A method for depigmenting melanoma tissue and its use

Abstract

The application discloses a method suitable for depigmentation of melanoma tissue in immunohistochemical staining, and the method comprises the following steps: after an antigen repairing step in the immunohistochemical staining process, a depigmentation operation is performed; a hydrogen peroxide methanol solution is used as a depigmentation solution; and the depigmentation is performed through heating incubation, wherein the heating temperature is 55 DEG C to 75 DEG C, the volume fraction of the hydrogen peroxide methanol solution is 5% to 15%, and the incubation time is 10 to 20 minutes. The method simplifies the depigmentation process of melanin, is high in efficiency, and does not affect the morphology of tissue cells and IHC staining while achieving depigmentation, and is suitable for depigmentation of melanin cells of melanoma tissue by using a full-automatic immunohistochemical staining instrument, and can realize automatic depigmentation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunohistochemistry, and particularly relates to a melanoma tissue depigmentation method and application. BACKGROUND

[0002] Malignant melanoma is a kind of tumor often encountered in pathological diagnosis. Melanin in melanocytes is mainly synthesized and secreted in a lysosome-related melanosome, and the process mainly depends on the product formed by tyrosinase catalyzing tyrosine and binding with the protein in the cytoplasm to form melanin particles. The melanin particles 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 after DAB coloration in immunohistochemical detection is very close to the color in clinical pathological diagnosis, so it is difficult to distinguish whether it is pigmentation or a positive result after conventional immunohistochemical DAB coloration.

[0004] The most commonly used depigmentation method at present is to immerse in 0.1% permanganate potassium solution for 2-4 hours, and then treat with 1% oxalic acid for 1 minute, and then wash with water for 5 minutes to achieve the purpose of removing the pigment in melanoma tissue. The existing method has the following disadvantages in the depigmentation process:

[0005] 1. The existing method needs at least 2-4 hours in the depigmentation process, which is not conducive to rapid results;

[0006] 2. The existing method mainly uses strong oxidizing agent to immerse and wash to remove the pigment, which has a relatively large damage to the morphology and structure of the tissue cells, and is not conducive to the subsequent H&E and IHC staining;

[0007] 3. The existing method needs to be depigmented first and then dyed, which needs to separate the two processes, which is more troublesome in actual work.

[0008] Therefore, it is necessary to provide a depigmentation solution and method which can save depigmentation time, do not damage the morphology and structure of the cells and tissues, and are simple to operate. SUMMARY

[0009] In order to solve at least one of the above problems, the application provides a melanoma tissue depigmentation method, kit and application, which simplifies the melanin depigmentation process, improves the efficiency, and the depigmentation result does not affect the morphology of the tissue cells, does not affect the IHC staining, and is suitable for full-automatic immunohistochemical staining instrument, and can realize the automation of depigmentation.

[0010] In order to achieve the above object, the present application adopts the following technical means: The first aspect of the present application provides a method for depigmentation of melanoma tissue in immunohistochemical staining. In the process of immunohistochemical staining, the depigmentation operation is performed after the antigen repair step.

[0011] In some embodiments of the present application, the depigmentation operation is performed by using hydrogen peroxide-methanol solution as the depigmentation liquid and by heating incubation. In some embodiments of the present application, the temperature of the heating incubation is 55-75℃.

[0012] In some embodiments of the present application, the hydrogen peroxide-methanol solution with a volume fraction of 5-15% is used as the depigmentation liquid. In some embodiments of the present application, the incubation time is 10-20 minutes. In some embodiments of the present application, the hydrogen peroxide-methanol solution with a volume fraction of 5-15% is used as the depigmentation liquid, and the incubation is performed at a temperature of 55-75℃ for 10-20 minutes. In some embodiments of the present application, preferably, the concentration of the hydrogen peroxide-methanol solution is 10% by volume. In some embodiments of the present application, preferably, the incubation temperature is 65℃. In some embodiments of the present application, preferably, the incubation time is 10 minutes.

[0013] The second aspect of the present application provides the use of hydrogen peroxide-methanol solution in the preparation of a product for diagnosing melanoma. The product is a diagnostic reagent or a kit.

[0014] In some embodiments of the present application, the volume fraction of the hydrogen peroxide-methanol solution is 5-15%.

[0015] In some embodiments of the present application, the product is used in the immunohistochemical staining of melanoma tissue by using a full-automatic immunohistochemical staining instrument.

