A calibration method for detecting tissue target protein expression results

Abstract

The present invention discloses a calibration method for detecting the expression result of a target protein in a tissue. The calibration method performs immunohistochemical staining of the same batch on a standard cell chip and a tissue slice to be tested to obtain a stained standard cell mass and a stained tissue slice, and then obtains the adaptation parameters of each stained standard cell mass and stained tissue slice by immunohistochemical analysis software, and then uses the algorithm of the present invention to obtain the relative score of each stained standard cell mass and stained tissue slice, and calibrates the expression result of the target protein in the stained tissue slice based on the relative score of each stained standard cell mass. The calibration method of the present invention can enable the same tissue to be tested to obtain accurate pathological scores on different detection platforms.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a calibration method for detecting expression results of a tissue target protein. Background Art

[0002] Immunohistochemistry (IHC) is an antibody-based technique used to detect protein expression in tissues where structure and organization have been preserved. IHC is often used in clinical settings to detect various pathogenic features in tissue samples, such as neoplasia, metastasis, infection, and inflammation, for diagnostic purposes and medication basis. For example, the prior art uses IHC technology to detect the protein expression and gene amplification status of human epidermal growth factor receptor-2 (HER2).

[0003] However, due to the differences in antibody clone numbers, antibody dilutions, antibodies themselves, color development times, and operating procedures used in different detection platforms during the immunohistochemical detection process, there are large differences in the results of immunohistochemical detection, which leads to differences in the interpretation of immunohistochemical results. In particular, it is difficult to control weak and medium-intensity coloring during the interpretation process, which in turn leads to inconsistent pathological scores for the same sample tissue. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides the following technical solutions:

[0005] Compared with the prior art, the present invention has the following beneficial effects:

[0006] 1) By setting up a standard control group in the same section or the same batch of stained sections, the pathological scoring deviation caused by different immunohistochemical conditions was avoided;

[0007] 2) The sections were digitized and identified and scored using immunohistochemistry software, avoiding variability in manual interpretation;

[0008] 3) The data obtained by the immunohistochemical analysis software is processed using the algorithm of the present invention, thereby avoiding the impact of the same target protein on different detection platforms due to equipment differences. The accurate pathological score of the tissue to be tested can be obtained by processing the adapted data with the algorithm of the present invention, thereby achieving the effect of calibrating the target protein expression results. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 It is a schematic diagram of the flow chart of the calibration method of the present invention;

[0010] Figure 2 A schematic diagram of a process for determining a standard cell mass in the calibration method of the present invention;

[0011] Figure 3 The HER2 standard cell cluster in Example 3;

[0012] Figure 4 The data processing interface of the immunohistochemical analysis platform in Example 3;

[0013] Figure 5 This is the result diagram of the HER2 tissue piece to be tested in Example 4 on the 1# detection platform;

[0014] Figure 6 The results of the 1# and 2# detection platforms in Example 4 and the corrected relative scores are shown. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Embodiment 1:

[0017] The present invention provides a calibration method for detecting the expression results of a target protein in a tissue, such as Figure 1 As shown, the following steps are included:

[0018] S1: preparing a standard cell chip expressing a target protein; obtaining tissues for which target protein expression results need to be detected, and processing these tissues to prepare tissue slices to be tested, wherein the standard cell chip has a plurality of standard cell clusters, and the expression levels of the target protein in the plurality of standard cell clusters correspond to the pathological scores of the target protein-related diseases one by one;

[0019] S2: The tissue slice to be tested and the standard cell chip are subjected to immunohistochemical staining from the same batch to obtain a stained tissue slice and a calibration cell chip respectively; at this time, the standard cell cluster in the calibration cell chip is referred to as a stained standard cell cluster;

[0020] Through the above processing, the staining intensity of the stained standard cell cluster can be used to calibrate the staining result of the tissue slice to be tested;

[0021] S3: Acquire the adaptation parameters of the stained tissue slice and each stained standard cell cluster in step S2, wherein the adaptation parameters include the ROI region cell count (region of interest, ROI region for short) of each stained standard cell cluster and stained tissue slice, DAB staining area and DAB staining average intensity;

