Preparation method of paraffin-embedded cell sample

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of a cell paraffin embedding sample. The method comprises the following steps: S1, immobilizing a cell sample with a formaldehyde solution, centrifuging, discarding a supernatant, and resuspending cells to obtain a cell suspension; s2, preparing an agarose solution, cooling to 50-65 DEG C, mixing the agarose solution with the cell suspension, and cooling and solidifying to form agarose blocks; and S3, dehydrating, transparentizing and soaking the agarose blocks, and then carrying out paraffin embedding, low-temperature solidification and slicing to obtain the paraffin-embedded cell sample. According to the method, parameters in the processes of fixing, transparentizing, embedding treatment and the like are specifically designed and optimized, all the parameters are matched with one another, the cell morphology, the structural integrity and the subsequent dyeing effect can be effectively ensured, and the uniformity of the cell slices can reach 95% or above; the problem that in a traditional method, the difference of negative and positive results of single slices at different positions of a wax block is large is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing a cell paraffin-embedded sample. Background Art

[0002] At present, the preparation method used when cultured tissue cells and exfoliated cells are detected by hematoxylin-eosin staining, special staining and immunohistochemical staining is mostly smear preparation, and this preparation method has many shortcomings. Untreated cultured cells and exfoliated cells have complete cell membranes, and the lipids on their cell membranes often cannot fully dissolve, which has affected the penetration of dyes and antibodies. Cells are often poorly colored, and the structure is not clear enough. Immunohistochemical staining often causes false negative results, and is sometimes difficult to determine the position of positive or negative and positive coloring. Although Triton X-100 can be used for pre-treatment to increase the permeability of cell membranes, its concentration, time, etc. are difficult to grasp. With formalin fixation, the cell paraffin embedding section after gradient ethanol dehydration and dimethylbenzene treatment can avoid the above-mentioned shortcomings, and the cells are concentrated, the density is high, and it is convenient to observe, and all cells can be collected.

[0003] Formalin-fixed paraffin-embedded samples are common biological materials in the medical field. Such samples have been widely used in research fields such as high-throughput sequencing, in situ hybridization, and immunohistochemistry. At present, there are about hundreds of millions of formalin-fixed paraffin-embedded tissue samples in the world, and these tissue samples have become one of the most valuable biological resources for disease diagnosis and scientific research. With the rapid development of genomic detection technology, especially the rapid development of next-generation sequencing technology (NGS), unprecedented detection capabilities have been provided at the cellular and molecular levels. Researchers can use this technical means to study formalin-fixed paraffin-embedded samples and look for disease-related gene mutations.

[0004] Currently, most formalin-fixed paraffin-embedded samples are tissue formalin-fixed paraffin-embedded samples. Although there are relevant data and literature on the preparation of cell formalin-fixed paraffin-embedded samples, they are mainly divided into two types: artificial dehydration by centrifugation and wrapping cells with a medium so that they can be subsequently dehydrated, embedded and sectioned in the form of tissue blocks. Artificial dehydration by centrifugation is complicated and time-consuming, and has high requirements on experimental personnel. In addition, only one type of cell or multiple mixtures of single cells with consistent mass and volume can be prepared. Because the preparation of multiple mixed cells of different sizes may cause uneven distribution of cells due to steps such as centrifugation, the positive and negative results of single slices at different positions of the wax block vary greatly, resulting in errors in the results of downstream in situ hybridization, immunohistochemistry and other experiments. The method of wrapping cells with a medium so that they can be subsequently dehydrated, embedded and sliced ​​in the form of tissue blocks is similar to the present invention, but the traditional method is to inject cells into the medium, and the medium is a substance such as agarose gelatin, which is melted by high temperature, and then the cells are injected into the center after the medium is cooled to a suitable temperature and the outer surface is slightly solidified; this method has very high requirements for operators, and too high temperature will cause inactivation of cell enzymes, destruction of lipids, destruction of nuclear division, production of coagulase to cause cell coagulation, protein denaturation, etc., and at high temperature, substances such as agarose have not yet solidified, and cells are very easy to penetrate out; when the temperature is too low, agarose solidifies over a large area inside, making it impossible to inject cells or the injected cells are extremely unevenly distributed, which will also make the single-slice section results at different positions of the wax block vary greatly, affecting downstream experiments; even if the temperature is selected appropriately, since substances such as agarose gelatin solidify quickly at room temperature, the operation of injecting cells also requires to be very quick and skilled. The two traditional methods of artificial dehydration by centrifugation and wrapping cells with a medium to allow them to be subsequently dehydrated, embedded and sectioned in the form of tissue blocks have many manual operation steps, high requirements, and are difficult to achieve mass production.

