Depigmentation method suitable for melanoma gene detection

Abstract

The invention discloses a depigmentation method suitable for melanoma gene detection, and belongs to the technical field of molecular pathology, the depigmentation method comprises the following steps: pre-treating a melanoma sample, adding into a first part of treating fluid, soaking at 80 DEG C for 20 minutes, adding into a second part of treating fluid, soaking at 80 DEG C for 10 minutes, washing with water, and airing; a preparation method of the first part of treating fluid comprises the following steps: diluting H2O2 with a Tris-HCl buffer solution until the volume concentration of H2O2 is 1%, and then adding NaCl until the mass concentration of NaCl is 1% to obtain the first part of treating fluid; according to the depigmentation method, rapid and efficient depigmentation can be achieved for melanoma with different melanin contents, the DNA integrity and amplification efficiency of the melanoma treated through the depigmentation method are high, and the problem of depigmentation is not prone to occurring.

Description

Technical Field

[0001] This invention relates to the field of molecular pathology technology, specifically to a depigmentation method suitable for melanoma gene detection. Background Technology

[0002] Melanoma, a highly aggressive malignant tumor originating from melanocytes of the neural crest, exhibits significant anatomical heterogeneity, affecting multiple tissues including the skin and its appendages, mucosal epithelium (gastrointestinal / respiratory / genitourinary tracts, etc.), the uvea of ​​the eye, and the pia mater. Melanoma contains numerous high-density brownish-black to dark brown pigment granules. During HE and immunohistochemical staining, these pigment granules can obscure the tumor's tissue structure, hindering accurate diagnosis; therefore, depigmentation treatment is necessary. These pigment granules are sulfur-containing, iron-free pigments formed by melanocytes. They are highly stable, insoluble in water and organic solvents, but can be dissolved in strong alkalis and bleached by strong oxidizing agents.

[0003] The current Clinical Practice Guidelines for the Pathological Diagnosis of Melanoma (2021 Edition) emphasize that gene testing based on clinically significant molecular targets such as C-KIT and PDGFRA—i.e., molecular diagnostic techniques—has become an important basis for guiding targeted therapy decisions and prognostic assessments. However, the large number of high-density brownish-to-dark brown pigment granules in melanoma can cause multiple molecular interference effects, including: first, deviations in nucleic acid quantification due to light absorption characteristics; second, covalent binding with DNA molecules, reducing nucleic acid purity; and third, competitive binding with Taq DNA polymerase, significantly inhibiting PCR amplification efficiency and thus increasing the risk of false negatives.

[0004] For melanoma, compared to methods that diagnose tumors based on histological morphology (such as HE staining and immunohistochemical staining), methods that diagnose tumors based on molecular diagnostic techniques have higher requirements for depigmentation treatment. Moreover, different depigmentation treatment methods have a greater impact on the DNA integrity and amplification efficiency of melanoma after depigmentation treatment. Zhang Yuping, Wang Hui, Li Xiufeng, Wan Jilan, Zhang Yunxiang. Exploration of the application value of rapid and efficient depigmentation methods in molecular pathological gene detection of melanoma. Chinese Journal of Pathology, July 2025, Vol. 54, No. 7. This article published a systematic evaluation of the impact of four depigmentation methods—H2O2 method, potassium permanganate method, Tris-HCl method, and PBS method—on DNA integrity and amplification efficiency by combining Sanger sequencing and PCR dual-platform verification. The results showed that the Tris-HCl method can efficiently and rapidly remove pigment from FFPE melanoma tissue. The operation procedure is simple and easy to promote and apply in laboratory and clinical testing. Moreover, it can obtain high-quality DNA in molecular detection, significantly improve the amplification efficiency of target genes, and produce clear sequencing peaks without interference from impurities, thus improving the accuracy and reliability of gene detection results. Meanwhile, the optimal conditions for the Tris-HCl method in depigmenting melanoma were determined, namely, diluting H2O2 to a concentration of 1% using Tris-HCl buffer (pH 10), heating at 70°C for 30 min for depigmentation, washing with distilled water for 3 min, and air-drying at room temperature.