[0022] S4: performing algorithm processing on the adaptation parameters described in step S3 to obtain relative scores of each standard cell cluster and the tissue slice to be tested, and calibrating the expression results of the target protein in the tissue slice to be tested based on the relative scores of each standard cell cluster;

[0023] The algorithm processing includes calculating the relative score of each standard cell cluster, the relative score of the tissue slice to be tested, and the calibration score interval of each standard cell cluster:

[0024] The relative score of the stained standard cell clusters was obtained by the following formula:

[0025]

[0026] Among them, C n Represents the relative score of the stained standard cell cluster with pathological score n; I n A represents the average intensity of the target protein in the stained standard cell cluster with a pathological score of n, that is, the average DAB staining intensity of the stained standard cell cluster with a pathological score of n in step S3; n represents the staining area of ​​the target protein in the stained standard cell cluster with a pathological score of n, that is, the DAB staining area of ​​the stained standard cell cluster with a pathological score of n in step S3; N n The number of cells in the stained standard cell cluster with a pathological score of n, that is, the cell count of the ROI area of ​​the stained standard cell cluster with a pathological score of n in step S3; n is a natural number starting from 0, and nmax refers to the maximum value of the pathological score. Generally speaking, the larger the pathological score value, the higher the expression level of the target protein and the darker the staining.

[0027] The relative score of the tissue slice to be tested is obtained by the following formula:

[0028]

[0029] Among them, C s Refers to the relative score of the tissue slice to be examined; I s represents the average intensity of the target protein on the tissue slice to be tested, that is, the average intensity of DAB staining of the stained tissue slice in step S3; A s represents the staining area of ​​the target protein in the tissue slice to be tested, i.e., the DAB staining area of ​​the stained tissue slice in step S3; s Represents the number of cells in the tissue slice to be tested, that is, the cell count of the ROI area of ​​the stained tissue slice in step S3;

[0030] The calibration score interval for each standard cell cluster is:

[0031] When n=0,

[0032]

[0033] When 0<n<nmax,

[0034]

[0035] When n = nmax,

[0036]

[0037] in, Represents the calibration score interval of the standard cell cluster with a pathological score of n; n is a natural number starting from 0, and nmax refers to the maximum value of the pathological score;

[0038] When the relative score C of the tissue to be examined s The calibration score interval belonging to a certain stained standard cell group When , the pathological score of the tissue slice to be tested is the same as the pathological score of the stained standard cell cluster, thereby completing the calibration of the target protein expression result in the tissue slice to be tested.

[0039] Furthermore, the preparation of the standard cell chip in step S1 includes the determination of a standard cell cluster, such as Figure 2 As shown, the determination of the standard cell cluster includes the following steps:

[0040] S11: screening multiple cell lines with different expression levels of the target protein according to the pathological score level of the target protein-related disease, and amplifying these cell lines to obtain multiple amplified cell clusters; preferably, the number of cells in each amplified cell cluster ranges from 100 million to 500 million;

[0041] S12: preparing a plurality of expanded cell clusters into an expanded cell cluster wax block, and then slicing and immunohistochemically staining the expanded cell cluster chip wax block to obtain a staining result of the expanded cell cluster;

[0042] S13: Interpret the staining results of each amplified cell cluster and determine the standard cell cluster based on the staining interpretation results of the amplified cell cluster.

[0043] Furthermore, the interpretation of the staining results of the amplified cell clusters refers to submitting the staining results of each amplified cell cluster in step S12 to multiple pathologists for pathological scoring interpretation according to the clinically relevant consensus or clinically relevant guidelines of the corresponding detection protein (target). If the interpretation result is situation (1), the pathologists have inconsistent staining interpretation results for each amplified cell cluster, and steps S11-S12 are repeated; if the staining interpretation result is situation (2), that is, the pathologists have completely consistent staining interpretation results for each amplified cell cluster, and the interpreted pathological scores are in one-to-one correspondence with each amplified cell cluster in S12, then the amplified cell cluster obtained by culturing the cell line is determined to be a standard cell cluster.

