caused by cerebral hemorrhage, are caused by alcoholism, drug poisoning, or blood glucose disorder.

4. The method of claim 2, wherein with respect to an expression level of the peripheral blood protein in a healthy subject or a subject with dizziness, syncope and coma that are not caused by cerebral hemorrhage, an up regulation in an expression level of the peripheral blood protein indicates a risk of cerebral hemorrhage; and the dizziness, syncope and coma that are not caused by cerebral hemorrhage, are caused by alcoholism, drug poisoning, or blood glucose disorder.

5. The application of claim 1, wherein the cerebral hemorrhage is acute cerebral hemorrhage.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

[0040] Technical solutions of this application will be described in detail below with reference to the experiments.

Significance of Leucine-Rich α2-Glycoprotein-1 (LRG1) Concentration Change in Peripheral Blood in Diagnosis of Cerebral Hemorrhage

Screening and Collection of Clinical Samples

[0041] 90 cases with cerebral hemorrhage in the past 2 years were collected, all of which were the initial onset. The gender, age blood pressure, blood fat, blood glucose, liver function, kidney function, four blood coagulation items and electrocardiogram (ECG) of the patients were recorded. All of the patients were confirmed by computed tomography (CT) scan or brain magnetic resonance imaging (MRI). Meanwhile, normal cases with matching age and gender were taken as a control group. Samples were collected as follows. 5 mL of venous blood was collected from individual patients collected, allowed to stand for 15 min and centrifuged at 3000 r/min for 15 min to collect a serum, which was transferred to a sterile cryopreservation tube and stored at −80° C. Similarly, 5 mL of venous serum was collected from the control group and treated through the above steps. Moreover, venous serum samples were collected from 50 cases with coma or dizziness caused by alcoholism, drug poisoning and blood glucose disorder for differential diagnosis.

Study on Plasma Samples of Patients

[0042] The serum and cells were separated with a high-speed centrifuge. Devices, materials and regents used herein included a homogenizer, a high-speed centrifuge, a 3 kDa ultrafiltration centrifuge tube and an inductively coupled plasma-mass spectrometry (ICP-MS). The water used herein was ultrapure water with a resistivity of 18.2 MΩ/cm. Pre-treatment of samples was performed as follows. The serum samples were taken out, thawed and determined for the LRG1 content at room temperature by an Elisa assay. Equipment used herein includes a fully automated biochemistry analyzers (DXC800, Beckman Coulter and AU400, Beckman Coulter), an electronic analytical balance (with a readability of 0.0001 g, Shimadzu), a microplate reader (imported), a Roche automatic electro-chemiluminescence immunoassay analyzer (Elecsys2010), a special protein analyzer (imported), a water purifier (MEDICA60), a hot water bath (DK-600), a low-temperature high-speed centrifuge (sigma), an ultralow-temperature refrigerator (Thermo Electron Corporation) and a low-temperature refrigerator (HFC350, Germany).

Sample Analysis

[0043] The samples were analyzed by the Elisa assay.

Statistical Analysis

[0044] The analysis results of individual groups were compared by T test using SPSS statistical software (P<0.05).

Results

[0045] The LRG1 level was determined with kits respectively made in Japan and Wuhan. The detection value obtained by the kit made in Japan was much higher than that obtained by the kit made in Wuhan, which could be explained by that the antibody binding sites of these two kits were different. The normal range was determined by the mean value plus or minus 2 standard deviations, and an upper limit was the mean value plus 2 standard deviations. An upper limit (CUTOFF value) of the LRG1 kit made in Japan (LRG1 Japan) was 1300 ng/mL, and an upper limit (CUTOFF value) of the LRG1 kit made in Wuhan (LRG1 Wuhan) was 80 ng/mL. 88 cases with cerebral hemorrhage and 40 normal cases were detected with the Japan kit, where 82 of the 88 cerebral hemorrhage cases exceeded the CUTOFF value, and 8 of the 40 normal cases exceeded the CUTOFF value. 90 cases with cerebral hemorrhage and 60 normal cases were detected with the Wuhan kit, where 83 of the 90 cerebral hemorrhage cases exceeded the CUTOFF value (7 false negative cases), and 16 of the 60 normal cases exceeded the CUTOFF value (false positive rate: 26%).

[0046] The LRG1 content in the peripheral blood of the three groups was shown in Table 1.

TABLE-US-00001 TABLE 1 The LRG1 content in peripheral blood of three groups of cases (X ± SD, with a unit of ng/mL) LRG1 LRG1 (Japan kit) (Wuhan kit) Normal people 1183 ± 59  60 ± 11 Patients with dizziness which is not caused 1185 ± 80  46 ± 9 by cerebral hemorrhage Patients with acute cerebral hemorrhage 1544 ± 68 145 ± 10

[0047] The LRG1 contents of the patients with acute cerebral hemorrhage were compared with the LRG1 contents of normal people and patients with a dizziness which was not caused by cerebral hemorrhage (P<0.01).

[0048] The sensitivity and specificity of LRG1 in the diagnosis of acute cerebral hemorrhage were shown in Table 2, where the sensitivity and specificity were calculated as follows:

[0049] sensitivity=the number of true positive cases/(the number of true positive cases+the number of false positive cases)×100%, namely, the accuracy for diagnosing the patients; and

[0050] specificity=the number of true negative cases/(the number of true negative cases+the number of false negative cases)×100%, namely, the accuracy for diagnosing the non-patients.

[0051] A false positive rate=the number of false positive cases/the number of gold standard negative cases.

[0052] A false negative rate=the number of false negative cases/the number of gold standard positive cases.

[0053] An upper limit (CUTOFF value) of a normal range determined by the LRG1 Japan kit was 1300 ng/mL, and an upper limit (CUTOFF value) of a normal range determined by the LRG1 Wuhan kit was 80 ng/mL. 88 cerebral hemorrhage cases and 40 normal cases were detected using the Japan kit, where 82 of the 88 cases exceeded the CUTOFF value, and 8 of the 40 normal cases exceeded the CUTOFF value. 90 cerebral hemorrhage cases and 60 normal cases were detected using the Wuhan kit, where 83 of the 90 cerebral hemorrhage cases exceeded the CUTOFF value, and 16 of the 60 normal cases exceeded the CUTOFF value (a false positive rate: 26%).

TABLE-US-00002 TABLE 2 Sensitivity and specificity of LRG1 in diagnosis of acute cerebral hemorrhage Positive False Negative False coincidence rate positive rate coincidence rate negative rate LRG1 93.2% 20% 80% 6.8% (Japan kit) LRG1 92.2% 26% 74% 7.8% (Wuhan kit)

TABLE-US-00003 TABLE 3 Concentration change of LRG1 in different stages of acute cerebral hemorrhage (X ± SD, with an unit of ng/mL) Normal Time after onset of acute cerebral hemorrhage/ h people 0.5 1 4 24 72 LRG1 1183 ± 1403 ± 1886 ± 1602 ± 1512 ± 1425 ± (Japan 59 85 121 82 113 105 kit) LRG1 60 ± 173 ± 182 ± 133 ± 123 ± 120 ± (Wuhan 11 22 24 23 21 11 kit)

[0054] Compared with the normal people, the acute cerebral hemorrhage cases experienced a significant increase in the LRG1 concentration at 0.5 h and 72 hours after the onset (P<0.05).