Dewaxing fluid and its use

Abstract

The present application relates to a kind of dewaxing liquid and its application, the dewaxing liquid includes dewaxing liquid I and dewaxing liquid II, wherein the mass ratio of 2~4:7 of methyl oleate: mineral oil is formed by dewaxing liquid I, the mass ratio of 2~4:1 of ethanol: cyclohexanone is formed by dewaxing liquid II.The dewaxing liquid of the present application is composed of environmentally friendly and safe ingredients, which can be soluble with ethanol and can dissolve paraffin wax, and the dewaxing effect is equivalent to xylene.Further, the above-mentioned dewaxing liquid is applied to pathological detection tissue dewaxing, which can avoid the harm to the body of the technician, reduce environmental pollution and save xylene purification cost.The cell structure observed under microscope after staining of the pathological section prepared by the present application has bright staining and clear structure, which is basically consistent with the result obtained by xylene dewaxing treatment, and has no effect on doctor reading and diagnosis result.

Description

Technical Field

[0001] This invention relates to the field of pathology technology, specifically to a dewaxing solution and its application. Background Technology

[0002] Hematoxylin and eosin are primarily used for staining cell tissues, a method known as HE staining, which is one of the most commonly used staining methods in paraffin sectioning. The cell nucleus, composed of acidic substances, has a strong affinity for basic dyes (hematoxylin); conversely, the cytoplasm, containing basic substances, has a stronger affinity for acidic dyes (eosin). Therefore, after staining cells or tissue sections with hematoxylin-eosin solution, the cell nucleus is stained a bright blue-purple by hematoxylin, while the cytoplasm, muscle fibers, collagen fibers, etc., appear red to varying degrees. HE staining is the most fundamental staining method in histology, embryology, and pathology teaching and research, and its application is extremely widespread.

[0003] The common steps in hematoxylin-eosin staining include: dewaxing, hydration, hematoxylin staining, differentiation, bluing, eosin staining, dehydration, clearing, and mounting. Before staining, the paraffin wax supporting the tissue must be removed from the paraffin sections; this process is called dewaxing. Dewaxing must be thorough; otherwise, uneven staining, punctate or patchy areas of unstained tissue, and unclear nuclear-cytoplasmic contrast will occur. Currently, the most commonly used dewaxing solution for paraffin sections is xylene. Xylene is also a traditional tissue clearing agent in pathological slide preparation, providing good clearing effects, but it easily causes tissue shrinkage, brittleness, and hardening, making sectioning difficult. Furthermore, xylene is a highly toxic, volatile solution with a strong, irritating odor. It is a neurotoxin that causes significant harm to the physiological systems of those exposed over a long period, producing target organ effects such as severe eye and upper respiratory tract irritation, conjunctival and pharyngeal congestion, dizziness, nausea, limb weakness, and confusion. It poses a serious threat to the health of pathology workers. Xylene volatilization into the air also easily causes environmental pollution, and benzene compounds are difficult to decompose, resulting in higher remediation costs. Therefore, finding alternatives to xylene has become an urgent problem to be solved. Summary of the Invention

[0004] Therefore, it is necessary to provide a dewaxing solution to address the above problems. Using this dewaxing solution for tissue dewaxing can completely avoid the use of xylene, thereby preventing harm to technicians, reducing environmental pollution, and saving on subsequent xylene cleanup costs.

[0005] A dewaxing solution includes dewaxing solution I and dewaxing solution II, wherein dewaxing solution I is composed of methyl oleate and mineral oil in a mass ratio of 2 to 4:7, and dewaxing solution II is composed of ethanol and cyclohexanone in a mass ratio of 2 to 4:1.

[0006] The aforementioned dewaxing solutions are composed of environmentally friendly and safe ingredients. They are miscible with ethanol and can dissolve paraffin. Dewaxing solution I, composed of methyl oleate and mineral oil, can effectively dissolve paraffin in tissue sections, resulting in excellent dewaxing. Simultaneously, it ensures that residual components are well-miscible with cyclohexanone and ethanol in dewaxing solution II, allowing them to be replaced by dewaxing solution II without affecting tissue morphology. Dewaxing solution II, composed of ethanol and cyclohexanone, avoids excessive dehydration of the tissue by ethanol and also allows for better compatibility with the components of dewaxing solution I. The presence of ethanol increases the polarity of dewaxing solution II, improving its overall compatibility with components in subsequent staining processes and preventing rapid changes in solvent physicochemical properties that could adversely affect the tissue. Ultimately, the combination of dewaxing solutions I and II achieves an environmentally friendly dewaxing effect comparable to or even better than xylene.