[0044] In addition, according to the requirements of the clinical pathology guidelines, for the target protein that needs to be gene amplified, in order to further ensure the accuracy of the interpretation result, after obtaining the interpretation result consistent with the immunohistochemical interpretation, there is also step S14: that is, after the multiple amplified cell group wax blocks in step S12 are sliced ​​again, the gene that can express the target protein is tested. In this embodiment, a DNA FISH in situ hybridization detection kit is used for detection, and the test results are fluorescently interpreted by a professional pathologist. If the fluorescence interpretation result is: there is no genetic mutation of the corresponding gene in the amplified cell group with the highest pathological score (the genetic mutation includes one or more of gene amplification and gene fusion), then steps S11-S13 are repeated; if the fluorescence interpretation result is: there is a genetic mutation of the corresponding gene in the amplified cell group with the highest pathological score, then the genetic mutation cell group obtained by culturing the cell line is determined to be a standard cell group. All standard cell group paraffin blocks corresponding to the pathological scores are combined into cell chip wax blocks to prepare standard cell chip paraffin blocks, and sliced ​​to obtain standard cell chips.

[0045] Step S2 requires that the tissue slice to be tested and the standard cell cluster chip be processed synchronously under the same conditions, thereby avoiding the difference in staining effect caused by differences in staining platform, antibody, and human operation, which specifically includes the following contents:

[0046] Place the standard cell chip in S1 and the tissue slice to be tested in this round of experiments on a slide (they can be placed on the same slide or on different slides at this time) and then conduct subsequent experiments in the same batch, and then perform immunohistochemical staining on the standard cell chip and the tissue slice to be tested in the same batch. By staining the standard cell chip and the tissue slice to be tested under the same immunohistochemical detection conditions, the staining results of each calibration cell group can be used as the calibration basis for the tissue slice to be tested.

[0047] In step S3 of this embodiment, the acquisition of the adaptation parameters specifically includes the following steps:

[0048] S31: Scanning the calibration cell chip and the stained tissue slice (the calibration cell chip and the stained tissue slice can be on the same glass slide or on different glass slides) to obtain a calibration cell chip scan image and a stained tissue slice scan image; importing the scanned calibration cell chip scan image and the stained tissue slice scan image into immunohistochemical analysis software;

[0049] S32: Using immunohistochemical analysis software, ROI area selection and quantitative identification are performed on each stained standard cell cluster area and the stained tissue slice scan image in the calibration cell chip scan image; wherein ROI area selection refers to identifying all areas or multi-point ROI areas on each stained standard cell cluster and stained tissue slice; and quantitatively identifying the cell nucleus on each stained standard cell cluster and stained tissue slice in these ROI areas, the area occupied by the target protein identified as positive (i.e., positive protein), and the expression intensity of the target protein to obtain quantitative identification data; wherein the expression intensity of the target protein is represented by the DAB value, and the grayscale value of the DAB staining intensity ranges from 0 to 255, indicating that the brown staining is from the weakest to the strongest; the positive protein refers to the target protein with a DAB value higher than a certain value;

[0050] S33: Use immunohistochemistry software to quantitatively analyze the data obtained by quantitative identification, so as to obtain the cell count, DAB staining area and DAB staining average intensity of each stained standard cell cluster and stained tissue slice ROI area respectively; wherein, the DAB staining area refers to the area occupied by the positive protein in the ROI region of each cell cluster.

[0051] Furthermore, when the ROI region selection is a multi-point ROI region selection, that is, multiple ROI regions with the same area are selected for each standard cell cluster and stained tissue slice for quantitative identification and then quantitative analysis, in this case, formula (1) refers to the average value of each adaptation parameter obtained by the multi-point ROI region of each stained standard cell cluster; formula (2) I s , A s 、N s It refers to the average value of each adaptation parameter obtained in the multi-point ROI area of ​​the stained tissue slice.

