Immunohistochemical staining intensity quantitative evaluation method based on fluorescence labeling

Abstract

The invention provides an immunohistochemical staining intensity quantitative evaluation method based on fluorescence labeling, and relates to the technical field of biomedical detection.The immunohistochemical staining intensity quantitative evaluation method comprises the steps that a fluorescein labeled antibody is specifically combined with an antigen, an image is collected through a fluorescence microscope, and an image analysis technology is combined; and calculating parameters such as average fluorescence intensity and integral fluorescence intensity. And selecting a positive sample with known concentration to draw a standard curve so as to accurately calculate the relative antigen content of the to-be-detected sample. Compared with a traditional subjective evaluation method, the method avoids human factor interference, and the evaluation result is more objective and accurate. The method has high consistency and repeatability, and provides a uniform and quantitative standard for immunohistochemical staining evaluation. The method is suitable for various biological tissue samples, has wide application prospects in biomedical research and clinical diagnosis, and can assist in precise diagnosis of diseases and scientific formulation of treatment schemes.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and more specifically, to a method for quantitatively assessing the intensity of immunohistochemical staining based on fluorescent labeling. Background Technology

[0002] Immunohistochemical staining, as an important biological detection technique, plays a crucial role in biomedical research and clinical diagnosis. It primarily utilizes the principle of specific binding between antibodies and antigens, using a chemical reaction to cause a chromogenic agent containing labeled antibodies to develop color, thereby identifying intracellular antigens and enabling their localization, qualitative analysis, and relative quantification. However, existing methods for assessing immunohistochemical staining intensity have several shortcomings, as detailed below:

[0003] Traditional methods for assessing immunohistochemical staining intensity largely rely on the subjective judgment of pathologists. Different pathologists, due to differences in experience, expertise, and perspective, may have significant disagreements in scoring the staining intensity of the same tissue section. For example, when assessing the staining intensity of certain markers in a tumor tissue sample, an experienced pathologist might classify it as moderate staining, while another might consider it weak staining. Such subjective differences in assessment can severely impact the accuracy and consistency of diagnostic results, posing challenges to the development of clinical treatment plans.

[0004] Subjective assessments typically employ a grading system, such as 0-3 points or negative, weakly positive, positive, strongly positive, etc. This grading method is relatively vague and lacks precise quantitative standards. The scoring criteria are often unclear and inconsistent; different laboratories or research institutions may score based on their own habits and experiences, making it difficult to effectively compare and communicate assessment results across different studies. This poses a significant obstacle for biomedical research and multi-center clinical studies that require substantial data support.

[0005] Traditional enzyme-labeled immunohistochemical staining methods use enzyme-labeled antibodies, which catalyze the color development of the substrate to reveal the antigen. However, the catalytic efficiency of the enzyme and the color development of the substrate have certain limitations, resulting in relatively low detection sensitivity. For some antigens with low expression levels, their presence may not be accurately detected, thus missing some important biological information.

[0006] The stability of chromogenic agents is poor: Commonly used chromogenic agents such as DAB are affected by various factors, such as development time, temperature, and pH value. The instability of these agents is relatively poor, easily leading to uneven color development and excessively dark background staining, affecting the accurate judgment of staining intensity. Furthermore, the color of the chromogenic agent gradually fades over time, which is not conducive to long-term preservation and subsequent retrospective analysis.

[0007] During immunohistochemical image acquisition, different microscope equipment and imaging parameters can lead to variations in image quality and fluorescence intensity. The lack of a unified image acquisition standard makes direct comparison and analysis of images acquired by different laboratories difficult. Furthermore, the image acquisition process may be affected by factors such as sample preparation and mounting, further reducing the accuracy and reliability of the images.

[0008] Existing image analysis algorithms suffer from limitations in processing immunohistochemical images, including incomplete removal of complex backgrounds and inaccurate signal segmentation. For example, in some tissue sections, due to complex cell morphology and dense tissue structure, image analysis algorithms may fail to accurately distinguish target antigen signals from surrounding background signals, leading to significant errors in fluorescence intensity measurement. Furthermore, their ability to process weak or overlapping signals is limited, hindering the accurate extraction and analysis of this crucial biological information.

[0009] Currently, there is a lack of a unified quality control system for assessing immunohistochemical staining intensity, with significant differences in operating procedures, reagent selection, and equipment between different laboratories. These differences lead to poor reproducibility and comparability of experimental results, affecting the application value of immunohistochemistry in clinical diagnosis and research. For example, the quality and titer of antibodies used in different laboratories may vary, resulting in inconsistent staining results; the calibration and maintenance of equipment can also affect the detection accuracy of fluorescence signals.

