Novel environment-friendly dewaxing transparent agent and preparation method thereof

Abstract

The invention discloses a novel environment-friendly dewaxing transparent agent, which is prepared from 70 to 95 parts of isoparaffin and 5 to 30 parts of D-limonene. The environment-friendly dewaxing and transparentizing agent has the beneficial effects that the problem that toxic and odorous xylene is used for dewaxing and transparentizing pathological tissue slices in the prior art is solved, formula components are simplified, the complexity of a preparation process is reduced, and the environment-friendly dewaxing and transparentizing agent is suitable for commercial large-scale production; the isoparaffin is a component which is low in toxicity, colorless, odorless and naturally degradable, is widely applied to products such as skin care products, has good solubility to paraffin and is mutually soluble with ethanol, and the D-limonene is derived from natural orange peel, is not chemically synthesized, has good wax dissolving performance and is free of toxic and side effects. The solvent can replace various chemical solvents, is well compatible with ethanol and neutral resin sealing adhesive, and is suitable for preparing an environment-friendly dewaxing transparent agent.

Description

Technical Field

[0001] This invention relates to the field of pathological tissue sectioning technology, specifically to a novel environmentally friendly dewaxing and clearing agent and its preparation method. Background Technology

[0002] Pathological samples are mainly divided into tissue samples and liquid samples. The pathological diagnosis of tissue samples generally uses paraffin sectioning techniques. Among them, HE staining is one of the most commonly used staining methods in paraffin sectioning. It is suitable for staining any animal tissue and can reveal the basic morphology and pathological lesions. It is also one of the most basic and universal techniques in scientific research and teaching in pathology, histology, embryology, etc. HE requires the following steps: tissue sampling, fixation, dehydration, clearing, paraffin infiltration, embedding, sectioning, baking, dewaxing, hydration, hematoxylin staining, washing, dehydration, eosin staining, clearing, and mounting. Dewaxing and clearing of tissues requires the use of dewaxing and clearing agents. If dewaxing and clearing are not thorough, it will directly lead to substandard slide quality, such as uneven staining, inconsistent nuclear-cytoplasmic contrast, and unclear nuclear structure. Pathological diagnosis of liquid samples often uses techniques such as smear preparation and thin-layer liquid-based cytology preparation. The staining method involved is generally Papanicolaou staining. Clearing before mounting also requires the use of clearing agents. The quality of the clearing effect is related to whether the nuclear cytoplasm is clear and transparent, especially for cancer cells or aggregated cells, which directly affects the accuracy of diagnosis.

[0003] In the existing technology, pathologists use xylene as a reagent for clearing and dewaxing. The advantages of xylene are that it has a good dewaxing and clearing effect, a large amount of wax dissolved, and a low price. The disadvantages of xylene are that it is a moderately toxic substance with high volatility, which can damage the human body's respiratory function, nerve function, immune function, reproductive function and hematopoietic function. It is also one of the main carcinogens and is not conducive to environmental protection.

[0004] Chinese invention patent application CN114964978A discloses "an environmentally friendly dewaxing liquid and its preparation method". This method uses a dewaxing liquid composed of isododecane, isoalkanes D40, methylcyclohexane, n-heptane, anhydrous ethanol, surfactant, cosolvent, isooctadecyl alcohol, and epoxy stearate to replace xylene. The dewaxing time is shorter than that of xylene, and the tissue does not shrink or become brittle after long-term immersion. However, its formula has many components and the transparency effect in liquid-based cytology slides is not good.

[0005] Chinese invention patent application CN106370491A discloses "environmentally friendly transparent dewaxing solution and its preparation method and its application in the preparation of pathological sections". The method uses a dewaxing solution composed of white oil, nonane, octane, heptane, castor oil, pentanol and hexanol to replace xylene for tissue pathology slide preparation. The prepared tissue staining slides will not damage tissue cells and tissue antigens. Although it meets environmental protection requirements, its slide preparation effect in liquid-based cytology is not good. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a novel environmentally friendly dewaxing and clearing agent and its preparation method. This solves the technical problem that existing environmentally friendly dewaxing liquid formulations have many components and complicated preparation processes. Moreover, they often use toxic and odorous xylene for dewaxing and clearing. Although they achieve good dewaxing and clearing effects in pathological slide preparation, their clearing effect in liquid-based cytology slide preparation is not ideal, and they cannot even be used for liquid-based cytology slide preparation.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a novel environmentally friendly dewaxing and clarifying agent and its preparation method, comprising:

[0009] It includes 70-95 parts of isoalkanes and 5-30 parts of D-limonene.