[0052] Embodiment 2:

[0053] This embodiment is different from embodiment 1 in that the target protein is ALK protein. This protein has four pathological scoring standards of 0, 1+, 2+, and 3+, corresponding to four standard cell clusters. The calibration process of the ALK protein expression results using the technical solution of the present invention is as follows:

[0054] S1: preparing a standard cell chip expressing ALK protein, wherein the standard cell chip has four standard cell clusters, and the expression levels of the four standard cell clusters for ALK protein are different and correspond to four pathological scores of 0, 1+, 2+, and 3+;

[0055] S2: Take the patient's tumor tissue, make the tumor tissue into a tissue slice to be tested, then perform subsequent processing on the tissue slice to be tested and the standard cell chip in the same batch, and finally perform immunohistochemical staining to obtain a stained tissue slice and a calibration cell chip. At this time, the standard cell cluster constituting the calibration cell chip is the stained standard cell cluster;

[0056] S3: Scanning the stained tissue slice and the calibration cell chip to obtain the corresponding scanned images, and then processing the scanned images using immunohistochemistry software to obtain the adaptation parameters of the stained tissue slice and each stained standard cell cluster;

[0057] S4: Substitute the above adaptation parameters into formulas (1) and (2) to obtain the relative scores C0, C1, C2, and C3 of the four stained standard cell clusters and the relative score C of the stained tissue slide. s Substitute the relative scores of the four stained standard cell clusters into formulas (3-1)-(3-3) to obtain the calibration score intervals of the four stained standard cell clusters: Then the relative score of ALK protein staining tissue slices was calculated. s Comparisons were made with four calibration score intervals, assuming that the staining

[0058] Relative score of tissue slices C s belong If the expression of ALK in the tissue slice is within the range of , then the pathological score of the ALK stained tissue slice is the same as the standard cell cluster score with a pathological score of 2+, that is, the pathological score of the ALK stained tissue slice is 2+, thereby completing the calibration of the target protein expression results in the tissue slice to be tested.

[0059] Embodiment 3:

[0060] The difference between this embodiment and embodiment 1 or 2 is that in this embodiment, the HER2 receptor protein is taken as an example to explain the technical solution of the present invention in more detail.

[0061] At present, the interpretation results of HER2 immunohistochemistry in my country are based on the Chinese Breast Cancer HER2 Detection Guidelines (2019 Edition) jointly issued by Chinese pathology experts and clinical experts. The interpretation standard of HER2 results is: a pathological score of 0 indicates no coloring or ≤10% of the invasive cancer cells present incomplete and weak cell membrane staining; a pathological score of 1+ indicates that >10% of the invasive cancer cells present incomplete and weak cell membrane staining; a pathological score of 2+ has two situations, the first is that >10% of the invasive cancer cells present weak-medium intensity complete cell membrane staining; the second is that ≤10% of the invasive cancer cells present strong and complete cell membrane staining; a pathological score of 3+ indicates that >10% of the invasive cancer cells present strong, complete and uniform cell membrane staining. Since the pathological score of HER2 has four levels, when the calibration method of the present invention is used to calibrate the expression results of the HER2 receptor protein in the test tissue, the expression level of the HER2 receptor protein in the standard cell group must correspond to the above-mentioned pathological score level.

[0062] Since the pathological scoring of HER2 has four levels, when the calibration method of the present invention is used to calibrate the expression results of HER2 receptor protein, the expression level of HER2 receptor protein by the standard cell group must correspond to the above pathological scoring levels one by one.

[0063] The calibration method for detecting tissue HER2 receptor protein expression in the present invention is as follows:

[0064] S1: preparing a standard cell chip expressing HER2 receptor protein, wherein the HER2 standard cell chip has a plurality of standard cell clusters that can express HER2 receptor protein, and the expression levels of HER2 receptor protein in the plurality of standard cell clusters correspond to the pathological scores of HER2-related diseases one by one, wherein the determination of the standard cell clusters comprises the following steps:

[0065] S11: According to the pathological scoring grades of HER2 receptor protein-related diseases, human tumor cell lines with four different expression levels of HER2 receptor protein are prepared for amplification culture to obtain four amplified cell clusters; and the number of cells in each amplified cell cluster ranges from 100 million to 500 million;

[0066] S12: The four expanded cell clusters are made into expanded cell cluster wax blocks, and then the expanded cell cluster wax blocks are subjected to paraffin sectioning, pasting and baking, section dewaxing and hydration sectioning to obtain expanded cell cluster sections, and then the expanded cell cluster sections are subjected to HER2 immunohistochemical staining using a staining machine and a HER2 immunohistochemical antibody with a clinically specified clone number; preferably, the expanded cell cluster wax block is a high-density expanded cell cluster wax block, and in this embodiment, a Roche Ventana fully automatic staining machine and a HER24B5 antibody are used for immunohistochemical staining;

