Heart biomarker combination protein for identifying sudden death of unknown cause and application thereof

Abstract

This invention relates to a combination of cardiac biomarker proteins for identifying sudden death from unknown causes and its applications. The invention utilizes formalin-fixed paraffin-embedded cardiac tissue sections, and identifies biomarker proteins (MYH6 and COX5B) using proteomics technology. Immunohistochemical staining and quantitative analysis of these two cardiac biomarker proteins (MYH6 and COX5B) are then performed to enable precise forensic identification of sudden death from unknown causes. This invention also provides a corresponding one-stop identification platform, specifically including immunohistochemical staining, quantitative analysis of staining intensity, and forensic software outputting the corresponding results. Experiments have confirmed that the aforementioned cardiac biomarker combination proteins MYH6 and COX5B have accurate identification value for sudden death from unknown causes and hold promise as precise indicators for identifying this condition. This invention opens up a new method for forensic identification of sudden death from unknown causes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a combination protein of cardiac biomarkers for identifying sudden death from unknown causes and its application. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death worldwide, causing approximately 17 million deaths annually, with about 25% being sudden cardiac death. Sudden cardiac death is defined as "an unexpected death in a seemingly healthy individual occurring within one hour of the onset of symptoms, or an unexpected death occurring within 24 hours without witnesses." While a standardized autopsy can identify the cause of death in some cases, in others, even after standardized procedures, the exact cause remains unexplained. These deaths are presumed to be due to pre-existing arrhythmias or hemodynamic abnormalities and are termed sudden unexplained death (SUD). Due to the lack of precise forensic strategies for identifying sudden unexplained death, only speculative investigations are possible in practice. The conclusions are often unconvincing to the parties involved, especially when such cases involve medication or special circumstances (such as detention centers or compulsory drug rehabilitation centers), which can lead to suspicion, endless complaints, public attention, and difficulties in judicial proceedings.

[0003] As one of the most commonly used methods for preserving tissue samples in forensic autopsy practice, formalin-fixed paraffin-embedded human tissue has advantages such as high stability and the ability to be preserved for a long time at room temperature. Using formalin-fixed paraffin-embedded human tissue, some biomarkers in pathological processes can be detected, addressing medical challenges. In particular, due to continuous technological advancements in recent years, high-quality proteins can now be extracted from formalin-fixed paraffin-embedded human tissue for biomarker research. Summary of the Invention

[0004] The purpose of this invention is to provide a combination of cardiac biomarker proteins for identifying sudden death from unknown causes and its applications. This invention utilizes formalin-fixed paraffin-embedded cardiac tissue sections, and identifies biomarker proteins (MYH6 and COX5B) using proteomics technology. Immunohistochemical staining and quantitative analysis of these two cardiac biomarker proteins (MYH6 and COX5B) are then performed to enable precise forensic identification of sudden death from unknown causes. This invention also provides a corresponding one-stop identification platform, specifically including immunohistochemical staining, quantitative analysis of staining intensity, and identification software outputting the corresponding results. Experiments have confirmed that the cardiac biomarker combination proteins MYH6 and COX5B have accurate identification value for sudden death from unknown causes and hold promise as precise indicators for identifying this condition. This invention opens up a new method for forensic identification of sudden death from unknown causes.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] The first objective of this invention is to provide a cardiac biomarker combination protein for sudden death of unknown cause, wherein the cardiac biomarker combination protein is a combination protein of MYH6 and COX5B.

[0007] The second objective of this invention is to provide an application of cardiac biomarker combinatorial proteins in the preparation of products for identifying sudden death from unknown causes.

[0008] Furthermore, the identification products include reagents or kits for identifying sudden death from unknown causes.

[0009] Furthermore, the reagents or kits described above include goat serum blocking solution, primary and secondary antibodies against cardiac biomarker combinatorial proteins MYH6 and COX5B, DAB chromogenic solution, and sodium citrate buffer.

[0010] The third objective of this invention is to provide a reagent for detecting the levels of cardiac biomarker combinatorial proteins MYH6 and COX5B in the preparation of products for identifying sudden death from unknown causes.

