CN116106537A

CN116106537A - Method and kit for staining breast cancer sample

Info

Publication number: CN116106537A
Application number: CN202310136460.6A
Authority: CN
Inventors: 孙景云; 王光杓; 范师恒; 陈龙昀; 吴东方; 高志博
Original assignee: Shenzhen Yuce Biotechnology Co ltd
Current assignee: Shenzhen Yuce Biotechnology Co ltd
Priority date: 2023-02-20
Filing date: 2023-02-20
Publication date: 2023-05-12

Abstract

A staining method and kit of breast cancer samples, the staining method includes: the staining step comprises the step of sequentially staining breast cancer samples to be detected by using corresponding primary antibodies according to the antigen sequence in the following antigen groups to obtain stained samples: CD20, CD8, FOXP3, CD4, CD56, panCK. The invention carries out multiple immunofluorescence detection on a single slice, can simultaneously present the expression condition of six antigen targets, and obviously improves the utilization rate of a single tissue sample, in particular to a precious sample.

Description

Method and kit for staining breast cancer sample

Technical Field

The invention relates to the technical field of fluorescence detection, in particular to a method and a kit for staining breast cancer samples.

Background

Breast Cancer (BC) is one of the most common malignant tumors in women. Symptoms of early breast cancer are not obvious, and local symptoms such as breast lump, breast skin abnormality and the like are often taken as main symptoms; advanced breast cancer can generate cancer cell distant metastasis, and the whole body multi-organ lesion appears, which directly threatens the life of patients. At present, the etiology of breast cancer is not clear, early screening work is paid attention to in the 80 s of the 20 th century for improving the survival rate, cytology and pathological diagnosis are mainly utilized, and the early screening work is combined with different treatment modes, so that invasive diseases are eradicated. The prognosis of breast cancer is closely related to the development stage of the disease, and the relative survival rate of breast cancer patients for 5 years is 89.9% according to the data of international cancer organization statistics, wherein the survival rate of in-situ cancer for 5 years is 98.8%, the survival rate of early invasive cancer for 5 years is 85.5%, and the survival rate of invasive cancer for 5 years with distant metastasis is only 27.4%. Biomarker detection is significant for prognosis and efficacy prediction of breast cancer patients, but the existing detection method and detection efficiency have limitations and disadvantages, so that more effective biomarkers need to be developed, a more efficient detection method is constructed, standardized detection is realized to save manpower, material resources and financial resources, and detection accuracy is improved to guide treatment.

Immunostaining techniques include: IHC (immunochemistry), ICC (immunochemistry) and IF (Immunofluorescence). Wherein IHC/IF detection is performed by using antibodies and fluorescent detection to study the localization, relative expression and activation of target proteins in fixed cells or tissues in formalin-fixed and paraffin-embedded (formalin fixation and paraffin embedding, FFPE) samples. Today, the technology of tumor molecular detection is mature gradually, and the information base of related research and clinical decision is also beginning to pay more attention to the coverage of tumor microenvironment. The complexity of tumor microenvironments requires that a single Zhang Zuzhi sample slice yield multiple information, improving the utilization of the sample slice, particularly the precious sample. The biggest limitation of traditional IHC/IF detection is the small number of co-stained targets (1-3 per patch) at a time, whereas accurate tumor assessment requires detection of multiple protein targets, requiring adequate histological specimens. In most practical cases, however, the biopsy sample of the patient fails to meet additional tests beyond the oncological histological typing, which results in missing the opportunity to obtain important diagnostic and prognostic information from the patient sample. Furthermore, even with sufficient samples to perform a series of consecutive tissue sections with conventional IHC/IF staining, correlation between proteins in a multicellular population study could not be accurately assessed. Therefore, the current IHC/IF detection method still has great limitation in acquiring information, and cannot account for all the situations of the complex TIME. Another major limitation of conventional IHC/IF is the high variability between observers, lack of standardized operation, and randomness; the result interpretation mainly adopts man-made qualitative or semi-quantitative analysis, has certain subjectivity, and has errors among different batches and among people. To reduce the subjective impact, there is currently an international consensus that requires a laboratory with a high level of experience of pathologists. In addition, combining the current research situation and the technical requirement, the standardization and the automation are the necessity of the development of the times, so that the difference among observers can be eliminated, and the detection efficiency is improved.

A recent technique of multi-labeled immunohistochemical staining/immunofluorescent staining (multiplex immunohistochemistry/immunofluorescenc e, mhic/IF) enabled the labeling of multiple biomarkers on a single tissue section, while obtaining multi-channel information on cell composition and spatial arrangement by post-analysis, and performing high-dimensional analysis of TIME. The technology is matched with quantitative analysis software, can automatically distinguish tumor and non-tumor tissues, objectively analyze a plurality of biomarkers and cell composition, functional state and cell-cell interaction, and has the advantages of high repeatability, high efficiency and high cost efficiency. Especially under the condition of rare histological specimens, the mIHC/IF detection can be used as a new detection method for selection, has wide application prospect in clinic, but the existing tumor immunotherapy efficacy evaluation lacks sufficient biomarkers.

Disclosure of Invention

According to a first aspect, in an embodiment, there is provided a method of staining a breast cancer sample, comprising:

the staining step comprises the step of sequentially staining breast cancer samples to be detected by using corresponding primary antibodies according to the antigen sequence in the following antigen groups to obtain stained samples:

CD20、CD8、FOXP3、CD4、CD56、panCK。

according to a second aspect, in an embodiment, there is provided an image obtained by the dyeing method according to any one of the first aspects.

According to a third aspect, in an embodiment, there is provided an image analysis method including: and (3) carrying out biomarker analysis calculation on the fluorescence expression data of the target area of the image in the second aspect to obtain a biomarker value of the sample.