[0005] Therefore, how to simplify the preparation process of formalin-fixed paraffin-embedded cell samples while ensuring or even improving the preparation quality (observation stability and accuracy) of the specimens and increasing the preparation yield has become an urgent problem to be solved in this field. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a cell paraffin-embedded sample.

[0007] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a method for preparing a paraffin-embedded cell sample, comprising the following steps:

[0009] S1. Fix the cell sample with a formaldehyde solution, centrifuge and discard the supernatant, and resuspend the cells to obtain a cell suspension;

[0010] S2. Prepare an agarose solution, cool it to 50-65°C, mix the agarose solution with the cell suspension, and cool it down to solidify to form an agarose block;

[0011] S3. The agarose block is subjected to conventional fixation, dehydration, transparency, and infiltration processes, and then embedded in paraffin, solidified at low temperature, and sliced ​​to obtain a cell-fixed paraffin-embedded sample.

[0012] In the preparation of formalin-fixed paraffin-embedded cell samples, the present invention creatively mixes agarose with cell suspension to obtain uniform cell agarose blocks. Compared with the traditional agarose injection method, the uniformity of cell sections at different positions in the paraffin block in the present method reaches more than 95%, and the cells in the single slices are evenly distributed, which solves the problem that the positive and negative results of single slices at different positions in the wax block in the traditional method are quite different, resulting in errors in the experimental results of downstream in situ hybridization, immunohistochemistry, etc.

[0013] Preferably, in step S1, the concentration of the formaldehyde aqueous solution is 4%, and the fixed time is 25 to 35 minutes.

[0014] Through experimental exploration, it was found that the present invention uses 4% formaldehyde aqueous solution to fix cell samples, which can effectively maintain cell morphology and structure, is easy to handle, and has good compatibility. After fixation for 25 to 35 minutes, the formaldehyde aqueous solution can quickly penetrate the cells and fix the internal structure of the cells, keeping the cell structure intact, which is convenient for subsequent staining and observation. However, if the concentration of formaldehyde aqueous solution is too high or the fixation time is too long, it may cause hardening of cell tissue, affect the quality of subsequent sections and the staining of the internal structure of the cells, while if the concentration is too low or the fixation time is too short, it may lead to insufficient fixation and incomplete cell structure. Therefore, the preferred fixative concentration and fixation time of the present invention have an important influence on ensuring that the cell morphology is maintained, the structure is intact, and the subsequent staining effect.

[0015] Preferably, in step S2, the concentration of the agarose solution is 2-3%.

[0016] Through experimental exploration, it was found that when the agarose solution adopts the preferred concentration range of the present invention, it can effectively ensure the structural support of the cells, avoid damage during the slicing process, and improve the quality and uniformity of the slices; while too high a concentration of the agarose solution may lead to low concentration of nucleic acid extracted from cell slices, reduced staining effect, and failure of FISH detection probes to hybridize; too low a concentration may affect the fixation of the internal structure of the cells, resulting in the loss of cell details, and even the inability of the agarose block to effectively solidify, which is not conducive to subsequent detection and analysis.

[0017] Preferably, in step S3, the dehydration process uses gradient ethanol dehydration, and the concentration of the ethanol is 70-100%.

[0018] Preferably, in step S3, the specific process of transparency is: treating with xylene for 45 to 60 minutes, and repeating the treatment 3 times.

[0019] Preferably, in step S3, the specific process of the soaking is: treating with a 60° C. paraffin solution for 45 to 60 minutes, and repeating the treatment three times.

[0020] Preferably, in step S3, the specific conditions for low-temperature solidification are: placing at -15 to -25°C for 30 to 60 minutes.