[0007] In one embodiment, the dewaxing solution I is composed of methyl oleate and mineral oil in a mass ratio of 3:7, and the dewaxing solution II is composed of ethanol and cyclohexanone in a mass ratio of 3:1.

[0008] The present invention also provides the application of a dewaxing solution in the dewaxing and staining of pathological tissues.

[0009] In one embodiment, the staining is hematoxylin-eosin staining.

[0010] This invention also provides a hematoxylin-eosin staining method, comprising the following steps:

[0011] (1) Take tissue sections, dewax them sequentially using the above-mentioned dewaxing solution I and dewaxing solution II, and rinse them;

[0012] (2) Stain with hematoxylin and rinse;

[0013] (3) Disintegrate with hydrochloric acid and alcohol, then rinse;

[0014] (4) Return to blue;

[0015] (5) Stain with eosin and rinse;

[0016] (6) Baking: Baking the slices at 65±5℃ for 1 to 3 minutes;

[0017] (7) Transparent;

[0018] (8) Cover the film.

[0019] The above dyeing method does not use xylene throughout the entire process, thus avoiding harm to technicians, reducing environmental pollution, and saving on xylene purification costs.

[0020] Furthermore, the present invention uses the aforementioned dewaxing solution for tissue staining, which takes less time than the traditional method using xylene for dewaxing, resulting in a shorter overall process time and effectively improving the efficiency of hematoxylin-eosin staining (HE staining). Simultaneously, baking the slides at 65±5℃ for 1–3 minutes before clearing achieves ideal clearing results. Moreover, compared to the xylene method which requires wetting followed by mounting, the present invention, due to the heating process during baking, allows for direct mounting, achieving both ideal clearing and subsequent mounting effects.

[0021] In traditional methods, xylene is used in the clearing and sealing processes. Xylene evaporates quickly and can easily cause tissue cracking. This method completely replaces xylene, thus avoiding the problem of tissue cracking.

[0022] In some embodiments, the dewaxing time in step (1) is 5±1 min, and the tissue is rinsed directly with water after dewaxing. Rinsing the sections directly with water after dewaxing eliminates the alcohol washing step following dewaxing with xylene, further shortening the overall staining time of the tissue.

[0023] In some embodiments, hematoxylin staining in step (2) lasts for 5-6 minutes; eosin staining in step (5) lasts for 30 seconds to 1 minute. The staining time can be appropriately increased or decreased depending on the staining results of hematoxylin and eosin.

[0024] In some embodiments, in step (3), the hydrochloric acid alcohol is prepared by mixing 36wt%-38wt% concentrated hydrochloric acid with 75% ethanol at a volume ratio of 0.5 to 1:99.

[0025] In some embodiments, in step (4), an ammonia solution is used for blueing. The ammonia solution is prepared by mixing ammonia solution with a mass-volume concentration of 25% and water at a volume ratio of 0.5 to 0.8:99. It is understood that warm water or tap water can also be used for rinsing and blueing.

[0026] In some embodiments, in step (7), turpentine is used for clearing treatment for 1 to 3 minutes; in step (8), the film is directly sealed with neutral resin.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The dewaxing solution of this invention is composed of environmentally friendly and safe ingredients. It is miscible with ethanol and can also dissolve paraffin. Its dewaxing effect is comparable to that of xylene.

[0029] When the above dewaxing solution is used in the tissue dewaxing process for pathological testing, it can avoid the physical harm of xylene to technicians, reduce environmental pollution, and save xylene purification costs.

[0030] The hematoxylin-eosin staining method of the present invention, on the one hand, uses the above-mentioned dewaxing solution, which has the advantages of being green, environmentally friendly and safe, and on the other hand, changes the staining steps. After dewaxing, the sections are directly rinsed with water, eliminating the alcohol washing step after dewaxing using the traditional xylene method. The whole process takes less time than conventional methods, effectively improving the efficiency of hematoxylin-eosin staining (HE staining).