[0011] A fourth objective of this invention is to provide an identification system for detecting sudden death of unknown cause, comprising:

[0012] Analysis unit: It contains substances for determining the intensity of immunohistochemical staining of the cardiac biomarker combination proteins MYH6 and COX5B as described in claim 1 in a sample;

[0013] An evaluation unit includes forensic software embedded with an algorithm for converting the immunohistochemical staining intensities of MYH6 and COX5B determined by the analysis unit into probability values, and capable of generating predictions of unexplained sudden death based on the probability values.

[0014] Furthermore, the sample was derived from a section of formalin-fixed paraffin-embedded heart tissue.

[0015] Furthermore, the formula for calculating the immunohistochemical staining intensity is as follows:

[0016] Immunohistochemical staining intensity = total IOD value / total tissue area

[0017] The total IOD value is the sum of the intensity values ​​of each pixel within the IHC-positive region.

[0018] Total tissue area: The area of ​​cells within the tissue boundary.

[0019] Furthermore, the identification software calculates the probability value of sudden death of unknown cause based on the logistic regression algorithm in IDLE, using the immunohistochemical staining intensity of MYH6 and COX5B.

[0020] Furthermore, in the evaluation unit containing the forensic software, when the probability value of the unexplained sudden death is greater than a preset value, the output prediction result is "unexplained sudden death"; when the probability value of the unexplained sudden death is less than the preset value, the output prediction result is "excluded as unexplained sudden death".

[0021] Furthermore, the preset value is 50%.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention provides the forensic identification application of the cardiac biomarker combinatorial proteins MYH6 and COX5B in sudden death from unknown causes, and develops a corresponding one-stop identification platform. Validation was conducted using the identification platform developed in this patented invention on 8 independent samples already identified as sudden death from unknown causes. The results showed that in 7 of the 8 cases, the platform accurately calculated a probability value close to 1 for sudden death from unknown causes, consistent with known causes of death, achieving an accuracy of 87.5%. This invention opens up a new method for forensic identification of sudden death from unknown causes. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the changes in MYH6 staining intensity in sudden death of unknown cause.

[0025] Figure 2 A schematic diagram showing the changes in staining intensity of COX5B in sudden death of unknown cause;

[0026] Figure 3 A schematic diagram illustrating the efficacy of MYH6 in identifying sudden death of unknown cause (SUD) using receiver operating characteristic (ROC) curves;

[0027] Figure 4 A schematic diagram illustrating the efficacy of COX5B in identifying sudden death of unknown cause (SUD) using receiver operating characteristic (ROC) curves;

[0028] Figure 5 The receiver operating characteristic (ROC) curve illustrates the efficacy of combining MHY6 and COX5B in identifying sudden death of unknown cause (SUD).

[0029] Figure 6 A schematic diagram of the immunohistochemical staining apparatus and operating procedures for cardiac biomarker combinatorial proteins (MYH6 and COX5B) to perform immunohistochemical staining on formalin-fixed paraffin-embedded cardiac tissue sections.

[0030] Figure 7 This is a schematic diagram illustrating the method and operating guidelines for quantitative analysis of immunohistochemical staining intensity using Image Pro Plus 6.0 software;

[0031] Figure 8 This is a schematic diagram of the corresponding identification software interface and operation guide. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0033] In the following examples, the antibody sources are as follows:

[0034] Rabbit anti-human polyclonal antibody MYH6 (Wuhan Sanying Biotechnology Co., Ltd.) and rabbit anti-human monoclonal antibody COX5B (Aibokang (Shanghai) Trading Co., Ltd.) used for immunohistochemical staining were purchased from biotechnology companies and diluted according to the manufacturer's instructions (MYH6, 1:50; COX5B, 1:500) for immunohistochemical staining of formalin-fixed paraffin-embedded heart tissue sections.