[0010] In multicenter biomedical studies, a high degree of consistency and comparability of experimental results from different research centers is required. However, due to the lack of standardization in existing immunohistochemical assessment methods, assessment results between different centers often vary significantly, making effective integration and analysis difficult. This limits the conduct and depth of multicenter studies and hinders the development of biomedical research. Therefore, a quantitative assessment method for immunohistochemical staining intensity based on fluorescent labeling is proposed. Summary of the Invention

[0011] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling, comprising the following steps: Step 1, Sample preparation: Select biological tissue samples to be tested, fix them in 4% paraformaldehyde solution for 24 hours, dehydrate them with gradient alcohol (70%, 80%, 90%, 95%, 100%) for 1 hour each, clear them with xylene twice for 30 minutes each time, and treat them with paraffin at 60°C three times for 1 hour each time to prepare tissue sections with a thickness of 4-6 μm. Perform routine dewaxing and hydration treatment on the sections, then perform antigen retrieval using citrate buffer pH 6.0, and heat at 95°C for 20 minutes;

[0012] Step 2: Use multispectral imaging or automated fluorescence microscopy to acquire tissue section images containing DAPI cell nuclei and target protein-specific fluorescent labels selected from FITC, Cy3, Cy5, Alexa Fluor 488 / 594 / 647, and Pan-Cadherin / Alexa Fluor 488 labeled cell membrane fluorescence channels. The image resolution is 0.2 μm / pixel to 0.3 μm / pixel.

[0013] Step 3: Perform background correction, Gaussian filtering (kernel size 3×3), median filtering (window size 5×5), and contrast-limited adaptive histogram equalization (CLAHE) on each fluorescence channel image sequentially.

[0014] Step 4, Immunoreaction: The specific antibody is conjugated to the solid-phase carrier at a dilution of 1:200 to form a solid-phase antibody. After incubation at 37°C for 1 hour, unbound antibodies are removed. Then, the test sample is added and incubated at 37°C for 1.5 hours to allow the protein antigen to form an antigen-antibody complex with the solid-phase antibody. The sample is washed three times with PBS buffer for 5 minutes each time. Then, fluorescently labeled antibody (fluorescein isothiocyanate) is added at a dilution of 1:300 and incubated at 37°C for 1 hour to allow it to specifically bind to the antigen and form an antibody-antigen-antibody complex. The sample is then washed three times with PBS buffer for 5 minutes each time.

[0015] Step 5, Fluorescence detection: Use a fluorescence microscope to observe the labeled sample under the conditions of excitation wavelength 490-500nm and emission wavelength 510-520nm to obtain fluorescence images;

[0016] Step 6, Intensity Quantification: The acquired fluorescence images are analyzed using image analysis software. Based on pixel values, the grayscale value range is set to 0-255. The immunohistochemical staining intensity is quantitatively assessed by measuring the average fluorescence intensity value of the positive staining area. At the same time, a negative control sample is set up without primary antibody. The average fluorescence intensity value of the negative control sample is used as the background value. The average fluorescence intensity value of the test sample is subtracted from the background value to obtain the actual staining intensity value.

[0017] As a preferred technical solution of the present invention, in the sample preparation step, the alcohol gradient used for dehydration is 70% alcohol for 1 hour, 80% alcohol for 1 hour, 90% alcohol for 1 hour, 95% alcohol for 1 hour, and 100% alcohol twice, each time for 1 hour, to ensure complete tissue dehydration.

[0018] As a preferred embodiment of the present invention, in the immune reaction step, when the specific antibody is linked to the solid-phase carrier, the incubation temperature is 37°C and the incubation time can be adjusted between 1 and 1.5 hours to ensure that the antibody is fully bound.

[0019] As a preferred technical solution of the present invention, in the immune reaction step, the dilution ratio of the fluorescently labeled antibody can be optimized and adjusted between 1:250 and 1:350 to achieve the best fluorescent labeling effect.

[0020] As a preferred technical solution of the present invention, in the fluorescence detection step, the exposure time of the fluorescence microscope can be adjusted between 50 and 200 milliseconds to obtain a clear and accurate fluorescence image.

[0021] As a preferred technical solution of the present invention, in the intensity quantification step, the image analysis software used is ImageJ software, which accurately distinguishes positive staining areas and background areas by setting a threshold, so as to improve the accuracy of quantitative assessment.

[0022] As a preferred technical solution of the present invention, in the sample preparation step, in addition to using citrate buffer (pH 6.0), antigen retrieval can also be performed using EDTA buffer (pH 8.0) and heated at 98°C for 30 minutes.

[0023] As a preferred embodiment of the present invention, in the immune reaction step, the concentration of the PBS buffer used for washing is 0.01M and the pH value is 7.2-7.4, so as to ensure the washing effect and maintain the activity of antibodies and antigens.

[0024] As a preferred technical solution of the present invention, when conducting multiple sample evaluations, an internal reference sample can be used. This internal reference sample is a standard sample with known staining intensity, which is operated simultaneously with the sample to be tested during each evaluation to correct for errors between different batches of experiments.

[0025] As a preferred technical solution of the present invention, when the fluorescence intensity in the sample is too high and exceeds the measurement range of the image analysis software, the sample can be appropriately diluted. The dilution ratio can be selected between 1:2 and 1:10 depending on the actual situation.

[0026] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention combines fluorescent labeling with image analysis: This method utilizes fluorescein-labeled antibodies that specifically bind to antigens, followed by imaging with a fluorescence microscope, enabling precise localization and detection of target antigens. Advanced image analysis techniques quantify the fluorescence signal, calculating specific parameters such as average fluorescence intensity and integrated fluorescence intensity, providing accurate quantitative data for antigen expression levels. For example, in the detection of tissue samples, traditional subjective assessment methods may only provide a rough staining intensity level, while the method of this invention can accurately determine the specific numerical value of the relative content of the target antigen, such as the relative content of Her-2 antigen accurate to the ng / mL level, providing a more accurate basis for disease diagnosis and research.

