Antigen protection type melanoma specimen decoloring kit and application thereof

Abstract

This invention discloses an antigen-protective melanoma specimen decolorization kit and its application, relating to the biological and medical fields. The kit consists of two separately packaged components: Component A is a lyophilized decolorizing agent powder composed of potassium permanganate, sodium acetate, and mannitol, with each component accounting for 0.1% of the total mass before lyophilization, 0.2% of the total mass of potassium permanganate, and 1.0% of the total mass of sodium acetate and mannitol, respectively. This antigen-protective melanoma specimen decolorization kit uses a lyophilized powder form for Component A and a 5x concentrated solution form for Component B, solving stability problems such as easy decomposition of potassium permanganate solution and easy crystallization of oxalate solution. Accelerated stability tests show that the pH value, effective concentration, and decolorization efficiency of the working solution after reconstitution / dilution do not change significantly, and the batch-to-batch CV value is <5%, meeting the quality control requirements of commercially available kits.

Description

Technical Field

[0001] This invention relates to biological and medical technologies, specifically to an antigen-protective melanoma specimen destaining kit and its application. Background Technology

[0002] Accurate pathological diagnosis of melanoma heavily relies on morphological observation and immunophenotypic analysis of FFPE tissue sections. However, the melanin-rich granules within tumor cells can obscure nuclear details in routine hematoxylin and eosin (H&E) staining, and in IHC staining using 3,3'-diaminobenzidine (DAB), their brownish color can easily be confused with positive signals, leading to diagnostic difficulties, false positives, or false negatives. Therefore, destaining melanin-rich specimens is a crucial pretreatment step to improve diagnostic accuracy.

[0003] In recent years, various methods have been developed for depigmenting melanin, mainly including chemical oxidation methods (such as potassium permanganate and hydrogen peroxide) and heterochromatic staining methods. However, while strong oxidants destroy melanin, they inevitably generate a large number of reactive oxygen species and cause drastic fluctuations in the pH of the treatment solution, thereby disrupting the three-dimensional conformation of proteins (antigens) and resulting in weakened or completely lost staining signals of key markers (such as S100, HMB45, and Melan-A) in subsequent IHC. Summary of the Invention

[0004] The purpose of this invention is to provide an antigen-protective melanoma specimen destaining kit and its application, in order to solve the problem in the prior art that a large number of reactive oxygen free radicals are generated, which leads to drastic fluctuations in the pH of the treatment solution, thereby destroying the three-dimensional conformation of proteins (antigens) and weakening or completely losing the staining signals of key markers (such as S100, HMB45, Melan-A) in IHC.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an antigen-protective melanoma specimen destaining kit, comprising separately packaged reagent A and reagent B:

[0006] Agent A is an oxidative decolorizing agent lyophilized powder, composed of potassium permanganate, sodium acetate, and mannitol, wherein the proportions of each component to the total mass before lyophilization are as follows: potassium permanganate 0.1%, sodium acetate 0.2%, and mannitol 1.0%.

[0007] Agent B is a 5-fold concentrated antigen-protective reducing solution. Its stock solution is composed of sodium oxalate, sodium citrate, poloxamer F68 and ascorbate palmitate, wherein the weight-volume concentration of each component in the stock solution is: sodium oxalate 1.5%, sodium citrate 1.0%, poloxamer F68 0.05%, and ascorbate palmitate 0.01%.

[0008] Furthermore, the working solution obtained after reconstitution of the lyophilized agent A has a pH value of 5.2 ± 0.1.

[0009] Furthermore, the working solution obtained by diluting the original solution of agent B by 5 times has a pH value of 4.5 ± 0.3.

[0010] Furthermore, the mass concentration ratio of poloxamer F68 to ascorbate palmitate in the working solution of agent B is (5±1):1.

[0011] A method for destaining melanoma FFPE tissue sections using the kit described above, comprising the following sequential steps:

[0012] S1. Redissolve the freeze-dried powder of agent A in sterile water to prepare an oxidation working solution;

[0013] S2. Immerse the tissue sections in the oxidation working solution and incubate at 36-40°C for 40-60 minutes;

[0014] S3. Wash the slides with buffer solution;

[0015] S4. Dilute the concentrated solution of agent B with sterile water to prepare a reducing working solution;

[0016] S5. Immerse the slices processed in step S3 into the reduction working solution and incubate at 18-28°C for 10-15 minutes.

