A depigmentation method suitable for melanoma gene detection

Abstract

The application 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: after pretreatment of a melanoma sample, the first treatment solution is added, and the sample is soaked at 80 DEG C for 20 min; the second treatment solution is added, and the sample is soaked at 80 DEG C for 10 min; the sample is washed with water and dried; the preparation method of the first treatment solution comprises the following steps: H2O2 is diluted to 1% of the volume concentration of H2O2 by using Tris-HCl buffer solution, then NaCl is added to 1% of the mass concentration of NaCl, and the first treatment solution is obtained; the depigmentation method can realize rapid and efficient depigmentation for melanomas with different melanin contents, the DNA integrity and amplification efficiency of the melanoma treated by using the depigmentation method are high, and the problem of sample loss is not prone to occurring.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular pathology, and particularly relates to a depigmentation method suitable for melanoma gene detection. BACKGROUND

[0002] As a highly invasive malignant tumor derived from neural crest melanocytes, melanoma has significant heterogeneity in anatomical distribution, which can involve skin and its appendages, mucosal epithelium (digestive tract / respiratory tract / urogenital tract, etc.), uveal and pia mater, and other tissues. Melanoma contains a large number of high-density brown to dark brown pigment granules. In HE and immunohistochemical staining, pigment granules can cover the tissue structure of the tumor, resulting in incorrect tumor diagnosis. Therefore, depigmentation is needed. Pigment granules are a kind of pigment formed by pigment mother cells, which do not contain iron but contain sulfur. They are very stable and insoluble in water and organic solvents, but can be dissolved in strong alkali and bleached by strong oxidizing agents.

[0003] The current "Guidelines for Clinical Practice of Pathological Diagnosis of Melanoma (2021 Edition)" emphasizes that gene detection based on C-KIT and PDGFRA and other molecular targets with clear clinical significance, i.e. molecular diagnostic techniques, has become an important basis for guiding targeted therapy decisions and prognosis evaluation. However, the large number of high-density brown to dark brown pigment granules in melanoma can cause multiple molecular interference effects, including: first, nucleic acid quantitative bias caused by light absorption characteristics; second, covalent binding with DNA molecules, reducing nucleic acid purity; third, competitive binding with Taq DNA polymerase, significantly inhibiting PCR amplification efficiency, and thus increasing the risk of false negatives.

[0004] For melanoma, the method of making tumor diagnosis based on molecular diagnostic techniques has higher requirements for depigmentation than the method of making tumor diagnosis based on histomorphology level (such as HE staining, immunohistochemical staining, etc.), and different depigmentation methods have greater influence on the DNA integrity and amplification efficiency of depigmented melanoma. Zhang YP, Wang H, Li XF, Wan JL, Zhang YX. Exploration of the application value of a rapid and efficient depigmentation method in the molecular pathological gene detection of melanoma. Chinese Journal of Pathology. 2025, July, Vol. 54, No. 7 discloses that by comparing four depigmentation methods of H2O2 method, potassium permanganate method, Tris-HCl method and PBS method, combined with Sanger sequencing and PCR double platform verification, the influence on DNA integrity and amplification efficiency is systematically evaluated, and the results show that the Tris-HCl method can efficiently and rapidly remove pigments in melanoma FFPE tissues, the operation process is simple, easy to popularize and apply in laboratory and clinical detection, and high-quality DNA can be obtained in molecular detection, which significantly improves the amplification efficiency of target genes, the sequencing peak chart is clear, and there is no interference of impurity peaks, which improves the accuracy and reliability of gene detection results. At the same time, the best conditions of Tris-HCl method in depigmentation of melanoma are determined, that is, after diluting H2O2 to a concentration of 1% with Tris-HCl buffer (pH value is 10), heating at 70℃ for 30min, washing with distilled water for 3min, and air drying at room temperature.