[0005] The aforementioned Tris-HCl method achieves rapid and efficient depigmentation of melanoma through a four-fold synergistic mechanism: first, HCl induces tyrosine acidolysis and oxidation; second, thermocatalysis of H2O2 generates hydroxyl radicals to enhance oxidation; third, a pH 10 buffer system maintains tissue acid-base balance; and fourth, heating at 70°C significantly improves depigmentation efficiency. However, the aforementioned Tris-HCl method has the following problems: First, Chen Yonghua, Lin Biyun, Li Xiaofang, Li Nanhong, Yuan Zhenxing. Effect of low-concentration hydrogen peroxide solution on tissue demelaninization at high temperatures. Journal of Clinical and Experimental Pathology. January 2020. The study published that higher temperatures and pH values ​​increase the likelihood of detachment. For the aforementioned Tris-HCl method, detachment occurs after depigmentation. The most common method to address this issue is to change the buffer system. The inventors attempted to change the Tris-HCl system to a Tris-citric acid system, but the depigmentation effect of the Tris-citric acid system was inferior to that of the Tris-HCl system.

[0006] Secondly, the article "Effect of EDTA-Hydrogen Peroxide Repair on Staining of Melanin-Rich Tumor Tissue" by Liu Dan, Liu Shengjun, Chen Yang, Ke Qi, and Wang Anqun, published in *Medical Theory and Practice*, Vol. 36, No. 24, 2023, discloses that H2O2 depigmentation is superior to potassium permanganate and other pigment staining methods. However, the problem of time-consuming methods remains unresolved. The depigmentation ability of H2O2 is closely related to temperature and treatment time; higher temperatures and longer treatment times result in more thorough depigmentation, but also more severe damage to tissue structure. This paper describes a repair method using boiling H2O2 in a 0.75% EDTA (pH 9) hydrogen peroxide solution for 20 minutes, achieving simple operation and uniform and thorough depigmentation, applicable regardless of melanin content. In contrast, the aforementioned Tris-HCl method, due to its low temperature, results in poor depigmentation in melanomas with high melanin content. Furthermore, the lack of EDTA repair affects the DNA integrity and amplification efficiency of the depigmented melanoma. The most common method to address this problem is to perform decolorization under boiling or microwave conditions, but both boiling and microwave conditions can exacerbate the problem of film separation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a depigmentation method suitable for melanoma gene detection. It can achieve rapid and efficient depigmentation for melanomas with different melanin contents. Melanomas treated with the depigmentation method of this invention have high DNA integrity and amplification efficiency, and are less prone to detachment problems.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A depigmentation method suitable for melanoma gene detection involves pre-treating a melanoma sample, adding it to a first treatment solution, soaking it at 80°C for 20 minutes, adding it to a second treatment solution, soaking it at 80°C for 10 minutes, washing it with water, and air-drying it. The pretreatment involved fixing the samples in 3.7% neutral formaldehyde, embedding them in paraffin, sectioning them into 4μm sections, preventing slide detachment, baking the slides at 65℃ for ≥1h, and then performing routine dewaxing. The first treatment solution was prepared by diluting H2O2 with Tris-HCl buffer to a volume concentration of 1%, and then adding NaCl to a mass concentration of 1% to obtain the first treatment solution. The second treatment solution was prepared by adding EDTA to Tris-HCl buffer until the concentration of EDTA was 1.2 mmol / L to obtain EDTA-containing Tris-HCl buffer, and then using EDTA-containing Tris-HCl buffer to dilute H2O2 to a volume concentration of 1% to obtain the second treatment solution. In the preparation of the first and second treatment solutions, the pH of the Tris-HCl buffer was 10 and the concentration of Tris was 100 mmol / L.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Sun Lijun, Huang Bin; Application of dual oxidation depigmentation method in immunohistochemical diagnosis of malignant melanoma. Chinese Journal of Cancer Prevention and Treatment. October 2012. This paper discloses a method that repairs and then oxidizes, enabling antigen repair and preventing flaking. Unlike this paper, this invention uses a first and second treatment solution for depigmentation. The first treatment solution does not use a highly oxidizing agent, reducing antigen damage and flaking. In addition, NaCl is added, which promotes the exposure of sulfur bonds in pigment particles, thereby promoting the oxidation of sulfur bonds by H2O2 and improving the efficiency of depigmentation. EDTA is added to the second treatment solution. EDTA has a repair function, enabling antigen repair during depigmentation and preventing flaking. By simultaneously performing oxidation and then oxidation repair, rapid and efficient depigmentation can be achieved for melanomas with different melanin contents, while ensuring DNA integrity and amplification efficiency, and minimizing flaking. (2) In the depigmentation method of the present invention, after depigmentation treatment of melanoma with a melanin content of 51-55%, the average comprehensive score of depigmentation and HE staining is 4, the average total DNA content is 14.65 μg, and the average proportion of DNA fragments >5000 bp is 60.2%. 260 / A 280 The average value is 1.92, A 260 / A 230 The average value was 2.14, and no detachment problem occurred; after depigmentation treatment of melanomas with a melanin content of 76-79%, the average combined score of depigmentation and HE staining was 4. Attached Figure Description