[0035] In the following embodiments, the case sources are as follows:

[0036] All cases were collected from forensic examinations routinely handled by institutions such as the Department of Forensic Medicine and Department of Pathology of the School of Basic Medical Sciences, Fudan University, and the Department of Forensic Medicine of the School of Basic Medical Sciences, Gannan Medical University, between 1997 and 2022. Detailed records were kept of basic case information (age, gender, medical history), the process of death (prodromal symptoms, survival time, location of death, etc.), and the post-mortem time interval (PMI, the time interval between death and medical examination). A total of 71 cases were collected, including the unexplained sudden death group (SUD, n=29), the CO intoxication group (CO intoxication, n=17), the mechanical injury death group (Fatal Injury, n=15), and the definite cause of death sudden cardiac death group (SCD, n=10). The CO intoxication death group and the mechanical injury death group served as negative controls, while the sudden cardiac death group served as a positive control.

[0037] In the following embodiments, the references are as follows:

[0038] [1]Tyanova S,Temu T,Cox J.The MaxQuant computational platform formass spectrometry-based shotgun proteomics.Nat Protoc.2016;11(12):2301-2319.doi:10.1038 / nprot.2016.136IF:14.8Q1.

[0039] [2]Zhou Y,Zhou B,Pache L,et al.Metascape provides a biologist-oriented resource for the analysis of systems-level datasets.Nat Commun.2019;10(1):1523.Published 2019Apr 3.doi:10.1038 / s41467-019-09234-6IF:16.6Q1.

[0040] [3]Li L,Ching WK,Liu ZP.Robust biomarker screening from geneexpression data by stable machine learning-recursive feature eliminationmethods.Comput Biol Chem.2022;100:107747.doi:10.1016 / j.compbiolchem.2022.107747.

[0041] [4]Wang J,Li X,Liu Z et al(2021)Second-generation antipsychoticsinduce cardiotoxicity by disrupting spliceosome signaling:Implications fromproteomic and transcriptomic analyses.Pharmacol Res 170:105714.doi:10.1016 / j.phrs.2021.105714.

[0042] [5]Li L,Dong X,Tu C et al(2019)Opposite effects of cannabinoid CB(1)and CB(2)receptors on antipsychotic clozapine-induced cardiotoxicity.Br JPharmacol 176:890-905.doi:10.1111 / bph.14591.

[0043] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0044] Example 1

[0045] In this embodiment, the biomarker proteins MYH6 and COX5B were identified using proteomics technology, as detailed below:

[0046] MYH6 and COX5B are cardiac biomarkers for sudden death of unknown cause identified through proteomics technology. Specifically, formalin-fixed paraffin-embedded heart samples from three cases of sudden cardiac death (SUD), three cases of carbon monoxide poisoning, and three cases of fatal injury were collected from the Department of Forensic Medicine, School of Basic Medical Sciences, Gannan Medical University, and label-free quantitative proteomics analysis was performed on them. [1] Candidate protein biomarkers were obtained from the quantitative results through differential expression analysis. This was then combined with bioinformatics... [2] Recursive feature elimination in machine learning [3] The above candidate biomarker proteins were screened using the method described above, and MYH6 and COX5B were identified as biomarker proteins for sudden death of unknown cause.

[0047] Example 2

[0048] like Figure 6 As shown in this embodiment, immunohistochemical staining was performed on formalin-fixed paraffin-embedded heart tissue sections from 71 cases. The specific steps are as follows:

[0049] (1) Human formalin-fixed paraffin-embedded heart tissue sections were dewaxed to water in gradient xylene and gradient ethanol, respectively.

[0050] (2) The dewaxed sections were placed in a microwave oven at 100°C and subjected to antigen retrieval in 0.01M sodium citrate buffer solution (pH=6) for 10 minutes, and then cooled at room temperature for half an hour.

[0051] (3) After blocking with goat serum for 60 minutes, add the corresponding diluted primary antibodies (anti-MYH6 antibody, anti-COX5B antibody) on the slide and incubate overnight at 4°C.

[0052] (4) On the second day, the slides were washed three times with phosphate-buffered saline (PBS) for 5 minutes each time, and HRP-labeled goat anti-rabbit IgG (secondary antibody, antibody against primary antibody) was added to the slides. After incubation at 37°C for 60 minutes, the slides were immunized in DAB chromogenic solution for 10 minutes.