[0067] S13: Interpret the staining results of the four amplified cell clusters after staining in step S12, and determine the standard cell cluster based on the staining interpretation results of the four amplified cell clusters. The specific steps are: the staining results of each HER2 amplified cell cluster in step S12 are handed over to multiple (such as more than three) pathologists to interpret the staining results of step S12 for pathological scoring, and the interpretation is based on the "Guidelines for HER2 Detection of Breast Cancer in China (2019 Edition)". In this embodiment, three pathologists specifically interpret the pathological scores; if the interpretation result is situation (1): that is, the pathological scores of the same amplified cell cluster by the three pathologists are inconsistent, then steps S11 and S12 are repeated; if the interpretation result is situation (2): that is, the pathological scores of the same amplified cell cluster by the three pathologists are consistent, and when the interpreted pathological scores of the four amplified cell clusters correspond to the pathological scores one by one, then the four amplified cell clusters obtained by culturing the corresponding four cell lines are standard cell clusters of HER2 receptor protein, and then according to the interpretation, the four amplified cell clusters are obtained by culturing the corresponding four cell lines. The four types of amplified cell clusters were numbered according to the pathological scores read, as shown in 3. When the cell cluster was not stained, the HER2 pathological score was 0, and the corresponding amplified cell cluster was marked as 1; when the cell membrane in the cell cluster was stained, discontinuously and weakly, the HER2 pathological score was 1+, and the corresponding amplified cell cluster was marked as 2; when the cell membrane in the cell cluster was stained continuously and strongly, the HER2 pathological score was 2+, and the corresponding amplified cell cluster was marked as 3; when the cell membrane in the cell cluster was stained continuously and the staining intensity was strong, the HER2 pathological score was 3+, and the corresponding amplified cell cluster was marked as 4;

[0068] S14: After the wax block of the amplified cell cluster corresponding to situation (2) in step S13 is subjected to routine processing procedures such as paraffin sectioning, it is tested using a DNA FISH in situ hybridization detection kit. This test uses the Abbott breast cancer HER-2 gene detection kit (fluorescence in situ hybridization method), and the test results are interpreted by a professional pathologist for fluorescence. If the fluorescence interpretation result is: there is no HER2 DNA gene amplification in the amplified cell cluster with a pathological score of 3+, i.e., cell cluster No. 4, then steps S11-S13 are repeated; if the fluorescence interpretation result is: there is HER2 DNA gene amplification in the amplified cell cluster with a pathological score of 3+, i.e., cell cluster No. 4, then the HER2 amplified cell cluster obtained by culturing the selected cell line is the HER2 standard cell cluster.

[0069] S2: Obtain and process the pathological tissue to obtain a tissue slice to be tested, place the HER2 standard cell chip and the tissue slice to be tested on the same glass slide or on different glass slides for subsequent processing and immunohistochemical staining of the same batch, and obtain a HER2 stained tissue slice and a HER2 calibration cell chip, and calibrate the staining intensity of the tissue slice to be tested by the staining intensity of the stained standard cell cluster in the calibration cell chip;

[0070] S3: Acquire the adaptation parameters of the HER2 stained tissue slice and the HER2 stained standard cell cluster in step S2, wherein the adaptation parameters include the ROI area cell count, DAB staining area and DAB staining average intensity of each stained standard cell cluster and stained tissue slice; the specific acquisition steps of the adaptation parameters include the following:

[0071] S31: Scanning the calibration cell chips and stained tissue slices in the same batch of staining to obtain scan images and stained tissue slice scan images, and importing the scan images into the immunohistochemical analysis platform (such as Figure 4 To make data recognition more comprehensive and accurate, the scans and stained tissue slice scans obtained are panoramic scans;

[0072] S32: using the immunohistochemical analysis platform to select and quantitatively identify the stained standard cell cluster regions in the stained tissue slice scans and the calibration cell chip scans;