[0010] Figure 1 HE staining image of the sample with the lowest overall score for depigmentation and HE staining in Example 1; Figure 2 HE staining image of the sample with the lowest overall score for depigmentation and HE staining in Comparative Example 1; Figure 3 HE staining image of the sample with the lowest overall score for depigmentation and HE staining in Comparative Example 2; Figure 4 HE staining image of the sample with the lowest overall score for depigmentation and HE staining in Comparative Example 3; Figure 5 HE staining image of the sample with the lowest overall score for depigmentation and HE staining in Comparative Example 4; Figure 6 HE staining image of the sample with the lowest overall score for depigmentation and HE staining in Comparative Example 5; Figure 7 This is an agarose gel electrophoresis image of the C-KIT and PDGFRA target genes after amplification, from the sample with the lowest overall score for depigmentation and HE staining in Example 1. In the figure, lane M is the marker, representing the size of the amplified product fragment; lane 1 is C-KITExon 9; lane 2 is C-KITExon 11; lane 3 is C-KITExon 13; lane 4 is C-KITExon 17; lane 5 is PDGFRAExon 12; lane 6 is PDGFRAExon 14; and lane 7 is PDGFRAExon 18. Figure 8 The image shows the agarose gel electrophoresis results of the C-KIT and PDGFRA target genes after amplification in the sample with the lowest combined score of depigmentation and HE staining in Comparative Example 1. In the figure, lane M is the marker, representing the size of the amplified product fragment; lane 1 is C-KITExon 9; lane 2 is C-KITExon 11; lane 3 is C-KITExon 13; lane 4 is C-KITExon 17; lane 5 is PDGFRAExon 12; lane 6 is PDGFRAExon 14; and lane 7 is PDGFRAExon 18. Figure 9 The image shows the agarose gel electrophoresis results of the C-KIT and PDGFRA target genes after amplification in the sample with the lowest combined score of depigmentation and HE staining in Comparative Example 2. In the figure, lane M is the marker, representing the size of the amplified product fragment; lane 1 is C-KITExon 9; lane 2 is C-KITExon 11; lane 3 is C-KITExon 13; lane 4 is C-KITExon 17; lane 5 is PDGFRAExon 12; lane 6 is PDGFRAExon 14; and lane 7 is PDGFRAExon 18. Figure 10 The image shows the agarose gel electrophoresis results of the C-KIT and PDGFRA target genes after amplification in the sample with the lowest combined score of depigmentation and HE staining in Comparative Example 3. In the figure, lane M is the marker, representing the size of the amplified product fragment; lane 1 is C-KITExon 9; lane 2 is C-KITExon 11; lane 3 is C-KITExon 13; lane 4 is C-KITExon 17; lane 5 is PDGFRAExon 12; lane 6 is PDGFRAExon 14; and lane 7 is PDGFRAExon 18. Figure 11 The image shows the agarose gel electrophoresis results of the C-KIT and PDGFRA target genes after amplification in the sample with the lowest combined score of depigmentation and HE staining in Comparative Example 4. In the figure, lane M is the marker, representing the size of the amplified product fragment; lane 1 is C-KITExon 9; lane 2 is C-KITExon 11; lane 3 is C-KITExon 13; lane 4 is C-KITExon 17; lane 5 is PDGFRAExon 12; lane 6 is PDGFRAExon 14; and lane 7 is PDGFRAExon 18. Figure 12 The image shows the agarose gel electrophoresis results of the C-KIT and PDGFRA target genes after amplification in the sample with the lowest combined score of depigmentation and HE staining in Comparative Example 5. In the figure, lane M is the marker, representing the size of the amplified product fragment; lane 1 is C-KITExon 9; lane 2 is C-KITExon 11; lane 3 is C-KITExon 13; lane 4 is C-KITExon 17; lane 5 is PDGFRAExon 12; lane 6 is PDGFRAExon 14; and lane 7 is PDGFRAExon 18. Detailed Implementation