[0005] The foregoing Tris-HCl method realizes rapid and efficient depigmentation of melanoma through a four-fold synergistic mechanism, which is specifically: the first fold is that HCl can induce tyrosine acid oxidation; the second fold is that heat catalyzes H2O2 to generate hydroxyl radicals to enhance oxidation; the third fold is that the buffer system with a pH value of 10 can maintain the acid-base balance of the tissue; and the fourth fold is that heating at 70℃ can significantly improve the depigmentation efficiency. However, the foregoing Tris-HCl method has the following problems:

[0006] Firstly, Chen YH, Lin BY, Li XF, Li NH, Yuan ZX. Effect of low concentration hydrogen peroxide solution on tissue depigmentation at high temperature. Journal of Clinical and Experimental Pathology. 2020, January discloses that the higher the temperature and pH value, the more likely the problem of tissue detachment occurs. For the foregoing Tris-HCl method, the problem of tissue detachment occurs after depigmentation. In view of this problem, the most commonly used method is to change the buffer system, and the inventors tried to change the Tris-HCl system to Tris-citric acid system, but the depigmentation effect of Tris-citric acid system is worse than that of Tris-HCl system.

[0007] Second, Liu Dan, Liu Shengjun, Chen Yang, Ke Qi, Wang Anqun. EDTA hydrogen peroxide repair on melanin-rich tumor tissue staining effect. Medical theory and practice. 2023, vol. 36, No. 24 discloses that H2O2 depigmentation is better than potassium permanganate and other pigment staining methods, but the problem of long time is not solved, the ability of H2O2 depigmentation is closely related to temperature and processing time, the higher the temperature and the longer the time, the more complete the depigmentation, but the more serious the damage to the tissue structure, the document repairs in the boiling H2O2 concentration of 0.75% EDTA (pH value is 9) hydrogen peroxide solution for 20 min, so as to realize the removal of melanin, simple operation, uniform and complete melanin depigmentation, suitable for any amount of melanin. For the aforementioned Tris-HCl method, due to the low temperature, the depigmentation effect is poor when the high melanin content melanoma is treated, and the DNA integrity and amplification efficiency of the melanoma after depigmentation treatment are affected. In view of the problem, the most commonly used method is to carry out depigmentation treatment under boiling or microwave conditions, but boiling and microwave conditions will aggravate the problem of depigmentation. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a depigmentation method suitable for melanoma gene detection, which can realize rapid and efficient depigmentation of melanomas with different melanin contents, and the DNA integrity and amplification efficiency of the melanoma treated by the depigmentation method of the present application are high, and the problem of depigmentation is not prone to occur.

[0009] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0010] A depigmentation method suitable for melanoma gene detection, after pretreatment of the melanoma sample, the first portion of the treatment liquid is added, soaked at 80℃ for 20min, the second portion of the treatment liquid is added, soaked at 80℃ for 10min, washed with water, and dried;

[0011] The pretreatment is to fix the sample with 3.7% neutral formaldehyde, paraffin embedding, 4μm section, anti-depiece piece, 65℃ baking piece≥1h, and conventional dewaxing;

[0012] The preparation method of the first portion of the treatment liquid is to dilute H2O2 to 1% of the volume concentration of H2O2 with Tris-HCl buffer, then add NaCl to 1% of the mass concentration of NaCl, and obtain the first portion of the treatment liquid;

[0013] The preparation method of the second treatment liquid is that EDTA is added into Tris-HCl buffer solution to obtain Tris-HCl buffer solution containing EDTA, and then H2O2 is diluted into the Tris-HCl buffer solution containing EDTA to obtain the second treatment liquid with a volume concentration of 1% of H2O2.

[0014] In the preparation of the first treatment liquid and the second treatment liquid, the pH value of the Tris-HCl buffer solution is 10, and the concentration of Tris is 100 mmol / L.