[0031] In addition, traditional methods use xylene in the clearing and mounting processes. Because xylene evaporates quickly, it can easily cause tissue to dry and crack. This method completely replaces xylene. When this dewaxing solution is used in conjunction with a xylene-free mounting process, such as the hematoxylin-eosin staining method of the present invention, the problem of tissue drying and cracking can be avoided.

[0032] The pathological sections prepared using the hematoxylin-eosin staining method of the present invention can be observed under a microscope with brightly stained and clearly structured cells, which is basically the same as the result obtained by xylene dewaxing. This method has no impact on doctors' reading and diagnosis results, and also avoids the possible problem of tissue drying and cracking. Attached Figure Description

[0033] Figure 1 In the figures, a and b are HE-stained samples of appendix tissue specimens prepared by the method of Example 4 and Comparative Example 1, respectively (magnification: 10×10).

[0034] Figure 2 In the figures, a and b are HE-stained samples of uterine fibroid specimens prepared by the method of Example 4 and Comparative Example 1, respectively (magnification: 10×10).

[0035] Figure 3 In the figures, a and b are HE-stained samples of endometrial specimen sections prepared by the method of Example 4 and Comparative Example 1, respectively (magnification: 10×10).

[0036] Figure 4 In the figures, a and b are HE-stained samples of prostatectomy specimen sections prepared by the method of Example 4 and Comparative Example 1, respectively (magnification: 10×10).

[0037] Figure 5 In the figures a, b, and c, HE-stained samples of thyroidectomy specimen sections prepared by dewaxing solution according to the method of Example 4 in Examples 1-3 of the present invention (magnification: 10×10);

[0038] Figure 6 In the figures, a and b are HE-stained samples of thyroidectomy specimen sections prepared by the methods of Example 4 and Example 5 of the present invention, respectively (magnification: 10×10).

[0039] Figure 7 In the figures, a and b are HE-stained samples of thyroid specimen sections prepared by the method of Example 4 and Comparative Example 2, respectively (magnification: 10×10). Detailed Implementation

[0040] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0042] Unless otherwise specified, all reagents used in the following examples are commercially available; and all methods used in the following examples are conventional methods.

[0043] Methyl oleate: purchased from Suzhou Fengbei Biotechnology Co., Ltd., model number 1107.

[0044] Mineral oil: purchased from Solarbiom (Beijing Solarbiom Technology Co., Ltd.), model number 8040.

[0045] 0.1% Hydrochloric Acid Ethanol: Mix 36wt%-38wt% concentrated hydrochloric acid with 75% ethanol at a volume ratio of 0.5 to 1:99 to obtain the solution.

[0046] 0.5% ammonia solution: Mix 25% ammonia solution with water at a volume ratio of 0.5 to 0.8:99.

[0047] Example 1

[0048] A dewaxing solution includes dewaxing solution I composed of methyl oleate and mineral oil in a mass ratio of 3:7, and dewaxing solution II composed of ethanol and cyclohexanone in a mass ratio of 3:1.

[0049] Example 2

[0050] A dewaxing solution, comprising dewaxing solution I composed of methyl oleate and mineral oil in a mass ratio of 2:7, and dewaxing solution II composed of ethanol and cyclohexanone in a mass ratio of 2:1.

[0051] Example 3

[0052] A dewaxing solution, comprising dewaxing solution I composed of methyl oleate and mineral oil in a mass ratio of 4:7, and dewaxing solution II composed of ethanol and cyclohexanone in a mass ratio of 4:1.

[0053] Example 4

[0054] A staining method using hematoxylin-eosin includes the following steps:

[0055] (1) Take tissue sections and dewax them for 5 minutes each using dewaxing solution I and dewaxing solution II in Example 1, and then rinse.

[0056] (2) Stain with hematoxylin for 5 minutes, then rinse.

[0057] (3) Differentiate with 0.1% hydrochloric acid ethanol for 2 seconds, rinse, and observe under a microscope.

[0058] (4) 0.5% ammonia solution turns blue in 2 seconds.