[0010] In some embodiments, the mass ratio of each component in the environmentally friendly dewaxing and clarifying agent is: 90 parts isoparaffin and 10 parts D-limonene.

[0011] In some embodiments, the isoalkane is an Isopar series isoalkane or a D series isoalkane.

[0012] In some embodiments, the Isopar series isoalkanes are one or more of Isopar C, Isopar E, Isopar L, Isopar G, Isopar H, and Isopar M.

[0013] In some embodiments, the D-series isoalkanes are one or more of isoalkanes D40, D60, D80, D100, and D130.

[0014] In some embodiments, the following steps are included:

[0015] S1. Weigh out isoparaffin and D-limonene according to the specified quantities;

[0016] S2. Pour the isoalkanes and D-limonene into a beaker in sequence and stir at 400-600 rpm for 10-15 minutes.

[0017] S3. Filter the mixed solution through a 0.45µm filter membrane. The resulting filtrate is the environmentally friendly dewaxing and clarifying agent.

[0018] Compared with existing technologies, this invention provides a novel environmentally friendly dewaxing and transparentizing agent and its preparation method. The components of the environmentally friendly dewaxing and transparentizing agent in this application are only two raw materials: isoparaffin and D-limonene. The formula is simple and the preparation process is simple and easy to operate, making it very suitable for large-scale commercial production. Moreover, both components of the environmentally friendly dewaxing and transparentizing agent are non-toxic and pollution-free environmentally friendly raw materials. Isoparaffin is clear, colorless, and odorless, contains almost no aromatics and sulfur, can be naturally degraded, and is harmless to the human body and water. Isoparaffin is low in toxicity and is not a carcinogen. It is widely used in skin care products, hair care products, sunscreens, cosmetics, makeup, makeup removers, and other products. Isoparaffin has good solubility in paraffin and is miscible with ethanol, making it very suitable for use in environmentally friendly dewaxing and transparentizing agents.

[0019] D-Limonene is a natural component found in orange peels, not a chemically synthesized product. It is non-toxic and harmless, and has become an important component in cleaning agent formulations. D-Limonene can replace various chemical solvents currently used in industrial cleaning, changing the current situation where chemical products are toxic and harmful.

[0020] In addition, D-limonene has excellent wax-dissolving properties and is compatible with ethanol and neutral resin mounting adhesives, making it very suitable for use as an environmentally friendly dewaxing and clearing agent. The environmentally friendly dewaxing and clearing agent in this application can be used for both dewaxing and clearing of pathological tissue sections and clearing of cytological slides, solving the problem that existing environmentally friendly dewaxing and clearing agents cannot be used for both purposes. At the same time, both raw materials of the environmentally friendly dewaxing and clearing agent are commercially available and produced in large quantities, and the raw materials are readily available and inexpensive. Attached Figure Description

[0021] Figure 1 This is a comparison image of the staining results of hamartomatous polyp tissue sections in Example 1;

[0022] Figure 2 This is a comparison image of the stained slides of tissue sections from nodular goiter with adenoma-like hyperplasia in Example 2;

[0023] Figure 3 This is a comparison image of the staining and slide preparation results of differentiated hepatocellular carcinoma tissue sections in Example 3;

[0024] Figure 4 This is a comparison image of the cervical exfoliated cell staining results from Example 1;

[0025] Figure 5 This is a comparison image of the cervical exfoliated cell staining slide results from Example 2;

[0026] Figure 6This is a comparison image of the cervical exfoliated cell staining results in Example 3;