[0021] The present invention specifically designs the parameters of the transparent procedure, permeation and embedding treatment for cell samples, and the parameters cooperate with each other. Any change in any parameter during the treatment process will affect the quality of the slice to varying degrees. The quality of the slice obtained can be up to standard by operating with the parameters within the limited range of the present invention. The quality of the slice obtained by using the preferred solution in the embodiment of the present invention is the best.

[0022] Preferably, the cell sample comprises at least one of human colon adenocarcinoma cells, breast cancer cells, human myeloid leukemia cells, human hepatoblastoma cells, human bladder transitional cell carcinoma cells, human malignant embryonic rhabdoid tumor cells and human monocytic leukemia cells.

[0023] Experimental investigations have found that the use of the preparation method of the present invention to embed tumor cell samples from different organs can effectively avoid the degradation of tumor cell nucleic acids while maintaining the integrity of cell morphology and structure, thereby ensuring subsequent high-quality molecular biological analysis of tumor cells.

[0024] In a second aspect, the present invention provides application of the preparation method in preparing paraffin-embedded cell samples.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) In the preparation of paraffin-embedded cell samples, the present invention creatively mixes agarose with cell suspension to obtain uniform cell agarose blocks. Compared with the traditional agarose injection method, the uniformity of cell sections at different positions in the paraffin block reaches more than 95%, and the cells in a single section are evenly distributed, which solves the problem that the results of single sections at different positions in the paraffin block in the traditional method are quite different, leading to errors in the results of downstream in situ hybridization, immunohistochemistry and other experiments;

[0027] (2) The present invention specifically designs and optimizes the parameters of the fixation, transparency and embedding processes for cell samples. The parameters cooperate with each other to effectively ensure the preservation of cell morphology, structural integrity and subsequent staining effect, significantly improve the quality of section specimens, and simplify the preparation process of embedded samples;

[0028] (3) The preparation method of the present invention can be applied to automatic dehydration equipment, which not only ensures the quality of the cell paraffin-embedded sample slices but also can achieve mass production using the automatic dehydration platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of hematoxylin-eosin staining of the slices prepared by the traditional formalin-fixed paraffin-embedded method;

[0030] Figure 2 This is a fluorescence in situ hybridization test result diagram of the light microscopy specimen prepared in Example 1;

[0031] Figure 3 is a 2D diagram of droplet distribution of the droplet digital PCR detection results of Example 2;

[0032] Figure 4 This is a graph showing the measured results of droplet digital PCR detection in Example 2;

[0033] Figure 5 This is the result of hematoxylin-eosin staining under an upright microscope at 100 times magnification in Example 3;

[0034] Figure 6 is a diagram of the hematoxylin-eosin staining result of the slices prepared in Example 3;

[0035] Figure 7 This is the result of fluorescence in situ hybridization detection of the fusion gene BRC-ABL under an upright microscope at 400 times magnification;

[0036] Figure 8 The fluorescence in situ hybridization detection result 1 under an upright microscope 1000 times in Example 4;

[0037] Fig. 9 The fluorescence in situ hybridization detection result 2 under an upright microscope 1000 times in Example 4;

[0038] Fig.10 This is a picture of the hematoxylin-eosin staining result of the slices prepared in the experimental group of Comparative Example 1;

[0039] Fig.11 This is a diagram showing the hematoxylin-eosin staining result of the slices prepared from the control group 1 in comparative example 1;

[0040] Fig.12 This is a diagram showing the hematoxylin-eosin staining result of the slices prepared from the control group 2 in comparative example 1;

[0041] Fig.13 This is a 2D image of droplet distribution of droplet digital PCR test results for human colon adenocarcinoma cells;

[0042] Fig.14This is a 2D map of droplet distribution of breast cancer cell droplet digital PCR test results;

[0043] Fig.15 This is a 2D diagram of the droplet distribution of the droplet digital PCR test results of human myeloid leukemia cells;

[0044] Fig.16 This is a 2D image of droplet distribution of droplet digital PCR test results of human hepatoblastoma cells;

[0045] Fig.17 This is a 2D image of the droplet distribution of the droplet digital PCR test results for human bladder transitional cell carcinoma;

[0046] Fig.18 This is a 2D diagram of droplet distribution of the droplet digital PCR test results of human malignant embryonic rhabdoid tumor cells;

[0047] Fig.19 This is a 2D diagram of droplet distribution of droplet digital PCR test results for human monocytic leukemia cells. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0050] Example 1