According to a fourth aspect, in one embodiment, there is provided a kit for detecting a breast cancer sample, comprising the following antibodies:

anti-CD20 monoclonal antibody paired Opal 520, anti-CD8 monoclonal antibody paired Opal 480, anti-FOXP3 monoclonal antibody paired Opal 570, anti-CD4 monoclonal antibody paired Opal 620, anti-CD 56 monoclonal antibody paired Opal 690, anti-panCK monoclonal antibody paired Opal 780.

According to the method and the kit for staining the breast cancer sample, multiple immunofluorescence detection is carried out on a single slice, the expression condition of six antigen targets can be presented simultaneously, and the utilization rate of the single tissue sample, especially the precious sample, is obviously improved.

In one embodiment, the method and kit of the present invention are used to analyze TILs (tumor infiltrating lymphocytes) in the immune microenvironment of breast cancer with excellent accuracy and precision.

In one embodiment, the invention facilitates the observation of interactions and symbiotic localization of target proteins, with significantly reduced demand for tissue samples; and can be applied to automatic dyeing machine, show improvement degree of accuracy and repeatability, simple and convenient high efficiency.

Drawings

FIG. 1 is a schematic diagram of the detection flow in example 1;

fig. 2.1 is a CD56 antibody: staining results plot of antibody dilution = 1:800;

fig. 2.2 is a CD56 antibody: staining results plot of antibody dilution = 1:1000 (30 min);

fig. 2.3 is a CD56 antibody: staining results plot of antibody dilution=1:1000 (60 min);

FIGS. 3.1 to 3.4 are graphs showing dye pair staining results of CD56 antibodies;

FIG. 4 is a graph of dye-paired MIF and SNR results for CD56 antibodies;

FIGS. 5.1 to 5.4 are graphs showing the results of staining for the number of different antibody repairs of CD56 antibody;

FIG. 6 is a graph of the results of MIF and SNR for various antibody repairs of CD56 antibodies;

FIG. 7 is a graph of dye-paired MIF and SNR results for CD4 antibodies;

FIG. 8 is a graph of the results of MIF and SNR for various antibody repairs of CD4 antibodies;

FIG. 9 is a graph of dye-paired MIF and SNR results for CD8 antibodies;

FIG. 10 is a graph of the results of MIF and SNR for various antibody repairs of CD8 antibodies;

FIG. 11 is a graph of dye-paired MIF and SNR results for CD20 antibodies;

FIG. 12 is a graph of the results of MIF and SNR for various antibody repairs of CD20 antibodies;

FIG. 13 is a graph of dye paired MIF and SNR results for FoxP3 antibodies;

FIG. 14 is a graph showing the results of MIF and SNR for various antibody repairs of FoxP3 antibody;

FIG. 15 is a graph of dye-paired MIF and SNR results for panCK antibodies;

FIG. 16 is a graph showing the results of MIF and SNR for various antibody repairs of panCK antibodies;

FIG. 17 is a graph showing the result of pre-experiment-1;

FIG. 18 is a graph showing the result of pre-experiment-2 staining;

FIG. 19 shows the result of staining (20X) in the breast cancer sample of example 1;

FIG. 20 is a graph showing the results of positive cell density correlation analysis of IHC and mIF (multiplex immunofluorescence) tests in breast cancer samples of example 1;

FIG. 21 is a graph showing the results of a positive cell proportion correlation analysis of IHC and mIF detection in breast cancer samples of example 1;

fig. 22 is the staining results of the repeated experiments in breast cancer samples of example 1.

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.

CD20、CD8、FOXP3、CD4、CD56、panCK。

in one embodiment, the antigen-corresponding primary antibody and the staining agent are paired in turn for staining as follows:

In one embodiment, the method comprises repeating the steps of antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, staining (i.e. dye incubation) and antibody removal according to the antigen sequence. Each round carries out antigen repair, blocking, primary antibody incubation, secondary antibody incubation, staining and antibody removal on one antigen.

In one embodiment, the secondary antibody used in the secondary antibody incubation is labeled with horseradish peroxidase.

In one embodiment, after staining the first 5 antigens, the sample is blocked by using a blocking solution, and then the 6 th antigen is sequentially subjected to primary antibody incubation, secondary antibody incubation, signal amplification staining, antibody removal, staining by using a corresponding stain, and staining by using a nuclear stain, so as to obtain a stained sample.

In one embodiment, the stain used in signal amplification staining comprises Opal TSA-DIG dye.

In one embodiment, the nuclear stain includes, but is not limited to, DAPI (i.e., 4', 6-diamidino-2-phenylindole, CAS registry number 47165-04-8).

In one embodiment, the breast cancer sample comprises a breast cancer tissue slice.

In one embodiment, the method further comprises a detection step, wherein the stained sample is subjected to continuous spectrum imaging and detection.

anti-CD20 monoclonal antibody, anti-CD8 monoclonal antibody, anti-FOXP3 monoclonal antibody, anti-CD4 monoclonal antibody, anti-CD 56 monoclonal antibody, anti-panCK monoclonal antibody.

In one embodiment, the kit further comprises a coloring agent, wherein the number of types of the coloring agent is consistent with the number of types of the antibody

In one embodiment, the stain comprises a fluorescent stain.

In one embodiment, the stain comprises:

Opal 520、Opal 480、Opal 570、Opal 620、Opal 690、Opal 780。

in one embodiment, the pairing relationship of the antibody and the staining agent is as follows:

In one embodiment, the fluorescent dye Opal TSA-DIG is also included.

In one embodiment, a nuclear stain is also included.

In one embodiment, the stain comprises an Opal TSA-DIG dye.