[0027] Figure 7 This is a comparison image of the results of cell staining preparation in the pleural and peritoneal fluid in Example 1;

[0028] Figure 8 This is a comparison image of the results of cell staining preparation in the pleural and peritoneal fluid in Example 2;

[0029] Figure 9 This is a comparison image of the results of cell staining preparation in the pleural and peritoneal fluid in Example 3. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] Example 1

[0032] This embodiment provides an environmentally friendly dewaxing and clarifying agent, which comprises, by weight, 90 parts of Isopar L and 10 parts of D-limonene;

[0033] The preparation method of the environmentally friendly dewaxing and clarifying agent includes the following steps:

[0034] Step 1: Weigh out 90 parts of Isopar L and 10 parts of D-limonene according to the specified quantities;

[0035] Step 2: Pour Isopar L and D-limonene into a beaker in sequence and stir at 400 rpm for 10 minutes;

[0036] Step 3: Filter the mixed solution through a 0.45µm filter membrane. The resulting filtrate is the environmentally friendly dewaxing and clarifying agent.

[0037] The method for using the environmentally friendly dewaxing and clearing agent of this invention in pathological tissue sections includes the following steps:

[0038] Step 1: Take the paraffin-embedded slide specimen, bake it at 60℃ for 30 minutes, and then immerse it in 3 vats of the environmentally friendly dewaxing and clearing agent of this invention for 5 minutes in each vat.

[0039] Step Two: After soaking in Step One, section the tissue. First, immerse the section in anhydrous ethanol for 1 minute, then transfer it to 95% ethanol for 2 minutes, and then to 75% ethanol for 2 minutes, completing a gradient hydration process from high to low concentration. Next, rinse with circulating water for 1 minute to fully hydrate the tissue and restore its hydrophilicity, providing a suitable environment for subsequent staining. Then, immerse the section in hematoxylin staining solution for 5 minutes to allow the cell nuclei to bind with the dye, turning them blue-purple and clearly displaying the morphology of the cell nuclei. After staining, rinse with circulating water for 2 minutes to remove unbound dye and prevent background interference. Then, immerse the section in a blueing solution for 2 minutes to adjust the pH value using a weakly alkaline solution, causing the cell nuclei to change from brownish-red to a bright blue, improving staining contrast and the clarity of microscopic observation. Rinse again with circulating water for 1 minute to stop the blueing reaction. Then... The slides were immersed in eosin staining solution for 3 minutes to stain the cytoplasm and extracellular matrix pink, creating a striking color contrast between the cell nucleus and cytoplasm. After staining, the slides were sequentially dehydrated using 75% ethanol (1 minute), 95% ethanol (1 minute), and anhydrous ethanol (1 minute) to gradually replace the water in the tissue, preparing for the clearing step. The slides were then immersed twice in an environmentally friendly dewaxing and clearing agent for 2 minutes each time. This agent's excellent solubility and light transmittance completely replaced the ethanol and achieved high tissue transparency, significantly improving light penetration. Finally, the slides were mounted with neutral resin glue to fix the tissue structure and prevent oxidation, fading, or contamination, creating complete pathological slide specimens. The staining quality, tissue structure clarity, cell morphology integrity, and background cleanliness were observed under an optical microscope to assess the slide preparation effect.

[0040] The application method of environmentally friendly dewaxing and clearing agents in gynecological liquid-based cytology slide preparation includes the following steps:

[0041] Step 1: Shake the bottle containing the cell preservation solution of exfoliated cervical cells on a shaker for 2 minutes;

[0042] Step 2: After shaking, use a pipette to take 1 ml of cervical exfoliated cell sample into the sedimentation chamber, add 0.5 ml of separation solution to the sedimentation chamber, and let it settle naturally for 10 min;

[0043] Step 3: Empty the liquid from the settling chamber and rinse the settling chamber 3 times with buffer solution, 1 minute each time;

[0044] Step 4: Discard the buffer solution in the settling chamber and add hematoxylin staining solution for 2 minutes;

[0045] Step 5: Discard the hematoxylin staining solution in the sedimentation chamber, and wash the sedimentation chamber 3 times with buffer solution, 1 minute each time;