[0015] Compared with the prior art, the beneficial effects of the present application are:

[0016] (1) Sun Lijun, Huang Bin; Dual oxidative melanin removal method in the application of immunohistochemical diagnosis of malignant melanoma. Chinese Journal of Cancer Prevention and Treatment. October 2012 disclosed that after repair and oxidation, antigen repair and avoidance of sheeting can be performed, which is different from the literature, the present application uses the first treatment liquid and the second treatment liquid to perform depigmentation treatment respectively, no high oxidative oxidizing agent is used in the first treatment liquid, which reduces the damage to the antigen and also reduces the sheeting problem, in addition, NaCl is additionally added, NaCl is beneficial to promote the exposure of sulfur-containing bonds in pigment particles, thereby promoting the oxidation of sulfur-containing bonds by H2O2, and improving the efficiency of depigmentation treatment, EDTA is additionally added in the second treatment liquid, EDTA has a repair effect, which can perform antigen repair while depigmentation, and also avoid the sheeting problem. Through the way of oxidation first and then oxidation repair, rapid and efficient depigmentation of melanoma with different melanin contents can be realized, and the DNA integrity and amplification efficiency are ensured, and the sheeting problem is not prone to occur;

[0017] (2) The depigmentation method of the present application, after the melanin content of the melanoma is 51-55%, the average value of the comprehensive score of depigmentation and HE staining is 4, the average value of the total amount of DNA is 14.65 μg, the average value of the proportion of DNA large fragments of >5000 bp is 60.2%, the average value of A 260 / A 280 value is 1.92, and the average value of A 260 / A 230 value is 2.14, and no sheeting problem occurs; after the melanin content of the melanoma is 76-79%, the average value of the comprehensive score of depigmentation and HE staining is 4. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 HE staining diagram of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0019] Figure 2 HE staining figure of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0020] Figure 3 HE staining figure of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0021] Figure 4 HE staining figure of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0022] Figure 5 HE staining figure of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0023] Figure 6 HE staining figure of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0024] Figure 7 Agarose gel electrophoresis figure of C-KIT target gene and PDGFRA target gene amplification of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0025] In the figure, M lane is Marker, representing the size of the amplification product fragment; 1 lane is C-KIT Exon 9; 2 lane is C-KIT Exon 11; 3 lane is C-KIT Exon 13; 4 lane is C-KIT Exon 17; 5 lane is PDGFRA Exon 12; 6 lane is PDGFRA Exon 14; 7 lane is PDGFRA Exon 18;

[0026] Figure 8 Agarose gel electrophoresis figure of C-KIT target gene and PDGFRA target gene amplification of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0027] In the figure, M lane is Marker, representing the size of the amplification product fragment; 1 lane is C-KIT Exon 9; 2 lane is C-KIT Exon 11; 3 lane is C-KIT Exon 13; 4 lane is C-KIT Exon 17; 5 lane is PDGFRA Exon 12; 6 lane is PDGFRA Exon 14; 7 lane is PDGFRA Exon 18;

[0028] Figure 9 Agarose gel electrophoresis figure of C-KIT target gene and PDGFRA target gene amplification of the sample with the lowest comprehensive score of depigmentation and HE staining in Example 1;

[0029] In the figure, M lane is Marker, representing the size of the amplified product fragment; 1 lane is C-KIT Exon 9; 2 lane is C-KIT Exon 11; 3 lane is C-KIT Exon 13; 4 lane is C-KIT Exon 17; 5 lane is PDGFRA Exon 12; 6 lane is PDGFRA Exon 14; 7 lane is PDGFRA Exon 18.

[0030] Figure 10 The agarose gel electrophoresis diagram of C-KIT target gene and PDGFRA target gene amplification of 1 sample with the lowest comprehensive score of depigmentation and HE staining in Comparative Example 3;

[0031] In the figure, M lane is Marker, representing the size of the amplified product fragment; 1 lane is C-KIT Exon 9; 2 lane is C-KIT Exon 11; 3 lane is C-KIT Exon 13; 4 lane is C-KIT Exon 17; 5 lane is PDGFRA Exon 12; 6 lane is PDGFRA Exon 14; 7 lane is PDGFRA Exon 18.

[0032] Figure 11 The agarose gel electrophoresis diagram of C-KIT target gene and PDGFRA target gene amplification of 1 sample with the lowest comprehensive score of depigmentation and HE staining in Comparative Example 4;

[0033] In the figure, M lane is Marker, representing the size of the amplified product fragment; 1 lane is C-KIT Exon 9; 2 lane is C-KIT Exon 11; 3 lane is C-KIT Exon 13; 4 lane is C-KIT Exon 17; 5 lane is PDGFRA Exon 12; 6 lane is PDGFRA Exon 14; 7 lane is PDGFRA Exon 18.

