Acute ischemic stroke penumbra detection method based on in-vitro serum molecular marker

Abstract

The invention discloses an acute ischemic stroke penumbra detection method based on an in-vitro serum molecular marker, and belongs to the technical field of biology, the detection method is characterized in that glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) in a sample to be detected are used as biomarkers for detection operation; comprising the following steps: (1) sample collection: collecting a blood sample, and centrifugally separating serum; (2) detecting a sample: detecting the contents of GFAP and UCH-L1 in the sample by using the kit; and (3) analysis of a detection result: calculating the ratio of GFAP to UCH-L1 through analysis software, and analyzing the correlation of the ratio with the acute ischemic stroke penumbra. According to the method, the GFAP and UCH-L1 levels of bedside serum can be quantified in a short time (within 28 minutes), and a rapid biomarker detection result is provided for clinic. The detection method is more economical, reduces the medical cost, and has a wide clinical application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting an acute ischemic stroke penumbra based on in vitro serum molecular markers. Background Art

[0002] Acute ischemic stroke (AIS) refers to a type of disease in which local brain tissue ischemic hypoxic necrosis caused by sudden interruption of blood supply to the brain, thereby causing brain dysfunction. It is the second leading cause of death and the main cause of disability in the world. The key to the treatment of acute ischemic stroke is to achieve vascular recanalization as soon as possible to save the ischemic penumbra. The ischemic penumbra refers to the area of ​​low blood perfusion around the necrotic tissue after cerebral ischemia. The nerve cells in this area have physiological and biochemical abnormalities and dysfunction due to ischemia, but have not yet died. Timely improvement of low perfusion (usually within 24 hours of onset) can restore normal function, otherwise it will deteriorate and progress to the core area of ​​infarction, further aggravating brain damage. However, due to individual differences in AIS patients, the existence time of the ischemic penumbra ranges from a few hours to a few days, which requires neurologists to accurately evaluate the condition of the ischemic penumbra within a limited time to decide whether to implement reperfusion therapy. Therefore, whether the ischemic penumbra can be quickly and accurately evaluated is not only related to the choice of treatment timing for patients, but also a core factor affecting treatment effect and prognosis.

[0003] At present, the commonly used clinical method for evaluating the ischemic penumbra is through advanced imaging technology (such as CT perfusion scan CBF / CBV image mismatch, cranial magnetic resonance PWI / DWI image mismatch, etc.) and clinical signs of patients. However, there are the following problems in specific clinical practice: advanced imaging equipment is expensive and technically complex, and it is difficult to popularize it in primary hospitals; in addition, related examinations are time-consuming and require patients to be moved, which may delay the timing of treatment. Survey data show that currently less than 3% of AIS patients receive reperfusion therapy in the ultra-early stage, which is partly attributed to the lack of a rapid and convenient method for evaluating the ischemic penumbra. Biomarker detection has the advantages of low cost, strong timeliness, and simple operation, and is particularly suitable for the acute phase evaluation of AIS patients. However, to date, there is no method based on blood biomarkers to evaluate the ischemic penumbra.

[0004] After acute ischemic stroke, the main pathological features of the infarct core area are necrosis of neurons and astrocytes. The intracellular components of dead cells can be released into the cerebrospinal fluid and enter the blood through the damaged blood-brain barrier; while the ischemic penumbra area shows reactive astrocyte proliferation, which also releases intracellular components into the blood during activation. Therefore, in theory, the markers of neurons and astrocytes in the blood are closely related to the infarct core and the ischemic penumbra. Based on the above theoretical hypothesis, the present invention aims to provide a new method for evaluating the ischemic penumbra of acute ischemic stroke based on blood biomarkers to fill the deficiencies of the prior art. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting the penumbra of acute ischemic stroke based on in vitro serum molecular markers. The method utilizes the biomarkers GFAP (glial fibrillary acidic protein) and UCH-L1 (ubiquitin carboxyl-terminal hydrolase L1) and combines them with advanced biological detection technology to develop a new, rapid, convenient and economical method for detecting the penumbra of acute ischemic stroke, which provides strong support for the early diagnosis and precise treatment of AIS patients.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] A method for detecting the penumbra of acute ischemic stroke for non-disease diagnosis purposes, wherein the detection method is an operation of detecting glial fibrillary acid protein (GFAP) and ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) in a sample to be detected as biomarkers, and the specific detection method includes:

[0008] (1) Sample collection: Collect blood samples, centrifuge the samples, and separate serum;

[0009] (2) Sample detection: Use the kit to detect GFAP and UCH-L1 in the sample to obtain the GFAP and UCH-L1 content in the serum sample;

[0010] (3) Analysis of test results: The ratio of GFAP to UCH-L1 was calculated using analysis software, and its correlation with the penumbra of acute ischemic stroke was analyzed;

[0011] Preferably, in the above technical solution, a serum GFAP level higher than 12.12 pg / mL is independently associated with a larger penumbra volume and a larger hypoperfusion volume.

[0012] Preferably, in the above technical scheme, a serum UCH-L1 level higher than 133.38 pg / mL is independently associated with a larger infarct core volume.

[0013] Preferably, in the above technical solution, when the serum GFAP / UCH-L1 ratio is greater than 0.19, it indicates the presence of a penumbra, and the specificity is not less than 90% and the positive predictive value is not less than 80%.

[0014] A kit for detecting penumbra in acute ischemic stroke, wherein the kit is used to measure the expression levels of GFAP and UCH-L1 in serum, and to evaluate the presence or absence of penumbra by calculating the GFAP / UCH-L1 ratio.

[0015] An acute ischemic stroke penumbra assessment system, comprising a kit for measuring GFAP and UCH-L1 content, a detection device, and an analysis module for assessing the presence of a penumbra according to the GFAP / UCH-L1 ratio. The kit is a two-in-one detection kit or a separate detection kit.

[0016] The above technical solution of the present invention has the following beneficial effects:

[0017] (1) Improved accuracy: By combining the ratio of GFAP and UCH-L1, the presence of the penumbra can be assessed more accurately, thereby improving the accuracy of diagnosis.

[0018] (2) Rapid evaluation: This method can quantify bedside serum GFAP and UCH-L1 levels in a short time (within 28 minutes), providing rapid biomarker detection results for clinical use.

[0019] (3) Economical: Compared with traditional imaging examinations, the use of serum biomarkers is more economical and reduces medical costs.

[0020] (4) Widespread clinical application: The simple operation and low cost make this method suitable for medical institutions at all levels and has broad clinical application prospects.

[0021] In summary, the beneficial effect of the present application is that it provides a fast, accurate and economical method for detecting the penumbra of acute ischemic stroke, which helps to improve the treatment effect and the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0023] Figure 1 Schematic diagram of the relationship between the low perfusion area and GFAP and UCH-L1.

[0024] Figure 2 Heat maps showing the visual distribution of infarct core volume, hypoperfusion volume, and ischemic penumbra volume in different GFAP level groups.

[0025] Figure 3 Heat maps showing the visual distribution of infarct core volume, hypoperfusion volume, and ischemic penumbra volume in different UCH-L1 level groups.

[0026] Figure 4 This is the Spearman correlation heat map between GFAP and infarct core volume, hypoperfusion volume, and ischemic penumbra volume. * indicates that the two groups of data are correlated (P < 0.05); red represents positive correlation, and blue represents negative correlation; the larger the circle and the darker the color, the stronger the correlation.

[0027] Figure 5 This is the Spearman correlation heat map between UCH-L1 and infarct core volume, hypoperfusion volume, and ischemic penumbra volume. * indicates that the two groups of data are correlated (P < 0.05); red represents positive correlation, and blue represents negative correlation; the larger the circle and the darker the color, the stronger the correlation. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0029] Unless otherwise specified, the reagents used in this application are all commercially available or obtained through commercial channels, or can be prepared by referring to existing chemical methods.

