Depigmentation method of melanoma tissue and application

Abstract

The invention discloses a method for depigmenting melanoma tissues in immunohistochemical staining, which comprises the following steps: in the immunohistochemical staining process, carrying out decoloring operation after an antigen repair step: heating and incubating by taking a hydrogen peroxide methanol solution as a decoloring solution for decoloring, the volume fraction of the hydrogen peroxide methanol solution is 5-15%, and the incubation time is 10-20 minutes. According to the method, the depigmentation process of melanin is simplified, the efficiency is high, the depigmentation result does not influence the form of tissue cells and IHC staining, meanwhile, the method is suitable for depigmentation of melanin cells of melanoma tissues by a full-automatic immunohistochemical staining instrument, and automation of depigmentation can be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of immunohistochemistry, and specifically relates to a depigmentation method for melanoma tissue and application thereof. Background Art

[0002] Malignant melanoma is a tumor that is often encountered in pathological diagnosis. Melanin in melanocytes is mainly synthesized and secreted in a lysosome-related melanosome. This process mainly relies on tyrosinase catalyzing the formation of tyrosine products and combining with proteins in the cytoplasm to form melanin particles, which are very stable and insoluble in water and most organic solvents.

[0003] Since the pigment in melanocytes is mainly red or yellow-black, the color in clinical pathological diagnosis is very close to the color after DAB development in immunohistochemical testing. Therefore, it is difficult to distinguish whether it is pigmentation or a positive result after conventional immunohistochemical DAB development.

[0004] The most commonly used depigmentation method is to soak in a 0.1% potassium permanganate solution, a strong oxidant, for 2-4 hours, then treat with 1% oxalic acid for 1 minute, and then wash with water for 5 minutes to remove the pigment in the melanoma tissue. The existing depigmentation method has the following disadvantages: 1. The existing method takes at least 2-4 hours in the depigmentation process, which is not conducive to rapid results; 2. The existing methods mainly use strong oxidants to remove pigments, which has a relatively large damage to the morphology and structure of tissue cells, which is not conducive to subsequent H&E and IHC staining; 3. The existing method requires depigmentation first and then dyeing, and the two processes need to be operated separately, which is troublesome in actual work.

[0005] Therefore, it is very necessary to provide a decolorizing solution and decolorizing method which can save decolorizing time, does not damage the morphology and structure of cell tissues, and is easy to operate. Summary of the invention

[0006] In order to solve at least one of the above problems, the present invention provides a depigmentation method, kit and application of melanoma tissue, which simplifies the depigmentation process of melanin and improves efficiency. The depigmentation result does not affect the morphology of tissue cells or IHC staining. At the same time, it is suitable for a fully automatic immunohistochemical staining instrument and can realize the automation of depigmentation.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical means: The first aspect of the present invention provides a method for depigmenting melanoma tissue in immunohistochemical staining, in which a depigmentation operation is performed after the antigen retrieval step during the immunohistochemical staining process.

[0008] In some embodiments of the present invention, the decolorization operation is performed by heating and incubating with a hydrogen peroxide methanol solution as a decolorization solution. In some embodiments of the present invention, the temperature of the heating and incubation is 55°C-75°C.

[0009] In some embodiments of the present invention, a 5%-15% hydrogen peroxide methanol solution by volume is used as a decolorizing solution; in some embodiments of the present invention, the incubation time is 10-20 minutes; in some embodiments of the present invention, a 5%-15% hydrogen peroxide methanol solution by volume is used as a decolorizing solution, and the decolorizing is carried out by incubation for 10-20 minutes at a temperature of 55°C-75°C. In some embodiments of the present invention, preferably, the concentration of the hydrogen peroxide methanol solution is 10% by volume. In some embodiments of the present invention, preferably, the incubation temperature is 65°C. In some embodiments of the present invention, preferably, the incubation time is 10 minutes.

[0010] The second aspect of the present invention provides the use of a hydrogen peroxide methanol solution in preparing a product for diagnosing melanoma, wherein the product is a diagnostic reagent or a kit.

[0011] In some embodiments of the present invention, the volume fraction of the hydrogen peroxide methanol solution is 5%-15%.

[0012] In some embodiments of the present invention, the product is used for immunohistochemical staining of melanoma tissue using a fully automatic immunohistochemical staining instrument.