[0017] S6. Wash the slide with buffer solution.

[0018] Furthermore, the incubation temperature in step S2 is 38±1℃.

[0019] Furthermore, the incubation temperature in step S5 is room temperature (22±3℃).

[0020] The application of an antigen-protective melanoma specimen destaining kit as described above in the preparation of formulations for the pathological diagnosis of melanoma, the application comprising destaining melanin-rich FFPE tissue sections prior to immunohistochemical staining, special staining, or nucleic acid extraction.

[0021] Compared with existing technologies, the antigen-protective melanoma specimen destaining kit and its application provided by this invention have the following beneficial effects:

[0022] Poloxamer F68, a nonionic block copolymer surfactant, primarily functions to stabilize the microenvironment of cell membranes and hydrophilic proteins, preventing their denaturation. Ascorbate palmitate, a lipid-soluble antioxidant, effectively penetrates and scavenges free radicals in the hydrophobic regions of tissues. In the stable, weakly acidic environment provided by the sodium citrate-sodium oxalate buffer system, the two achieve spatial synergistic protection of membrane stability in the hydrophilic region and targeted antioxidant protection in the hydrophobic region.

[0023] Agent A is a lyophilized powder, and Agent B is a 5x concentrated solution. This solves the stability problems such as easy decomposition of potassium permanganate solution and easy crystallization of oxalate solution. Accelerated stability tests show that the pH value, effective concentration and decolorization efficiency of the working solution after reconstitution / dilution do not change significantly, and the batch-to-batch CV value is <5%, which meets the quality control requirements of commercial kits.

[0024] Samples treated with the kit of this invention not only achieve high-quality IHC staining, but also have better protection of protein and nucleic acid integrity. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0026] Figure 1 This is a graph showing the consistency analysis of Melan-A staining results after three different batches of the kit from this invention were used to treat the same sample.

[0027] Figure 2 This is a flowchart illustrating a method for decolorizing melanoma FFPE tissue sections using an antigen-protected melanoma specimen decolorization kit, as provided in an embodiment of the present invention. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] As attached Figure 1 and Figure 2 As shown:

[0030] Example 1:

[0031] This invention provides an antigen-protective melanoma specimen destaining kit, comprising separately packaged reagent A and reagent B:

[0032] Agent A is an oxidizing decolorizing agent lyophilized powder, composed of potassium permanganate, sodium acetate, and mannitol, wherein the proportions of each component to the total mass before lyophilization are as follows: potassium permanganate 0.1%, sodium acetate 0.2%, and mannitol 1.0%.

[0033] Agent B is a 5-fold concentrated antigen-protective reducing solution. Its stock solution consists of sodium oxalate, sodium citrate, poloxamer F68, and ascorbate palmitate. The weight-volume concentrations of each component in the stock solution are: sodium oxalate 1.5%, sodium citrate 1.0%, poloxamer F68 0.05%, and ascorbate palmitate 0.01%.

[0034] Potassium permanganate in the lyophilized powder of the oxidizing decolorizing agent (A) oxidizes melanin granules in tissue sections into soluble, colorless products. Sodium acetate provides a buffer system to maintain a stable, weakly acidic environment required for the oxidation process, while mannitol acts as a lyophilization protectant and free radical scavenger, maintaining the stability of potassium permanganate during lyophilization and storage and reducing excessive damage to tissue antigens by the oxidant during decolorization. Subsequently, sodium oxalate in the antigen-protective reducing solution (5-fold concentrated) of agent B reduces and removes residual potassium permanganate and manganese dioxide precipitates. Sodium citrate further provides a buffer and chelates metal ions, while poloxamer F68 and ascorbyl palmitate work synergistically. The former acts as a surfactant to maintain membrane structure stability and reduce non-specific adsorption, while the latter acts as a lipid-soluble antioxidant to directly protect tissue antigens, especially lipid-related antigenic epitopes, from oxidative damage. This process efficiently removes melanin while maximizing the preservation of antigen immunogenicity, providing a clear background and reliable antigen signals for subsequent immunohistochemical and other detection methods.

[0035] The working solution obtained after reconstitution of the lyophilized powder A has a pH value of 5.2 ± 0.1.