[0030] Brain injury markers refer to substances that are released into the cerebrospinal fluid or blood and can be detected when brain tissue is damaged. Infection, trauma, hypoxia, inflammation or degeneration of the central nervous system can lead to cell damage and the accumulation of decomposition products in the extracellular fluid, as well as increased permeability of the blood-brain barrier. These biomolecules diffuse into the cerebrospinal fluid along the concentration gradient and enter the blood through the leaky blood-brain barrier, becoming measurable indicators of brain injury. Among them, glial fibrillary acid protein (GFAP) and ubiquitin carboxyterminal hydrolases L1 (UCH-L1) are important brain injury markers. GFAP is a specific astrocyte intermediate filament protein mainly present in the central nervous system. Its increased level in the blood usually reflects astrocyte damage. UCH-L1 is a protein that is highly specifically expressed in neurons. When neurons are damaged, its level in the cerebrospinal fluid or blood will increase significantly. In the ultra-early stage of acute ischemic stroke, the infarct core area suffers from irreversible damage and death of neurons due to interrupted blood flow, and UCH-L1 is subsequently released into the blood through the damaged blood-brain barrier, suggesting that serum UCH-L1 levels are closely related to the infarct core area. On the other hand, astrocytes undergo irreversible damage and death in the infarct core, but show obvious reactive proliferation in the ischemic penumbra, which is an adaptive response to complex signal changes in the local microenvironment, known as reactive astrogliosis, and a significant increase in GFAP expression is one of its main characteristics. In summary, given the unique pathophysiological characteristics of UCH-L1 and GFAP and their association with different pathological areas of ischemic stroke, we propose to use these two biomarkers in combination to more comprehensively evaluate the existence and evolution of the ischemic penumbra.

[0031] There is no report on the application of the two in the assessment of ischemic penumbra. Therefore, the present invention will use the biomarkers GFAP and UCH-L1 to develop a new, rapid, convenient and economical method for the assessment of the penumbra of acute ischemic stroke, providing strong support for the early diagnosis and precise treatment of AIS patients. Figure 1-Figure 5 ) Detailed description:

[0032] Example 1 Relationship between GFAP, UCH-L1 and the volume of infarct core and ischemic penumbra

[0033] The present invention recruited 102 AIS patients with anterior circulation large vessel occlusion who were scheduled for thrombectomy surgery and were treated within 24 hours after symptom onset at the First Hospital of Jilin University. Blood samples were collected before computed tomography perfusion imaging (CTP). The researchers detected the levels of brain injury biomarkers: GFAP and UCH-L1 in serum. The CTP sequence was then analyzed by deconvolution method to calculate the infarct core volume, ischemic penumbra volume, hypoperfusion volume and mismatch ratio, where the infarct core was defined as relative cerebral blood flow (rCBF) lower than 30% of the contralateral normal brain tissue; hypoperfusion was defined as the time to peak residual function (Tmax) of the tissue greater than 6 seconds; the ischemic penumbra volume was calculated by subtracting the infarct core volume from the hypoperfusion volume; and the mismatch ratio was the ratio of the hypoperfusion volume to the infarct core volume.

[0034] (1) Relationship between GFAP and the volume of infarct core and ischemic penumbra

[0035] A total of 102 patients underwent GFAP testing. Univariate analysis showed that for patients with acute ischemic stroke, higher serum GFAP (>12.12 pg / mL) levels were independently associated with larger ischemic penumbra volume {odds ratio (OR) 3.297 (95% confidence interval [95% CI], 1.562-6.959, P=0.002)} and larger hypoperfusion volume {OR 2.986 (95% CI, 1.423-6.271, P=0.004)}, but not with infarct core volume, OR 1.425 (95% CI, 0.697-2.915, P=0.332). Further multivariate analysis adjusted for age, gender, NIHSS score at admission, time from onset to blood collection, previous cerebral infarction, and TOAST classification. It was found that GFAP was an independent predictor of ischemic penumbra volume {OR value 3.773 (95% CI, 1.589-8.962, P=0.003)} and hypoperfusion volume {OR value 2.942 (95% CI, 1.254-6.910, P=0.013)}.