[0027] This invention establishes a clear correlation between fluorescence intensity parameters and antigen concentration by plotting a standard curve using positive samples of known concentrations. This standardized procedure allows the antigen content of the sample to be accurately calculated from the standard curve, effectively reducing experimental errors and improving the accuracy and reliability of the evaluation results. Traditional immunohistochemical staining evaluation methods often rely on the subjective judgment of pathologists, and differences in scoring between different doctors may affect the consistency and objectivity of the evaluation results. The method of this invention is based on objective fluorescence signal detection and image analysis algorithms, and operates entirely according to established procedures and standards, free from the interference of subjective factors of the evaluator, providing uniform and objective evaluation results for different samples. In the evaluation of 50 tissue samples, the Kappa coefficient of subjective scores from three pathologists was 0.72, indicating some differences; while the evaluation results of the method of this invention showed high consistency and reproducibility.

[0028] This invention presents immunohistochemical staining intensity using specific numerical parameters, such as mean fluorescence intensity and integrated fluorescence intensity, making the evaluation results more intuitive and clear. These quantified data facilitate comparison and communication between different studies, contributing to the standardization and normalization of biomedical research. This method is applicable to the immunohistochemical staining evaluation of various biological tissue samples and can be widely applied in multiple biomedical research fields such as oncology, neuroscience, and immunology. In tumor research, it helps researchers gain a deeper understanding of the expression levels of tumor markers, providing important evidence for early diagnosis, prognostic assessment, and personalized treatment of tumors. In neuroscience research, it can be used to detect changes in the expression of neurotransmitters, nerve growth factors, and other substances, helping to reveal the pathogenesis of nervous system diseases.

[0029] In clinical diagnosis, the method of this invention can provide doctors with more accurate and detailed diagnostic information, assisting in the formulation of more reasonable treatment plans. For example, in the diagnosis of breast cancer, by accurately detecting the expression levels of key antigens such as Her-2, doctors can determine whether a patient is suitable for targeted therapy, improving the effectiveness and specificity of treatment. This invention uses computer software for image analysis, enabling rapid and efficient processing of large amounts of fluorescence image data. Compared with traditional manual observation and scoring methods, it significantly shortens the evaluation time and improves work efficiency. When processing images of 50 tissue samples, the method of this invention, with the help of image analysis software, can complete the extraction of fluorescence signals and intensity calculation in a short time, while traditional methods may require pathologists to spend a lot of time observing and scoring each sample individually.

[0030] This invention has the potential to achieve high-throughput detection, enabling simultaneous processing and analysis of multiple samples. By optimizing the experimental workflow and image acquisition system, detection efficiency can be further improved to meet the needs of large-scale clinical screening and scientific research experiments. This invention introduces an internal reference sample for calibration, effectively eliminating potential systematic errors between different batches of experiments and ensuring the stability and comparability of experimental results. During multiple sample evaluations, the internal reference sample and the test sample are operated on simultaneously. By comparing the actual fluorescence intensity of the internal reference sample with known values, the experimental results are adjusted and corrected, improving the accuracy and reliability of the evaluation results.

[0031] This invention employs rigorous quality control at every stage of the evaluation process, including sample preparation, immunoassay, fluorescence detection, and image analysis. Real-time monitoring and analysis of experimental data allows for the timely detection and handling of potential errors and anomalies, ensuring the quality of the evaluation results. For example, if the sample fluorescence intensity is too high and exceeds the measurement range, the sample can be diluted and retested to ensure the accuracy of the evaluation results. Attached Figure Description

[0032] Figure 1 Data parameter diagram provided for this invention;

[0033] Figure 2 Data parameter diagram provided for this invention;

[0034] Figure 3 Data parameter diagram provided for this invention;

[0035] Figure 4 The sample preparation and slicing process flowchart provided by this invention;

[0036] Figure 5 A diagram showing the setting of fluorescence image acquisition parameters provided by this invention;

[0037] Figure 6 The flowchart of the immune reaction and staining process provided by this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0039] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0040] Example 1: A method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling, comprising the following steps: Step 1, Sample preparation: Select biological tissue samples to be tested, fix them in 4% paraformaldehyde solution for 24 hours, dehydrate them with gradient alcohol (70%, 80%, 90%, 95%, 100%) for 1 hour each, clear them with xylene twice for 30 minutes each time, and treat them with paraffin at 60°C three times for 1 hour each time to prepare tissue sections with a thickness of 4-6 μm. Perform routine dewaxing and hydration treatment on the sections, then use citrate buffer (pH 6.0) for antigen retrieval and heat them at 95°C for 20 minutes;

[0041] Step 2: Use multispectral imaging or automated fluorescence microscopy to acquire tissue section images containing DAPI cell nuclei and target protein-specific fluorescent labels selected from FITC, Cy3, Cy5, Alexa Fluor 488 / 594 / 647, and Pan-Cadherin / Alexa Fluor 488 labeled cell membrane fluorescence channels. The image resolution is 0.2 μm / pixel to 0.3 μm / pixel.

[0042] Step 3: Perform background correction, Gaussian filtering (kernel size 3×3), median filtering (window size 5×5), and contrast-limited adaptive histogram equalization (CLAHE) on each fluorescence channel image sequentially.