[0036] This pH value ensures that the oxidation reaction of potassium permanganate on melanin particles proceeds at a moderate rate, achieving effective decolorization while avoiding excessively vigorous oxidation or the formation of large amounts of manganese dioxide precipitate under overly acidic or alkaline conditions. This reduces irreversible damage to tissue microstructure and protein antigens caused by excessive oxidation or precipitate adhesion, creating favorable conditions for subsequent reduction steps.

[0037] The working solution obtained by diluting the original solution of agent B by 5 times has a pH value of 4.5 ± 0.3.

[0038] The pH value is maintained by sodium oxalate and sodium citrate. This weakly acidic environment is conducive to the efficient reduction of residual potassium permanganate and manganese dioxide by sodium oxalate, converting them into soluble manganese ions for elution. On the other hand, it can inhibit some adverse side reactions that may occur in the reduction step. At the same time, this pH range is compatible with the suitable conditions for antigen preservation. Combined with the protective components in the formula, it can further stabilize tissue antigens and ensure that the immunoreactivity of antigens is not impaired during the removal of oxidant residues and precipitates.

[0039] The mass concentration ratio of poloxamer F68 to ascorbate palmitate in the working solution of agent B is (5±1):1.

[0040] Poloxamer F68, as a nonionic surfactant, primarily forms a protective layer at the tissue-fluid interface through its amphiphilic structure, stabilizing cell membrane structure and reducing nonspecific protein denaturation. Ascorbyl palmitate, as a lipid-soluble antioxidant, can effectively penetrate into the lipid region of tissues, neutralizing lipid peroxidation free radicals and protecting lipid-related antigens. By controlling the concentrations of both within a specific ratio, the optimal balance between surfactant protection and lipid-soluble antioxidant protection can be ensured, thereby providing comprehensive and balanced protection for tissue antigens during the reduction step and avoiding decreased protective efficacy or potential interference caused by excessive or insufficient amounts of a single component.

[0041] A method for destaining melanoma FFPE tissue sections using a kit includes the following sequential steps:

[0042] S1. Redissolve the freeze-dried powder of agent A in sterile water to prepare an oxidation working solution;

[0043] S2. Immerse the tissue sections in the oxidizing working solution and incubate at 36-40℃ for 40-60 minutes;

[0044] S3. Wash the slides with buffer solution;

[0045] S4. Dilute the concentrated solution of agent B with sterile water to prepare a reducing working solution;

[0046] S5. Immerse the slices processed in step S3 into the reducing working solution and incubate at 18-28℃ for 10-15 minutes.

[0047] S6. Wash the slide with buffer solution.

[0048] First, in steps S1 and S2, the reconstituted Agent A oxidizing working solution is applied to the tissue sections under gentle heating conditions of 36 to 40 degrees Celsius. This combination of temperature and time promotes the complete oxidation and dissolution of melanin granules by potassium permanganate. The buffer washing in step S3 aims to remove most of the oxidizing agent and reaction products. Subsequently, in steps S4 and S5, the diluted Agent B reducing working solution is incubated at near room temperature conditions of 18 to 28 degrees Celsius. These gentle conditions facilitate the effective removal of residual oxidizing agents and precipitates by reducing agents such as sodium oxalate, while maximizing the antigenic protective functions of poloxamer F68 and ascorbyl palmitate. The final washing step S6 thoroughly removes all destaining reagent residues, ultimately yielding tissue sections with destaining melanin and well-preserved antigens, ready for subsequent staining or extraction.

[0049] The incubation temperature in step S2 is 38±1℃.

[0050] Precisely controlling the temperature of the oxidative decolorization process within a narrow range is to achieve the optimal balance between decolorization efficiency and antigen protection. This temperature is slightly higher than room temperature, which can effectively accelerate the redox reaction kinetics between potassium permanganate and melanin, ensuring that sufficient decolorization is completed within the specified incubation time of 40 to 60 minutes, while avoiding the risk of runaway oxidation reaction, thermal denaturation of tissue antigens, or destruction of tissue morphology due to excessively high temperatures.

[0051] The incubation temperature in step S5 is room temperature (22±3℃).

[0052] Preparation of Agent A (oxidative decolorizing lyophilized powder):

[0053] Weigh out 0.4g of potassium permanganate, 0.8g of sodium acetate, and 4.0g of mannitol, and place them in a clean beaker;

[0054] Add approximately 390 mL of sterile water for injection filtered through a 0.22 μm filter membrane, and stir at 500 rpm using a magnetic stirrer (IKA RCTbasic) until completely dissolved;

[0055] The pH was precisely adjusted to 5.20 with 1M acetic acid solution, transferred to a 500 mL volumetric flask, and brought to a final volume of 400 mL with sterile water for injection. The solution was then aseptically filtered through a 0.22 μm polyethersulfone (PES) membrane.