[0051] This embodiment provides a method for preparing a paraffin-embedded sample of cells with EGFR gene L858R mutation, comprising the following steps:

[0052] 1. Fixation: After obtaining the cell culture medium, centrifuge at 3000rpm for 5min, remove the supernatant, add PBS to mix, centrifuge at 3000rpm for 5min, remove the supernatant, repeat once; add fixative (4% neutral formaldehyde solution) to the cell pellet, mix, place at 4℃ for 30min, centrifuge at 3000rpm for 5min, remove the supernatant, add 5mL PBS to mix;

[0053] According to the mutation abundance of 20%, the mutant cells and wild-type cells were mixed evenly in proportion, centrifuged at 3000 rpm for 5 min, the supernatant was removed, and the remaining 3 mL was mixed and resuspended in the cell pellet, which was preheated in a 60°C water bath for cell preparation;

[0054] 2. Cells aggregate into blocks: Prepare 2% agarose solution with sterile water, dissolve at 100°C, cool to 55°C, add 0.5 mL of 2% agarose solution to 3 mL of cell suspension, mix well, and solidify at room temperature to make cell agarose blocks;

[0055] 3. Dehydration, transparency, and penetration: The solidified agarose block is placed in a sample box and dehydrated, transparentized, and penetrated using an automatic vacuum tissue dehydrator according to the following procedures (Table 1); before the automatic dehydrator runs the dehydration program according to the set time, the cell agarose block can be soaked in the reagent in step 1 and wait for dehydration. The soaking time must be less than 12 hours; the automatic dehydrator can set the program end time. If the end time is too late, there will be a blank period before the program starts. You can use a liquid that does not affect the sample (4% formalin) for soaking;

[0056] Table 1 Dehydration, transparency, and soaking procedures

[0057] step Reagents Time (minutes) temperature 1 Formalin (mass concentration 4%) - Room temperature 2 Deionized water 2 Room temperature 3 70% ethanol 45 Room temperature 4 80% ethanol 50 Room temperature 5 95% ethanol 50 Room temperature 6 100% ethanol 60 Room temperature 7 100% ethanol 90 Room temperature 8 100% ethanol 60 Room temperature 9 Xylene 45 35℃ 10 Xylene 45 35℃ 11 Xylene 45 35℃ 12 paraffin 60 60℃ 13 paraffin 60 60℃ 14 paraffin 60 60℃

[0058] 4. Embedding: After the procedure is completed, take out the agarose block, inject about 2 / 3 of the wax liquid into the embedding box, clamp the dehydrated, transparent and soaked cell agarose block into the embedding box, place it on the freezing table, and gently press it with tweezers while solidifying, so that the cell agarose is in the wax and close to the bottom of the wax mold. Then put it into the sample box and inject an appropriate amount of wax liquid and place it on the freezing table to solidify for 30 minutes at a temperature of -20°C; demold after the wax is completely solidified, and trim the wax block appropriately;

[0059] 5. Paraffin sectioning: insert the solidified and demolded wax block into the slicer slot, trim the wax block, and then slice it;

[0060] 6. For routine pathological hematoxylin-eosin staining, immunohistochemistry, and fluorescence in situ hybridization, the slice thickness is 5 μm. The cut wax slices are gently picked up with tweezers and placed on the water surface at room temperature for separation. The wax slices with inappropriate thickness are removed and placed on the 37°C water surface to flatten the cell agarose block. The wax slices are picked up so that they are in the center of the glass slide. The glass slides are then placed in the corresponding glass slide racks for baking. After the baking is completed until the paraffin is dissolved, the downstream experiments such as routine pathological hematoxylin-eosin staining, immunohistochemistry, and fluorescence in situ hybridization are continued.

[0061] 7. For sections used for molecular biology experiments after nucleic acid extraction, the section thickness is 15 μm. Slice slowly and adjust the slicing speed gradually to make the paraffin sections into wax rolls. Put the cut wax rolls into centrifuge tubes and use the paraffin-embedded tissue DNA extraction kit for extraction. The extracted nucleic acids are used for subsequent molecular biology experiments.