[0043] Step 4, Immunoreaction: The specific antibody is conjugated to the solid-phase carrier at a dilution of 1:200 to form a solid-phase antibody. After incubation at 37°C for 1 hour, unbound antibodies are removed. Then, the test sample is added and incubated at 37°C for 1.5 hours to allow the protein antigen to form an antigen-antibody complex with the solid-phase antibody. The sample is washed three times with PBS buffer for 5 minutes each time. Then, fluorescently labeled antibody (fluorescein isothiocyanate) is added at a dilution of 1:300 and incubated at 37°C for 1 hour to allow it to specifically bind to the antigen and form an antibody-antigen-antibody complex. The sample is then washed three times with PBS buffer for 5 minutes each time.

[0044] Step 5, Fluorescence detection: Use a fluorescence microscope to observe the labeled sample under the conditions of excitation wavelength 490-500nm and emission wavelength 510-520nm to obtain fluorescence images;

[0045] Step 6, Intensity Quantification: The acquired fluorescence images are analyzed using image analysis software. Based on pixel values, the grayscale value range is set to 0-255. The immunohistochemical staining intensity is quantitatively assessed by measuring the average fluorescence intensity value of the positive staining area. At the same time, a negative control sample is set up without primary antibody. The average fluorescence intensity value of the negative control sample is used as the background value. The average fluorescence intensity value of the test sample is subtracted from the background value to obtain the actual staining intensity value.

[0046] In the sample preparation steps, the alcohol gradient used for dehydration is 70% alcohol for 1 hour, 80% alcohol for 1 hour, 90% alcohol for 1 hour, 95% alcohol for 1 hour, and 100% alcohol twice, each time for 1 hour, to ensure complete tissue dehydration.

[0047] During the immune response, when the specific antibody is linked to the solid-phase carrier, the incubation temperature is 37°C, and the incubation time can be adjusted between 1 and 1.5 hours to ensure that the antibody binds fully.

[0048] During the immunization process, the dilution ratio of the fluorescently labeled antibody can be optimized between 1:250 and 1:350 to achieve the best fluorescent labeling effect.

[0049] In the fluorescence detection step, the exposure time of the fluorescence microscope can be adjusted between 50 and 200 milliseconds to obtain clear and accurate fluorescence images.

[0050] In the intensity quantification step, ImageJ software was used for image analysis. By setting thresholds, positive staining areas and background areas were accurately distinguished to improve the accuracy of quantitative assessment.

[0051] In the sample preparation steps, in addition to using citrate buffer (pH 6.0), EDTA buffer (pH 8.0) can also be used for antigen retrieval, and antigen retrieval can be performed by heating at 98°C for 30 minutes.

[0052] In the immunization reaction step, the concentration of PBS buffer used for washing is 0.01M, and the pH value is 7.2-7.4, in order to ensure the washing effect and maintain the activity of antibodies and antigens.

[0053] When conducting multiple sample evaluations, an internal reference sample can be used. This internal reference sample is a standard sample with known staining intensity. It is operated on simultaneously with the sample to be tested during each evaluation to correct for errors between different batches of experiments.

[0054] When the fluorescence intensity in the sample is too high and exceeds the measurement range of the image analysis software, the sample can be appropriately diluted. The dilution ratio can be selected between 1:2 and 1:10 depending on the actual situation.

[0055] The working principle of the fluorescently labeled immunohistochemical staining intensity quantitative assessment method: The core principle of the fluorescently labeled immunohistochemical staining intensity quantitative assessment method proposed in this invention is to utilize the specific binding reaction between antigen and antibody, and with the help of the characteristics of fluorescent labels, combined with advanced image analysis technology, to achieve accurate and objective quantitative assessment of immunohistochemical staining intensity. The working principle of each step of this method is explained in detail below.

[0056] After selecting the biological tissue samples to be tested, they were first fixed in a 4% paraformaldehyde solution for 24 hours. Paraformaldehyde can cross-link tissue proteins, thereby maintaining the tissue's morphology and structure, preventing autolysis and putrefaction, and providing a stable sample basis for subsequent processing. Subsequently, a gradient alcohol dehydration process was performed, with the alcohol concentration gradually increasing from 70% to 100%. This is because different concentrations of alcohol have different dehydrating effects on tissues; gradient dehydration allows the tissue to gradually lose water while avoiding shrinkage and deformation due to excessively rapid dehydration. After xylene clearing treatment, xylene replaces the alcohol in the tissue, making it transparent and facilitating paraffin infiltration. Paraffin impregnation completely saturates the tissue with paraffin to create tissue blocks with a certain degree of hardness, allowing for the cutting of uniformly thick tissue sections (4-6 μm).

[0057] Antigen retrieval: After dewaxing and hydration, antigen retrieval is necessary. During tissue fixation, antigenic epitopes may be masked, making it difficult for antibodies to bind. Antigen retrieval is performed by heating the sample at 95°C for 20 minutes in citrate buffer (pH 6.0). The high temperature and specific buffer environment re-expose the antigenic epitopes, improving the efficiency of antigen-antibody binding.

[0058] Solid-phase antibody formation: Specific antibodies are conjugated to a solid-phase carrier at a dilution ratio of 1:200 to form solid-phase antibodies. This process is based on the chemical properties of the antibody, using a specific chemical conjugation method to ensure the antibody adheres firmly to the solid-phase carrier. Incubation at 37°C for 1 hour promotes the binding reaction between the antibody and the solid-phase carrier; 37°C is close to the physiological temperature of the human body and is the optimal temperature for most biochemical reactions.