[0016] In some embodiments of the present application, after the antigen repair step, the hydrogen peroxide-methanol solution with a volume fraction of 5-15% is used as the depigmentation liquid, and the depigmentation is performed by heating incubation. In some embodiments of the present application, the heating is performed to a temperature of 55-75℃, and the incubation is performed for 10-20 minutes.

[0017] Advantages of the present application

[0018] Compared with the prior art, the present application has the following beneficial effects: the present application provides a new depigmentation method, which optimizes the concentration, temperature and incubation time of the hydrogen peroxide methanol solution used in the existing full-automatic immunohistochemical staining process, so as to achieve the depigmentation of the pigment in the melanoma tissue while blocking the endogenous peroxidase in the on-board immunohistochemical staining process. The depigmentation process is integrated into the immunohistochemical staining step of the full-automatic immunohistochemical staining equipment, so that the depigmentation and immunohistochemical staining are completed in one process, without affecting the normal immunohistochemical staining process and results. The problems of the morphological influence of the pre-depigmentation and the strong oxidant depigmentation on the tissue cells of the melanoma tissue are solved. In this way, the time of depigmentation is saved, the steps of immunohistochemical staining of the tissue are simplified, good immunohistochemical staining results are obtained, the automation of the immunohistochemical staining of the melanoma tissue is realized, and the work efficiency is improved. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The depigmentation effects under different temperature incubation conditions in the embodiment 1 of the present application are shown;

[0020] Figure 2 The staining results under different treatment conditions of different concentrations of depigmentation solution in the embodiment 2 of the present application are shown;

[0021] Figure 3 The staining results under different treatment time conditions in the embodiment 3 of the present application are shown;

[0022] Figure 4 The staining results under different treatment method conditions in the embodiment 5 of the present application are shown. DETAILED DESCRIPTION

[0023] The following examples are presented to demonstrate preferred embodiments of the present application. Those skilled in the art will appreciate that the technology disclosed represents the inventors' current knowledge of the technology that can be used to practice the present application and, as such, is considered to be the preferred way of practicing the present application. However, those skilled in the art will appreciate from the present description that many modifications of the particular embodiments disclosed herein can be made without departing from the spirit or scope of the present application.

[0024] 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 this application belongs. The materials, methods, and examples provided herein are illustrative only and are not intended to be limiting on the scope of the application. One skilled in the art will recognize many methods and materials as being suitable for the practice of this application.

[0025] The technical solutions of the patent will be further described in detail in combination with specific embodiments.

[0026] Reagent materials

[0027] The reagent materials used in the examples in the present application are shown in Table 1 below.

[0028] Table 1 Reagent materials

[0029]

[0030] Example 1 Incubation temperature screening

[0031] During the experiment, the inventor accidentally turned on the heating mode in the closed process, and found that the malignant skin melanoma had a certain degree of depigmentation. Therefore, the conventional 3% hydrogen peroxide methanol solution was used to block at different temperatures on the automatic immunohistochemical staining instrument, and the depigmentation effect of 3% hydrogen peroxide methanol solution on melanoma pigment under the conditions of scheme 1: 45℃, scheme 2: 55℃, scheme 3: 65℃, scheme 4: 75℃ and scheme 5: 85℃ was compared (no DAB coloration was added in this stage).

[0032] The operation steps are as follows:

[0033] (a) Set the steps and time of the staining process on the automatic immunohistochemical staining instrument;

[0034] (b) Load the melanoma slides into the automatic immunohistochemical staining instrument;

[0035] (c) Perform depigmentation staining on the automatic immunohistochemical staining instrument according to the process in Table 2 below,

[0036] Table 2 Automatic immunohistochemical staining instrument working process

[0037]

[0038] The test results are shown in Table 3. Figure 1

[0039] The results show that the conventional immunohistochemical blocking 3% hydrogen peroxide methanol solution is used to study whether there is a depigmentation effect on melanoma at different heating temperatures (instrument heating temperature deviation ±1℃), and it is found that there is no depigmentation effect at 45℃, the depigmentation effect is not obvious at 55℃, there is a certain depigmentation effect at 65℃-75℃, and when the incubation temperature reaches 85℃, the tissue has serious depigmentation.

