Application of cell spheres in construction of human breast cancer immunohistochemical quality control product

Abstract

The invention provides application of cell spheres in construction of a human breast cancer immunohistochemical quality control product, and belongs to the technical field of pathological examination. The immunohistochemical quality control product solves the problems that tumor cells in the immunohistochemical quality control product in the prior art are distributed in a scattered manner, lack of a morphological structure and have significant difference from a human breast cancer tissue sample to be detected; the quality control sheet manufactured by the method is more stable, can ensure that the forms of tissues on all used quality control sheets are kept consistent, can be mass-produced, does not have ethical problems, can provide breast cancer ER, PR, HER2 and Ki67 immunohistochemical quality control products for daily detection of clinical pathology departments, simplifies the procedure of setting immunohistochemical control in clinical work, and improves the clinical work efficiency. And the quality control requirements of human breast cancer immunohistochemical detection can be met only by a single wax block, and the method is convenient and stable.

Description

Technical Field

[0001] The invention relates to the technical field of pathological examination, and in particular to the application of cell spheres in constructing human breast cancer immunohistochemistry quality control products. Background Art

[0002] Immunohistochemistry-based detection of ER, PR, HER2 and Ki67 protein expression levels in breast cancer is the key to guiding breast cancer treatment. Using known commercial antibodies to detect ER, PR, HER2 and Ki67 protein expression in breast cancer paraffin tissue samples is a routine diagnosis, and the accuracy of the test will affect the patient's treatment plan decision and prognosis.

[0003] Due to the batch-to-batch differences of antibodies and related reagents, or the influence of immunohistochemical operators, immunohistochemical quality control tissues need to be routinely set up during the immunohistochemical detection of ER, PR, HER2 and Ki67 in breast cancer to ensure the correct immunohistochemical process. Through the quality control tissue, workers can observe the spatial localization and subcellular localization of the stained tissue, and evaluate the difference between the actual staining intensity and the preset intensity.

[0004] At present, the commonly used ER, PR, HER2 and Ki67 immunohistochemical quality control tissues include human breast cancer tissue samples and human breast cancer cell lines. These two types of quality control tissues have their own advantages and disadvantages. The use of human breast cancer tissue samples as quality control samples has a limited number of tissue samples that can be cut after selection, resulting in the need to replace quality control tissues during a large number of uses, resulting in differences in quality control tissues used in clinical samples; on the other hand, due to the heterogeneity of ER, PR, HER2 and Ki67 expression in human breast cancer tissue samples, the protein levels of ER, PR, HER2 and Ki67 detected on each tissue section may be unstable. Although the use of human breast cancer cell lines as quality control tissues can ensure the consistency of ER, PR, HER2 and Ki67 expression levels on each section and meet the demand for quality control tissues in clinical work, the tumor cells in this type of quality control tissue are scattered and lack morphological structure, which is significantly different from the human breast cancer tissue samples to be tested.

[0005] Chinese patent CN104569397A discloses a breast cancer detection quality control product and its preparation method, especially a quality control product for HER2 gene amplification FISH detection and its preparation method, belonging to the fields of clinical laboratory science, pathology and biotechnology. A breast cancer detection quality control product is a paraffin-embedded specimen slice made from a cultured breast cancer cell line. The advantages of this invention are: the quality control product made from a breast cancer cell line cultured in vitro can be used as a substitute for the quality control product made from real tissue samples, with a large sample size and high repeatability, and different HER2 gene levels can be detected by selecting different cell lines, the preparation method is simple, can be mass-produced, easy to store, and can be used for indoor quality control and inter-laboratory quality evaluation.

[0006] However, the above patented technology does not solve the defects of quality control products in the prior art. Summary of the invention

[0007] The purpose of the present invention is to provide an application of cell spheres in constructing human breast cancer immunohistochemistry quality control products, which can provide breast cancer ER, PR, HER2 and Ki67 immunohistochemistry quality control products for routine detection in clinical pathology departments.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an application of a cell sphere in constructing a human breast cancer immunohistochemistry quality control product. The cell sphere is used to directly prepare a wax block, and the wax block is used as a human breast cancer immunohistochemistry quality control product after being sliced.

[0009] Preferably, the cell sphere is prepared by a preparation method comprising the following steps: The cells were cultured as cell spheres with cell-cell adhesion.

[0010] Preferably, the cell sphere is derived from a human breast cancer cell line.

[0011] Preferably, the human breast cancer cell line includes BT474, BT20, T47D or HCC1806.

[0012] The present invention also provides a method for constructing a human breast cancer immunohistochemistry quality control product, comprising the following steps: The human breast cancer cell spheres with intercellular adhesion were fixed, molded, dehydrated, transparent, wax-impregnated, embedded and sliced ​​in sequence to obtain human breast cancer immunohistochemistry quality control products.