[0034] Figure 12 The agarose gel electrophoresis diagram of C-KIT target gene and PDGFRA target gene amplification of 1 sample with the lowest comprehensive score of depigmentation and HE staining in Comparative Example 5;

[0035] In the figure, M lane is Marker, representing the size of the amplified product fragment; 1 lane is C-KIT Exon 9; 2 lane is C-KIT Exon 11; 3 lane is C-KIT Exon 13; 4 lane is C-KIT Exon 17; 5 lane is PDGFRA Exon 12; 6 lane is PDGFRA Exon 14; 7 lane is PDGFRA Exon 18. DETAILED DESCRIPTION

[0036] In order to have a clearer understanding of the technical features, objectives and effects of the present application, the specific embodiments of the present application are described below.

[0037] Twenty cases of melanoma samples diagnosed in the Department of Pathology of Weifang People's Hospital from 2023 to 2024 were collected, including 12 male cases and 8 female cases. HE staining sections showed clear tumor cells and melanin under a microscope. The proportion of melanin content in tumor tissue was 51% in 4 cases, 52% in 3 cases, 53% in 6 cases, 54% in 4 cases, and 55% in 3 cases. The ages of the patients were 64 years old in 6 cases, 65 years old in 10 cases, 66 years old in 1 case, and 67 years old in 3 cases. The sites of the disease were 3 cases of plantar lumps, 5 cases of nasal lumps, 4 cases of ear lumps, 5 cases of thumb lumps, and 3 cases of thigh skin lumps. Then, the 20 samples were evenly divided into 6 parts, and then the depigmentation method in Example 1 and Comparative Examples 1-5 was used for depigmentation treatment.

[0038] Example 1

[0039] A depigmentation method suitable for melanoma gene detection, specifically comprising the following steps:

[0040] 1. Pretreatment: According to the method disclosed in Zhang YP, Wang H, Li XF, Wan JL, Zhang YX. Exploration of the application value of rapid and efficient depigmentation method in melanoma molecular pathological gene detection. Chinese Journal of Pathology. 2025, Vol. 54, No. 7, 3.7% neutral formaldehyde fixation was performed on 20 samples, paraffin embedding, 4 μm section, 65 ℃ baking for 1 h, xylene dewaxing, anhydrous ethanol removal of xylene, to obtain pretreated samples;

[0041] 2. Depigmentation treatment: the pretreated samples were treated with the following method:

[0042] The first treatment solution was heated to 80℃, the pretreated sample was added, and the sample was soaked at 80℃ for 20min, the sample was taken out, the second treatment solution was heated to 80℃, the sample was added, and the sample was soaked at 80℃ for 10min, the sample was taken out, distilled water was washed for 3min, and the sample was dried at room temperature, to obtain the depigmented melanoma;

[0043] The preparation method of the first treatment solution is as follows: dilute the 30% H2O2 aqueous solution to 1% H2O2 by volume concentration with Tris-HCl buffer solution with pH value of 10 and Tris concentration of 100 mmol / L, then add NaCl to a mass concentration of 1% NaCl to obtain the first treatment solution;

[0044] 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.

[0045] 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 230 The 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.

[0046] Comparative Example 1

[0047] A depigmentation method suitable for melanoma gene detection specifically includes the following steps:

[0048] 1. Preprocessing: Twenty samples were preprocessed according to the method in Example 1 to obtain preprocessed samples;

[0049] 2. Depigmentation treatment: The pretreated samples were depigmented according to the following methods:

[0050] 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.

[0051] 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.

[0052] The preparation method of the second treatment solution is: adding EDTA into Tris-HCl buffer solution with pH value of 10 and Tris concentration of 100 mmol / L until the concentration of EDTA is 1.2 mmol / L to obtain Tris-HCl buffer solution containing EDTA, and then diluting 30% H2O2 aqueous solution to 1% H2O2 by volume concentration using the Tris-HCl buffer solution containing EDTA to obtain the second treatment solution.