[0059] Antigen-antibody complex formation: After the test sample is added, the protein antigens in the sample and the solid-phase antibody are incubated at 37°C for 1.5 hours to form antigen-antibody complexes. This is based on the principle of specific binding between antigens and antibodies; the antibody's antigen-binding site can recognize and precisely bind to specific epitopes of the antigen, forming a stable complex. Washing with PBS buffer can remove unbound sample components and reduce interference from non-specific binding.

[0060] Fluorescein-labeled antibody binding: After adding fluorescently labeled antibody (fluorescein isothiocyanate, diluted 1:300), incubate at 37°C for 1 hour. The fluorescein-labeled antibody binds to the antigen in the antigen-antibody complex, forming an antibody-antigen-antibody complex. Fluorescein isothiocyanate emits fluorescence under specific excitation light, and the intensity of the fluorescence can indirectly reflect the antigen content.

[0061] Fluorescence excitation and emission are observed using a fluorescence microscope at excitation wavelengths of 490-500 nm and emission wavelengths of 510-520 nm. Fluorescein isothiocyanate absorbs energy at a specific excitation wavelength, transitioning from the ground state to an excited state. When it returns to the ground state, it emits fluorescence at a specific wavelength. The optical system of the fluorescence microscope provides suitable excitation light and collects and detects the emitted light, allowing us to observe the fluorescence signal in the sample.

[0062] Fluorescence microscopy converts fluorescence signals in a sample into images, which provide a clear view of the distribution of antigens within the sample. Acquiring images from multiple different fields of view allows for a more comprehensive reflection of the overall sample condition, avoiding errors caused by localized differences.

[0063] Preprocessing operations such as denoising, enhancement, and registration are performed on the acquired fluorescence images. Denoising (e.g., median filtering) removes noise interference from the image, which may be caused by the microscope's optical system, environmental factors, or impurities in the sample itself. Image enhancement (e.g., histogram equalization) improves image contrast and brightness, making the fluorescence signal clearer and facilitating subsequent analysis. Image registration for different channels ensures accurate spatial correspondence of different fluorescent labeling signals, improving the accuracy of analysis. Based on the DAPI-stained cell nucleus images, the Otsu algorithm is used to determine the cell nucleus region, which serves as a reference range for the tissue region. This is because the cell nucleus has distinct characteristics in the sample; accurate segmentation of the cell nucleus region can define the approximate extent of the tissue. Within the tissue region, an adaptive threshold segmentation method is used to segment the fluorescently labeled target antigen signal image, extracting the fluorescence signal region. Adaptive threshold segmentation automatically adjusts the threshold according to the local features of the image; compared to fixed threshold segmentation, it better adapts to image inhomogeneities and improves the accuracy of signal extraction.

[0064] Intensity Calculation and Quantitative Assessment: Parameters such as the average fluorescence intensity and integrated fluorescence intensity of the extracted fluorescence signal region are calculated. The average fluorescence intensity reflects the average brightness per pixel within the fluorescence signal region, while the integrated fluorescence intensity considers both the area and average brightness of the fluorescence signal region. When establishing a standard curve, positive samples of known concentrations are processed in the same way to obtain fluorescence intensity parameters for samples of different concentrations. Since there is a linear relationship between antigen concentration and fluorescence intensity, a standard curve is plotted with sample concentration on the x-axis and the corresponding fluorescence intensity parameters on the y-axis. Substituting the fluorescence intensity parameters of the sample to be tested into the standard curve allows for the determination of the relative content of the target antigen in the sample, thus enabling a quantitative assessment of the immunohistochemical staining intensity.

[0065] The working process of a method for quantitatively assessing the intensity of immunohistochemical staining based on fluorescent labels. Sample preparation stage: Select the biological tissue sample to be tested and quickly fix it in 4% paraformaldehyde solution for 24 hours. Paraformaldehyde can cross-link tissue proteins, maintain the morphology and structure of tissue cells, prevent tissue autolysis and putrefaction, and ensure the integrity and antigenicity of the tissue during subsequent processing.

[0066] The fixed tissue was sequentially immersed in alcohol of different concentrations for gradient dehydration: 70% alcohol for 1 hour, 80% alcohol for 1 hour, 90% alcohol for 1 hour, 95% alcohol for 1 hour, and 100% alcohol twice, each time for 1 hour. Gradient dehydration allows the tissue to gradually lose water, preventing shrinkage and deformation due to excessively rapid dehydration, and preparing it for subsequent clearing and paraffin impregnation steps.

[0067] After dehydration, the tissue was cleared in xylene twice, 30 minutes each time. Xylene replaces the alcohol in the tissue, making it transparent and facilitating paraffin infiltration. The tissue was then immersed in paraffin at 60°C three times, one hour each time, to ensure complete paraffin penetration and create tissue blocks of suitable hardness. The paraffin-impregnated tissue blocks were then sectioned into 4-6 μm thick sections using a microtome and placed on glass slides in preparation for subsequent staining and analysis.

[0068] The slides containing the tissue sections were placed in xylene for dewaxing, and then sequentially hydrated with a gradient of alcohols (100%, 95%, 90%, 80%, 70%) to transition the tissue sections from a paraffin environment to an aqueous environment, which facilitates subsequent antigen retrieval and antibody binding.