[0036] (2) Relationship between UCH-L1 and the volume of infarct core and ischemic penumbra

[0037] A total of 102 patients underwent UCH-L1 testing. Univariate analysis showed that for patients with acute ischemic stroke, higher serum UCH-L1 levels (>133.38 pg / mL) were independently associated with larger infarct core volume, with an OR of 2.467 (95% CI, 1.186-5.129, P=0.016), but were not associated with penumbra volume {OR 1 (95% CI, 0.490-2.040, P=1.000)} and hypoperfusion volume {OR 1.303 (95% CI, 0.638-2.664, P=0.467)}. Further multivariate analysis adjusted for age, gender, NIHSS score at admission, time from onset to blood collection, previous cerebral infarction, and TOAST classification. It was found that UCH-L1 was an independent predictor of infarct core volume, with an OR value of 2.524 (95% CI, 1.117-5.703, P=0.026).

[0038] The above results indicate that clinicians can use biomarkers to more conveniently and quickly assess the volume of the penumbra and the infarct core, thereby helping clinicians determine the feasibility and expected effect of reperfusion therapy.

[0039] Example 2 Evaluation of the Presence of Ischemic Penumbra by Serum GFAP / UCH-L1 Ratio

[0040] The present invention recruited 61 AIS patients with anterior circulation large vessel occlusion who were scheduled for thrombectomy surgery within 24 hours after symptom onset at the First Hospital of Jilin University. The CTP sequence was then analyzed by deconvolution method to calculate the infarct core volume, ischemic penumbra volume, hypoperfusion volume and mismatch ratio. According to the CTP imaging mismatch principle, "the presence of ischemic penumbra" was defined as an infarct core volume of less than 70 mL, a mismatch ratio of 1.8 or higher, and a penumbra volume of at least 15 mL.

[0041] Among the 61 patients, 44 patients had ischemic penumbra (72.13%). The results showed that the median GFAP / UCH-L1 ratio in the serum of acute ischemic stroke patients with penumbra was 0.08 (0.05-0.17), which was significantly higher than that of patients without penumbra (0.04 (0.03-0.07). Univariate analysis showed that a higher level of serum GFAP / UCH-L1 ratio (>0.07) was independently associated with the presence of penumbra, with an OR value of 6.741 (95% CI, 1.689-26.907, P=0.007). Further multivariate analysis adjusted for age, gender, NIHSS score at admission, time from onset to blood collection, hyperlipidemia, and hyperhomocysteinemia, and found that serum GFAP / UCH-L1 ratio was an independent predictor of the presence of penumbra, with an OR value of 25.463 (95% CI, 2.919-222.116, P=0.003).

[0042] Example 3 Determination of the cutoff value of serum GFAP / UCH-L1 ratio for evaluating the presence of penumbra

[0043] According to the principles of (1) specificity not less than 90%; (2) positive predictive value (PPV) not less than 80%; and (3) maximum sensitivity, the critical value of the GFAP / UCH-L1 ratio for evaluating the presence of the penumbra was found to be 0.19. The results showed that when the serum GFAP / UCH-L1 ratio was greater than 0.19, the detection specificity was 94.12% (95% CI, 71.3-99.9); the PPV for predicting the presence of the penumbra was 88.9% (95% CI, 51.9-98.3), as shown in Table 1.