[0013] Preferably, the fixation uses 5-20% neutral buffered formalin as the fixative.

[0014] Preferably, the fixed time is more than 20 hours.

[0015] Preferably, the encapsulating material used in the molding is agarose.

[0016] Preferably, the slice has a thickness of 3 to 5 μm.

[0017] The present invention also provides a human breast cancer immunohistochemistry quality control product obtained by the above construction method.

[0018] Beneficial effects of the present invention: The present invention confirms that cell spheres made from human breast cancer cell lines can effectively replace human breast cancer samples and quality control films made from cell lines, and the staining effect is not inferior to that of human breast cancer samples and quality control films made from cell lines. The materials are easily available in the market and can be used for commercial production.

[0019] The present invention uses 10% neutral buffered formalin fixative and a tissue dehydrator to complete the production of quality control products according to the production requirements of pathological tissue. The operation of quality control products can be completed in the production of conventional pathological tissues without the need for additional procedures, which is simple and easy.

[0020] The quality control product of the present invention can be used in the process of conventional breast cancer ER, PR, HER2 and Ki67 immunohistochemical detection, and can be machine-stained or manually operated together with human breast cancer samples to be tested, which is beneficial to quality monitoring during the detection process.

[0021] In the detection of HER2 in human breast cancer, it is necessary to evaluate the staining intensity, membrane integrity and positive ratio. In the present invention, the quality control product has four tissues with different staining results, and the preset 3+ stained tissue is a strong and complete cell membrane staining, with a ratio of 100%; the 2+ stained tissue is a medium-intensity complete cell membrane staining, with a ratio of 100%; the 1+ stained tissue is a weak incomplete cell membrane staining, with a ratio greater than 10%; the 0-stained tissue is a non-stained cell, with a ratio of 100%. The preset staining results are fully in line with the guidelines for the detection of HER2 in human breast cancer (Arch Pathol Lab Med (2023) 147 (9): 993–1000.). The four gradient staining results in the present invention can meet the clinical detection needs of HER2 in human breast cancer.

[0022] For ER detection in human breast cancer, it is often necessary to exclude false positives and false negatives caused by improper staining procedures. In clinical practice, normal breast ductal epithelium is commonly used as an ER positive internal control, the germinal center of the tonsil is used as a positive external control, and the mantle zone B cells of the tonsil are used as a negative external control (J Clin Oncol. 2020 Apr 20;38(12):1346-1366.). In the detection of PR in human breast cancer, similar to ER, it is also necessary to exclude false positives and false negatives caused by improper staining procedures. Endocervical columnar epithelial cells are commonly used as positive controls in clinical practice, but there is a lack of suitable negative control tissues. In the present invention, we provide suitable cell spheres as positive and negative controls for ER and PR staining. For the detection of Ki67 in human breast cancer, there is currently a lack of suitable control tissues. In the present invention, we provide suitable cell spheres as Ki67 low expression controls and high expression controls.

[0023] The human breast cancer ER, PR, HER2 and Ki67 immunohistochemical quality control tissues provided in the present invention can simplify the procedure of setting up immunohistochemical controls in clinical work. Only a single wax block is needed to meet the quality control needs of human breast cancer immunohistochemical detection, which is convenient and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Make a flow chart for the quality control product in the present invention; Figure 2 This is a diagram of cell sphere collection in a specific embodiment of the present invention; Figure 3 This is a diagram of a cell sphere molding process according to a specific embodiment of the present invention; Figure 4 A quality control product diagram produced for a specific embodiment of the present invention; Figure 5 The HER2 staining of the quality control product in a specific embodiment of the present invention is compared with the staining of the quality control product made from the patient's breast cancer tissue and the human breast cancer cell line; Figure 6 This is a staining diagram of ER and PR of quality control products in a specific embodiment of the present invention; Figure 7 This is a staining diagram of Ki67, a quality control product in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1 This embodiment is a quality control product based on a commercial cell line. This quality control product can be grown in batches and supplied in large quantities. At the same time, this type of quality control product is a wax block based on a standardized pathological tissue processing flow, which can meet the production needs of standardized quality control films.

[0027] refer to Figure 1 The quality control product production process includes the cultivation of commercial human breast cancer cells and the collection and fixation of cell spheres. After the fixed cell spheres are molded, they enter the standardized paraffin tissue production process. The produced wax blocks include quality control tissues with four different HER2 expression levels and two different ER, PR and Ki67 expression levels.

[0028] The commercial human breast cancer cell line used in the present invention is more stable than the existing commonly used human breast cancer tissue from patients, and can meet the needs of long-term and large-scale uniform use of quality control products. At the same time, the quality control product has a similar tissue structure to human breast cancer tissue, and the staining interpretation of ER, PR, HER2 and Ki67 is more accurate.