[0062] Example 2

[0063] This example uses digital PCR detection to verify the formalin-fixed paraffin-embedded wax roll of EGFR gene L858R site mutation cells prepared in Example 1. The specific method is as follows:

[0064] 1. Randomly select several wax rolls cut from the front, middle and back parts of the wax block in Example 1, put them into centrifuge tubes respectively, and extract them using a paraffin-embedded tissue DNA extraction kit (centrifuge column type, Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number DP340);

[0065] 2. After extraction, the nucleic acid was analyzed by Bio-rad ddPCR TM Supermix for Probes (no dUTP) reagent and Bio-rad QX200 instrument were used for digital PCR detection. The amplification system and procedure were as follows:

[0066] Table 2 EGFR gene L858R site mutation reaction system

[0067] Components Volume (μL) <![CDATA[ddPCR TM Supermix for Probes]]> 10 L858R-F(10μM) 0.9 L858R-R(10μM) 0.9 L858R-P1(10μM) 0.7 L858R-P2 (10μM) 0.7 water 4.8 template 2 Total volume 20

[0068] Table 3 PCR amplification conditions for EGFR gene L858R mutation

[0069]

[0070] Table 4 EGFR gene L858R site mutation digital PCR results

[0071]

[0072] From the results in Table 4, it can be seen that the coefficient of variation of the mutant concentration logarithm, the wild-type concentration logarithm and the mutation abundance are all less than 5%, indicating that the positive and negative cells of the cell-fixed paraffin-embedded wax roll prepared by the method of the present invention are evenly distributed.

[0073] Example 3

[0074] This example uses the EGFR gene L858R site mutation cell fixed paraffin embedded sections prepared in Example 1 for hematoxylin-eosin staining, and the specific method is as follows:

[0075] 1. Take the slices that have been baked and dewax them in xylene for 5 minutes, then change to fresh xylene and dewax them for 5 minutes, and then rehydrate them in graded alcohols: anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, and then distilled water for 2 minutes;

[0076] 2. Staining: Place the treated sections in hematoxylin staining solution for 5 minutes, differentiate in 1% hydrochloric acid aqueous solution for 5 seconds, soak in tap water for blueing for 5 minutes, soak in distilled water for washing for 20 seconds, place in 95% ethanol for 5 seconds, and place in eosin staining solution for 1 minute;

[0077] 3. Sealing: Dehydrate the stained sections in 95% ethanol for 2 minutes, then use fresh 95% ethanol to dehydrate for another 2 minutes; place in xylene to make it transparent for 5 minutes, then use fresh xylene to make it transparent for another 5 minutes, seal the sections with neutral gum, and observe the cells under an optical microscope;

[0078] Depend on Figure 5 and Figure 6 The results showed that the formalin-fixed paraffin-embedded cell samples prepared by this method could be used for hematoxylin-eosin staining, and the cells in the sections were evenly distributed and the single cells were intact.

[0079] Example 4

[0080] This example uses the EGFR gene L858R site mutation cell fixed paraffin-embedded sections prepared in Example 1 to perform fluorescence in situ hybridization detection, specifically as follows:

[0081] 1. Take the slices that have been baked in a 65℃ constant temperature oven for 24 hours, put them in room temperature xylene for 15 minutes, and then replace them with fresh xylene at room temperature for 15 minutes;

[0082] 2. Take out the slide and place it in room temperature anhydrous ethanol for 10 minutes, then place it in room temperature anhydrous ethanol, 90% ethanol, and 70% ethanol for 3 minutes each;

[0083] 3. Take out the slide, put it in deionized water at room temperature for 3 minutes, absorb excess water with lint-free paper towels, boil it in 100℃ deionized water for 25 minutes, and then dry it at room temperature;

[0084] 4. Add appropriate amount of pepsin reaction solution to the sample area and digest for 10 minutes;

[0085] 5. Shake off excess liquid, place the slide in room temperature 2×SSC for 5 min, and replace with fresh 2×SSC for 5 min;

[0086] 6. Take out the slide and place it in 70% ethanol, 90% ethanol, and anhydrous ethanol at room temperature for 3 minutes each, and dry it at room temperature;

[0087] 7. Take out the hybridization solution, shake and mix, centrifuge instantly, add 10μL of hybridization solution to the hybridization area, quickly cover with a cover slip, press lightly to evenly distribute the hybridization solution to avoid bubbles, and seal the slide along the edge of the cover slip with rubber glue to completely cover the contact area between the cover slip and the slide;