[0011] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0012] Twenty melanoma samples diagnosed between 2023 and 2024 at the Department of Pathology, Weifang People's Hospital were collected. Of these, 12 were male and 8 were female. HE-stained sections showed clearly visible tumor cells and melanin under a microscope. The melanin content in the tumor tissue was as follows: 4 cases 51%, 3 cases 52%, 6 cases 53%, 4 cases 54%, and 3 cases 55%. The patients' ages were: 6 cases 64 years, 10 cases 65 years, 1 case 66 years, and 3 cases 67 years. The lesions were located in the following locations: 3 cases of plantar masses, 5 cases of nasal masses, 4 cases of ear masses, 5 cases of thumb masses, and 3 cases of thigh skin masses. The 20 samples were then divided into 6 equal parts and depigmented according to the methods described in Example 1 and Comparative Examples 1-5.

[0013] Example 1 A depigmentation method suitable for melanoma gene detection specifically includes the following steps: 1. Pretreatment: Following the method published in Zhang Yuping, Wang Hui, Li Xiufeng, Wan Jilan, Zhang Yunxiang. Exploration of the application value of rapid and efficient depigmentation method in molecular pathological gene detection of melanoma. Chinese Journal of Pathology. July 2025, Vol. 54, No. 7, all 20 samples were fixed in 3.7% neutral formaldehyde, embedded in paraffin, sectioned at 4μm, baked at 65℃ for 1h, dewaxed with xylene, and xylene removed with anhydrous ethanol to obtain pretreated samples; 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods: The first treatment solution was heated to 80°C, and the pretreated sample was added. The sample was soaked at 80°C for 20 minutes. The sample was then removed. The second treatment solution was heated to 80°C, and the sample was added. The sample was soaked at 80°C for 10 minutes. The sample was then removed, washed with distilled water for 3 minutes, and air-dried at room temperature to obtain depigmented melanoma. The first treatment solution was prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% with a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L, and then adding NaCl to a mass concentration of 1% to obtain the first treatment solution. The second treatment solution was prepared as follows: EDTA was added to a Tris-HCl buffer with a pH of 10 and a Tris concentration of 100 mmol / L until the EDTA concentration was 1.2 mmol / L, to obtain a Tris-HCl buffer containing EDTA. Then, the 30% H2O2 aqueous solution was diluted with the Tris-HCl buffer containing EDTA to a volume concentration of 1% to obtain the second treatment solution.