In one embodiment, the kit further comprises at least one of an antigen retrieval solution, a horseradish peroxidase-labeled secondary antibody, a blocking solution, and an anti-fluorescence quencher.

In one embodiment, the present invention provides a kit for full-automatic multicolor immunofluorescence detection of breast cancer samples and a staining method. The invention develops a detection kit through a multiple immunofluorescence technology, and utilizes an image recognition system (such as an InFormv 2.3 and a HALO pathology image analysis system) to obtain tissue and cell phenotype information, fluorescence intensity information of a corresponding target and the like to analyze TILs of a breast cancer immune microenvironment, so that the detection accuracy and precision of the kit are verified to meet scientific research detection requirements.

In one embodiment, the invention mainly solves the technical problems that the tumor immunotherapy efficacy evaluation lacks enough biomarkers and how to evaluate the immunotherapy efficacy in a larger scale by related indexes of tumor immune microenvironment; meanwhile, the requirement of tumor immune microenvironment analysis by a multiple immune fluorescence technology in clinical pathology work and scientific research is well met, and doctors and scientific researchers are efficiently assisted to complete the analysis of various immune histochemical indexes after immune histochemical multiple marking.

In one embodiment, the invention provides a full-automatic multicolor immunofluorescence detection kit and a staining method for breast cancer samples. The kit can simultaneously express six antibodies on one tissue slice, can provide more target protein information and co-localization conditions for pathologists, and can greatly improve the dyeing efficiency and save samples; meanwhile, the problems of low repeatability, long time and the like caused by manual dyeing can be avoided; the kit can be applied to a full-automatic sheet dyeing machine and has wide application prospect.

Example 1

The embodiment provides a full-automatic multicolor immunofluorescence detection kit for breast cancer samples, which comprises the following components: peroxidase blocking agent, primary antibody reagent, secondary antibody reagent and fluorescent dye; the anti-reagent comprises a rabbit anti-human CD4 monoclonal antibody, a mouse anti-human CD8 monoclonal antibody, a rabbit anti-human CD20 monoclonal antibody, a rabbit anti-human CD56 monoclonal antibody, a rabbit anti-human FOXP3 monoclonal antibody and a rabbit anti-human panCK monoclonal antibody; the secondary antibody reagent comprises an HRP (horseradish peroxidase) labeled anti-mouse and anti-rabbit mixture. The key materials involved in the full-automatic multicolor immunofluorescence detection are shown in Table 1.

TABLE 1 Key bill of materials

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The method comprises the steps of obtaining a breast cancer tissue sample from each subject, preparing a tissue section, performing multiple immunohistochemical staining treatment (such as Bond RX automatic staining instrument of Leica company) on the breast cancer tissue sample, and obtaining a corresponding immunohistochemical microscopic panorama through an imaging scanning instrument (such as Vectra polar is spectral type quantitative pathological analysis system of Akoya company).

The method is suitable for a full-automatic immunohistochemical staining machine, and the staining procedure comprises the following steps:

table 26 mIF staining procedure for dyes

Table 36 dyeing procedure for mIF yin-ginseng samples with dyes

The accuracy and the precision of the embodiment are qualified through a performance verification experiment. Sample H by pathologist&E, judging a dyeing result, wherein a sample meets the quality control requirement; the staining results revealed that subcellular localization was correct (CD 4, CD8, CD20, CD56 signals were localized to the cell membrane, panCK signal to the cytoplasm, FOXP3 signal to the nucleus); the accuracy verification result shows that the density of positive cells and the proportion correlation coefficient are both larger than 0.6, and the acceptance criterion is met. The results of the precision verification showed that when the positive cell density (over all Denit)y) is greater than 100/mm ² The c.v. average distribution is about 0.08 to 0.2; when the positive cell density is less than 100/mm ² The c.v. average distribution is about 0.17 to 0.38, meeting acceptance criteria.

The present invention will be further described with reference to specific steps, but the detection samples in this embodiment are not limited to breast cancer and tonsil samples, and only breast cancer samples are used as development targets during development, and performance verification experiments are performed by selecting breast cancer samples.

As shown in fig. 1, a schematic diagram of a detection flow is shown, and specific steps are as follows:

1. full-automatic multicolor immunofluorescence detection kit for preparing breast cancer sample

The embodiment provides a full-automatic multicolor immunofluorescence detection kit for breast cancer samples, which is developed as follows:

in the methodology development experiments, each antibody was optimized. The antibody comprises: CD4, CD8, CD20, CD56, FOXP3, panCK. The combination of the 6 antibodies is selected from the characteristics of the three-stage breast cancer and the inspection targets of the current medicine application.

The early stage of antibody test and experimental condition fumbling, including links such as preliminary verification of antibody characteristics, fumbling of antibody concentration, pairing of antibody dyes, fumbling of antibody repair times, preliminary experiments of mIF experiments, and the like, finally determines specific antibodies and experimental conditions.

The determination of the mIF condition includes: co-dyeing, channel selection, dyeing round selection and overall dyeing effect.

The method comprises the following specific steps:

(1) Antibody validation experiments: confirming whether the antibody is usable by using an IHC experiment; and primarily determining a ginseng sample;

(2) Concentration resistance fumbling experiments: and (3) carrying out proper concentration adjustment by combining the concentration recommended by the antibody specification, setting 2-3 concentrations, and developing an IHC experiment to find the primary antibody concentration. For example: antibody Anti-CD68 (KP 1) -Abcam, recommended antibody concentration of 1:100 (antibody: antibody dilution=1:100 by volume), set 1: 50. 1: 100. 1:200, performing IHC experiments;

(3) Antibody acid-base repair experiments: and (3) combining the repair conditions recommended by the antibody instruction, confirming ER1 or ER2, carrying out IHC experiments based on the common conditions of 95 ℃ for 20min, and adjusting according to the experimental results. For example: adjusting ER1 to ER2; the condition parameters are adjusted to 95 ℃ for 20min;95 ℃ for 40min;95℃for 80min.