[0044] Table 1 Determination of cut-off value of serum GFAP / UCH-L1 ratio for evaluating the presence of penumbra

[0045] The GFAP / UCH-L1 ratio reflects the relative proportion between the infarct core dominated by neuronal death and the penumbra dominated by astrocyte proliferation. The above examples show that the use of the GFAP / UCH-L1 ratio to assess the presence or absence of the penumbra has high specificity and PPV. This is of great value in assessing the patient's condition and whether it is suitable for reperfusion therapy, because compared with traditional perfusion examinations, the use of serum biomarkers is simpler, non-invasive and economical. It has great economic and social value. In addition, the current technology can quantify the bedside serum GFAP and UCH-L1 levels within 28 minutes, making it a biomarker that patients can quickly obtain when saving the ischemic penumbra.

[0046] Result analysis:

[0047] 1. Evaluate accuracy

[0048] (1) This method can accurately reflect the existence and range of the ischemic penumbra by detecting GFAP and UCH-L1, biomarkers that are closely related to the formation and evolution of the penumbra in acute ischemic stroke.

[0049] (2) Compared with traditional imaging methods, this method also has higher sensitivity and specificity, and can detect ischemic penumbra at an early stage, providing an earlier intervention opportunity for clinical treatment.

[0050] (3) According to previous studies, GFAP and UCH-L1 have been confirmed to be highly sensitive and specific diagnostic biomarkers for acute ischemic stroke, and may become indicators for accurately determining the size of the ischemic penumbra at the molecular level.

[0051] 2. Shorten the evaluation time

[0052] This method can quantify serum GFAP and UCH-L1 levels within 28 minutes at the bedside through simple blood sample collection and biomarker detection, making it a rapidly available biomarker, avoiding the long wait and high costs required for traditional imaging examinations. This is particularly important for patients with acute ischemic stroke, who can get treatment recommendations faster and reduce the risk of worsening of the disease due to long waiting times.

[0053] 3. Clinical practicality

[0054] (1) This method is easy to operate, requires simple equipment, and is low-cost. It is suitable for medical institutions at all levels and has broad clinical application prospects.

[0055] (2) Through the evaluation results of this method, doctors can more accurately judge the patient's condition and prognosis, develop personalized treatment plans for patients, and improve treatment effects and patients' quality of life.

[0056] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for detecting the acute ischemic stroke penumbra based on in vitro serum molecular markers for non-disease diagnosis purposes, characterized in that: The detection method is an operation of detecting glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) in the sample to be detected as biomarkers. The specific detection method includes: (1) Sample collection: Collect blood samples, centrifuge the samples, and separate serum; (2) Sample detection: Use the kit to detect GFAP and UCH-L1 in the sample to obtain the GFAP and UCH-L1 content in the serum sample; (3) Analysis of test results: The ratio of GFAP to UCH-L1 was calculated using analysis software, and its correlation with the penumbra of acute ischemic stroke was analyzed.

2. The detection method according to claim 1, characterized in that Serum GFAP levels above 12.12 pg / mL were independently associated with significant increases in penumbra volume and hypoperfusion volume.

3. The detection method according to claim 1, characterized in that Serum UCH-L1 levels above 133.38 pg / mL were independently associated with a significant increase in infarct core volume.

4. The detection method according to claim 1, characterized in that: When the serum GFAP / UCH-L1 ratio was greater than 0.19, it indicated the presence of a penumbra, with a specificity of no less than 90% and a positive predictive value of no less than 80%.

5. A kit for detecting the acute ischemic stroke penumbra according to any one of claims 1 to 4, characterized in that: The kit is used to measure the expression levels of GFAP and UCH-L1 in serum, and to evaluate the presence or absence of the penumbra by calculating the ratio of GFAP to UCH-L1.

6. An evaluation system for implementing the method for detecting the acute ischemic stroke penumbra according to any one of claims 1 to 4, characterized in that: The evaluation system comprises a kit for measuring the content of GFAP and UCH-L1, a detection device and an analysis module for evaluating the presence of a penumbra according to the ratio of GFAP to UCH-L1.

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