[0053] (5) Finally, stain with hematoxylin for 3 minutes to visualize the cell nuclei. Staining of all target proteins was performed on serial sections of the same tissue block in each case to maintain consistency.

[0054] Example 3

[0055] This embodiment quantitatively analyzes the staining intensity of the immunohistochemically stained cardiac tissue sections obtained in Example 2. Five microscopic fields of view of each section at 200× were randomly selected using a microscopic imaging system (Leica microscopy system, Wetzlar, Germany). The subsequent analysis was performed using Image Pro Plus 6.0 (MediaCybernetics, Rockville, Maryland, USA).

[0056] For wider application and reliability of results, strict criteria were defined for selecting the areas to be analyzed: only images covering cardiac tissue were selected, and tissues including fat and blood vessels were excluded whenever possible. Overlapping tissues or contaminated backgrounds were also excluded from the analysis.

[0057] S1. For each protein, the staining intensity of each region is initially expressed as a total IOD value, which is obtained by summing the intensity values ​​of each pixel in the target field of view and reflects the color intensity and positivity (percentage).

[0058] S2. To further reduce the impact of tissue area on the total IOD value, the average staining intensity is used as the final indicator, and the calculation formula is as follows:

[0059] Average staining intensity of each region = total IOD value / total tissue area

[0060] In the formula,

[0061] Total IOD value: The sum of the intensity values ​​of each pixel within the IHC-positive region.

[0062] Total tissue area: The area of ​​cells within the tissue boundary, excluding non-tissue spaces such as gaps.

[0063] S3. Import five images of each protein from each case into Image Pro Plus 6.0 software. The analysis of each image can be basically divided into four steps:

[0064] S3-1. Calibrate optical density. Click the "Measure / Calibrate / Intensity calibration" command in sequence, and select the "Std.Optical Density" mode. Then click the "Options / Incident Level Image" button to calibrate the blank area of ​​the background region.

[0065] S3-2. Select the measurement values ​​and set up the data collector. Add options including "Area" and "IOD values" to the measurement table via Count / Size. Add "Image name," "IOD(SUM)," and "Area(SUM)" to the data collector.

[0066] S3-3, Color Selection. Use the color extraction tool to set and adjust color selection in "Color Cube Based" mode. Select the positive areas for each protein and the entire tissue area separately under immunohistochemical staining. To ensure consistency, use the same color selection file for the same batch of sections stained with IHC for each protein.

[0067] S3-4, Calculation Results. Open the image and the corresponding color selection file. The IOD value and the area of ​​the selected region will be displayed in the data collector using the "Count" function. The staining intensity of each region is equal to the IOD value of that region divided by its area.

[0068] like Figure 7 As shown, the specific steps of the quantitative analysis method for staining intensity are as follows:

[0069] (1) After selecting the image to be measured, click “Measure / Calibrate / Optical Density” in sequence to open the calibration optical density panel;

[0070] (2) After selecting the “Std.optical Density” calibration mode in ①, click “Options” in ②;

[0071] (3) After clicking the “Image” button shown in the figure, select the blank area in the slice to complete the optical density calibration;

[0072] (4) Click the “Measure / Automatic Count / Measure” button in sequence to open the “Count / Size” panel;

[0073] (5) Add “Area” and “IOD values” to the “Measure” table in section ①.

[0074] (6) Click “Measurement / Data Collector” to open “Data Collector”;

[0075] (7) In the “Data Collector”, select “Name”, “Area” (Sum) and “IOD” (Sum) in the right-hand selection bar, and then in the “Count / Size” panel (e.g.) Figure 7 (5) Click ② “Select Colors” in the middle to open the “Segmentation” panel;

[0076] (8) In the “Segmentation” panel, click the color picker icon ① to select the target area. The color selection file of the selected target area can be saved and retrieved in ②;

[0077] (9) In the “Count / Size” panel (e.g.) Figure 7 (5) After clicking the “Count” button in ③, click “Collect Now” in the “DataCollector” panel to read the IOD value and area value of the slice in the selected area in the Data List.