(4) Antibody dye pairing experiments: panel (CD 4, CD8, CD20, CD56, FOXP3, panCK) is a six-label seven-color mIF experiment, planned to use

channels

480, 520, 570, 620, 690, 780, recommended paired channels in conjunction with antibody specifications and related references, 3-7 channel antibody dye paired IF experiments were performed on different antibodies; and carrying out fluorescence intensity statistics on experimental results, calculating a signal to noise ratio, and confirming a better pairing channel of the antibody. For example: the antibody Anti-CD68 (KP 1) -Abcam is set to carry out IF pairing experiments with 7 different channels of 480, 520, 570, 620, 650, 690, 780 and the like, and the results show that the signal to noise ratio of the channels of 650, 780 and 570 is higher, and in the subsequent mIF experiment condition optimization, the 3 channels are preferably selected to carry out the pairing of the antibody dye.

In the early stage, the performance of antibody repair times is firstly fumbling, and for antibodies with fewer repair times and better dyeing effect, the antibodies are firstly dyed; for antibodies exhibiting better staining effect for more repair times, post staining in panel; wherein the signal to noise ratio is calculated as a measurement basis. In addition, the dye condition is finally determined by debugging for a plurality of times in the mIF condition fumbling.

For dyeing pairing selection, in the process of antibody development, we all performed 3-8 unequal dyeing channel IF tests, and calculated signal-to-noise ratios to measure the priority of channel selection; and then, carrying out multiple adjustment in the subsequent mIF experiment, avoiding cross color, better showing the positive dyeing effect and determining the dyeing condition.

(5) Antigen retrieval frequency experiment: panel (CD 4, CD8, CD20, CD56, FOXP3, panCK) is a six-label seven-color mIF experiment, antigen retrieval is 7 times, and DAPI staining is the last time; performing IF experiments after performing antigen repair on different antibodies for 1-6 times, and selecting 1, 3 and 5 rounds for repair experiments; and (3) carrying out fluorescence intensity statistics on experimental results, calculating a signal to noise ratio, and confirming the superior repair turn of the antibody. For example: antibody Anti-CD68 (K P1) -Abcam, setting 1, 3, 5 times of repair and then incubating the primary antibody for performing the IF experiment of repair times, and arranging the antibody in the optimal repair time for performing the experiment according to the signal-to-noise ratio result and the subsequent mIF experiment condition optimization.

(6) mIF pre-experiment: summarizing the antibody characteristics according to the experimental results, determining primary antibody concentration, acid-base repair conditions, antibody pairing dye and antigen repair rounds, and primarily determining mIF experimental conditions; and optimizing mIF conditions according to experimental results until the effects of no nonspecific dyeing, no cross-dyeing after spectrum resolution and the like are achieved.

A. The screening procedure for each antibody was as follows:

1. experimental condition screening of CD56 antibodies

CD56 antibody concentration screening the following antibody reagents were used: anti-NCAM1/CD56[ CAL53] (ab 237708) -Abcam.

Dilution ratio of antibody fuzzing: fig. 2.1 is a CD56 antibody: staining results for antibody dilutions = 1:800 (30 min), figure 2.2 is a CD56 antibody: staining results for antibody dilutions = 1:1000 (30 min), fig. 2.3 is a CD56 antibody: staining results for antibody dilutions=1:1000 (60 min). Thus, the protocol corresponding to fig. 2.2, CD56 antibody, was selected: antibody dilution=1:1000 (30 min).

Antibody dye pairing (intestinal cancer sample 21R4111 SLZA): the staining results are shown in FIGS. 3.1 to 3.4, and the MIF and SNR (signal to noise ratio) results are shown in Table 4 and FIG. 4.

In the case of the sample, the sample is a sample of a tissue such as human brain, glioma, pancreatic and pancreatic cancer tissue, mouse brain tissue, rat brain tissue, etc., and the strength of the expression site of the relevant gene is combined, based on the reference of the antibody specification, so that a sample available in a laboratory is selected in the specific experiment. For example, if there are many intestinal cancer samples, the sample is selected.

TABLE 4 Table 4

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					CD56	480:6.07、620:94.82、690:17.63	480>690>620	620>690>480	690

Number of antibody repairs (21R 4111 SLZA): the staining results are shown in FIGS. 5.1 to 5.4, and the MIF and SNR (signal to noise ratio) results are shown in Table 5 and FIG. 6.

TABLE 5

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD56
	1 time, 3 times, 5 times: 58.72	5 times>3 times>1 time	5 times>3 times>1 time	Can all be

2. Experimental condition screening of CD4 antibodies

Using recombinant Anti-CD4 antibody [ EPR6855] (ab 133616) -Abcam, the dilution ratio included 1: 500. 1: 1000. 1:2000.

CD4 dye pairing: MIF and SNR results are shown in table 6 and fig. 7.

TABLE 6

CD4 antibody repair times: MIF and SNR results are shown in table 7 and fig. 8.

TABLE 7

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD4	226.17 for 1 time, 24.47 for 3 times, 5 times: 25.71	3 times>5 times	3 times>5 times	3

3. Experimental condition screening of CD8 antibodies

CD8 antibody concentration:

reagent: CD8 Monoclonal Antibody (4B 11) (MA 1-80231) -Thermo; concentration: 1:50, 1: 100. 1:200.

reagent: recombinant Anti-CD8 alpha antibody [ EP1150Y ] (ab 93278) -Abcam; concentration: 1:2000. 1: 4000. 1:8000.

