An aqueous deparaffinizing reagent and a method for preparing the same

Abstract

This invention provides an aqueous dewaxing reagent comprising the following components by weight percentage: 0.3-6% surfactant and 0.5-3% composite penetration enhancer, with the balance being water. The composite penetration enhancer is composed of limonene and terpineol, wherein the mass of terpineol is not less than 10% of the total mass of the composite penetration enhancer. The preparation method involves mixing the surfactant, composite penetration enhancer, and water. This invention utilizes a composite penetration enhancer composed of limonene and terpineol, which significantly improves dewaxing speed, reduces temperature dependence, widens the operating temperature window, reduces tissue damage, provides better protection against antigens, and is beneficial for IHC staining.

Description

Technical Field

[0001] This invention relates to the field of pathological tissue sectioning technology, and in particular to an aqueous dewaxing reagent for tissue sections and its preparation method. Background Technology

[0002] In pathological diagnosis and research, the staining analysis of tissue sections is a crucial basis for the early diagnosis and qualitative analysis of cancer. Paraffin embedding of tissues is widely used due to its excellent morphological preservation; however, before immunohistochemical staining and hematoxylin-eosin staining, the paraffin in the tissue must be completely removed, i.e., dewaxing. Traditional dewaxing processes commonly use xylene as a dewaxing agent. However, xylene has significant toxicity and volatility, and can not only paralyze the central nervous system and irritate mucous membranes, but long-term exposure can also lead to immune dysfunction, dermatitis, and anemia in practitioners. In addition, xylene easily causes tissue shrinkage and hardening, affecting the quality of the sections, and the toxic gases and waste liquids generated during its use also pose a serious threat to the laboratory environment.

[0003] To avoid using xylene, Chinese patent CN115505463A discloses an aqueous dewaxing agent that uses a system of nonionic and anionic surfactants combined with water as a matrix, aiming to reduce toxicity and environmental hazards. This technology replaces xylene-based dewaxing agents to some extent, but its system still has the following limitations: First, the synergistic effect of its surfactant combination is limited, resulting in insufficient dewaxing efficiency for fatty or dense tissues, and the presence of a "cloud point" phenomenon limits the operating temperature window to 70-80℃. Improper temperature control can easily cause solution stratification or a decrease in dewaxing effect. Second, the antioxidants used have poor stability; their antioxidant properties easily decay during repeated heating and use, affecting the product's lifespan. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an aqueous dewaxing reagent and its preparation method, so as to solve the above-mentioned technical problems existing in the prior art.

[0005] This invention is achieved using the following technical solution: A first aspect of the present invention is to provide an aqueous dewaxing agent comprising the following components by weight percentage: 0.3-6% surfactant and 0.5-3% composite penetration enhancer, with the balance being water, wherein the composite penetration enhancer is composed of limonene and terpineol, wherein the mass of terpineol is not less than 10% of the total mass of the composite penetration enhancer.

[0006] Preferably, the composite penetration enhancer contains 0.4-2.5% D-limonene and 0.1-0.5% α-terpineol. More preferably, the mass ratio of limonene to terpineol is 2:1-4:1.

[0007] More preferably, the limonene is selected from D-limonene or L-limonene.

[0008] More preferably, the terpineol is selected from α-terpineol, β-terpineol, or γ-terpineol.

[0009] Preferably, the surfactant is composed of a nonionic surfactant and an anionic surfactant. The amounts of the two surfactants are 0.15-3.5% of the nonionic surfactant and 0.15-2.5% of the anionic surfactant, respectively.

[0010] The nonionic surfactant comprises polyoxyethylene-8-octylphenyl ether (CAS: 9036-19-5) and at least one C8-C16 alkyl glucoside. The C8-C16 alkyl glucoside may be selected from, but is not limited to, one or more of octyl glucoside, decyl glucoside, lauryl glucoside, tetradecyl glucoside, and hexadecyl glucoside. The weight percentages of polyoxyethylene-8-octylphenyl ether and C8-C16 alkyl glucoside in the nonionic surfactant are 0.1-2.5% and 0.05-1% respectively.

[0011] The anionic surfactant comprises at least one C10-C16 sodium alcohol polyoxyethylene ether sulfate and at least one C10-C16 sodium alkylbenzene sulfonate. The C10-C16 sodium alcohol polyoxyethylene ether sulfate (AES) can be represented by the following general chemical formula: RO(CH2CH2O). n SO3Na, wherein R is a C10-C16 alkyl group, n=1-15, and may be selected from one or more of the compounds of the above general formula; sodium C10-C16 alkylbenzenesulfonate may be selected from, but not limited to, sodium undecylbenzenesulfonate, sodium dodecylbenzenesulfonate, and sodium hexadecylbenzenesulfonate, or any one or more. The weight amounts of sodium C10-C16 alcohol polyoxyethylene ether sulfate and sodium C10-C16 alkylbenzenesulfonate in the anionic surfactant are 0.1-2.0% and 0.05-0.5%, respectively.

[0012] The inventors unexpectedly discovered that using a composite penetration enhancer composed of limonene and terpineol can significantly improve dewaxing speed and reduce tissue damage. The specific mechanism is not yet fully understood, but it may be due to two factors: First, terpenoids and surfactants coexist in the aqueous phase and can be adsorbed together at the paraffin-water interface. Due to their excellent penetration ability and the reduced interfacial tension by the surfactant, terpenoids allow the aqueous dewaxing agent to quickly wet the tissue section surface and penetrate between the paraffin and the tissue. The terpenoids first penetrate into the internal network structure of the paraffin, causing swelling and softening, disrupting its dense crystalline structure. The surfactant then synergistically dissolves the paraffin, achieving rapid exfoliation. Second, limonene and terpineol can produce a synergistic effect. Limonene first penetrates and swells the paraffin structure, while terpineol then penetrates the paraffin-tissue interface. Simultaneously, it combines with the surfactant to construct a stable microemulsion system, thereby significantly improving the dewaxing speed, preventing paraffin redeposition, and widening the effective temperature window.