[0078] Note: This demo software has a Chinese language plugin installed, and some buttons are displayed in Chinese. Please refer to actual operation for the most accurate representation. The sliced ​​images and color selection areas shown in this demo are for demonstration purposes only and have no practical significance.

[0079] After analyzing each region, the staining intensity of each protein in the myocardial tissue was calculated as the sum of the staining intensities of the five regions divided by 5.

[0080] S4. Statistical Analysis

[0081] Unless otherwise stated, data are presented as mean ± standard error (SEM). For continuous variables, the Student's-St. test or nonparametric Mann-Whitney test was used for comparisons between two groups, and one-way ANOVA or nonparametric tests were used for comparisons among multiple groups. Receiver operating characteristic (ROC) curve analysis was used to assess the sensitivity and specificity of each group, and the area under the curve (AUC) was calculated and analyzed. Graphs were created using GraphPad Prism 8.0 (LaGiolla, CA, USA). A p-value < 0.05 was considered statistically significant under two-tailed conditions.

[0082] Example 4

[0083] In this embodiment, the probability of sudden death of unknown cause is calculated by using identification software to determine the staining intensity of MYH6 and COX5B obtained in Example 3.

[0084] To improve practicality, a software was developed based on the logistic regression algorithm in IDLE (Python 3.11, 64-bit) to analyze the immunohistochemical staining intensity results of MYH6 and COX5B proteins in formalin-fixed paraffin-embedded heart tissue sections from the above 71 cases, and to develop an identification software for calculating the probability of sudden death from unknown causes.

[0085] Logistic regression, also known as logistic regression analysis, is a generalized linear regression model. Logistic regression estimates the probability of an event based on a given dataset of independent variables. Since the result is a probability, the dependent variable ranges between 0 and 1. In the results, cases with a probability of 1 are classified as sudden unexplained death (SUD), and cases with a probability of 0 are classified as the control group. The higher the probability (from 0 to 1, i.e., from the control group to the SUD group), the higher the risk of SUD. By inputting the MYH6 and COX5B staining intensities of new samples into forensic software, the probability value of sudden unexplained death is calculated.

[0086] like Figure 8 As shown, the forensic software interface includes two parameters and one button. The two parameters are the immunohistochemical staining intensities of MYH6 and COX5B in formalin-fixed paraffin-embedded heart tissue sections, and the button is the Calculate button. When using the forensic software, input the corresponding MYH6 and COX5B staining intensities of myocardial tissue into the two parameter boxes, and click the Calculate button. The forensic software will calculate and immediately output the probability value of unexplained sudden death using the formula described above. The specific steps are as follows:

[0087] (1) The original interface of the identification software;

[0088] (2) Enter the corresponding staining intensity values ​​in the MYH6 and COX5B parameter boxes respectively, and click the "Calculate" button;

[0089] (3) The software calculates the probability value of sudden death of unknown cause and displays it after RISK (%).

[0090] Note: The data in the figure is for demonstration purposes only.

[0091] The staining intensity values ​​of MYH6 and COX5B in myocardial sections were obtained using Image Pro Plus 6.0 software, and then... Figure 8 Enter the information in the corresponding text box, and then click the "Calculate" button to get the final calculation result.

[0092] Example 5

[0093] This embodiment utilizes a one-stop identification platform for accurate identification of sudden death from unknown causes. The platform includes immunohistochemical staining devices and operating procedures for the aforementioned cardiac biomarker combination proteins MYH6 and COX5B. [4，5] ( Figure 6 ), Quantitative analysis methods and operating guidelines for staining intensity ( Figure 7 ), immunohistochemical staining kits and corresponding identification software ( Figure 8 The aforementioned identification platform has the function of accurately identifying sudden deaths of unknown cause.

[0094] The immunohistochemical staining apparatus includes a series of devices for immunohistochemical staining of formalin-fixed paraffin-embedded human heart tissue, such as a dewaxing tank, a dehydration tank, an endogenous peroxidase removal agent, a blocking solution, and an antibody incubation box.