[0056] Dispense the filtrate into 10 mL neutral borosilicate glass vials at a rate of 4.0 mL per vial (corresponding to a final preparation of 400 mL working solution), and partially press the rubber stopper.

[0057] Placed in a freeze dryer (Christ Alpha 2-4 LDplus), the freeze-drying process was performed as follows: pre-freezing to -45°C and holding for 3 hours; first stage drying: shelf temperature increased to -25°C, vacuum maintained at 0.1 mbar, drying for 30 hours; second stage desorption drying: shelf temperature slowly increased to 25°C and held for 10 hours. The freeze-dried cake was then sealed with a cap to obtain a uniform purple freeze-dried cake.

[0058] Preparation of Agent B (5× Antigen Protective Reducing Concentrate):

[0059] Weigh out 12.0g of sodium oxalate, 8.0g of sodium citrate, and 0.4g of pharmaceutical grade poloxamer F68;

[0060] First, dissolve sodium citrate in 700 mL of sterile water for injection. Then, add sodium oxalate and poloxamer F68 in sequence while stirring until completely clear.

[0061] Separately dissolve 0.08 g of ascorbate palmitate in 2 mL of anhydrous ethanol. While stirring at 800 rpm, slowly add the ethanol solution dropwise to the above aqueous solution. Dilute to 800 mL with sterile water for injection; the pH of the solution at this point is approximately 4.5.

[0062] Sterilize by filtration through a 0.22 μm PES membrane, dispense 80 mL of each into 100 mL brown amber glass bottles, fill with high-purity nitrogen, and immediately seal with butyl rubber stoppers and aluminum-plastic caps. Store away from light.

[0063] Example 2:

[0064] This embodiment is basically the same as the previous embodiment, except that an antigen-protective melanoma specimen destaining kit is used in the preparation of a formulation for the pathological diagnosis of melanoma. The application includes destaining melanin-rich FFPE tissue sections before immunohistochemical staining, special staining, or nucleic acid extraction.

[0065] The kit first removes melanin granules from melanin-rich FFPE tissue sections that severely interfere with optical microscopy. Its unique two-step formulation and process protect the integrity of antigenic epitopes and nucleic acids in the tissue while decolorizing. This allows the decolorized sections to be directly used for immunohistochemical staining to detect specific tumor markers, for special staining to reveal other tissue structures, or for nucleic acid extraction for molecular detection, thereby significantly improving the accuracy and reliability of pathological diagnosis. Therefore, this kit is a key component in the preparation of pretreatment agents for assisting in the pathological diagnosis of melanoma.

[0066] Decolorization effect, antigen protection effect and molecular compatibility verification

[0067] Samples: Three cases of pathologically confirmed cutaneous malignant melanoma FFPE tissue blocks containing a large amount of melanin were selected, and 18 serial sections (4 μm thick) were prepared from each case.

[0068] Grouping and processing:

[0069] Group 1 (unbleached control): Subsequent staining was performed directly.

[0070] Group 2 (traditional method control): oxidation with 0.25% potassium permanganate solution for 20 min, followed by reduction with 1% oxalic acid for 2 min.

[0071] Group 3: Strictly follow the instructions. Work solution A (prepared from reagent A in Example 1) was incubated at 38°C for 50 min and washed 3 times with PBS; work solution B (prepared from reagent B in Example 1) was incubated at room temperature for 12 min and washed 3 times with PBS.

[0072] Tests and Results:

[0073] IHC staining: Each slide was stained with HMB45 (clone number HMB45), Melan-A (clone number A103), and S100 (clone number EP32) using a Leica BOND-III fully automated staining system and DAB staining system. The staining was performed in a double-blind manner by two senior pathologists.

[0074] Results: Group 3 showed the strongest specific signal intensity and the lowest background staining in all antibody stainings, significantly better than Group 2. Group 2 showed obvious signal attenuation due to antigen damage.

[0075] Image analysis and quantification: Ten high-power fields (400X) were randomly selected from HMB45 stained sections using the pathological image analysis system (Image-Pro Plus 10.0) to measure the mean optical density (MOD) of the positive areas.