[0013] In some embodiments of the present invention, after the antigen repair step, a 5%-15% volume fraction hydrogen peroxide methanol solution is used as a decolorizing solution and heated and incubated for decolorization. In some embodiments of the present invention, the decolorization is carried out by heating to 55°C-75°C and incubating for 10-20 minutes.

[0014] Beneficial Effects of the Invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a new depigmentation method, which utilizes the hydrogen peroxide methanol solution used in the existing fully automatic immunohistochemical staining process, optimizes its concentration, temperature and incubation time, so as to achieve depigmentation of the pigment in the melanoma tissue while blocking the endogenous peroxidase during the onboard immunohistochemical staining process, integrates the depigmentation process into the immunohistochemical staining step of the fully automatic immunohistochemical staining equipment, and achieves depigmentation and immunohistochemical staining in one process, without affecting the normal immunohistochemical staining process and results, solves the problem that melanoma tissue needs to be pre-depigmented and the morphological effect of strong oxidant depigmentation on tissue cells, thus saving depigmentation time and simplifying the immunohistochemical staining steps of this type of tissue, and also achieving good immunohistochemical staining results, realizing the automation of immunohistochemical staining of melanoma tissue, and improving work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The decolorization effect under different temperature incubation conditions in Example 1 of the present invention is shown; Figure 2 The staining results under different treatment conditions of different concentrations of decolorizing solution in Example 2 of the present invention are shown; Figure 3 The dyeing results under different treatment time conditions in Example 3 of the present invention are shown; Figure 4 The dyeing results under different treatment conditions in Example 5 of the present invention are shown. DETAILED DESCRIPTION

[0016] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the disclosed techniques represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0018] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0019] Reagents and Materials The reagent materials used in the examples of this application are shown in Table 1 below.

[0020] Table 1 Reagents and Materials

[0021] Example 1 Incubation Temperature Screening During the experiment, the inventor accidentally turned on the heating mode in the blocking process, and found that malignant skin melanoma had a certain degree of decolorization. Therefore, a conventional 3% hydrogen peroxide methanol solution was used for blocking at different temperatures on a fully automatic immunohistochemical staining instrument, and the decolorization effect of 3% hydrogen peroxide methanol solution on melanoma pigment was compared under five different temperature conditions: scheme 1: 45°C, scheme 2: 55°C, scheme 3: 65°C, scheme 4: 75°C, and scheme 5: 85°C (DAB was not added for color development at this stage).

[0022] The steps are as follows: (a) Set the steps and time of the staining process on the fully automatic immunohistochemical staining instrument; (b) Loading the melanoma slide onto the fully automatic immunohistochemical staining instrument; (c) Depigmentation staining was performed on a fully automatic immunohistochemical staining instrument according to the process in Table 2 below, Table 2 Workflow of fully automatic immunohistochemical staining instrument

[0023] Test results such as Figure 1 shown.

[0024] The results showed that: using 3% hydrogen peroxide methanol solution used for routine immunohistochemistry sealing, the study of whether it has a depigmentation effect on melanoma at different heating temperatures (instrument heating temperature control deviation ±1°C) found that there was no depigmentation effect at 45°C, the depigmentation effect was not obvious at 55°C, there was a certain depigmentation effect between 65°C and 75°C, and when the incubation temperature reached 85°C, the tissue underwent severe depigmentation.

[0025] Example 2 Hydrogen peroxide concentration screening A malignant skin melanoma with more melanin deposition was selected as a sample, and hydrogen peroxide methanol solutions were prepared according to different hydrogen peroxide concentrations. The depigmentation effects of different hydrogen peroxide concentrations on melanoma were compared on a fully automatic immunohistochemical staining instrument. In this embodiment, DAB was not added for color development, and only hematoxylin was used for staining. The incubation temperature of the depigmentation solution was 65° C. for 20 minutes. 100 mL of hydrogen peroxide methanol solutions of different concentrations were prepared, and the preparation scheme is shown in Table 3.

[0026] Table 3 Preparation of hydrogen peroxide methanol solutions of different concentrations

[0027] Follow the steps below to conduct the experiment: (a) Use the LYNX480 fully automatic immunohistochemical staining instrument produced by Tuling (Hangzhou) Biopharmaceutical Co., Ltd. to set the steps and time required for the staining process; (b) Loading a melanoma sample slide without prior decolorization treatment onto the fully automatic immunohistochemical staining instrument; (c) Depigmentation staining was performed on a fully automatic immunohistochemical staining instrument according to the process in Table 4 below.