[0095] The immunohistochemical staining kit contains goat serum blocking solution for immunohistochemical staining of formalin-fixed paraffin-embedded heart tissue sections, primary and secondary antibodies for cardiac biomarker proteins, DAB chromogenic solution, sodium citrate buffer (ready-to-use dry powder), and instructions for use.

[0096] The identification software only requires inputting the staining intensity results of MYH6 and COX5B to generate a probability value for sudden death of unknown cause with a single click. A probability value close to 1 indicates sudden death of unknown cause. A probability value much less than 1 excludes sudden death of unknown cause.

[0097] Example 6: Significant Changes in Myocardial MYH6 and COX5B in Cases of Sudden Unexplained Death (SUD)

[0098] This embodiment was used to study the changes in staining intensity of MYH6 and COX5B in sudden death of unknown cause. This invention collected 29 cases of sudden death of unknown cause, using 17 cases of CO poisoning and 15 cases of mechanical injury as negative controls, and 10 cases of sudden cardiac death with a clear cause as positive controls. Immunohistochemical staining and corresponding quantitative detection analyses were performed.

[0099] Immunohistochemical staining and quantitative analysis showed that the staining intensity of the two cardiac biomarker combination proteins, MYH6 and COX5B, in hearts of unexplained sudden death was not significantly different from the positive control, but both were significantly different from the negative control. Specifically, MYH6 was significantly elevated in unexplained sudden death compared to the negative control (P<0.001). Figure 1 COX5B levels were significantly lower in unexplained sudden death compared to the negative control (P<0.01). Figure 2This indicates that MYH6 and COX5GB are significantly altered in the myocardium of SUD cases.

[0100] like Figure 1 and Figure 2 As shown, compared with the negative control groups (CO intoxication and fatal injury), the myocardial MYH6 level was significantly increased and COX5B was significantly decreased in the sudden death from unknown cause (SUD) group. The sudden cardiac death (SCD) group with a known cause of death served as a positive control. (ns, no significance; *, p<0.05; **, p<0.01; ***, p<0.001).

[0101] Example 7: The combined use of myocardial MYH6 and COX5B showed better identification effect for SUD than using either alone.

[0102] In this embodiment, the identification effects of MYH6 alone, COX5B alone, and the combination of MYH6 and COX5B on SUD in the myocardium are demonstrated respectively; the area under the curve (AUC), identification sensitivity, and specificity under the three conditions are analyzed using ROC.

[0103] Table 1. Identification performance of MYH6 alone, COX5B alone, and the combination of MYH6 and COX5B on SUD.

[0104]

[0105] like Figure 3-5 As shown in Table 1, the receiver operating characteristic (ROC) curves demonstrate the efficacy of MYH6 (A), COX5B (B), and the combination of MHY6 and COX5B (C) in identifying sudden death from unknown causes (SUD) (D).

[0106] The results showed that the area under the curve (AUC) for MYH6 was 0.8028 (95% CI: 0.6907–0.9149, p = 0.000049465). Figure 3 The area under the curve (AUC) generated by COX5B was 0.8211 (95% CI: 0.7185–0.9248, p = 0.000016801). Figure 4 The area under the curve (AUC) generated by combining MYH6 and COX5B was 0.9073 (95% CI: 0.8297–0.9849, p = 4.8E-08). Figure 5 ).

[0107] As shown in Table 1, the sensitivity of MYH6 in identifying sudden death of unknown cause was 0.8621, and the specificity was 0.6562. The sensitivity of COX5B in identifying sudden death of unknown cause was 0.7241, and the specificity was 0.7812. The combined sensitivity of MYH6 and COX5B in identifying sudden death of unknown cause was 0.8966, and the specificity was 0.8437.

[0108] Using this forensic platform to examine formalin-fixed paraffin-embedded heart tissue sections, the ROC curve analysis results for identifying sudden death of unknown cause were as follows: MYH6 showed a sensitivity of 0.8621, a specificity of 0.6562, and an area under the curve (AUC) of 0.8028 (95% CI: 0.6907-0.9149, p = 0.000049465); COX5B showed a sensitivity of 0. The specificity of 7241 was 0.7812, and the area under the curve (AUC) was 0.8211 (95% CI: 0.7185-0.9248, p = 0.000016801); the sensitivity of MYH6 and COX5B combined in identifying sudden death of unknown cause was 0.8966, the specificity was 0.8437, and the AUC was 0.9073 (95% CI: 0.8297-0.9849, p = 4.8E-08).