CD8 dye pairing: MIF and SNR results are shown in table 8 and fig. 9.

TABLE 8

CD8 antibody repair times: MIF and SNR results are shown in table 9 and fig. 10.

TABLE 9

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD8
	1 time 4.23, 3 times 5.54, 5 times: 3.3	5 times>3 times>1 time	5 times>3 times>1 time	3、5

4. Experimental condition screening of CD20 antibodies

Reagent: CD20 (E7B 7T)

Rabit mAb (48750S) -Cell Signaling Technology; concentration: 1:400 (30 min), 1:300 (60 min).

Reagent: recombinant Anti-CD20 antibody [ EP459Y ] (ab 78237) -Abcam; concentration: 1: 50. 1:150 (20 min, 40 min), 1:200.

CD20 dye pairing: MIF and SNR results are shown in table 10 and fig. 11.

Table 10

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					CD20	480:5、570:62.99、690:36.14	480>690>570	480>570>690	480

CD20 antibody repair times: MIF and SNR results are shown in table 11 and fig. 12.

TABLE 11

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD20
	1 time, 3 times, 5 times: 58.72	5 times>3 times>1 time	5 times>3 times>1 time	Can all be

5. Experimental condition screening of FOXP3 antibodies

Reagent: foxP3 (D6O 8R) Rabbit mAb (12653S) -Cell Signaling Technology; concentration: 1:800.

Reagent: anti-FOXP3 Anti-body [ EPR22102-37] (ab 215206) -Abcam; concentration: 1: 25. 1: 50. 1:250.

FOXP3 dye pairing: MIF and SNR results are shown in table 12 and fig. 13.

Table 12

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					FoxP3	480:5.01、570、690:54.57	480>670>690	480>570>690	480、570

FOXP3 antibody repair times: MIF and SNR results are shown in table 13 and fig. 14.

TABLE 13

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					FoxP3
	1 time 70.8, 3 times 69.8 and 5 times:69.8	5 times>3 times>1 time	3 times>5 times>1 time	3>5

6. Experimental condition screening of panCK antibodies

Reagent: anti-pan Cytokeratin antibody [ KRT/1877R ] (ab 234297) -Abcam; concentration: 1:200. 1:400.

reagent: pan-Cytokeratin antibody (AE 1/AE 3) (sc-81714) -Santa Cruz Biotechnology; concentration: 1: 300. 1: 600. 1:900.

pairing panCK dyes: MIF and SNR results are shown in table 14 and fig. 15.

TABLE 14

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					panCK	480:6.65、520:20.56、650:7.07	620>650>690>480	650>480>690>620	(650)480

Number of panCK antibody repairs: MIF and SNR results are shown in table 15 and fig. 16.

TABLE 15

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					panCK	54.72 times, 3 times 29.84 times, 5 times: 39.52	5 times>3 times>1 time	3 times>5 times>1 time	Can all be

B. mIF Pre-experiment

Mif pre-experimental conditions: pre-experiment-1, experimental conditions are shown in Table 16. The experimental results are shown in table 16 and fig. 17. The experimental sample used in fig. 17 was breast cancer sample 2100996FZZA. Results: 780 does not develop color; pre-experiment adjustment: the samples were replaced.

Table 16

Mif pre-experimental conditions: pre-experiment-2, experimental conditions are shown in Table 17. The experimental results are shown in table 17 and fig. 18. The experimental sample used in fig. 18 was breast cancer sample 2100996FZZA.

TABLE 17

Mif pre-experimental conditions: breast cancer sample validation experiment

And (3) performing automatic dyeing by using Bond RX, and performing multiple mIF condition optimization, wherein the finally determined dyeing program is as follows.

Table 18 experimental parameters

Fig. 19 is a graph of the results of validation of breast cancer samples.

2. Application of the kit in a full-automatic sheet dyeing machine and a dyeing program. The method comprises the following steps:

the kit is applied to breast cancer tissue detection, tonsil tissues are used as yang ginseng, multiple immunohistochemical staining is implemented, and a used instrument is a BOND R X full-automatic sheet dyeing machine.

(1) Taking one of breast cancer and tonsil paraffin tissue, respectively preparing 1 paraffin tissue slice (experimental slice) for the breast cancer, 2 paraffin tissue slices (yin shen and Yang Can slice) for the tonsil paraffin tissue, attaching a sample label to the sample with the thickness of 3 micrometers, and placing the sample in a slice box for standby in a refrigerator at the temperature of 4 ℃;

(2) And opening the electrothermal blowing drying box, and preheating the electrothermal blowing drying box to 65 ℃ in advance. Placing the slices into a slice rack, and placing the slice rack into an electrothermal blowing drying oven at 65 ℃ for 3 hours. And (3) observing the wax on the surface of the tissue by naked eyes to thoroughly melt, and filling in an experiment record table.

(3) Reagent preparation: taking out the primary antibody, the secondary antibody, the dye and the sealing liquid from the refrigerator at the temperature of 4 ℃, wrapping a test tube with tinfoil paper for the reagent needing to be protected from light, placing the test tube on ice for standby, and placing the test tube in a centrifuge for short centrifugation.

a. Antibody preparation: taking out the antibody stock solution, and placing the antibody stock solution in a centrifugal machine for short centrifugation for later use. In a burette (Titration Kit), a working solution was prepared from an antibody diluent at the dilution ratio shown in the following table, a test tube was wrapped with a tinfoil paper, and the outside of the tube was labeled with the dye name, preparation time, and preparation personnel.