[0028] Table 4 Immunohistochemistry process

[0029] Test results: The depigmentation effects of Schemes 1-4 are shown in Table 5 and Table 6. Figure 2 shown.

[0030] Table 5 Depigmentation results

[0031] The results showed that 10%-15% hydrogen peroxide methanol solution could completely remove the pigment when heated and incubated at 65°C for 20 minutes.

[0032] Example 3 Incubation time screening A melanoma tissue sample with high melanin deposition was selected and depigmented using 10% hydrogen peroxide methanol solution. The staining process of Example 2 was followed except for the incubation time of the depigmentation solution. The incubation time of the depigmentation solution was 3 minutes, 5 minutes, 10 minutes and 20 minutes, respectively.

[0033] The effect of different incubation times of 10% hydrogen peroxide methanol solution at 65°C on the depigmentation effect was tested using this example. The test results are shown in Tables 6 and Figure 3 shown.

[0034] Table 6 Test results

[0035] The results showed that 10% hydrogen peroxide methanol solution can completely remove the pigment by incubation at 65°C for 10-20 minutes. In order to save time, 10 minutes is preferred.

[0036] Example 4 Effect of heating conditions on depigmentation effect A melanoma tissue sample with high melanin deposition was selected and treated with 10% and 15% hydrogen peroxide methanol solutions for 10 minutes respectively without heating for depigmentation verification. The rest of the immunohistochemical process was the same as in Example 2.

[0037] The results showed that without heating, the pigmentation of 10% hydrogen peroxide methanol solution and 15% hydrogen peroxide methanol solution remained basically unchanged.

[0038] Example 5 IHC Validation Rabbit anti-human cytokeratin (broad spectrum) antibody reagent was selected as the primary antibody for detection.

[0039] Two samples of the same malignant melanoma tissue were taken for experiments: Scheme 1: The staining method of the present application was used for the staining experiment. The malignant melanoma tissue sample was directly put on the machine for decolorization and staining by incubating with 10% hydrogen peroxide methanol solution at 65°C for 10 minutes.

[0040] Solution 2: Use the traditional method to carry out the dyeing experiment, soak in 0.1% potassium permanganate solution for 2 hours, then use 1% oxalic acid solution for 1 minute to decolorize, and then dye on the machine; The specific steps are shown in Table 7 below.

[0041] Table 7 Experimental procedures for different schemes

[0042] In the above table, 11-1 is the blocking + decolorization step, and 11-2 is the blocking step.

[0043] The staining results are shown in Table 8 and Figure 4 shown.

[0044] Table 8 Detection results of different schemes The results showed that the morphology of tissue cells after decolorizing melanin by blocking endogenous peroxidase with 10% hydrogen peroxide methanol solution during immunohistochemical staining was significantly better than that of traditional methods. The IHC staining effect was also better than that of traditional methods. There was no need for pre-decolorization or strong oxidant decolorization of tissue cells, which reduced the number of operation steps and saved about 2 hours.

[0045] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A method for depigmenting melanoma tissue in immunohistochemical staining, characterized in that: During immunohistochemical staining, destaining is performed after the antigen retrieval step.

2. A method for depigmenting melanoma tissue in immunohistochemical staining according to claim 1, characterized in that: The decolorization operation uses a hydrogen peroxide methanol solution as a decolorization solution and heats and incubates to perform decolorization.

3. The method for depigmenting melanoma tissue in immunohistochemical staining according to claim 2, characterized in that: The heating temperature is 55°C-75°C.

4. The method for depigmenting melanoma tissue in immunohistochemical staining according to claim 2, characterized in that: The volume fraction of the hydrogen peroxide methanol solution is 5%-15%.

5. The method for depigmenting melanoma tissue in immunohistochemical staining according to claim 2, characterized in that: The incubation time is 10-20 minutes.

6. Application of hydrogen peroxide methanol solution in the preparation of products for diagnosing melanoma.

7. The use according to claim 6, characterized in that: The volume fraction of the hydrogen peroxide methanol solution is 5%-15%.

8. The use according to claim 6, characterized in that: It is applied in immunohistochemical staining of melanoma tissue using a fully automatic immunohistochemical staining instrument.

9. The use according to claim 8, characterized in that: After the repair step, decolorization is performed by heating and incubation using a 5% to 15% volume fraction of hydrogen peroxide methanol solution as a decolorization solution.

10. The use according to claim 9, characterized in that: Heat to 55°C-75°C and incubate for 10-20 minutes.

Images