[0046] Step 6: Pour out the buffer solution in the settling chamber, add rinsing solution to the settling chamber, let stand for 1 minute, and repeat 3 times;

[0047] Step 7: Discard the rinsing solution in the settling chamber, add EAOG or EA50 staining solution to the settling chamber, and stain for 2 minutes;

[0048] Step 8: Discard the EAOG or EA50 staining solution in the settling chamber, and rinse the settling chamber 3 times with rinsing solution, 1 minute each time;

[0049] Step 9: Discard the rinsing liquid in the settling chamber, and add the environmentally friendly dewaxing and clarifying agent of this invention for 5 minutes;

[0050] Step 10: Discard the environmentally friendly dewaxing and clarifying agent in the settling chamber, remove the settling chamber, seal the slide with neutral resin glue, and observe the slide preparation results under an optical microscope.

[0051] The method for using environmentally friendly dewaxing and clearing agents in non-gynecological liquid-based cytology slide preparation includes the following steps:

[0052] Step 1: Take 2ml of pleural and peritoneal fluid sample and let it settle in the sedimentation chamber for 10 minutes;

[0053] Step 2: Pour out the liquid from the settling chamber and rinse the settling chamber 3 times with buffer solution, 1 minute each time;

[0054] Step 3: Discard the buffer solution in the settling chamber and add hematoxylin staining solution for 30 seconds;

[0055] Step 4: Discard the hematoxylin staining solution in the sedimentation chamber, and wash the sedimentation chamber 3 times with buffer solution, 1 minute each time;

[0056] Step 5: Pour out the buffer solution in the settling chamber, add rinsing solution to the settling chamber, let stand for 1 minute, and repeat 3 times;

[0057] Step 6: Discard the rinsing solution in the settling chamber, add EAOG or EA50 staining solution to the settling chamber, and stain for 30 seconds;

[0058] Step 7: Discard the EAOG or EA50 staining solution in the settling chamber, and rinse the settling chamber 3 times with rinsing solution, 1 minute each time;

[0059] Step 8: Discard the rinsing liquid in the settling chamber, and add the environmentally friendly dewaxing and clarifying agent of this invention for 5 minutes;

[0060] Step 9: Discard the environmentally friendly dewaxing and clarifying agent in the settling chamber, remove the settling chamber, seal the slide with neutral resin glue, and observe the slide preparation results under an optical microscope;

[0061] Wherein 1a is a tissue staining section prepared by xylene treatment in the control example, 1b is a tissue staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention, 4a is a gynecological cytology staining section prepared by xylene in the control example, 4b is a gynecological cytology staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention, 7a is a non-gynecological pleural and peritoneal fluid cytology staining section prepared by xylene treatment in the control example, and 7b is a non-gynecological pleural and peritoneal fluid cytology staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention.

[0062] Example 2

[0063] This embodiment provides an environmentally friendly dewaxing and clarifying agent, which comprises, by weight, the following:

[0064] 35 parts Isopar C, 35 parts Isopar E, and 30 parts D-limonene;

[0065] The preparation method of the environmentally friendly dewaxing and clarifying agent includes the following steps:

[0066] Step 1: Weigh out 35 parts of Isopar C, 35 parts of Isopar E, and 30 parts of D-limonene according to the specified quantities;

[0067] Step 2: Pour Isopar C, Isopar E, and D-limonene into a beaker in sequence and stir at 600 rpm for 15 minutes;

[0068] The mixed solution is filtered through a 0.45µm filter membrane, and the resulting filtrate is the environmentally friendly dewaxing and clarifying agent.

[0069] The method for using the environmentally friendly dewaxing and clearing agent of this invention in pathological tissue sections includes the following steps:

[0070] Step 1: Take a paraffin section specimen, bake it at 70℃ for 20 minutes, and then immerse it in 3 vats of the environmentally friendly dewaxing and clearing agent of this invention for 10 minutes in each vat.