Table 18 preparation of working solution for primary antibody

b. Dye preparation:

(1) preparing dye storage liquid, taking out antibody and Opal dye, wrapping a test tube with tinfoil paper, and placing in a centrifuge for short centrifugation. Add 75. Mu.L of DMSO solution to each of Opa L480, opal 520, opal 570, opal 620, opal 690, TSA-DIG dye tubes; 300 μl of deionized water solution was added to the Opal780 dye tube. After the dye tube is uniformly mixed up and down for 10 times, the dye tube is put into a centrifuge for short centrifugation after the reagent is completely dissolved.

(2) Taking out the dye storage liquid, and putting the dye storage liquid into a centrifugal machine for short centrifugation for standby. In a burette (Titration Kit), a working solution was prepared from a diluent in accordance with the dilution ratio shown in the following table, a test tube was wrapped with tinfoil paper, the dye name was marked outside the tube, and the preparation time was measured.

Table 19 dye working solution formulation

c. Preparing a secondary antibody: the secondary antibody was pipetted into a burette (calculation formula for the amount of secondary antibody reagent: 1618+150×7n (μl), where n is the number of sections, 1618 μl is the dead cartridge volume of 30mL of the kit, and 150 μl is the amount per section).

d. Preparing a sealing liquid: the blocking solution was pipetted into a burette (formula for blocking solution usage: 1618+150×7n (μl), where n is the number of slices, 1618 μl is the dead cartridge volume of 30mL of the kit, and 150 μl is the usage per slice).

(4) Setting a dyeing program:

a. detecting samples and setting a yang ginseng sample program: clicking on "program settings" see table below for specific parameters.

Table 20mIF program

The conventional step of immunohistochemistry is to carry out antigen retrieval, acid retrieval or alkali retrieval; however, we also adjusted for poor continuous staining of antibodies, such as antibody panCK: ER1,95 ℃,20min+20min, equivalent to 2 consecutive repairs; however, the antibody is usually repaired for 20min for 1 time, but the antibody is optimized, the effect of 40min for single repair is poor, and the parameters of 20min+20min two-round repair are finally confirmed through multiple fumbling.

b. Setting a yin ginseng sample program: clicking on "program settings" see table below for specific parameters.

TABLE 21mIF yin ginseng sample procedure

(5) The detection system and the reagent rack filled with the antibody and the dye are put into a BOND RX instrument, and a dyeing program is started:

(6) The right-click slide rack directly starts to dye, and the dyeing start time and the dyeing end time are displayed below the slide rack, so that an experiment record table is filled.

(7) Sealing piece:

a. after dyeing is finished, a 'loading/unloading button' below the slide frame is pressed, the locking state of the slide frame is released, the slide frame is taken out, the primary antibody and the secondary antibody box are sealed by a sealing film or a cover, the primary antibody and the secondary antibody box are put into a refrigerator at the temperature of 4 ℃ for preservation, TSA-DIG, opal780 and DAPI dyes are directly poured into a waste liquid bottle, and other Opal dyes can be continuously used in the working solution validity period.

b. Sections were removed from the slide rack and the universal slide cover was removed (Bond Universal Covertiles). Except for the tissue, the water trace on the slice was wiped off with dust free paper.

c. 1-2 drops of the anti-fluorescence quenching agent are dripped into each slice for sealing, so that the anti-fluorescence quenching agent is ensured to cover tissues without bubbles, and meanwhile, the anti-fluorescence quenching agent needs to be prevented from overflowing. And (3) placing the slices in a storage plate for storage, keeping the slices away from light at room temperature for 30min, and placing the slices in a refrigerator at 4 ℃ for keeping the slices away from light for scanning after the anti-fluorescence quenching agent is solidified.

(8) Scanning slice: the slices were scanned in full slices using a Vectra polar instrument, setup procedure. After the end of the scan, the positive control and negative control samples were ensured to have the expected staining effect. Positive control expected staining effect: positive signals were seen in the samples (cell membrane localization was CD4, CD8, CD20, CD56, cytoplasmic localization was panCK, nuclear localization was FoxP3, no obvious cross-color, and uniform staining). Fig. 19 is the staining results (20X) in breast cancer samples, negative control expected staining effect: no significant positive signal was seen with the samples.

(9) Data analysis: the data is uploaded to the shared folder, and the path is the designated project folder. And then carrying out data processing according to analysis requirements.

3. Performance verification experimental methods and results of the kit applied in breast cancer tissues.

The specific experiment is as follows:

(1) Sample:

to evaluate the Accuracy of polychromatic immunofluorescence results (Accuracy), 3 FFPE samples from BC were examined, each sample selected from 10 serial sections for performance verification, 6 for IHC monochromatic immunohistochemistry, 3 for panel mIF, 1 for H & E. For each experiment, 2 serial sections of normal human tonsil tissue were taken for control experiments, 1 for the yang reference control experiment and 1 for the IgG isotype control, all specimens were obtained from domestic tissue chip service company.

To evaluate the Precision (Precision) of polychromatic immunofluorescence results, BC 3 samples were tested, each sample being at least 10 serial sections, wherein: 9 per sample were used for the reproducibility experiments and 1 slice was used for H & E staining. For each experiment, 2 serial sections of normal human tonsil tissue were taken for control experiments, 1 for the yang reference control experiment and 1 for the IgG isotype control, all specimens were obtained from domestic tissue chip service company.

(2) HE quality control: and judging the HE dyeing result of the sections according to the HE reading result fed back by the pathologist, wherein the HE dyeing result judging standard is shown in the following table.

Table 22HE quality control Standard

* And (3) injection: all five parameters are within the reference range, and can be judged as the level sample, and if one parameter does not accord with the level sample, the next level sample is judged. Risk samples are typically not taken, but may be tried on-line, but the end result may be worse than a qualified sample.