[0040] Example 2 Hydrogen peroxide concentration screening

[0041] ​Select a case of more melanin deposition of malignant skin melanoma as a sample, according to different hydrogen peroxide concentration to prepare hydrogen peroxide methanol solution, in the automatic immunohistochemical staining instrument, compare the effect of different hydrogen peroxide concentration on melanin depigmentation, this embodiment does not add DAB coloration, only use hematoxylin staining, the incubation temperature of depigmentation solution is 65 DEG C, and the time is 20 minutes; 100 mL of different concentration of hydrogen peroxide methanol solution is prepared, and the preparation scheme is shown in table 3.

[0042] Table 3 hydrogen peroxide methanol solution with different concentrations

[0043]

[0044] According to the following operation steps:

[0045] (a) adopt LYNX480 full-automatic immunohistochemical staining instrument of Huglog (Hangzhou) biological medicine Co., Ltd., set the steps and time of the staining process needed;

[0046] (b) the melanoma sample slide without pre-decoloring treatment is loaded into the full-automatic immunohistochemical staining instrument;

[0047] (c) according to the following table 4 process, the depigmentation staining is carried out on the full-automatic immunohistochemical staining instrument.

[0048] Table 4 immunohistochemical process

[0049]

[0050] The detection results are shown in table 5 and as follows: Figure 2

[0051] Table 5 depigmentation results

[0052]

[0053] The results show that: 10%-15% hydrogen peroxide methanol solution can completely remove the pigment under the condition of heating incubation at 65 DEG C for 20 minutes.

[0054] Example 3 incubation time screening

[0055] Select a case of more melanin deposition of melanoma tissue sample, use 10% hydrogen peroxide methanol solution to carry out depigmentation verification, except that the depigmentation solution incubation time is different, the rest is according to the staining process of example 2, the depigmentation solution incubation time is 3 minutes, 5 minutes, 10 minutes and 20 minutes respectively.

[0056] The effect of 10% hydrogen peroxide methanol solution on depigmentation under the condition of different incubation time at 65 DEG C is detected by using this embodiment, and the detection results are shown in table 6 and Figure 3 ​shown.

[0057] Table 6 detection results

[0058]

[0059] The results show that 10% hydrogen peroxide methanol solution can completely remove pigments at 65°C for 10-20 minutes. In order to save time, 10 minutes is preferred.

[0060] Example 4 Influence of heating conditions on depigmentation effect

[0061] Select a melanoma tissue sample with more melanin deposition, treat it with 10% and 15% hydrogen peroxide methanol solution for 10 minutes without heating, and verify the depigmentation. The rest of the immunohistochemical process is the same as Example 2.

[0062] The results show that 10% hydrogen peroxide methanol solution and 15% hydrogen peroxide methanol solution have no change in pigmentation without heating.

[0063] Example 5 IHC verification

[0064] Select rabbit anti-human cytokeratin (broad spectrum) antibody reagent as the primary antibody for detection.

[0065] The same malignant melanoma tissue sample was taken for two experiments:

[0066] Scheme 1: Use the staining method of the present application for staining experiment, directly put the malignant melanoma tissue sample into the machine for 10% hydrogen peroxide methanol solution incubation at 65°C for 10 minutes for depigmentation and staining.

[0067] Scheme 2: Use the traditional method for staining experiment, soak in 0.1% potassium permanganate solution for 2 hours, then treat with 1% oxalic acid solution for 1 minute for depigmentation, and then put into the machine for staining.

[0068] The specific steps are shown in Table 7.

[0069] Table 7 Experimental process of different schemes

[0070]

[0071] In the above table, 11-1 is the blocking + depigmentation step, and 11-2 is the blocking step.

[0072] The staining results are shown in Table 8 and Figure 4 shown.

[0073] Table 8 Detection results of different schemes

[0074] The results show that the cell morphology of the tissue after the endogenous peroxidase is blocked and the melanin is decolorized by using 10% hydrogen peroxide methanol solution in the process of immunohistochemical staining is obviously better than that of the traditional method, and the IHC staining effect is also better than that of the traditional method, without the need of pre-decoloring and strong oxidant decoloring of the tissue cells, thereby reducing the operation steps and saving about 2 hours of time.

[0075] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits disclosed herein.

Claims

1. A method for depigmenting melanoma tissue during immunohistochemical staining, characterized in that, In the immunohistochemical staining process, hydrogen peroxide methanol solution is used for heating and incubation in the destaining and blocking steps after the antigen retrieval step. This process blocks endogenous peroxidase while destaining the pigment in melanoma tissue. The heating temperature is 65℃-75℃; the volume fraction of the hydrogen peroxide methanol solution is 10%-15%; and the incubation time is 10-20 minutes.

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