[0069] The slide was placed in citrate buffer (pH 6.0) and heated at 95°C for 20 minutes to perform antigen retrieval. During tissue fixation, antigenic epitopes may be masked; antigen retrieval can re-expose the antigenic epitopes, enhancing the binding ability of antigens and antibodies.

[0070] Immunoreaction Phase: Specific antibodies are conjugated to a solid-phase carrier at a 1:200 dilution to form a solid-phase antibody. Incubation at 37°C for 1 hour promotes antibody binding to the solid-phase carrier. Unbound antibodies are then removed to reduce interference from non-specific binding. The test sample is added to the solid-phase antibody and incubated at 37°C for 1.5 hours to allow the protein antigens in the sample to fully bind with the solid-phase antibody, forming an antigen-antibody complex. The sample is washed three times with 0.01M PBS buffer (pH 7.2-7.4) for 5 minutes each time to remove unbound sample components. Fluorescently labeled antibody (fluorescein isothiocyanate, diluted 1:300) is added and incubated at 37°C for 1 hour to allow the fluorescein-labeled antibody to bind to the antigen in the antigen-antibody complex, forming an antibody-antigen-antibody complex. The sample is washed three more times with PBS buffer for 5 minutes each time to remove unbound fluorescein-labeled antibody.

[0071] Fluorescence detection stage: Using a fluorescence microscope, set the excitation wavelength to 490-500 nm and the emission wavelength to 510-520 nm to suit the fluorescence characteristics of fluorescein isothiocyanate. Adjust the microscope exposure time appropriately, within the range of 50-200 ms, depending on the sample conditions to obtain clear fluorescence images. Acquire images of the fluorescently labeled samples, taking at least five images from different fields of view for each sample to ensure a comprehensive reflection of the staining condition. The acquired image resolution should not be lower than the set pixel value to guarantee image clarity and the accuracy of subsequent analysis.

[0072] Image analysis and intensity quantification stage: The acquired raw images undergo denoising processing. Median filtering can be used to remove noise interference and smooth the image. Histogram equalization and other image enhancement operations are performed to improve image contrast and brightness, highlighting the fluorescence signal. Image registration is performed on different channels, including the fluorescently labeled target antigen channel and the DAPI channel, to ensure accurate spatial correspondence between the target antigen signal and the cell nuclear signal, facilitating subsequent signal extraction and analysis.

[0073] Based on DAPI-stained cell nuclear images, the Otsu algorithm was used to determine the nuclear region, which was then used as a reference range for the tissue region. Within the tissue region, an adaptive thresholding method was used to segment the fluorescently labeled target antigen signal image and extract the fluorescent signal region. Adaptive thresholding can automatically adjust the threshold according to the local features of the image, better adapting to image inhomogeneity and improving the accuracy of signal extraction.

[0074] The average fluorescence intensity and integrated fluorescence intensity of the extracted fluorescence signal region are calculated. The formula for calculating the average fluorescence intensity is: Average fluorescence intensity = Total gray value of the fluorescence signal region / Number of pixels in the fluorescence signal region; the formula for calculating the integrated fluorescence intensity is: Integrated fluorescence intensity = Average fluorescence intensity × Area of ​​the fluorescence signal region.

[0075] Positive samples of known concentrations were subjected to the same treatment to obtain fluorescence intensity parameters for samples of different concentrations. A standard curve was plotted with sample concentration on the x-axis and the corresponding fluorescence intensity parameters on the y-axis. The fluorescence intensity parameters of the samples to be tested were substituted into the standard curve to obtain the relative content of the target antigen in the samples, thereby achieving a quantitative assessment of the immunohistochemical staining intensity.

[0076] When conducting multiple sample evaluations, an internal reference sample (a standard sample with known staining intensity) is introduced. During each evaluation, the internal reference sample and the sample to be tested are operated on simultaneously. By comparing the actual fluorescence intensity of the internal reference sample with the known value, possible errors between different batches of experiments are corrected, thereby improving the accuracy and reliability of the evaluation results.

[0077] When the fluorescence intensity in a sample is too high and exceeds the measurement range of the image analysis software, the sample should be appropriately diluted. The dilution ratio should be selected between 1:2 and 1:10, depending on the actual situation. Then, the immunoassay and fluorescence detection steps should be repeated to bring the fluorescence intensity within a measurable range, ensuring the accuracy of the evaluation results.

[0078] Test case

[0079] I. Experimental Objective:

[0080] The accuracy, reliability, and practicality of the quantitative assessment method for immunohistochemical staining intensity based on fluorescent labeling proposed in this invention are verified. The method is compared with traditional subjective assessment methods to evaluate its application value in biomedical research and clinical diagnosis.

[0081] II. Experimental Materials and Equipment:

[0082] (I) Experimental Materials:

[0083] Biological tissue samples: Fifty tissue samples were selected from surgically removed breast cancer patients, and were pathologically confirmed. Ten positive tissue samples with known concentrations were also selected for plotting a standard curve.

[0084] Reagents: 4% paraformaldehyde solution, gradient alcohols (70%, 80%, 90%, 95%, 100%), xylene, paraffin, citrate buffer (pH 6.0), specific anti-human Her-2 antibody, FITC-labeled secondary antibody, PBS buffer, DAPI staining solution, blocking solution (PBS buffer containing 10% goat serum).

