A detection method for the penumbra of acute ischemic stroke based on in vitro serum molecular markers

Abstract

The present invention discloses a method for detecting the penumbra of acute ischemic stroke based on in vitro serum molecular markers, belonging to the field of biotechnology. This detection method is an operation of detecting glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in a sample to be detected as biomarkers, including: (1) Sample collection: Collecting a blood sample and centrifuging to separate the serum; (2) Sample detection: Using a kit to detect the contents of GFAP and UCH-L1 in the sample; (3) Analysis of detection results: Calculating the ratio of GFAP to UCH-L1 through analysis software and analyzing its correlation with the penumbra of acute ischemic stroke. This method can quantify the levels of bedside serum GFAP and UCH-L1 within a short time (within 28 minutes), providing rapid biomarker detection results for clinical practice. This detection method is more economical, reduces medical costs, and has broad clinical application prospects.

Description

Technical Field

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

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

[0003] Currently, the commonly used methods for evaluating the ischemic penumbra in clinical practice are through advanced imaging techniques (such as CBF / CBV image mismatch in CT perfusion scan, PWI / DWI image mismatch in head magnetic resonance, etc.) and the clinical signs of patients. However, the following problems exist in specific clinical practices: Advanced imaging equipment is expensive and the technology is complex, making it difficult to popularize in primary hospitals; in addition, the relevant examinations are time-consuming and require the patient to be moved, which may delay the treatment time. Survey data shows that currently, less than 3% of AIS patients receive reperfusion therapy in the ultra-early stage, which is to some extent 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, simple operation, etc., and is particularly suitable for the acute-phase evaluation of AIS patients. However, so far, there is no method for evaluating the ischemic penumbra based on blood biomarkers.

[0004] After acute ischemic stroke, the main pathological feature of the infarct core is the 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. In the ischemic penumbra, there is reactive astrogliosis, and intracellular components are also released into the blood during its activation process. Therefore, theoretically, the markers of neurons and astrocytes in the blood are closely related to the infarct core and 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 existing technology. 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 ex vivo serum molecular markers. By using the biomarkers GFAP (glial fibrillary acidic protein) and UCH-L1 (ubiquitin carboxyl-terminal hydrolase L1) and combining advanced biological detection techniques, a new, rapid, convenient, and economical method for detecting the penumbra of acute ischemic stroke is developed, providing 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-diagnostic purposes, wherein the detection method is an operation of detecting glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) in a sample to be detected as biomarkers. The specific detection method includes:

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

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

[0010] (3) Analysis of detection results: Calculate the ratio of GFAP to UCH-L1 through analysis software and analyze its correlation with the penumbra of acute ischemic stroke;

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

[0012] Preferably, in the above technical solution, a serum UCH-L1 level higher than 133.38 pg / mL is independently correlated 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 the penumbra of acute ischemic stroke, which is used to measure the expression levels of GFAP and UCH-L1 in serum, and evaluate the presence or absence of the penumbra by calculating the GFAP / UCH-L1 ratio.

[0015] An evaluation system for the penumbra of acute ischemic stroke, the evaluation system includes a kit for measuring the contents of GFAP and UCH-L1, a detection device, and an analysis module for evaluating the presence of the 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 evaluated more precisely, improving the accuracy of diagnosis.

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

[0019] (3) Economy: Compared with traditional imaging examinations, using serum biomarkers is more economical, reducing medical costs.

[0020] (4) Wide clinical application: The simple operation and low cost make this method applicable to medical institutions at all levels, with broad clinical application prospects.

[0021] In summary, the beneficial effect of this application is to provide a rapid, 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 drawings incorporated in the specification and constituting a part of the specification illustrate embodiments of the present invention and, together with the description, are used to explain the principles of the present invention.

[0023] Figure 1 It is a schematic diagram of the relationship between the hypoperfused area and GFAP and UCH-L1.

[0024] Figure 2 It is a heat map of the visual distribution of the infarct core volume, hypoperfused volume, and ischemic penumbra volume in different GFAP level groups.

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

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

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

[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: unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions, and numerical values set forth in these embodiments do not limit the scope of the present invention.