[0071] Step Two: The tissue sections dewaxed in Step One are then stained sequentially: First, the sections are immersed in anhydrous ethanol for 2 minutes, then transferred to 95% ethanol for 1 minute, and then to 75% ethanol for 1 minute, completing a gradual hydration process from high to low concentrations. Next, the sections are rinsed in circulating water for 1 minute to ensure thorough hydration and provide a suitable environment for subsequent staining. Then, the sections are immersed in hematoxylin staining solution for 5 minutes to clearly stain the nuclear structures. After staining, the sections are rinsed with circulating water for 1 minute to remove unbound dye. Finally, the sections are immersed in a blueing solution for 1 minute to change the nuclear color from brownish-red to a bright blue, improving staining contrast and microscopic appearance. Observe the effect; rinse again with circulating water for 1 minute to stop the blueing reaction; then immerse the section in eosin staining solution for 2 minutes to make the cytoplasm and extracellular matrix uniformly pink; after staining, sequentially immerse in 75% ethanol for 2 minutes, 95% ethanol for 2 minutes, and anhydrous ethanol for 2 minutes for gradient dehydration to ensure that the water inside the tissue is completely replaced; then immerse the section twice in environmentally friendly dewaxing and clearing agent for 2 minutes each time to fully replace the ethanol in the tissue and achieve tissue transparency, enhancing light penetration; finally, use neutral resin glue to mount the section to prepare a complete paraffin section specimen, and observe its staining quality, tissue structure clarity, and background cleanliness under an optical microscope.

[0072] The application method of environmentally friendly dewaxing and clearing agents in gynecological liquid-based cytology slide preparation includes the following steps:

[0073] Step 1: Shake the bottle containing the cell preservation solution of exfoliated cervical cells on a shaker for 2 minutes;

[0074] Step 2: After shaking, use a pipette to take 1 ml of cervical exfoliated cell sample into the sedimentation chamber, add 0.5 ml of separation solution to the sedimentation chamber, and let it settle naturally for 15 min;

[0075] Step 3: Empty the liquid from the settling chamber and rinse the settling chamber 3 times with buffer solution, 1 minute each time;

[0076] Step 4: Discard the buffer solution in the settling chamber and add hematoxylin staining solution to stain for 3 minutes;

[0077] Step 5: Discard the hematoxylin staining solution in the sedimentation chamber, and wash the sedimentation chamber 3 times with buffer solution, 1 minute each time;

[0078] Step 6: Pour out the buffer solution in the settling chamber, add rinsing solution to the settling chamber, let stand for 1 minute, and repeat 3 times;

[0079] Step 7: Discard the rinsing solution in the settling chamber, add EAOG or EA50 staining solution to the settling chamber, and stain for 3 minutes;

[0080] Step 8: Discard the EAOG or EA50 staining solution in the settling chamber, and rinse the settling chamber 3 times with rinsing solution, 1 minute each time;

[0081] Step 9: Discard the rinsing liquid in the settling chamber, and add the environmentally friendly dewaxing and clarifying agent of this invention for 3 minutes;

[0082] Step 10: Discard the environmentally friendly dewaxing and clarifying agent in the settling chamber, remove the settling chamber, seal the slide with neutral resin glue, and observe the slide preparation results under an optical microscope.

[0083] The method for using environmentally friendly dewaxing and clearing agents in non-gynecological liquid-based cytology slide preparation includes the following steps:

[0084] Step 1: Take 2ml of pleural and peritoneal fluid sample and let it settle in the sedimentation chamber for 10 minutes;

[0085] Step 2: Pour out the liquid from the settling chamber and rinse the settling chamber 3 times with buffer solution, 1 minute each time;

[0086] Step 3: Discard the buffer solution in the settling chamber and add hematoxylin staining solution to stain for 1 minute;

[0087] Step 4: Discard the hematoxylin staining solution in the sedimentation chamber, and wash the sedimentation chamber 3 times with buffer solution, 1 minute each time;

[0088] Step 5: Pour out the buffer solution in the settling chamber, add rinsing solution to the settling chamber, let stand for 1 minute, and repeat 3 times;