[0014] Following the methods and scoring criteria published in Zhang Yuping, Wang Hui, Li Xiufeng, Wan Jilan, and Zhang Yunxiang, "An Exploration of the Application Value of Rapid and Efficient Depigmentation Method in Molecular Pathological Gene Detection of Melanoma," Chinese Journal of Pathology, July 2025, Vol. 54, No. 7, all depigmented melanomas were subjected to HE staining, DNA extraction and quality comparison, C-KIT gene Sanger sequencing, and PDGFRA gene Sanger sequencing. HE staining patterns, the combined score of depigmentation and HE staining (i.e., the average of the depigmentation score and the HE staining score), total DNA amount, the proportion of DNA fragments >5000bp, and A... 260 / A 280 Value, A 260 / A 230The values, agarose gel electrophoresis images of C-KIT target gene amplification, and agarose gel electrophoresis images of PDGFRA target gene amplification were statistically analyzed, and the number of detachment problems was recorded.

[0015] Comparative Example 1 A depigmentation method suitable for melanoma gene detection specifically includes the following steps: 1. Preprocessing: Twenty samples were preprocessed according to the method in Example 1 to obtain preprocessed samples; 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods: The first treatment solution was heated to 80°C, and the pretreated sample was added. The sample was soaked at 80°C for 20 minutes. The sample was then removed. The second treatment solution was heated to 80°C, and the sample was added. The sample was soaked at 80°C for 10 minutes. The sample was then removed, washed with distilled water for 3 minutes, and air-dried at room temperature to obtain depigmented melanoma. The first treatment solution was prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% with a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L, thus obtaining the first treatment solution. The second treatment solution was prepared as follows: EDTA was added to a Tris-HCl buffer with a pH of 10 and a Tris concentration of 100 mmol / L until the EDTA concentration was 1.2 mmol / L, to obtain a Tris-HCl buffer containing EDTA. Then, the 30% H2O2 aqueous solution was diluted with the Tris-HCl buffer containing EDTA to a volume concentration of 1% to obtain the second treatment solution.

[0016] The detection and scoring were performed according to the detection and statistical methods in Example 1, and the results were statistically analyzed.

[0017] Comparative Example 2 A depigmentation method suitable for melanoma gene detection specifically includes the following steps: 1. Preprocessing: Twenty samples were preprocessed according to the method in Example 1 to obtain preprocessed samples; 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods: The first treatment solution was heated to 80°C, and the pretreated sample was added. The sample was soaked at 80°C for 10 minutes. The sample was then removed. The second treatment solution was heated to 80°C, and the sample was added. The sample was soaked at 80°C for 20 minutes. The sample was then removed, washed with distilled water for 3 minutes, and air-dried at room temperature to obtain depigmented melanoma. The first treatment solution was prepared by adding EDTA to a Tris-HCl buffer with a pH of 10 and a Tris concentration of 100 mmol / L until the EDTA concentration was 1.2 mmol / L, to obtain a Tris-HCl buffer containing EDTA. Then, the 30% H2O2 aqueous solution was diluted with the Tris-HCl buffer containing EDTA to a volume concentration of 1% to obtain the second treatment solution. The second treatment solution was prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% using a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L, and then adding NaCl to a mass concentration of 1% to obtain the first treatment solution.

[0018] The detection and scoring were performed according to the detection and statistical methods in Example 1, and the results were statistically analyzed.

[0019] Comparative Example 3 A depigmentation method suitable for melanoma gene detection specifically includes the following steps: 1. Preprocessing: Twenty samples were preprocessed according to the method in Example 1 to obtain preprocessed samples; 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods: The first treatment solution was heated to 80°C, and the pretreated sample was added. The sample was soaked at 80°C for 20 minutes. The sample was then removed. The second treatment solution was heated to 80°C, and the sample was added. The sample was soaked at 80°C for 10 minutes. The sample was then removed, washed with distilled water for 3 minutes, and air-dried at room temperature to obtain depigmented melanoma. The first treatment solution was prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% with a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L, and then adding NaCl to a mass concentration of 1% to obtain the first treatment solution. The second treatment solution was prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% with a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L, thus obtaining the second treatment solution.