[0073] The analysis platform processes Figure 4 As shown, when performing region selection, the immunohistochemical analysis platform selects ROI regions of the same size for the four HER2 stained standard cell clusters respectively; and multi-point ROI region selection is performed on the stained tissue slice to ensure that the quantitatively selected region can reflect the overall HER2 staining data of the tissue slice to be tested. Preferably, at least three ROI regions of the same size are selected for the stained tissue slice, and each ROI region selected for the stained tissue slice is required to have the same area as the ROI region selected for the stained standard cell cluster; in addition, the number of ROI regions selected for the stained tissue slice can be adjusted according to actual conditions. In this embodiment, one ROI region is selected for each stained standard cell cluster, and three ROI regions are selected for the stained tissue slice as an example; when performing quantitative identification, the immunohistochemical analysis platform identifies the selected region, and the identification includes identifying the cell nuclei on each stained standard cell cluster and the stained tissue slice, the target protein identified as positive, that is, the area occupied by the positive protein, and the expression intensity of the target protein, so as to obtain quantitative identification data.

[0074] The coloring intensity of the target protein in the recognition software is represented by the DAB value, so whether the target protein is positive is based on the DAB value, but the DAB value of the positive protein is related to the specific target protein and the staining antibody. Therefore, in this embodiment, according to the characteristics of the HER2 receptor protein and staining experience, it is set that when the DAB value is higher than 55, the HER2 receptor protein is identified as a positive protein;

[0075] S33: Quantitatively analyzing the quantitative identification data obtained by the immunohistochemical analysis platform, thereby obtaining the ROI regional cell count, DAB staining area and DAB staining average intensity of each stained standard cell cluster and the ROI regional cell count, DAB staining area and DAB staining average intensity of the stained tissue slice;

[0076] S34: Since the pathological scores of HER2-related diseases have four levels: 0, 1+, 2+, and 3+, nmax=3 here. Substituting the value of n into formula (1) and formulas (3-1)-(3-3) in Example 1, the following relative score formulas for the four stained standard cell clusters and the calibration score intervals for the four stained standard cell clusters can be obtained:

[0077] The calculation formula for the staining score of the stained standard cell cluster with a pathological score of 0 is:

[0078]

[0079] The calibration score interval of the stained standard cell cluster with a pathological score of 0 is:

[0080]

[0081] The calculation formula for the staining score of the stained standard cell cluster with a pathological score of 1+ is:

[0082]

[0083] The calibration score interval for the stained standard cell clusters with a pathology score of 1+ is:

[0084]

[0085] The calculation formula for the staining score of the stained standard cell cluster with a pathological score of 2+ is:

[0086]

[0087] The calibration score interval for the stained standard cell clusters with a pathology score of 2+ is:

[0088]

[0089] The calculation formula for the staining score of the stained standard cell cluster with a pathological score of 3+ is:

[0090]

[0091] The calibration score interval for the stained standard cell clusters with a pathology score of 3+ is:

[0092]

[0093] Then, the specific values ​​of the adaptation parameters of each stained standard cell cluster obtained in step S3 are respectively substituted into the above eight formulas (4-1)-(7-1), so as to obtain the relative scores of the four HER2 receptor protein stained standard cell clusters and the calibration score intervals of each stained standard cell cluster. At the same time, the adaptation parameters of the stained tissue slice obtained in step S34 of this embodiment are substituted into formula (2) in embodiment 1 to obtain the relative score of the stained tissue slice.

[0094] Finally, C s The values ​​are compared with each calibration score interval. s Value falls into When C s Value falls into When the calibration score of the target protein expression result in the tissue slice to be tested is 1+; C s Value falls into When the calibration score of the target protein expression result in the tissue slice to be tested is 2+; C s Value falls into When , the calibration score of the target protein expression result in the tissue slice to be tested is 3+.

[0095] Embodiment 4:

[0096] The difference between this embodiment and embodiment 1, 2 or 3 is that in this embodiment, the tissue slice to be tested is subjected to immunohistochemical detection on two different detection platforms to detect the expression of HER2 protein in the tissue slice to be tested, and then the calibration method of the present invention is used to process the data of the stained tissue slices and each stained standard cell cluster obtained by the two detection platforms using the algorithm of the present invention; the immunohistochemical staining results of the 1# detection platform and the 2# detection platform are as follows: Figure 5 and Figure 6 shown.