[0088] 8. Place the slide in the hybridizer, denature at 85°C for 5 min, and hybridize at 37°C for 18 h;

[0089] 9. Turn off the hybridizer, take out the slide, gently peel off the rubber glue, remove the cover glass, and place the slide in 2×SSC at 37°C for 10 minutes;

[0090] 10. Remove the slide and place it in 37°C 0.1% NP-40 / 2×SSC for 5 min;

[0091] 11. Take out the slides, put them in 70% ethanol at room temperature for 3 minutes, and then dry them naturally in the dark;

[0092] 12. At room temperature, add 10 μL of DAPI counterstain onto the coverslip. Place the slide with the target area facing downward on the coverslip, press gently to avoid creating bubbles, and observe under a fluorescence microscope in the dark.

[0093] Depend on Figure 7-9 The results show that the formalin-fixed paraffin-embedded cell samples prepared by the method of the present invention can be used for fluorescence in situ hybridization detection, and the cells in the sections are evenly distributed and the single cells are intact.

[0094] Example 5

[0095] This example uses KRAS gene G12D site mutant cells to verify the effects of different parameters in the steps of the preparation method of the present invention, such as agarose concentration, temperature when agarose is mixed with cells, paraffin block solidification temperature and solidification time, on paraffin sections.

[0096] 1. Take 24 tubes of cells with the same number, refer to the method described in Example 1, prepare 24 paraffin blocks according to the parameters of the 24 experimental groups in Table 6, and cut 3 slices from each block for detection;

[0097] Table 5 Experimental parameters

[0098] Classification parameter Agarose concentration A1: 2%, A2: 3% Temperature when agarose is mixed with cells B1: 50℃, B2: 60℃, B3: 65℃ Paraffin block solidification temperature C1: -15℃, C2: -25℃ Paraffin block solidification time D1: 30min, D2: 60min

[0099] Table 6 Experimental design

[0100]

[0101]

[0102] 2. After nucleic acid extraction using the reagents described in Example 2, digital PCR detection was performed, and the amplification system and procedure are shown in Table 2-3;

[0103] 3. The digital PCR results of KRAS gene G12D site mutation are as follows:

[0104] Table 7 Digital PCR results of KRAS gene G12D site mutation

[0105]

[0106]

[0107] From the results in Table 7, it can be seen that within the parameter conditions of the present invention, using the same concentration level of cells and following the procedures of experimental groups 1 to 24 for sample preparation, wax blocks with the same effect can be obtained, indicating that the parameter ranges in the steps of agarose concentration, agarose mixing cell temperature, paraffin block solidification temperature and solidification time in the preparation method of the present invention are applicable.

[0108] Comparative Example 1

[0109] This comparative example uses KRAS gene G12D site mutation cells to verify the effects of different transparent and permeabilization procedures on formalin-fixed paraffin-embedded cell samples. The specific method is as follows:

[0110] 1. Three cell agarose blocks with the same number of cells were prepared using the method described in Example 1, and then dehydrated, transparentized and infiltrated using an automatic vacuum tissue dehydrator according to the following different procedures; the experimental group followed the dehydration, transparentization and infiltration procedures of Example 1, the control group 1 reduced the xylene transparent step in the original procedure of Example 1, and the control group 2 reduced the infiltration procedure. The specific procedures are shown in Table 8;

[0111] Table 8

[0112]

[0113] 2. The samples were embedded and sliced ​​using the method described in Example 1. Six slices were cut from each of the three wax blocks, three of which were tested immediately, and three were placed in a 37°C environment for 7 days before being taken out for testing;

[0114] 3. Nucleic acid extraction was performed using the reagents described in Example 2. The extracted nucleic acid was detected by fluorescence spectrophotometry using Qubit 1×dsDNA HS (high sensitivity) quantitative kit (Thermo Fisher) and Qubit4 fluorometer (Thermo Fisher). The degradation rate of the paraffin section was calculated using the following formula: degradation rate = (nucleic acid concentration on day 0 - nucleic acid concentration on day 7) / nucleic acid concentration on day 0 × 100%. The results are shown in Table 9:

[0115] Table 9 Fluorescence spectrophotometry detection results

[0116]

[0117] From the results in Table 9, it can be seen that the mean nucleic acid concentration on the 0th day of detection was control group 1> experimental group> control group 2. After being placed at 37°C for 7 days, the mean nucleic acid concentration was experimental group> control group 1> control group 2. Comparing the nucleic acid degradation rates of the three groups of samples, control group 1> control group 2> experimental group. The initial test and nucleic acid degradation rate results of control group 2 were the worst; although the initial test concentration of control group 1 was the highest, its nucleic acid degradation rate was much higher than that of the other two groups of samples, and its stability was poor; the nucleic acid degradation rate of the experimental group samples was the lowest, that is, the samples prepared using the transparent and permeation procedures of Example 1 were more stable.

[0118] This comparative example also used the three groups of KRAS gene G12D site mutation cells prepared above to perform hematoxylin-eosin staining according to the method described in Example 3. Figure 10-12 The results show that the single cells in the experimental group slices are more complete and the cells are more evenly distributed, indicating that the cell formalin-fixed paraffin-embedded samples prepared using the transparent and permeation procedures of Example 1 have better effects and the single cells are more complete.

[0119] Comparative Example 2

[0120] This comparative example uses KRAS gene G12D site mutation cells to verify the effects of different fixatives and concentrations, fixation time, and agarose concentration on formalin-fixed paraffin-embedded cell samples. The specific differences between the preparation method and Example 1 are shown in Table 10:

[0121] Table 10

[0122]

[0123] The agarose blocks prepared in the control groups 3-8 were dehydrated, transparentized and infiltrated by an automatic vacuum tissue dehydrator according to the procedure described in Example 1, and then the samples were embedded and sliced. Six slices were cut from each of the six wax blocks, three slices were tested immediately, and three slices were placed in a 37°C environment for 7 days before being taken out for testing; then nucleic acid was extracted using the reagents described in Example 2, and the extracted nucleic acid was detected by fluorescence spectrophotometry using Qubit 1×dsDNA HS (high sensitivity) quantitative kit (Thermo Fisher) and Qubit4 fluorometer (Thermo Fisher), and the degradation rate of the paraffin sections was calculated using the following formula: degradation rate = (nucleic acid concentration on day 0 - nucleic acid concentration on day 7) / nucleic acid concentration on day 0 × 100%, and the average value of the data of the three slices was finally taken. The specific results are shown in Table 11.

[0124] Table 11

[0125]

[0126]

[0127] From the results in Table 11, it can be seen that the nucleic acid degradation rate of the slice sample prepared by the technical solution of the present invention is the lowest, and the sample is more stable. When the formalin solution was used for cell fixation in the control group 3, the fixative could not effectively penetrate within a short fixation time, resulting in that the cell nucleic acid could not be effectively protected, the nucleic acid degradation rate increased compared with Example 1, and the stability decreased; in the control group 4, 10% formaldehyde solution was used for cell fixation, and the nucleic acid degradation rate also increased. Excessively high concentrations of formaldehyde solution fixatives may cause excessive cross-linking of proteins and nucleic acids, thereby causing denaturation and degradation of nucleic acids. In addition, high concentrations of fixatives may prevent the nucleic acids inside the cells from effectively contacting the fixatives, thereby reducing the protective effect of nucleic acids; in the control groups 5-6, the cell fixation When the fixing time exceeds the specified range of the present invention, the nucleic acid degradation rate increases significantly. If the fixing time is too short, it may lead to insufficient crosslinking, which cannot effectively protect the nucleic acid and increase the risk of degradation. If the fixing time is too long, it may lead to excessive crosslinking, resulting in changes in the spatial structure of the nucleic acid, and even making the nucleic acid unable to be effectively extracted or used. Control group 7 uses a 1% agarose solution to prepare agarose blocks. The concentration of the agarose solution is too low, resulting in the inability of the agarose block to effectively solidify. The agarose solution concentration of control group 8 is too high, which may affect the fixation of the internal structure of the cell, thereby resulting in the inability to effectively protect the cell nucleic acid and the increase in the nucleic acid degradation rate. The present invention optimizes the concentration of the fixative, the fixing time, and the agarose concentration, and various parameters cooperate with each other to ensure the stability of the nucleic acid and the effectiveness of subsequent analysis.