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

[0030] Brain injury markers refer to substances that can be detected in cerebrospinal fluid or blood when brain tissue is damaged. Central nervous system infections, traumas, hypoxia, inflammations, or degenerations can lead to cell damage and the accumulation of breakdown products in the extracellular fluid, as well as an 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 acidic protein (GFAP) and ubiquitin carboxyterminal hydrolases L1 (UCH-L1) are important brain injury markers. As a specific intermediate filament protein mainly present in the central nervous system, an increase in its blood level usually reflects the damage of astrocytes. UCH-L1 is a protein highly specifically expressed in neurons. When neurons are damaged, its level in cerebrospinal fluid or blood will increase significantly. In the ultra-early stage of acute ischemic stroke, due to the interruption of blood flow in the infarct core area, neurons suffer irreversible damage and death. Subsequently, UCH-L1 is released into the blood through the damaged blood-brain barrier, indicating a close relationship between serum UCH-L1 level and the infarct core area. On the other hand, astrocytes suffer irreversible damage and death in the infarct core, but show obvious reactive hyperplasia in the ischemic penumbra, which is an adaptive response to complex signal changes in the local microenvironment, known as reactive astrogliosis. A significant increase in GFAP expression level is one of its main characteristics. In summary, in view of the unique pathophysiological characteristics of UCH-L1 and GFAP and their relevance to different pathological regions of ischemic stroke, we propose to jointly apply these two biomarkers to more comprehensively evaluate the presence and evolution of the ischemic penumbra.

[0031] There is currently no report on the application of the two in the evaluation of the ischemic penumbra. Therefore, the present invention will utilize the biomarkers GFAP and UCH-L1 to develop a new, rapid, convenient, and economical method for evaluating the ischemic penumbra of acute ischemic stroke, providing strong support for the early diagnosis and precise treatment of AIS patients. The following is a detailed description by way of examples (please also refer to Figures 1 - 5 )

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

[0033] A total of 102 patients with acute ischemic stroke (AIS) with large vessel occlusion in the anterior circulation who were scheduled for thrombectomy and presented within 24 hours after symptom onset were recruited from the First Hospital of Jilin University. Blood samples were collected before computed tomography perfusion imaging (CTP). The researchers measured the levels of brain injury biomarkers, glial fibrillary acidic protein (GFAP) and ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), in the serum. Subsequently, CTP sequences were analyzed using deconvolution method to calculate the infarct core volume, ischemic penumbra volume, hypoperfused volume, and mismatch ratio. The infarct core was defined as a relative cerebral blood flow (rCBF) of less than 30% of the contralateral normal brain tissue; hypoperfusion was defined as a tissue residual time to peak (Tmax) of greater than 6 seconds; the ischemic penumbra volume was calculated as the hypoperfused volume minus the infarct core volume; and the mismatch ratio was the ratio of the hypoperfused volume to the infarct core volume.

[0034] (1) Relationship between GFAP and infarct core and ischemic penumbra volumes

[0035] A total of 102 patients underwent GFAP measurement. Univariate analysis revealed that in patients with acute ischemic stroke, higher serum GFAP levels (>12.12 pg / mL) were independently associated with a larger ischemic penumbra volume {odds ratio (OR) 3.297 (95% confidence interval [95% CI], 1.562 - 6.959, P = 0.002)} and a larger hypoperfused volume {OR 2.986 (95% CI, 1.423 - 6.271, P = 0.004)}, but not with the infarct core volume, OR 1.425 (95% CI, 0.697 - 2.915, P = 0.332). Further multivariate analysis, after adjusting for age, gender, National Institutes of Health Stroke Scale (NIHSS) score at admission, time from onset to blood sampling, previous cerebral infarction, and Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification, showed that GFAP was an independent predictor of ischemic penumbra volume {OR 3.773 (95% CI, 1.589 - 8.962, P = 0.003)} and hypoperfused volume {OR 2.942 (95% CI, 1.254 - 6.910, P = 0.013)}.

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

[0037] A total of 102 patients underwent UCH-L1 detection. Through univariate analysis, it was found that for patients with acute ischemic stroke, a higher serum UCH-L1 (>133.38 pg / mL) level was independently associated with a larger infarct core volume, with an OR value of 2.467 (95% CI, 1.186 - 5.129, P = 0.016). However, it was not related to the penumbra volume {OR value 1 (95% CI, 0.490 - 2.040, P = 1.000)} and the hypoperfused volume {OR value 1.303 (95% CI, 0.638 - 2.664, P = 0.467)}. Further multivariate analysis, after adjusting for age, gender, NIHSS score at admission, time from onset to blood sampling, previous cerebral infarction, and TOAST classification, 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 more conveniently, quickly, and rapidly evaluate the volumes of the penumbra and infarct core through biomarkers, thus helping clinicians judge the feasibility and expected effects of reperfusion therapy.