[0059] (5) Stain with eosin for 1 minute, then rinse.

[0060] (6) Bake the slices in a 65℃ oven for 2 minutes.

[0061] (7) Turpentine oil clearing agent clears in 2 minutes.

[0062] (8) Neutral resin sealing.

[0063] Example 5

[0064] A staining method using hematoxylin-eosin includes the following steps:

[0065] (1) Take tissue sections and dewax them for 5 minutes each using dewaxing solution I and dewaxing solution II in Example 1, and then rinse.

[0066] (2) Stain with hematoxylin for 5 minutes, then rinse.

[0067] (3) Differentiate with 0.1% hydrochloric acid ethanol for 2 seconds, rinse, and observe under a microscope.

[0068] (4) 0.5% ammonia solution turns blue in 2 seconds.

[0069] (5) Stain with eosin for 1 minute, then rinse.

[0070] (6) Dehydrate with 85%, 95%, and 95% ethanol for 10 seconds each, and dehydrate with 100% ethanol in three separate dehydration sessions, each lasting 1 minute.

[0071] (7) Turpentine oil clearing agent clears in 2 minutes.

[0072] (8) Neutral resin sealing.

[0073] Comparative Example 1

[0074] A staining method using hematoxylin-eosin (HE staining) is provided, wherein the staining reagent is prepared using Sigma raw materials, and the organic solvents such as ethanol are commercially available. The staining method includes the following steps:

[0075] (1) Take tissue sections and dewax them three times with xylene dewaxing solution for 5 minutes each time.

[0076] (2) Wash twice with 100% ethanol for 1 minute each time, and wash for 1 minute each with 95%, 85%, and 75% ethanol, then rinse with water.

[0077] (3) Stain with hematoxylin for 5 minutes, then rinse.

[0078] (4) Differentiate with 0.1% hydrochloric acid alcohol for 2 seconds (controlled under a microscope), then rinse.

[0079] (5) 0.5% ammonia solution turns blue in 2 seconds.

[0080] (6) Stain with eosin for 1 minute, then rinse.

[0081] (7) Dehydrate with 85%, 95%, and 95% ethanol for 10 seconds each, and dehydrate with 100% ethanol three times for 1 minute each time.

[0082] (8) Xylene is cleared three times, one minute each time.

[0083] (9) Mount the sheet with xylene-wetted neutral resin.

[0084] Comparative Example 2

[0085] A dewaxing solution is basically the same as that in Example 1, except that dewaxing solution I is composed of methyl oleate and dewaxing solution II is composed of ethanol.

[0086] Experimental Example 1

[0087] The stained samples of each histopathological section prepared using the method of Example 4 and the stained samples of each histopathological section prepared using the method of Comparative Example 1 were observed simultaneously under a microscope. The results of each experiment are as follows. Figure 1-4 and Table 1 below:

[0088] Table 1. Comparison of staining effects with traditional methods

[0089]

[0090]

[0091] As shown in Table 1 above, the dewaxing solution of Example 1, when applied to the hematoxylin-eosin staining (HE staining) method (Example 4), has a similar staining effect on tissues as the conventional xylene-based hematoxylin-eosin staining (HE staining) method (Comparative Example 1). The dewaxing and clearing effects on large specimens, puncture specimens, and other specimens are consistent.

[0092] However, compared to Comparative Example 1, the method in Example 1 does not use xylene, a common substance, thus avoiding harm to technicians and environmental pollution. Furthermore, the improved dyeing method has a shorter dyeing time, effectively improving dyeing efficiency. This invention's dyeing method, by changing the dewaxing process and the post-dyeing clearing process, achieves the advantages of ensuring accurate dyeing results while eliminating the need for xylene, shortening dyeing time, and effectively improving dyeing efficiency.

[0093] Example 2

[0094] This example compares the effects of dewaxing solutions with different formulations in Examples 2 and 3 on the staining effect. Specifically, the dewaxing solutions from Examples 2 and 3 were used, and staining was performed using hematoxylin-eosin staining as described in Example 4. The results are as follows: Figure 5 As shown in Table 2 below.