[0128] Effect example

[0129] This effect example is to use the method of the present invention to embed and slice tumor cell samples from different organs to verify whether the tumor cells of different organs are suitable for the method of the present invention.

[0130] Table 12 Cell sample information

[0131] serial number name Mutation site 1 Human colon adenocarcinoma cells KRAS、PIK3CA 2 Breast cancer cells KRAS、BRAF 3 Human myeloid leukemia cells ABL 4 Human hepatoblastoma cells NRAS 5 Human bladder transitional cell carcinoma KRAS 6 Human malignant embryonic rhabdoid tumor cells NRAS 7 Human monocytic leukemia cells NRAS

[0132] 1. Take one tube of each cell shown in Table 12, and prepare 7 paraffin blocks according to the method described in Example 1, cut each into 6 slices, 3 slices are tested immediately, and 3 slices are placed in a 37°C environment for 7 days before being taken out for testing;

[0133] 2. Digital PCR detection was performed according to the method of Example 2. The reaction system and amplification conditions were shown in Tables 2-3. The extracted nucleic acid was detected by fluorescence spectrophotometry using Qubit 1×dsDNA HS (high sensitivity) quantification kit (Thermo Fisher) and Qubit4 fluorometer (Thermo Fisher), and the nucleic acid degradation rate was calculated. The specific results are shown in Table 13.

[0134] Table 13 Fluorescence spectrophotometry detection results

[0135] Sample / Result (ng / μL) 37℃ 0 days 7 days at 37°C Nucleic acid degradation rate Human colon adenocarcinoma cells 11.01 9.06 17.71％ Breast cancer cells 13.55 11.49 15.20％ Human myeloid leukemia cells 12.30 10.28 16.42％ Human hepatoblastoma cells 14.75 12.0 18.64％ Human bladder transitional cell carcinoma 10.60 8.99 15.19％ Human malignant embryonic rhabdoid tumor cells 13.95 11.77 15.63％ Human monocytic leukemia cells 8.33 7.01 15.85％

[0136] Depend on Figure 13-19 It can be seen that the paraffin sections prepared by the present invention can be detected normally; as shown in Table 13, after being placed at 37° C. for 7 days, the nucleic acid degradation rate of the samples was less than 20%, and the stability was good.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a paraffin-embedded cell sample, characterized in that: The following steps are involved: S1. Fix the cell sample with formaldehyde solution, centrifuge and discard the supernatant, resuspend the cells to obtain a cell suspension; S2. Prepare an agarose solution, cool it to 50-65°C, mix the agarose solution with the cell suspension, and cool it down to solidify to form an agarose block; S3. The agarose block is dehydrated, transparentized, and infiltrated, then embedded in paraffin, solidified at low temperature, and sliced ​​to obtain a cell paraffin-embedded sample.

2. The preparation method according to claim 1, characterized in that In the step S1, the concentration of the formaldehyde solution is 4%, and the fixing time is 25 to 35 minutes.

3. The preparation method according to claim 1, characterized in that: In step S2, the concentration of the agarose solution is 2-3%.

4. The preparation method according to claim 1, characterized in that: In step S3, the dehydration process uses gradient ethanol dehydration, and the concentration of the ethanol is 70-100%.

5. The preparation method according to claim 1, characterized in that: In step S3, the specific process of the transparency is: using xylene to treat for 45 to 60 minutes, and repeating the treatment three times.

6. The preparation method according to claim 1, characterized in that: In step S3, the specific process of the soaking is: using a 60° C. paraffin solution for 45 to 60 minutes, and repeating the treatment three times.

7. The preparation method according to claim 1, characterized in that: In step S3, the specific conditions for the low-temperature solidification are: placing at -15 to -25°C for 30 to 60 minutes.

8. The preparation method according to claim 1, characterized in that: The cell sample includes at least one of human colon adenocarcinoma cells, breast cancer cells, human myeloid leukemia cells, human hepatoblastoma cells, human bladder transitional cell carcinoma cells, human malignant embryonic rhabdoid tumor cells and human monocytic leukemia cells.

9. Use of the preparation method according to any one of claims 1 to 8 in preparing cell paraffin-embedded samples.

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