[0039] Example 2 Evaluation of the presence of ischemic penumbra by the serum GFAP / UCH-L1 ratio

[0040] In this invention, 61 AIS patients with large vessel occlusion in the anterior circulation who were scheduled for thrombectomy and presented within 24 hours after symptom onset were recruited from the First Hospital of Jilin University. Subsequently, CTP sequences were analyzed by deconvolution method to calculate the infarct core volume, ischemic penumbra volume, hypoperfused volume, and mismatch ratio. According to the CTP imaging mismatch principle, "the presence of ischemic penumbra" was defined as an infarct core volume 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%). It was found that in the serum of acute ischemic stroke patients with penumbra, the median GFAP / UCH-L1 ratio was 0.08 (0.05 - 0.17), which was significantly higher than that of patients without penumbra, 0.04 (0.03 - 0.07). Through univariate analysis, it was found 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, after adjusting for age, gender, NIHSS score at admission, time from onset to blood sampling, hyperlipidemia, and hyperhomocysteinemia, it was found that the 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 cut-off value for evaluating the presence of penumbra using the serum GFAP / UCH-L1 ratio

[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 cut-off value for evaluating the presence of penumbra using the GFAP / UCH-L1 ratio 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 penumbra was 88.9% (95% CI, 51.9 - 98.3), as shown in Table 1.

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

[0045]

[0046] The GFAP / UCH-L1 ratio reflects the relative proportion between the infarct core mainly composed of neuronal death and the penumbra mainly composed of astrocyte proliferation. The above examples show that using the GFAP / UCH-L1 ratio to evaluate the presence of penumbra has high specificity and PPV. This has extremely high value for evaluating the patient's condition and whether they are suitable for reperfusion therapy, because compared with traditional perfusion examinations, using serum biomarkers is simpler, non-invasive, and more economical. It has great economic and social value. In addition, the current technology can quantify the levels of serum GFAP and UCH-L1 at the bedside within 28 minutes, making it a biomarker that can be quickly obtained for patients in saving the ischemic penumbra.

[0047] Result analysis:

[0048] 1. Evaluation accuracy

[0049] (1) By detecting the biomarkers GFAP and UCH-L1, which are closely related to the formation and evolution of the penumbra in acute ischemic stroke, this method can accurately reflect the presence and extent of the ischemic penumbra.

[0050] (2) Compared with traditional imaging methods, this method also has high sensitivity and specificity, can detect the ischemic penumbra at an early stage, and provides an earlier intervention opportunity for clinical treatment.

[0051] (3) According to previous studies, GFAP and UCH-L1 have been confirmed as highly sensitive and specific diagnostic biomarkers for acute ischemic stroke, and may become indicators for accurately judging the size of the ischemic penumbra at the molecular level.

[0052] 2. Shorten the evaluation time

[0053] By simple blood sample collection and biomarker detection, this method can quantify the serum levels of GFAP and UCH-L1 within 28 minutes at the bedside, making them rapidly available biomarkers, avoiding the long waiting time and high cost required for traditional imaging examinations. This is particularly important for patients with acute ischemic stroke, enabling them to obtain treatment recommendations faster and reducing the risk of disease progression due to excessive waiting time.

[0054] 3. Clinical practicability

[0055] (1) This method is easy to operate, with simple equipment and low cost, applicable to medical institutions at all levels, and has broad clinical application prospects.

[0056] (2) Through the evaluation results of this method, doctors can more accurately judge the patient's condition and prognosis, formulate personalized treatment plans for patients, and improve the treatment effect and the quality of life of patients.

[0057] Although the present invention has been disclosed above by way of examples, it is not intended to limit the present invention. Any person skilled in the art can make various different selections and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is defined by the claims and their equivalent forms.

Claims

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

2. The detection method according to claim 1, wherein A serum GFAP level higher than 12.12 pg / mL is independently associated with a significant increase in the penumbra volume and the hypoperfused volume.

3. The detection method according to claim 1, wherein A serum UCH-L1 level higher than 133.38 pg / mL is independently associated with a significant increase in the infarct core volume.

4. An evaluation system for implementing the detection method of the penumbra of acute ischemic stroke according to any one of claims 1-3, characterized in that, The evaluation system includes a kit for measuring the contents 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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