[0097] contrast Figure 5 , Figure 6 and Figure 3 It can be seen that the staining intensity of the same stained standard cell clusters and stained tissue slides in the 1# and 2# detection platforms is significantly different. It can be seen that the staining effects of the same cell clusters will be very different due to differences in antibody clone numbers, antibody dilutions, color development times, etc. on different detection platforms. If manual interpretation is used at this time, the pathological scores of the same sample tissue on different detection platforms will be greatly different.

[0098] By adopting the calibration method of the present invention, consistent pathological scoring results can be obtained for the same sample tissue when tested on different testing platforms.

[0099] After the calibration method of the present invention is used to complete the quantitative identification of the stained tissue slices and each stained standard cell cluster of the two platforms using the PVB PWSA histochemical AI software, the stained tissue slices and each stained standard cell cluster are processed according to the adaptation parameters output by the Finovavir PAR-BF analysis platform using the algorithm of the present invention to obtain the relative scores of the four stained standard cell clusters and the relative score of one stained tissue slice, and the results are shown in Table 1.

[0100] Table 1 Relative scores of stained standard cell clusters and stained tissue slides

[0101]

[0102] The relative scores of each stained standard cell cluster were processed by algorithm using formulas (3-1)-(3-3) in step S4 of Example 3 to obtain the calibration score range of each stained standard cell cluster on the two detection platforms. The results are shown in Table 2.

[0103] Table 2 Correction score range of stained standard cell clusters

[0104]

[0105] As can be seen from Table 2, the relative scores of the stained tissue slides of the two detection platforms all fall within the calibration score range of the stained standard cell group with a pathological score of 1+, and the pathological score of the stained tissue slide is obtained to be 1+, so the calibration result of the HER2 receptor protein expression result of the tissue slide to be tested is 1+. It can be seen that although the same tissue to be tested is subjected to immunohistochemical detection on different detection platforms, the calibration method of the present invention can obtain stable and reliable pathological score results.

[0106] It should be noted that the technical features in the above-mentioned embodiments 1 to 4 can be combined in any way, and the combined technical solutions all belong to the protection scope of the present application. In this article, terms such as "including", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of further restrictions, the elements defined by the sentence "including one..." do not exclude the existence of other identical elements in the process, method, article or equipment including the elements.

[0107] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A calibration method for detecting the expression results of a target protein in a tissue, characterized in that: The following steps are involved: S1: preparing a standard cell chip expressing a target protein, obtaining and processing a tissue to be tested, and preparing a tissue slice to be tested, wherein the standard cell chip has a plurality of standard cell clusters, and the expression levels of the plurality of standard cell clusters for the same target protein correspond to the pathological scores of the disease related to the target protein; S2: The tissue slice to be tested and the standard cell chip are subjected to immunohistochemical staining from the same batch to obtain a stained tissue slice and a calibration cell chip respectively; at this time, the standard cell cluster in the calibration cell chip is referred to as a stained standard cell cluster; S3: respectively obtaining the adaptation parameters of the stained standard cell cluster and the stained tissue slice in step S2; the adaptation parameters include ROI area cell count, DAB staining area and DAB staining average intensity; S4: performing algorithm processing on the adaptation parameters described in step S3 to obtain the relative scores of each stained standard cell cluster and the relative scores of the stained tissue slices in this batch of immunohistochemical staining experiments, and calibrating the expression results of the target protein in the stained tissue slices based on the relative scores of each stained standard cell cluster; The expression results of the target protein in the stained tissue slice are calibrated based on the relative scores of each stained standard cell group, including the following steps: When the relative score of the stained tissue slice belongs to the calibration score interval of a certain stained standard cell cluster, it is considered that the pathological score of the stained tissue slice is the same as the pathological score of the stained standard cell cluster, thereby completing the calibration of the target protein expression result of the tissue to be tested.