[0095] Table 2. Effect of adjusting the composition ratio of dewaxing solution on dyeing effect

[0096]

[0097] From the above table 2 and Figure 5 It can be seen that the dewaxing solutions of Examples 2 and 3, when used in the hematoxylin-eosin staining (HE staining) method, can still achieve the expected staining effect on tissues and can be used for subsequent pathological examination. However, due to the adjustment of the composition ratio of the dewaxing solution, its staining effect is reduced compared with the dewaxing solution of Example 1.

[0098] Experimental Example 3

[0099] The stained samples of each histopathological section prepared using the method of Example 4 and the stained samples of each histopathological section prepared using the method of Example 5 were observed simultaneously under a microscope. The results of each experiment are as follows. Figure 6 and Table 3 below:

[0100] Table 3. The effect of adjusting the staining steps on the staining effect.

[0101]

[0102] From the above table 3 and Figure 6It can be seen that the hematoxylin-eosin staining (HE staining) method in Example 4 omits the step of dehydration with gradient ethanol and directly bakes the slide after eosin staining. The staining effect on tissues is comparable to that of the existing conventional step-by-step ethanol dehydration staining method (Example 5), and the dewaxing and clearing effects on large specimens, puncture specimens and other specimens are consistent.

[0103] However, it saves time, reduces operational complexity, and achieves better staining results.

[0104] Experiment Example 4

[0105] The stained samples of each histopathological section prepared using the method of Example 4, and the stained samples of each histopathological section prepared using the dewaxing agent of Comparative Example 2 and the method of Example 4, were simultaneously observed under a microscope. The results of each experiment are as follows: Figure 7 and Table 4 below:

[0106] Table 4. Effects of dewaxing solution composition adjustment on dyeing effect

[0107]

[0108]

[0109] From the above table 4 and Figure 7 It can be seen that the dewaxing solution of Comparative Example 2, when used in the hematoxylin-eosin staining (HE staining) method, has a significantly reduced effect on tissue staining, which can no longer meet the expected requirements and cannot be used for subsequent pathological examination.

[0110] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0111] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A dewaxing solution, characterized in that, It includes dewaxing solution I and dewaxing solution II, wherein dewaxing solution I is composed of methyl oleate and mineral oil in a mass ratio of 3:7, and dewaxing solution II is composed of ethanol and cyclohexanone in a mass ratio of 3:

1.

2. The application of the dewaxing solution according to claim 1 in the dewaxing and staining of pathological tissues.

3. The application of the dewaxing solution according to claim 2 in the dewaxing and staining of pathological tissues, characterized in that, The staining was hematoxylin-eosin staining.

4. A hematoxylin-eosin staining method, characterized in that, Includes the following steps: (1) Take tissue sections and dewax them sequentially using the dewaxing solution I and dewaxing solution II as described in claim 1. Rinse them, with the dewaxing time being 5±1 min. After dewaxing, rinse directly with water. (2) Stain with hematoxylin and rinse; (3) Disintegrate with hydrochloric acid and alcohol, then rinse; (4) Return to blue; (5) Stain with eosin and rinse; (6) Baking: Baking the slices at 65±5℃ for 1 to 3 minutes; (7) Transparent; (8) Cover the film.

5. The staining method according to claim 4, which is a hematoxylin-eosin staining method, is characterized in that, In step (2), hematoxylin staining lasts for 5-6 minutes; in step (5), eosin staining lasts for 30 seconds to 1 minute.

6. The staining method according to claim 4, which is a hematoxylin-eosin staining method, is characterized in that, In step (3), the hydrochloric acid alcohol is prepared by mixing 36wt%-38wt% concentrated hydrochloric acid with 75% ethanol at a volume ratio of 0.5 to 1:

99.

7. The staining method according to claim 4, which is a hematoxylin-eosin staining method, is characterized in that, In step (4), an ammonia solution is used for blueing. The ammonia solution is prepared by mixing ammonia solution with a mass-volume concentration of 25% and water at a volume ratio of 0.5 to 0.8:

99.

8. The staining method according to claim 4 is a hematoxylin-eosin staining method, characterized in that, In step (7), turpentine is used for clearing treatment for 1 to 3 minutes; in step (8), neutral resin is used for direct sealing.

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