(3) Accuracy analysis:

a. at least 3 samples were tested to assess accuracy.

b. The single IHC results serve as references for mIF.

c. In the analysis of the results, IHC or mIF staining was scanned using a Vectra polar scanner. Full scan and digital imaging analysis will be performed on 3 samples. The scanned image of the mIF was analyzed by imaging HALO software (or equivalent software) and then the cell density and proportion of each antibody staining of antibodies (CD 4, CD8, CD20, CD56, FOXP3, panCK) in a single mIF was compared to the cell density and proportion of a single IHC (at least 5 regions of interest (Region of Interest, ROI) were selected for analysis per sample).

d. The staining results for each slide will be shown as positive cell count per square millimeter for each marker and as percentage of positive cells in total cells. The results of the IHC or stain same label are ranked. (1) Loading an image of a single IHC to the HALO; (2) the density of positive cells stained with each antibody was calculated and formulated as: positive cell count/image area. (3) At least 5 ROIs were randomly selected, the positive cell densities of individual IHCs and mifs in each ROI were ranked, and then the spearman correlation coefficient Rs for each antibody was calculated.

e. The correlation of individual IHC and mIF is detected using the Szelman correlation coefficient Rs. The strength of correlation is described using Rs: 0.20-0.39, "weak"; 0.40-0.59, "medium"; 0.60-1.0, "strong". Accuracy studies can only be validated when the Rs value is 0.6 or more. Only by using data of at least 15 ROIs, the accuracy assessment can be verified when the Rs value is between 0.6 and 1.0.

(4) Precision analysis:

f. for accurate assessment, FFPE samples of 3 tumors will be subjected to a repeat experiment in at least three batches, with 3 sections per patient per experiment for antibody panel detection. And each experiment required 1 slice for the yang reference control detection and 1 slice for the isotype control detection.

g. Three batches evaluating the accuracy of the assay were performed by at least two different personnel for at least two days to cover as many variables as possible.

h. The stained sections were scanned by Vectra polar or equivalent methods. The 9 slices in each sample were subjected to full slice scanning and digital imaging analysis. The HALO or equivalent software analyzes the images and then compares the staining results of one batch with the staining results of other batches. (1) An image of the mIF of one sample of each batch is loaded to the HALO. (2) The density and percentage of positive cells stained for each antibody was formulated: positive cell count/image area and percentage of positive cells in total cells. (3) The c.v. value for each antibody staining was calculated.

i. The positive cell density and ratio (positive cell count/image area percentage) of each antibody were calculated to give c.v. values. Only when c.v. is less than or equal to 0.2, the precision assessment will be considered as passing; if C.V >0.2, we will maintain the machine stable, control laboratory environmental conditions, ensure more stable experimental conditions, repeat the experiment again, and then calculate c.v. values using the data of the repeated experiment; the slice scan results are excluded from the calculation because the slice cannot yield a valid result for any reason. In addition, the evaluation of the batch including the sample is repeated, and the c.v. value is recalculated in the repeated experimental results.

(5) Verification result:

a. the sample meets the quality control requirement: the pathologist interpreted the H & E staining results of the samples as shown in the following table.

Table 23 sample H & E quality control results

Sequence number

Sample numbering

Sample type

Accuracy of

Precision of

H&E quality control

Number of nucleated cells

1

2100996FZZA

Breast cancer

√

/

Qualified product

＞5000

2

22R3639SLZA

Breast cancer

√

Qualified product

＞5000

3

22R3642SLZA

Breast cancer

√

Qualified product

＞5000

4

22R3984SLZA

Breast cancer

/

√

Qualified product

＞5000

b. Dyeing result: in breast cancer samples meeting quality control requirements, automated staining was performed with Bond RX using Vectra polar panoramic scanning. The staining results revealed that subcellular localization was correct (cell membrane localization included CD4, CD8, CD20, CD56, cytoplasmic localization panCK, nuclear localization FoxP 3). Analysis of staining accuracy: 3 tumor FFPE samples, 3 sections each were taken for mIF detection and 6 sections for IHC detection. The image is analyzed by HALO, and statistical analysis is performed, the results are shown in fig. 3 and 4, fig. 3 is the positive cell density correlation analysis result of IHC and mIF detection in a breast cancer sample, and fig. 4 is the positive cell proportion correlation analysis result of IHC and mIF detection in a breast cancer sample. According to the data source: positive cell density and positive cell proportion of breast cancer samples IHC and mIF detection.

c. Correlation analysis: the correlation of IHC and mIF is calculated using the Szelman correlation coefficient R. Intensity of the correlation is described using R: 0.20-0.39, "weak"; 0.40-0.59, "medium"; 0.60-1.0, "strong". As shown in FIG. 20 and FIG. 21, the expression of 6 proteins of CD4, CD8, CD20, CD56, FOXP3 and panCK was examined using IHC and mIF, and the value of the Szechwan correlation coefficient R was 0.6 or more, thus passing the accuracy test.

d. Precision analysis: FFPE samples of 3 tumors will be subjected to repeated experiments in 3 batches, 3 sections per patient per experiment for detection, while one for positive control and 1 section for negative control per experiment. Using HALO, we performed analytical processing on the images, as well as statistical analysis (fig. 22, table 24). The c.v. value of positive cell density (positive cell count/percent image area) was calculated for each antibody and the average value was calculated (table 24). When the positive cell Density (Overall Density) is greater than 100/mm ² When C.V. is less than or equal to 0.2, the precision evaluation is carried out; when the positive cell density is less than 100/mm ² C.V. is less than or equal to 0.4 by precision evaluation. Since the c.v. satisfies the corresponding conditions, the precision verification was passed.