2. The calibration method according to claim 1, characterized in that: The algorithm processing includes: The relative score of each stained standard cell cluster is calculated according to formula (1), the relative score of the stained tissue slice is calculated according to formula (2), and the calibration score interval of each stained standard cell cluster is calculated according to formulas (3-1)-(3-2): Relative score of stained standard cell clusters: Relative scoring of stained tissue sections: The calibration score interval formula for each stained standard cell cluster is: When n=0, When 0<n<nmax, When n = nmax, Among them, C n Represents the relative score of the stained standard cell group with pathological score n, I n represents the average DAB staining intensity of the stained standard cell cluster with pathological score n in step S3, A n represents the DAB staining area of ​​the stained standard cell cluster with a pathological score of n in step S3, N n Represents the cell count of the ROI area of ​​the stained standard cell cluster with a pathological score of n in step S3; C s Represents the relative score of the stained tissue slice, I s represents the average intensity of DAB staining of the tissue sections stained in step S3, A s represents the DAB staining area of ​​the tissue slice stained in step S3, N s The cell count of the ROI area representing the stained tissue slice described in step S3; Represents the calibration score interval of the stained standard cell cluster with a pathological score of n; n is a natural number starting from 0, and nmax refers to the maximum value of the pathological score.

3. The calibration method according to claim 1, characterized in that: The preparation of the standard cell chip includes determining a standard cell cluster, and the determination of the standard cell cluster includes the following steps: S11: screening multiple cell lines with different expression levels of the target protein according to the pathological scores of the target protein-related diseases, and amplifying these cell lines to obtain multiple amplified cell clusters; S12: preparing a plurality of the expanded cell clusters into expanded cell cluster wax blocks, and then slicing and immunohistochemically staining the expanded cell cluster wax blocks to obtain staining results of each expanded cell cluster; S13: Interpreting the staining results of each of the amplified cell clusters to obtain a staining interpretation result, and determining a standard cell cluster according to the staining interpretation result of the amplified cell clusters.

4. The calibration method according to claim 3, characterized in that: The interpretation of the staining results in step S13 refers to submitting the staining results of each of the amplified cell clusters in step S12 to multiple pathologists for pathological scoring interpretation. If the interpretation result is situation (1): the pathological scores of each amplified cell cluster by each pathologist are inconsistent or the pathological scores are not in one-to-one correspondence with each amplified cell cluster, repeat steps S11-S12; if the staining interpretation result is situation (2): the interpretation results of each amplified cell cluster by each pathologist are consistent, and the pathological scores are in one-to-one correspondence with each amplified cell cluster in S12, then the amplified cell cluster cultured by the cell line is determined to be a standard cell cluster.

5. The calibration method according to claim 4, characterized in that: The determination of the standard cell cluster also includes step S14: performing genetic testing on the amplified cell cluster with the highest pathological score obtained in the situation (2) in step S13; when the test result is that there is no genetic mutation of the gene corresponding to the target protein expressed in the amplified cell cluster with the highest pathological score, repeating steps S11-S13; if the test result is that there is a genetic mutation of the gene corresponding to the target protein expressed in the amplified cell cluster with the highest pathological score, determining that the amplified cell cluster obtained by culturing the cell line is the standard cell cluster.

6. The calibration method according to claim 1, characterized in that: The acquisition of the adaptation parameters in step S3 includes the following steps: S31: scanning the calibration cell chip and the stained tissue slice to obtain a calibration cell chip scan image and a stained tissue slice scan image; S32: using immunohistochemical analysis software, performing ROI area selection and quantitative identification on each stained standard cell cluster area in the calibration cell chip scan and the stained tissue slice scan, respectively, to obtain quantitative identification data, wherein the ROI area refers to a region of interest; S33: The quantitative identification data are quantitatively analyzed using the immunohistochemical analysis software to obtain the cell count, DAB staining area and DAB staining average intensity of the ROI region of each stained standard cell cluster and stained tissue slice.

7. The calibration method according to claim 6, characterized in that: The quantitative identification refers to selecting a ROI area of ​​the same size for each of the stained standard cell clusters and the stained tissue slices for identification.

8. The calibration method according to claim 7, characterized in that The quantitative identification includes identifying the cell nuclei, the area occupied by positive proteins and the expression intensity of target proteins on the stained standard cell clusters and stained tissue slices. The positive proteins refer to stained proteins with DAB intensity higher than a certain value.

9. The calibration method according to claim 8, characterized in that: When multi-point ROI region selection is adopted, at least three ROI regions are selected for the stained tissue slice for identification.

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