[0053] According to the detection and statistical method in Example 1, detection and scoring were performed, and the statistical results were obtained.

[0054] Comparative Example 2

[0055] A depigmentation method suitable for melanoma gene detection, specifically comprising the following steps:

[0056] 1. Pretreatment: 20 samples were pretreated according to the method of Example 1 to obtain pretreated samples;

[0057] 2. Depigmentation treatment: the pretreated samples were respectively subjected to depigmentation treatment according to the following method:

[0058] The first treatment solution was heated to 80℃, the pretreated sample was added, and the sample was soaked at 80℃ for 10 min, then the sample was taken out, the second treatment solution was heated to 80℃, the sample was added, and the sample was soaked at 80℃ for 20 min, then the sample was taken out, distilled water was washed for 3 min, and the sample was dried at room temperature to obtain depigmented melanoma;

[0059] The preparation method of the first treatment solution is: adding EDTA into Tris-HCl buffer solution with pH value of 10 and Tris concentration of 100 mmol / L until the concentration of EDTA is 1.2 mmol / L to obtain Tris-HCl buffer solution containing EDTA, and then diluting 30% H2O2 aqueous solution to 1% H2O2 by volume concentration using the Tris-HCl buffer solution containing EDTA to obtain the second treatment solution;

[0060] The preparation method of the second treatment solution is: diluting 30% H2O2 aqueous solution to 1% H2O2 by volume concentration using Tris-HCl buffer solution with pH value of 10 and Tris concentration of 100 mmol / L, and then adding NaCl to a mass concentration of 1% to obtain the first treatment solution.

[0061] According to the detection and statistical method in Example 1, detection and scoring were performed, and the statistical results were obtained.

[0062] Comparative Example 3

[0063] A depigmentation method suitable for melanoma gene detection, specifically comprising the following steps:

[0064] 1. Pretreatment: 20 samples were pretreated according to the method of Example 1 to obtain pretreated samples;

[0065] 2. Depigmentation treatment: the pretreated samples were respectively treated according to the following method:

[0066] The first treatment solution was heated to 80℃, the pretreated sample was added, soaked at 80℃ for 20min, the sample was taken out, the second treatment solution was heated to 80℃, the sample was added, soaked at 80℃ for 10min, the sample was taken out, washed with distilled water for 3min, and dried at room temperature to obtain depigmented melanoma;

[0067] The preparation method of the first treatment solution is as follows: dilute 30% H2O2 aqueous solution to 1% H2O2 volume concentration with Tris-HCl buffer solution with pH value of 10 and Tris concentration of 100mmol / L, then add NaCl to 1% NaCl mass concentration to obtain the first treatment solution;

[0068] The preparation method of the second treatment solution is as follows: dilute 30% H2O2 aqueous solution to 1% H2O2 volume concentration with Tris-HCl buffer solution with pH value of 10 and Tris concentration of 100mmol / L to obtain the second treatment solution.

[0069] According to the detection and statistical method in Example 1, detection and scoring were performed, and the statistical results were obtained.

[0070] Comparative Example 4

[0071] A depigmentation method suitable for melanoma gene detection, specifically comprising the following steps:

[0072] 1. Pretreatment: 20 samples were pretreated according to the method of Example 1 to obtain pretreated samples;

[0073] 2. Depigmentation treatment: the pretreated samples were respectively treated according to the following method:

[0074] The treatment solution was heated to 80℃, the pretreated sample was added, soaked at 80℃ for 30min, the sample was taken out, washed with distilled water for 3min, and dried at room temperature to obtain depigmented melanoma;

[0075] The preparation method of the treatment liquid is: diluting a 30% H2O2 aqueous solution with a Tris-HCl buffer solution with a pH value of 10 and a Tris concentration of 100 mmol / L to a volume concentration of 1% of H2O2 to obtain the treatment liquid.

[0076] According to the detection and statistical method in Example 1, detection and scoring were performed, and the statistical results were obtained.