[0076] Results: The MOD value of group 3 (mean ± SD: 0.52 ± 0.05) was significantly higher than that of group 1 (0.21 ± 0.08, p < 0.01) and group 2 (0.28 ± 0.06, p < 0.01). This quantitative data constitutes the core part of the SGS test report.

[0077] Nucleic acid extraction and quality analysis: Take another slice from each group and extract total RNA using the FFPE RNA extraction kit (Qiagen). Detect RNA integrity (RIN) using an Agilent 2100 bioanalyzer.

[0078] Results: The average RIN value of RNA extracted from group 3 was 6.5, which was significantly higher than that of group 2 (3.8), indicating that the method of the present invention causes less damage to nucleic acids and is compatible with downstream molecular detection.

[0079] Comparative example:

[0080] To demonstrate the synergistic effect of poloxamer F68 and ascorbate palmitate, the following reducing working solution was prepared (the treatment conditions for solution A were the same as those in group 3 of Example 2):

[0081] Comparative Example 1: Basic reducing solution (0.3% sodium oxalate + 0.2% sodium citrate).

[0082] Comparative Example 2: Base reducing solution + 0.01% poloxamer F68.

[0083] Comparative Example 3: Basic reducing solution + 0.002% ascorbyl palmitate.

[0084] Comparative Example 4: Base reducing solution + 0.01% poloxamer F68 + 0.002% ascorbate palmitate.

[0085] HMB45IHC staining and MOD value determination were performed on the same melanoma sample after processing.

[0086] Results: The MOD value of Comparative Example 4 was significantly higher than that of Comparative Examples 2 and 3 (p < 0.01), while the MOD values ​​of the latter two were higher than those of Comparative Example 1. This data strongly confirms that the addition of the two protective components produced a synergistic effect beyond simple addition.

[0087] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. An antigen-protective melanoma specimen decoloring kit, characterized by, Includes separately packaged Agent A and Agent B: Agent A is an oxidative decolorizing agent lyophilized powder, composed of potassium permanganate, sodium acetate, and mannitol, wherein the proportions of each component to the total mass before lyophilization are as follows: potassium permanganate 0.1%, sodium acetate 0.2%, and mannitol 1.0%. Agent B is a 5-fold concentrated antigen-protective reducing solution. Its stock solution is composed of sodium oxalate, sodium citrate, poloxamer F68 and ascorbate palmitate, wherein the weight-volume concentration of each component in the stock solution is: sodium oxalate 1.5%, sodium citrate 1.0%, poloxamer F68 0.05%, and ascorbate palmitate 0.01%.

2. The antigen-protected melanoma specimen decolorizing kit according to claim 1, characterized by, The working solution obtained after reconstitution of the lyophilized powder A has a pH value of 5.2 ± 0.

1.

3. The antigen-protected melanoma specimen decolorizing kit according to claim 1, characterized by, The working solution obtained by diluting the original solution of agent B by 5 times has a pH value of 4.5 ± 0.

3.

4. The antigen-protected melanoma specimen decolorizing kit according to claim 1, characterized by, The mass concentration ratio of poloxamer F68 to ascorbate palmitate in the working solution of agent B is (5±1):

1.

5. A method for destaining melanoma FFPE tissue sections using the kit described in any one of claims 1-4, characterized in that, Includes the following sequential steps: S1. Redissolve the freeze-dried powder of agent A in sterile water to prepare an oxidation working solution; S2. Immerse the tissue sections in the oxidation working solution and incubate at 36-40°C for 40-60 minutes; S3. Wash the slides with buffer solution; S4. Dilute the concentrated solution of agent B with sterile water to prepare a reducing working solution; S5. Immerse the slices treated in step S3 into the reducing working solution and incubate at 18-28°C for 10-15 minutes. S6. Wash the slide with buffer solution.

6. A method for destaining melanoma FFPE tissue sections using a kit according to claim 5, characterized in that, The incubation temperature in step S2 is 38±1℃.

7. A method for destaining melanoma FFPE tissue sections using a kit according to claim 5, characterized in that, The incubation temperature in step S5 is room temperature (22±3℃).

8. The use of the antigen-protective melanoma specimen destaining kit as described in any one of claims 1-4 in the preparation of a formulation for the pathological diagnosis of melanoma, characterized in that, The application includes destaining melanin-rich FFPE tissue sections before immunohistochemical staining, special staining, or nucleic acid extraction.

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