[0089] Step 6: Discard the rinsing solution in the settling chamber, add EAOG or EA50 staining solution to the settling chamber, and stain for 1 minute;

[0090] Step 7: Discard the EAOG or EA50 staining solution in the settling chamber, and rinse the settling chamber 3 times with rinsing solution, 1 minute each time;

[0091] Step 8: Discard the rinsing liquid in the settling chamber, and add the environmentally friendly dewaxing and clarifying agent of this invention for 3 minutes;

[0092] Step 9: Discard the environmentally friendly dewaxing and clarifying agent in the settling chamber, remove the settling chamber, seal the slide with neutral resin glue, and observe the slide preparation results under an optical microscope;

[0093] Wherein 2a is a tissue staining section prepared by xylene treatment in the control example, 2b is a tissue staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention, 5a is a gynecological cytology staining section prepared by xylene treatment in the control example, 5b is a gynecological cytology staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention, 8a is a non-gynecological pleural and peritoneal fluid cytology staining section prepared by xylene treatment in the control example, and 8b is a non-gynecological pleural and peritoneal fluid cytology staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention.

[0094] Example 3

[0095] This embodiment provides an environmentally friendly dewaxing and clarifying agent, which comprises, by weight, the following:

[0096] 95 parts of isoparaffin D80 and 5 parts of D-limonene;

[0097] The preparation method of the environmentally friendly dewaxing and clarifying agent includes the following steps:

[0098] Step 1: Weigh out 95 parts of isoparaffin D80 and 5 parts of D-limonene according to the specified quantities;

[0099] Step 2: Pour the isoalkanes and D-limonene into a beaker in sequence, and stir at 500 rpm for 12 minutes;

[0100] Step 3: Filter the mixed solution through a 0.45µm filter membrane. The resulting filtrate is the environmentally friendly dewaxing and clarifying agent.

[0101] The method for using the environmentally friendly dewaxing and clearing agent of this invention in pathological tissue sections includes the following steps:

[0102] Step 1: Take a paraffin section specimen, bake it at 65℃ for 20 minutes, and then immerse it in 3 tanks of the environmentally friendly dewaxing and clearing agent of this invention for 6 minutes each.

[0103] Step Two: The tissue sections dewaxed in Step One are subjected to a series of treatments including gradient hydration, staining, clearing, and mounting to complete the entire paraffin section preparation process. The specific steps are as follows: First, immerse the dewaxed sections in anhydrous ethanol for 2 minutes; then transfer them to 95% ethanol for 1 minute for further hydration; next, immerse them in 75% ethanol for 1 minute to complete the gradient hydration process from high to low concentrations, avoiding tissue structure damage due to excessively rapid hydration. Finally, rinse the sections in circulating water for 1 minute to ensure thorough tissue hydration. To restore its hydrophilicity and provide a favorable reaction environment for subsequent staining steps, the sections are then immersed in hematoxylin staining solution for 10 minutes. This allows the cell nuclei to specifically bind to hematoxylin, turning them blue-purple and clearly revealing the morphology and structure of the cell nuclei. After staining, the sections are rinsed with circulating water for 1 minute to remove unbound dye and prevent background staining interference. Subsequently, the sections are immersed in a blueing solution for 2 minutes. The pH is adjusted using a weakly alkaline solution to change the staining of the cell nuclei from brownish-red to a bright blue, improving staining contrast and the clarity of microscopic observation. Rinse again with circulating water for 1 minute to stop the blueing reaction and remove any residual blueing solution. Next, immerse the sections in eosin staining solution for 3 minutes to stain the cytoplasm, extracellular matrix, and certain organelles (non-nuclear structures) pink, creating a clear color contrast between the nucleus and cytoplasm and enhancing the identification of tissue structures. After staining, perform gradient alcohol dehydration: first immerse the sections in 75% ethanol for 2 minutes, then in 95% ethanol for 2 minutes, and finally in anhydrous ethanol for 2 minutes, gradually replacing the water in the tissue to ensure complete dehydration and transparency. After preparing the necessary steps, the slides are immersed twice in an environmentally friendly dewaxing and clearing agent for 3 minutes each time. This agent utilizes the good solubility and light transmittance of the agent to completely replace the ethanol in the tissue and make the tissue highly transparent, significantly improving light transmission ability and facilitating observation under an optical microscope. Finally, the slides are mounted with neutral resin glue to fix the tissue structure and prevent oxidation, fading or contamination, thus preparing a complete pathological slide specimen. The staining effect, tissue structure clarity, cell morphology integrity and background cleanliness are observed under an optical microscope to evaluate the quality of the slide preparation.