[0109] Experimental results show that the combined use of MHY6 and COX5B has better identification performance than using MHY6 and COX5B alone, and can be used for accurate identification of sudden death of unknown cause.

[0110] Example 8 uses the developed platform for identifying sudden death of unknown cause to identify independent samples, achieving an accuracy rate of 87.5%.

[0111] Eight independent cases of sudden death of unknown cause, submitted by the forensic medicine department of another institution, were used. Formalin-fixed paraffin-embedded cardiac tissue sections from these samples underwent immunohistochemical and quantitative staining intensity analysis on this platform using the same method. The results were then input into the forensic software for probability calculation. The verification results are shown in Table 2. The probabilities of death as sudden death of unknown cause in the eight cases were 99.99992%, 85.58653%, 90.87899%, 99.79527%, 99.38843%, 98.87138%, 99.70640%, and 0.04246%, respectively. Except for the eighth case, the calculated probabilities of sudden death of unknown cause for the other seven cases were close to 1, thus achieving an accurate identification rate of 87.5%.

[0112] Table 2 shows the accurate identification results of the applied forensic platform for 8 cases of sudden death from unknown causes.

[0113]

[0114] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A cardiac biomarker combination protein for sudden unexplained death, characterized by, The cardiac biomarker combinatorial protein is a combination of MYH6 and COX5B.

2. The application of the cardiac biomarker combinatorial protein as described in claim 1 in the preparation of products for identifying sudden death from unknown causes.

3. The use of the cardiac biomarker combination protein according to claim 2 in the preparation of a product for the identification of sudden unexplained death, characterized in that, The identification products include reagents or kits used to identify sudden death from unknown causes.

4. The use of the cardiac biomarker combination protein according to claim 3 in the preparation of a product for identifying sudden unexplained death, characterized in that, The reagents or kits include goat serum blocking solution, primary and secondary antibodies for cardiac biomarker combinatorial proteins MYH6 and COX5B, DAB chromogenic solution, and sodium citrate buffer.

5. The application of a reagent for detecting the levels of cardiac biomarker combinatorial proteins MYH6 and COX5B in the preparation of products for identifying sudden death from unknown causes.

6. An identification system for identifying sudden unexplained death, characterized in that include: Analysis unit: It contains substances for determining the intensity of immunohistochemical staining of the cardiac biomarker combination proteins MYH6 and COX5B as described in claim 1 in a sample; An evaluation unit includes forensic software embedded with an algorithm for converting the immunohistochemical staining intensities of MYH6 and COX5B determined by the analysis unit into probability values, and capable of generating predictions of unexplained sudden death based on the probability values.

7. The identification system for identifying sudden unexplained death according to claim 6, wherein The sample was derived from a section of formalin-fixed paraffin-embedded heart tissue.

8. A forensic system for identifying sudden death of unknown cause according to claim 6, characterized in that, The formula for calculating the immunohistochemical staining intensity is as follows: Immunohistochemical staining intensity = total IOD value / total tissue area The total IOD value is the sum of the intensity values ​​of each pixel within the IHC-positive region. Total tissue area: The area of ​​cells within the tissue boundary.

9. A forensic system for identifying sudden death of unknown cause according to claim 8, characterized in that, The identification software calculates the probability value of sudden death of unknown cause based on the logistic regression algorithm in IDLE, using the immunohistochemical staining intensity of MYH6 and COX5B.

10. The identification system for identifying sudden unexplained death according to claim 9, wherein, In the evaluation unit containing forensic software, when the probability value of the unexplained sudden death is greater than a preset value, the output prediction result is "unexplained sudden death"; when the probability value of the unexplained sudden death is less than the preset value, the output prediction result is "excluded as unexplained sudden death".

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