[0020] The detection and scoring were performed according to the detection and statistical methods in Example 1, and the results were statistically analyzed.

[0021] Comparative Example 4 A depigmentation method suitable for melanoma gene detection specifically includes the following steps: 1. Preprocessing: Twenty samples were preprocessed according to the method in Example 1 to obtain preprocessed samples; 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods: The treatment solution was heated to 80°C, the pretreated sample was added, and the sample was soaked at 80°C for 30 minutes. The sample was then removed, washed with distilled water for 3 minutes, and air-dried at room temperature to obtain depigmented melanoma. The treatment solution is prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% with a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L.

[0022] The detection and scoring were performed according to the detection and statistical methods in Example 1, and the results were statistically analyzed.

[0023] Comparative Example 5 A depigmentation method suitable for melanoma gene detection specifically includes the following steps: 1. Preprocessing: Twenty samples were preprocessed according to the method in Example 1 to obtain preprocessed samples; 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods: The treatment solution was heated to 70°C, the pretreated sample was added, and the sample was soaked at 70°C for 30 minutes. The sample was then removed, washed with distilled water for 3 minutes, and air-dried at room temperature to obtain depigmented melanoma. The treatment solution is prepared by diluting a 30% H2O2 aqueous solution to a volume concentration of 1% with a Tris-HCl buffer solution with a pH of 10 and a Tris concentration of 100 mmol / L.

[0024] The detection and scoring were performed according to the detection and statistical methods in Example 1, and the results were statistically analyzed.

[0025] Test Example 1 From the depigmented melanomas obtained in Example 1 and Comparative Examples 1-5, the sample with the lowest combined score of depigmentation and HE staining was selected. Its HE staining images are shown below. Figure 1-6 ;Depend on Figure 1-6 It can be seen that the HE staining effect of Example 1, Comparative Example 3, Comparative Example 4, and Comparative Example 5 is better.

[0026] Test Example 2 The average combined score of depigmentation and HE staining, the average total DNA amount (the average total DNA amount before depigmentation treatment was 15.31 μg), the average percentage of DNA fragments >5000 bp, and A in 20 samples from Examples 1 and Comparative Examples 1-5 were calculated. 260 / A 280 The average value of A (before depigmentation treatment) 260 / A 280 The average value is 1.59), A 260 / A 230 The average value of A (before depigmentation treatment) 260 / A 230 The average value is 0.89, and the statistical results of the number of cases with film detachment are as follows:

[0027] A 260 / A 280 Value and A 260 / A 230 The value is an indicator of nucleic acid purity. A pure sample has an A value of [value missing]. 260 / A 280 A value greater than 1.8 (DNA) or 2.0 (RNA) indicates that A 260 / A 280 A value below 1.8 or 2.0 indicates the presence of proteins or phenolic substances in the sample. A pure sample has an A value... 260 / A 230 If the value is greater than 2.0, then A 260 / A 280 A value below 2.0 indicates the presence of contaminants in the sample, such as carbohydrates, salts (guanidine salts), etc.