[0085] Consumables: glass slides, coverslips, pipette tips, centrifuge tubes, etc.

[0086] (II) Test Equipment:

[0087] Microscopes: Fluorescence microscope (with excitation wavelength of 490-500nm and emission wavelength of 510-520nm), confocal microscope.

[0088] Image acquisition system: a high-resolution camera and image acquisition software that are compatible with the microscope.

[0089] Incubator: Used for sample incubation, allowing for precise temperature control.

[0090] Tissue slicer: Used to prepare tissue sections.

[0091] Centrifuge: Used for centrifugal processing of reagents.

[0092] Computer: Equipped with image analysis software (such as ImageJ).

[0093] III. Experimental Methods:

[0094] (I) Sample Preparation

[0095] Fifty tissue samples and ten positive samples with known concentrations were fixed in 4% paraformaldehyde solution for 24 hours.

[0096] After gradient alcohol dehydration (70%, 80%, 90%, 95%, and 100% alcohol for 1 hour each, with 100% alcohol treatment twice), xylene clearing treatment (twice, 30 minutes each time), and paraffin infiltration (60℃, 3 times, 1 hour each time), tissue sections with a thickness of 5 μm were prepared.

[0097] The slides were placed on a glass slide and subjected to routine dewaxing and hydration treatments. Then, antigen retrieval was performed by heating at 95°C for 20 minutes with citrate buffer (pH 6.0).

[0098] (II) Immune response:

[0099] The tissue sections were blocked with blocking solution and incubated at room temperature for 30 minutes.

[0100] Add specific anti-human Her-2 antibody (dilution ratio 1:200) and incubate overnight at 4°C.

[0101] Rinse the slides three times with PBS buffer, five minutes each time.

[0102] Add FITC-labeled secondary antibody (dilution ratio 1:300) and incubate at room temperature in the dark for 1.5 hours.

[0103] Rinse the slides three more times with PBS buffer, 5 minutes each time.

[0104] Add a mounting medium containing DAPI and cover with a coverslip.

[0105] (III) Image Acquisition:

[0106] A fluorescence microscope was used, with the excitation wavelength set to 490-500 nm and the emission wavelength to 510-520 nm. The exposure time was adjusted between 50-200 ms depending on the sample. Five images of each sample were acquired from different fields of view, with a resolution of 1024×1024 pixels.

[0107] (iv) Image analysis and quantitative assessment:

[0108] The acquired image is preprocessed according to the method of the present invention (median filtering for noise reduction, histogram equalization for enhancement, and channel registration).

[0109] Based on the DAPI-stained cell nucleus images, the Otsu algorithm was used to determine the cell nucleus region. Within this region, an adaptive threshold segmentation method was used to segment the FITC channel image and extract the fluorescence signal region.

[0110] Calculate the average fluorescence intensity and integrated fluorescence intensity of the extracted fluorescence signal region.

[0111] A standard curve was plotted with the concentration of a known positive sample as the x-axis and the corresponding fluorescence intensity parameter as the y-axis.

[0112] The fluorescence intensity parameters of 50 test samples were substituted into the standard curve to obtain the relative content of Her-2 in the test samples.

[0113] (V) Comparison of Traditional Subjective Evaluation Methods:

[0114] Three experienced pathologists were invited to evaluate the immunohistochemical staining intensity of 50 samples using a traditional subjective scoring method (0-3 points, 0 points: no staining; 1 point: weak staining; 2 points: moderate staining; 3 points: strong staining).

[0115] IV. Test Results

[0116] (I) Results of Standard Curve Plotting:

[0117] A standard curve was plotted based on the fluorescence intensity parameters of 10 positive samples with known concentrations. The results showed a good linear relationship between sample concentration and fluorescence intensity parameters (R0). 2 =0.95), indicating that the method of the present invention can accurately reflect the correspondence between antigen concentration and fluorescence intensity within a certain range.

[0118] (II) Quantitative evaluation results of the method of the present invention:

[0119] Quantitative assessments were performed on 50 tissue samples to obtain the relative content of Her-2 in each sample. Significant differences in fluorescence intensity parameters were observed among the different samples, reflecting variations in Her-2 expression levels.

[0120] (III) Results of Traditional Subjective Evaluation Methods:

[0121] There were some differences in the subjective ratings of the 50 samples by the three pathologists. The consistency of the ratings was assessed by the Kappa coefficient, which was 0.72, indicating that the consistency of the ratings among the doctors was moderate.

[0122] (iv) Correlation analysis of the two methods:

[0123] A correlation analysis was performed between the quantitative results obtained by the method of this invention and the results of traditional subjective rating. The results showed a significant positive correlation between the two (r = 0.85, P < 0.01), indicating that the method of this invention and the traditional subjective assessment method have a certain degree of consistency. However, the method of this invention can provide more accurate quantitative data, while the traditional subjective rating method can only provide relatively vague ratings.

[0124] V. Experimental Conclusions:

[0125] (a) Accuracy and Reliability:

[0126] The immunohistochemical staining intensity quantitative assessment method based on fluorescence labeling of this invention can accurately convert fluorescence intensity parameters into the relative content of target antigens in the sample by establishing a standard curve. The good linearity of the standard curve proves the accuracy of the method. The stability of the results of repeated experiments indicates that the method has high reliability.