[0104] The application method of environmentally friendly dewaxing and clearing agents in gynecological liquid-based cytology slide preparation includes the following steps:

[0105] Step 1: Shake the bottle containing the cell preservation solution of exfoliated cervical cells on a shaker for 2 minutes;

[0106] Step 2: After shaking, use a pipette to take 1 ml of cervical exfoliated cell sample into the sedimentation chamber, add 0.5 ml of separation solution to the sedimentation chamber, and let it settle naturally for 12 min;

[0107] Step 3: Empty the liquid from the settling chamber and rinse the settling chamber 3 times with buffer solution, 1 minute each time;

[0108] Step 4: Discard the buffer solution in the settling chamber and add hematoxylin staining solution to stain for 3 minutes;

[0109] Step 5: Discard the hematoxylin staining solution in the sedimentation chamber, and wash the sedimentation chamber 3 times with buffer solution, 1 minute each time;

[0110] Step 6: Pour out the buffer solution in the settling chamber, add rinsing solution to the settling chamber, let stand for 1 minute, and repeat 3 times;

[0111] Step 7: Discard the rinsing solution in the settling chamber, add EAOG or EA50 staining solution to the settling chamber, and stain for 3 minutes;

[0112] Step 8: Discard the EAOG or EA50 staining solution in the settling chamber, and rinse the settling chamber 3 times with rinsing solution, 1 minute each time;

[0113] Step 9: Discard the rinsing liquid in the settling chamber, and add the environmentally friendly dewaxing and clarifying agent of this invention for 3 minutes;

[0114] Step 10: Discard the environmentally friendly dewaxing and clarifying agent in the settling chamber, remove the settling chamber, seal the slide with neutral resin glue, and observe the slide preparation results under an optical microscope.

[0115] The method for using environmentally friendly dewaxing and clearing agents in non-gynecological liquid-based cytology slide preparation includes the following steps:

[0116] Step 1: Take 2ml of pleural and peritoneal fluid sample and let it settle in the sedimentation chamber for 10 minutes;

[0117] Step 2: Pour out the liquid from the settling chamber and rinse the settling chamber 3 times with buffer solution, 1 minute each time;

[0118] Step 3: Discard the buffer solution in the settling chamber and add hematoxylin staining solution for 40 seconds;

[0119] Step 4: Discard the hematoxylin staining solution in the sedimentation chamber, and wash the sedimentation chamber 3 times with buffer solution, 1 minute each time;

[0120] Step 5: Pour out the buffer solution in the settling chamber, add rinsing solution to the settling chamber, let stand for 1 minute, and repeat 3 times;

[0121] Step 6: Discard the rinsing solution in the settling chamber, add EAOG or EA50 staining solution to the settling chamber, and stain for 40 seconds;

[0122] Step 7: Discard the EAOG or EA50 staining solution in the settling chamber, and rinse the settling chamber 3 times with rinsing solution, 1 minute each time;

[0123] Step 8: Discard the rinsing liquid in the settling chamber, and add the environmentally friendly dewaxing and clarifying agent of this invention for 4 minutes;

[0124] Step 9: Discard the environmentally friendly dewaxing and clarifying agent in the settling chamber, remove the settling chamber, seal the slide with neutral resin glue, and observe the slide preparation results under an optical microscope;

[0125] Wherein 3a is a tissue staining section prepared by xylene treatment in the control example, 3b is a tissue staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention, 6a is a gynecological cytology staining section prepared by xylene treatment in the control example, 6b is a gynecological cytology staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention, 9a is a non-gynecological pleural and peritoneal fluid cytology staining section prepared by xylene treatment in the control example, and 9b is a non-gynecological pleural and peritoneal fluid cytology staining section prepared by the environmentally friendly dewaxing and clearing agent of the present invention.