[0029] refer to Figure 2 The key step in making quality control products is to make the cells in the ordinary cultured human breast cancer cell line adhere to each other and grow to form cell spheres that are not easy to scatter. This invention uses a shaker to make cell spheres that are not easy to scatter. This type of cell sphere is not easy to scatter during centrifugation, fixation and wax block production. In tissue staining, the tissue morphology structure is similar to that of in vitro tissue samples. Compared with quality control films made using patient tissue samples, quality control films made from cell spheres are more stable, which can ensure that the tissue morphology on all quality control films used remains consistent. Quality control films can be mass-produced, and there are no ethical issues.

[0030] refer to Figure 3 After the collected cell spheres are centrifuged and aggregated, they are molded with agarose, so that the cell spheres are not easy to scatter during the process of making paraffin tissue. This molding method is simple and easy, and does not affect the tissue fixation effect. When the cell amount is constant, the density of the cell spheres in the quality control film produced each time can be guaranteed to be consistent, reducing batch differences and being more suitable for commercial production.

[0031] refer to Figure 4 There are four quality control tissues in the present invention, all of which are produced in one wax block, which is convenient for making quality control films and can meet the needs of quality control films under different staining conditions. In the present invention, the wax block production process can be completely carried out according to the conventional tissue wax block production process, without the need to set up separate programs or conditions, and can completely simulate the wax block production process close to that of in vitro tissue samples.

[0032] refer to Figure 5, (AD) The human breast cancer HER2 quality control film of the present invention contains four different modes of HER2 staining. According to the human breast cancer HER2 detection guide (Arch Pathol Lab Med (2023) 147 (9): 993–1000.), the preset 3+ stained tissue is strong and complete cell membrane staining, with a ratio of 100%; 2+ stained tissue is medium-intensity complete cell membrane staining, with a ratio of 100%; 1+ stained tissue is weak and incomplete cell membrane staining, with a ratio greater than 10%; 0 stained tissue is unstained cells, with a ratio of 100%. It can be seen from this figure that the tissues and cells in the quality control film adhere to each other, which is close to the tumor cell growth pattern in the patient tissue sample, making it convenient for users to use this quality control film to pay attention to abnormal conditions in the HER2 staining process.

[0033] The quality control sheet of the present invention has a similar tissue morphology to the patient's breast cancer tissue sample (IL), and the cells adhere to each other and grow; compared with the currently commonly used cell line (EH) control, the quality control sheet of the present invention has the same staining effect, and even has a better effect in the evaluation of low expression.

[0034] refer to Figure 6 , (A and B) Negative and positive staining in the human breast cancer ER quality control film of the present invention. It can be seen that there is no nuclear staining in the negative quality control film; 50% of the cell nuclei are strongly stained in the positive quality control film. (C and D) Negative and positive staining in the human breast cancer PR quality control film of the present invention. It can be seen that there is no nuclear staining in the negative quality control film; 80% of the cell nuclei are strongly stained in the positive quality control film.

[0035] refer to Figure 7 , (A) Low expression staining in the human breast cancer Ki67 quality control sheet of the present invention, with an expression rate of 15%. (B) High expression staining in the human breast cancer Ki67 quality control sheet of the present invention, with an expression rate of 90%.

[0036] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of cell spheres in constructing human breast cancer immunohistochemistry quality control products, characterized in that: The cell sphere is used to directly prepare a wax block, and the wax block is used as a human breast cancer immunohistochemistry quality control product after being sliced.

2. The use according to claim 1, characterized in that: The cell sphere is prepared by a preparation method comprising the following steps: The cells were cultured as cell spheres with cell-cell adhesion.

3. The use according to claim 1 or 2, characterized in that: The cell spheres are derived from a human breast cancer cell line.

4. The use according to claim 3, characterized in that: The human breast cancer cell lines include BT474, BT20, T47D or HCC1806.

5. A method for constructing a human breast cancer immunohistochemistry quality control product, characterized in that: The following steps are involved: The human breast cancer cell spheres with intercellular adhesion were fixed, molded, dehydrated, transparent, wax-impregnated, embedded and sliced ​​in sequence to obtain human breast cancer immunohistochemistry quality control products.

6. The construction method according to claim 5, characterized in that: The fixation adopts 5-20% neutral buffered formalin as the fixative.

7. The construction method according to claim 6, characterized in that: The fixed time is more than 20 hours.

8. The construction method according to claim 5, characterized in that: The packaging material used in the molding is agarose.

9. The construction method according to claim 5, characterized in that: The thickness of the slice is 3-5 μm.

10. A human breast cancer immunohistochemistry quality control product obtained by the construction method according to any one of claims 5 to 9.

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