FIG. 22 shows the staining results of repeated experiments in breast cancer samples (200X; rows 1 to 9 are duplicate slides 1 to 9, respectively; columns 1 to 8 are panoramas, DAPI, FOXP3, CD56, CD8, CD4, panCK, and CD20, respectively). In FIG. 22, "100 μm" indicates that the scale corresponds to a distance of 100. Mu.m.

Table 24 results of precision verification of mIF detection in breast cancer samples

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Verification conclusion: sample H by pathologist&E, judging a dyeing result, wherein a sample meets the quality control requirement; the staining results revealed that subcellular localization was correct (CD 4, CD8, CD20, CD56 signals were localized to the cell membrane, panCK signal to the cytoplasm, FOXP3 signal to the nucleus); the accuracy verification result shows that the density of positive cells and the proportion correlation coefficient are both larger than 0.6, and the acceptance criterion is met. The results of the precision verification show that when the positive cell Density (Overall Density) is greater than 100/mm ² The c.v. average distribution is about 0.08 to 0.2; when the positive cell density is less than 100/mm ² The c.v. average distribution is about 0.17 to 0.38, meeting acceptance criteria. The performance verification is accepted by the test, and can be used for detecting breast cancer samples.

In one embodiment, the present invention provides a multiplex immunofluorescence kit and staining method for breast cancer samples. The kit provided by the invention comprises the following components: peroxidase blocking agent, primary antibody reagent, secondary antibody reagent and fluorescent dye; the anti-reagent comprises a rabbit anti-human CD4 monoclonal antibody, a mouse anti-human CD8 monoclonal antibody, a rabbit anti-human CD20 monoclonal antibody, a rabbit anti-human CD56 monoclonal antibody, a rabbit anti-human FOXP3 monoclonal antibody and a rabbit anti-human panCK monoclonal antibody; the secondary antibody reagent comprises an HRP-labeled anti-mouse and anti-rabbit mixture. The kit can simultaneously present the expression conditions of six antigen targets on one tissue sample slice, is convenient for observing the interaction and symbiotic positioning of target proteins, and remarkably reduces the demand on the tissue samples; the kit can be applied to an automatic dyeing machine, and is simple and efficient, and accuracy and repeatability are remarkably improved.

In one embodiment, the operation technology of the full-automatic multicolor immunofluorescence detection kit for breast cancer samples provided by the invention realizes automatic detection, performs multiple immunofluorescence experiments on a single slice, can simultaneously present the expression conditions of six antigen targets, and greatly improves the utilization rate of a single tissue sample, in particular to a precious sample.

In one embodiment, the sample is saved for the patient, so that the sample has enough quantity to be used as other detection items.

In one embodiment, the automated staining can perform high-throughput testing, standardized operation is realized, 30 pathological sections can be detected simultaneously at most in a single time, the defects of long manual operation time and large error are avoided, and a simple and efficient detection method is provided for clinical pathologists.

In one embodiment, the kit achieves automated detection of standardized operations, which have been validated for performance, with high accuracy and precision.

In one embodiment, the kit detection method reduces the requirements of the traditional detection method on the tissue samples, the puncture samples can also meet the requirements, and the problem of poor dyeing repeatability of the micro tissue samples is avoided.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims

1. A method of staining a breast cancer sample, comprising:

CD20、CD8、FOXP3、CD4、CD56、panCK。

2. the method of claim 1, wherein the primary antibody corresponding to the antigen and the staining agent are paired in turn for staining as follows:

3. The staining method of claim 1 comprising repeating antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, staining, and antibody removal in the order of antigens;

optionally, the secondary antibody used in the secondary antibody incubation is labeled with horseradish peroxidase.

4. The staining method according to claim 1, wherein after staining the first 5 antigens, the sample is blocked by using a blocking solution, and then the 6 th antigen is sequentially subjected to primary antibody incubation, secondary antibody incubation, signal amplification staining, antibody removal, staining by using a corresponding stain, and staining by using a nuclear stain, so as to obtain a stained sample;

optionally, the stain used in signal amplification staining comprises Opal TSA-DIG dye;

optionally, the nuclear stain comprises DAPI.

5. The method of staining of claim 1, wherein the breast cancer sample comprises a breast cancer tissue section.

6. The method of claim 1, further comprising the step of detecting, imaging and detecting the stained sample in a continuous spectrum.

7. An image obtained by the dyeing method according to any one of claims 1 to 6.

8. An image analysis method, comprising: biomarker analysis calculations are performed on the fluorescence expression data of the target region of the image of claim 7 to obtain a biomarker value for the sample.

9. A kit for detecting a breast cancer sample, comprising the following antibodies:

10. The kit of claim 9, further comprising a staining agent, wherein the number of staining agent species corresponds to the number of antibody species;

optionally, the stain comprises a fluorescent stain;

optionally, the staining agent comprises:

Opal 520、Opal 480、Opal 570、Opal 620、Opal 690、Opal 780；

alternatively, the pairing relationship of the antibody and the staining agent is as follows:

anti-CD20 monoclonal antibody paired Opal 520, anti-CD8 monoclonal antibody paired Opal 480, anti-FOXP3 monoclonal antibody paired Opal 570, anti-CD4 monoclonal antibody paired Opal 620, anti-CD 56 monoclonal antibody paired Opal 690, anti-panCK monoclonal antibody paired Opal 780;

optionally, the fluorescent dye Opal TSA-DIG is also included;

optionally, a nuclear stain is also included;

optionally, the stain comprises an Opal TSA-DIG dye;

optionally, the kit further comprises at least one of an antigen retrieval liquid, a horseradish peroxidase-labeled secondary antibody, a blocking liquid, and an anti-fluorescence quencher.