[0077] Comparative Example 5

[0078] A depigmentation method suitable for melanoma gene detection, specifically comprising the following steps:

[0079] 1. Pretreatment: 20 samples were pretreated according to the method of Example 1 to obtain pretreated samples;

[0080] 2. Depigmentation treatment: the pretreated samples were respectively subjected to depigmentation treatment according to the following method:

[0081] The treatment liquid was heated to 70℃, the pretreated sample was added, and the sample was soaked at 70℃ for 30min, then the sample was taken out, washed with distilled water for 3min, and dried at room temperature to obtain the depigmented melanoma;

[0082] The preparation method of the treatment liquid is: diluting a 30% H2O2 aqueous solution with a Tris-HCl buffer solution with a pH value of 10 and a Tris concentration of 100 mmol / L to a volume concentration of 1% of H2O2 to obtain the treatment liquid.

[0083] According to the detection and statistical method in Example 1, detection and scoring were performed, and the statistical results were obtained.

[0084] Test Example 1

[0085] From the depigmented melanomas obtained in Example 1 and Comparative Examples 1-5, 1 sample with the lowest comprehensive score of depigmentation and HE staining was selected, and the HE staining images thereof are shown in Figures 1-6 ; it can be seen from Figures 1-6 that the HE staining effects of Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5 are better.

[0086] Test Example 2

[0087] The average value of the comprehensive score of depigmentation and HE staining, the average value of the total amount of DNA (the average value of the total amount of DNA before depigmentation treatment is 15.31 μg), the average value of the proportion of DNA large fragments of >5000bp, the average value of A 260 / A 280 value (the average value of A 260 / A 280The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 230 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 230 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows:

[0088]

[0089] The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 230 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 230 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows:

[0090] From the results in the above table, it can be seen that the comprehensive effect of Example 1 is the best. Compared with Example 1, Comparative Example 1 does not add NaCl in the preparation of the first treatment solution, the average value of the comprehensive score of depigmentation and HE staining, the average value of A 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 230 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 230 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 260 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows: 280 The average value of A is 1.59, and the statistical results of the number of pieces with depigmentation problems are as follows:260 / A 230 The average values of the values are lower than those of Example 1. In addition, the samples of Comparative Examples 2-5 all have the problem of flaking.

[0091] Test Example 3

[0092] One sample with the lowest comprehensive score of depigmentation and HE staining was selected from the melanomas after depigmentation obtained from Example 1 and Comparative Examples 1-5. The agarose gel electrophoresis chart of the C-KIT target gene and PDGFRA target gene after amplification after C-KIT gene Sanger sequencing and PDGFRA gene Sanger sequencing of the sample is shown in Figures 7-12 . It can be seen that the agarose gel electrophoresis chart results of Example 1 and Comparative Example 1 are optimal. Figures 7-12

[0093] Test Example 4

[0094] The methods of Example 1 and Comparative Examples 1-5 were respectively applied to the depigmentation treatment of melanoma samples with high melanin content, specifically:

[0095] Twenty melanoma samples diagnosed by the Department of Pathology of Weifang People's Hospital from 2023 to 2024 were collected, including 11 male samples and 9 female samples. The tumor cells and melanin were clearly visible under the microscope of HE staining sections, and the proportion of melanin content in tumor tissue was 76% in 3 samples, 77% in 5 samples, 78% in 9 samples, and 79% in 3 samples. The ages of the patients were 65 years old in 7 samples, 66 years old in 2 samples, 67 years old in 10 samples, and 68 years old in 1 sample. The sites of the disease were 4 samples of plantar masses, 2 samples of nasal masses, 5 samples of ear masses, 8 samples of thumb masses, and 1 sample of thigh skin masses. Then, the 20 samples were evenly divided into 6 parts, and the depigmentation methods in Example 1 and Comparative Examples 1-5 were respectively applied to the depigmentation treatment. The average values of the comprehensive scores of depigmentation and HE staining of the 20 samples are as follows:

[0096]

[0097] After combining the results of Test Example 2 and the results in the above table, it can be seen that the average values of the comprehensive scores of depigmentation and HE staining of Comparative Example 1, Comparative Example 2, Comparative Example 4, and Comparative Example 5 are 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