[0127] (II) Objectivity and Repeatability:

[0128] Compared with traditional subjective evaluation methods, the method of this invention avoids the interference of human factors and evaluates based on quantitative fluorescence intensity parameters, resulting in more objective and reproducible results. Different experimenters using this method show a high degree of consistency in their results.

[0129] (III) Application Value:

[0130] The method of this invention has significant application value in biomedical research and clinical diagnosis. In research, it provides researchers with accurate quantitative data on antigen expression, contributing to a deeper understanding of the mechanisms underlying disease development. In clinical diagnosis, it assists doctors in more accurately predicting disease prognosis and developing treatment plans, improving diagnostic accuracy and treatment effectiveness.

[0131] In summary, the immunohistochemical staining intensity quantitative assessment method based on fluorescent labeling of the present invention is an accurate, reliable, and objective assessment method with broad application prospects.

[0132] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. A method for quantitatively assessing the intensity of immunohistochemical staining based on fluorescent labeling, characterized in that, Includes the following steps: Step 1, Sample Preparation: Select biological tissue samples to be tested, fix them in 4% paraformaldehyde solution for 24 hours, dehydrate them with gradient alcohol (70%, 80%, 90%, 95%, 100%) for 1 hour each, clear them with xylene twice for 30 minutes each time, and immerse them in paraffin at 60℃ three times for 1 hour each time to prepare tissue sections with a thickness of 4-6 μm. Perform routine dewaxing and hydration treatment on the sections, and then perform antigen retrieval using citrate buffer (pH 6.0) and heat at 95℃ for 20 minutes. Step 2: Use multispectral imaging or automated fluorescence microscopy to acquire tissue section images containing DAPI cell nuclei and target protein-specific fluorescent labels selected from FITC, Cy3, Cy5, Alexa Fluor 488 / 594 / 647, and Pan-Cadherin / Alexa Fluor488 labeled cell membrane fluorescence channels, with an image resolution of 0.2 μm / pixel to 0.3 μm / pixel; Step 3: Perform background correction, Gaussian filtering (kernel size 3×3), median filtering (window size 5×5), and contrast-limited adaptive histogram equalization (CLAHE) on each fluorescence channel image sequentially. Step 4, Immunoreaction: The specific antibody is conjugated to the solid-phase carrier at a dilution of 1:200 to form a solid-phase antibody. After incubation at 37°C for 1 hour, unbound antibodies are removed. Then, the test sample is added and incubated at 37°C for 1.5 hours to allow the protein antigen to form an antigen-antibody complex with the solid-phase antibody. The sample is washed three times with PBS buffer for 5 minutes each time. Then, fluorescently labeled antibody (fluorescein isothiocyanate) is added at a dilution of 1:300 and incubated at 37°C for 1 hour to allow it to specifically bind to the antigen and form an antibody-antigen-antibody complex. The sample is then washed three times with PBS buffer for 5 minutes each time. Step 5, Fluorescence detection: Use a fluorescence microscope to observe the labeled sample under the conditions of excitation wavelength 490-500nm and emission wavelength 510-520nm to obtain fluorescence images; Step 6, Intensity Quantification: The acquired fluorescence images are analyzed using image analysis software. Based on pixel values, the grayscale value range is set to 0-255. The immunohistochemical staining intensity is quantitatively assessed by measuring the average fluorescence intensity value of the positive staining area. At the same time, a negative control sample is set up without primary antibody. The average fluorescence intensity value of the negative control sample is used as the background value. The average fluorescence intensity value of the test sample is subtracted from the background value to obtain the actual staining intensity value.

2. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the sample preparation steps, the alcohol gradient used for dehydration is 70% alcohol for 1 hour, 80% alcohol for 1 hour, 90% alcohol for 1 hour, 95% alcohol for 1 hour, and 100% alcohol twice, each time for 1 hour, so that the tissue is completely dehydrated.

3. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the immune reaction step, when the specific antibody is linked to the solid-phase carrier, the incubation temperature is 37°C and the incubation time is adjusted between 1 and 1.5 hours.

4. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the immune reaction step, the dilution ratio of the fluorescently labeled antibody can be optimized and adjusted between 1:250 and 1:

350.

5. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the fluorescence detection step, the exposure time of the fluorescence microscope can be adjusted between 50 and 200 milliseconds.

6. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the intensity quantification step, ImageJ software is used for image analysis, and a threshold is set to accurately distinguish between positive staining areas and background areas.

7. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the sample preparation steps, in addition to using citrate buffer (pH 6.0), antigen retrieval can also be performed using EDTA buffer (pH 8.0), with heating at 98°C for 30 minutes.

8. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, In the immune reaction step, the concentration of the PBS buffer used for washing is 0.01M, and the pH value is 7.2-7.

4.

9. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, When conducting multiple sample evaluations, an internal reference sample is used. The internal reference sample is a standard sample with known staining intensity. It is operated on simultaneously with the sample to be tested during each evaluation and is then calibrated.

10. The method for quantitative assessment of immunohistochemical staining intensity based on fluorescent labeling according to claim 1, characterized in that, If the fluorescence intensity in the sample is too high and exceeds the measurement range of the image analysis software, the sample should be diluted. The dilution ratio should be between 1:2 and 1:10.

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