[0126] The environmentally friendly dewaxing and transparentizing agent of the present invention used in the dewaxing and transparentizing steps of Examples 1, 2 and 3 was replaced with xylene, while the other steps and parameters were the same. Using this as a control, the dewaxing and transparentizing effects of xylene and the environmentally friendly dewaxing and transparentizing agent of the present invention were compared.

[0127] The stained pathological tissue sections, gynecological and non-gynecological cytological slides were observed under a microscope, and the results were as follows: Figure 1-9 As shown;

[0128] from Figure 1-3 It can be seen that: after dewaxing pathological tissues using the environmentally friendly dewaxing and clearing solution of this invention, HE staining and clearing are performed. The dewaxing is thorough, the background is clean, the nuclear cytoplasm staining is uniform and bright, the cell nuclei and cytoplasm are clear, the layers are distinct, and the dewaxing and clearing effect is good. There is no significant difference compared with the traditional xylene dewaxing and clearing effect, and it can achieve the effect of xylene.

[0129] from Figure 4-6 It can be seen that: when gynecological cervical exfoliated cells are prepared by Papanicolaou staining and then cleared using the environmentally friendly dewaxing and clearing solution of this invention, the entire slide is brightly stained, the cytoplasm and cell nucleus are clearly stained, the cell structure is clear and easy to identify, the background is clean and bright, and the clearing effect is good. There is no significant difference compared with the traditional xylene dewaxing and clearing effect, and it can achieve the effect of xylene.

[0130] from Figure 7-9 It can be seen that: when non-gynecological pleural and peritoneal fluid cells are prepared by Papanicolaou staining and cleared using the environmentally friendly dewaxing and clearing solution of this invention, the entire slide is brightly stained, the nuclear membrane, nuclear chromatin and nucleolus are clear, the background is clean, and the clearing effect is good. There is no significant difference compared with the clearing effect of traditional xylene, and it can achieve the clearing effect of xylene.

[0131] Therefore, the environmentally friendly dewaxing and clearing agent of this invention can replace xylene and can be used for dewaxing and clearing of pathological tissue sections, as well as for clearing of gynecological and non-gynecological cytology slides.

[0132] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A novel environment-friendly dewaxing and transparent agent, characterized in that: The isoparaffin is 70-95 parts, and the D-limonene is 5-30 parts. ​ 2. A novel environment-friendly dewaxing and transparent agent according to claim 1, characterized in that: The mass ratio of each component in the environment-friendly dewaxing transparent agent is: isoparaffin 90 parts, and D-limonene 10 parts.

3. A novel environment-friendly dewaxing and transparent agent according to claim 1, characterized in that: The isoparaffin is Isopar series isoparaffin or D series isoparaffin.

4. A novel environment-friendly dewaxing and transparent agent according to claim 3, characterized in that: The Isopar series isoparaffin is one or more of Isopar C, Isopar E, Isopar L, Isopar G, Isopar H, and Isopar M.

5. A novel environment-friendly dewaxing and transparent agent according to claim 3, characterized in that: The D series isoparaffin is one or more of isoparaffin D40, isoparaffin D60, isoparaffin D80, isoparaffin D100, and isoparaffin D130.

6. A method for preparing a novel environment-friendly dewaxing and transparent agent, which is suitable for the novel environment-friendly dewaxing and transparent agent as claimed in any one of claims 1-5, characterized in that, The method comprises the following steps: S1, respectively weighing the isoparaffin and the D-limonene according to the metering; S2, sequentially pouring the isoparaffin and the D-limonene into a beaker, and stirring at a speed of 400-600 revolutions per minute for 10-15 minutes; S3, filtering the mixed solution through a 0.45 µm filter membrane, and the obtained filtrate is the environment-friendly dewaxing transparent agent.

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