[0028] The results in the table above show that Example 1 has the best overall effect. Compared with Example 1, Comparative Example 1 did not add NaCl in the preparation of the first treatment solution, and the average value of the comprehensive score of depigmentation and HE staining, A 260 / A 280 The average value, A 260 / A 230 The average values ​​were all lower than those in Example 1; in Comparative Example 2, the order of use of the first and second treatment solutions was changed compared to Example 1, and the average values ​​of the combined scores of depigmentation and HE staining, the average total DNA amount, the average percentage of DNA fragments >5000bp, and A were all lower than those in Example 1. 260 / A 280 The average value, A 260 / A 230The average values ​​of the values ​​were all lower than those of Example 1; compared with Example 1, no EDTA was added in the preparation of the second treatment solution in Comparative Example 3, and the average values ​​of the total DNA amount and the proportion of DNA fragments >5000bp were lower than those of Example 1; Comparative Examples 4 and 5 were both treated with only Tris-HCl buffer containing H2O2, and the average values ​​of the total DNA amount, the proportion of DNA fragments >5000bp, and A in Comparative Examples 4 and 5 were lower than those of Example 1; 260 / A 280 The average value, A 260 / A 230 The average values ​​were all lower than those in Example 1. Furthermore, Comparative Examples 2-5 all exhibited sheet detachment issues.

[0029] Test Example 3 From the depigmented melanomas obtained in Examples 1 and Comparative Examples 1-5, the sample with the lowest combined score of depigmentation and HE staining was selected. Agarose gel electrophoresis images of the sample after C-KIT gene Sanger sequencing, PDGFRA gene Sanger sequencing, and C-KIT and PDGFRA gene amplification are shown below. Figure 7-12 .Depend on Figure 7-12 It can be seen that the agarose gel electrophoresis results of Example 1 and Comparative Example 1 are the best.

[0030] Test Example 4 The methods of Example 1 and Comparative Examples 1-5 were applied to the depigmentation treatment of melanoma samples with high melanin content, specifically as follows: Twenty melanoma samples diagnosed between 2023 and 2024 at the Department of Pathology, Weifang People's Hospital were collected. Among them, 11 were male and 9 were female. HE-stained sections showed clearly visible tumor cells and melanin under a microscope. The melanin content in the tumor tissue was as follows: 76% in 3 cases, 77% in 5 cases, 78% in 9 cases, and 79% in 3 cases. The patients' ages were: 65 years in 7 cases, 66 years in 2 cases, 67 years in 10 cases, and 68 years in 1 case. The lesions were located in the following locations: plantar masses in 4 cases, nasal masses in 2 cases, ear masses in 5 cases, thumb masses in 8 cases, and thigh skin masses in 1 case. The 20 samples were then divided into 6 equal parts and depigmented according to the methods described in Example 1 and Comparative Examples 1-5. The average combined score of depigmentation and HE staining for the 20 samples is as follows:

[0031] Combining the results of test example 2 with the results in the table above, it can be seen that the average combined score of depigmentation and HE staining in comparative examples 1, 2, 4, and 5 is greatly affected by the melanin content in melanoma.

Claims

1. A depigmentation method suitable for melanoma gene detection, characterized in that, After pretreatment of melanoma samples, they were added to the first treatment solution and soaked at 80°C for 20 minutes. Then they were added to the second treatment solution and soaked at 80°C for 10 minutes. After washing with water, they were air-dried. The first treatment solution was prepared by diluting H2O2 with Tris-HCl buffer to a volume concentration of 1%, and then adding NaCl to a mass concentration of 1% to obtain the first treatment solution. The second treatment solution was prepared by adding EDTA to Tris-HCl buffer until the concentration of EDTA was 1.2 mmol / L to obtain EDTA-containing Tris-HCl buffer, and then using EDTA-containing Tris-HCl buffer to dilute H2O2 to a volume concentration of 1% to obtain the second treatment solution.

2. The depigmentation method for melanoma gene detection according to claim 1, characterized in that, The pretreatment involved fixing the samples in 3.7% neutral formaldehyde, embedding them in paraffin, sectioning them into 4μm sections, preventing slide detachment, baking the slides at 65℃ for ≥1 hour, and then performing routine dewaxing.

3. The depigmentation method for melanoma gene detection according to claim 1, characterized in that, In the preparation of the first and second treatment solutions, the pH of the Tris-HCl buffer was 10 and the concentration of Tris was 100 mmol / L.

Images