[0013] As a preferred technical solution, the aqueous dewaxing reagent of the present invention further includes the following components in weight percentage: 0.05-0.4% antioxidant, 0.02-0.3% preservative, and 0.05-0.2% pH buffer.

[0014] The antioxidant can be selected from commonly used substances in the art, including oil-soluble antioxidants and water-soluble antioxidants, such as, but not limited to, phytic acid and vitamin E. Preferably, the antioxidant is a composition of phytic acid and vitamin E, with the weight amounts of phytic acid being 0.04-0.3% and vitamin E being 0.01-0.1%.

[0015] The preservative can be a commonly used substance in the art, such as, but not limited to, benzyl alcohol, pentylene glycol, etc.

[0016] The pH buffer can be any commonly used substance in the art, such as, but not limited to, Tris-HCl buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, etc.

[0017] A second aspect of the present invention is to provide a method for preparing an aqueous dewaxing agent, comprising the following steps: mixing a surfactant, a composite penetration enhancer and water.

[0018] Preferably, the preparation method of the aqueous dewaxing reagent includes the following steps: stirring surfactant, composite penetration enhancer, antioxidant, pH buffer and water evenly and then emulsifying, then adding preservative and stirring evenly to obtain aqueous dewaxing reagent.

[0019] As a preferred technical solution, the preparation method of the aqueous dewaxing agent includes the following steps: S1. Mix the nonionic surfactant, composite penetration enhancer and oil-soluble antioxidant at 40-50℃ and stir until clear and homogeneous to obtain an oil phase solution; S2. Dissolve the anionic surfactant, pH buffer and water-soluble antioxidant in distilled water, heat to 50-60℃ and stir until completely dissolved to obtain an aqueous solution; S3. Add the oil phase solution to the aqueous phase solution and stir continuously at high speed for 10-15 minutes to form a uniform milky white microemulsion. S4. After cooling to room temperature, add preservative and stir evenly to obtain the aqueous dewaxing reagent.

[0020] The oil-soluble antioxidant is preferably vitamin E, and the water-soluble antioxidant is preferably phytic acid.

[0021] Preferably, the stirring speed in step S3 is 5000-10000 rpm.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The water-based dewaxing reagent provided by the present invention introduces terpene compounds with specific components, which first penetrate the paraffin slices through their rapid penetration and swelling effect. Combined with surfactants, it achieves rapid dewaxing from the inside out, and the dewaxing time can be shortened to 30-50% of the prior art. It has excellent dewaxing effect, especially for thick slices and dense tissues with high fat content.

[0023] (2) By introducing terpenoid compounds with specific components, the dependence on temperature is reduced, the operating temperature window is widened, tissue damage is reduced, and the problem of paraffin being re-precipitated and adsorbed on the tissue due to temperature changes or excessive concentration after being removed from the tissue is effectively avoided.

[0024] (3) The water-based dewaxing reagent of the present invention has a lower operating temperature, which can reduce heat damage. The faster dewaxing speed significantly shortens the exposure time of tissue in high temperature solution, making the tissue morphology more complete and the cell structure clearer after dewaxing. It has a better protective effect on antigens and is beneficial for IHC staining. Detailed Implementation

[0025] The following description is provided to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples and are not intended to limit the scope of the invention; other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.

[0026] Unless otherwise specified, all instruments and raw materials used in this invention are conventional commercial instruments or materials. Specifically, the C12-C14 sodium polyoxyethylene ether sulfate is selected from Wuhan Xinyang Ruihe Chemical Technology Co., Ltd., product CAS number 68511-39-7. Sodium C10~C12 alcohol polyoxyethylene ether sulfate is selected from Wuhan Xinyang Ruihe Chemical Technology Co., Ltd., CAS No. 68585-34-2; Sodium C14~C16 alcohol polyoxyethylene ether sulfate is selected from Wuhan Xinyang Ruihe Chemical Technology Co., Ltd., with CAS number 68081-91-4.

[0027] Unless otherwise mentioned, all detection indicators involved in the embodiments of this invention are performed using conventional detection methods in the art. Specifically, dewaxing time, tissue integrity, antigen preservation, and background cleanliness are measured according to the detection methods described in *Clinical Technical Operation Specifications: Pathology Volume*, Chinese Medical Association, People's Military Medical Publishing House, 2007.

[0028] Example 1 An aqueous dewaxing agent comprises the following components in weight percentage: 1.2% polyoxyethylene-8-octylanisole, 0.6% C12 alkyl glucoside, 0.6% sodium C12-C14 alcohol polyoxyethylene ether sulfate, 0.5% sodium C12 alkylbenzene sulfonate, 1.5% D-limonene, 0.3% α-terpineol, 0.15% phytic acid, 0.05% vitamin E, 0.15% benzyl alcohol, 0.1% Trip-HCl buffer, and the balance being distilled water.

[0029] The preparation method of the aqueous dewaxing agent includes the following steps: S1. Polyoxyethylene-8-octylanisole, C12 alkyl glucoside, D-limonene, α-terpineol and vitamin E are mixed at 45°C and stirred until clear and homogeneous to obtain an oil phase solution. S2. Dissolve sodium C12~C14 alcohol polyoxyethylene ether sulfate, sodium C12 alkylbenzene sulfonate, Trip-HCl buffer and phytic acid in distilled water, heat to 60°C and stir until completely dissolved to obtain an aqueous solution. S3. At 8000 rpm, the oil phase solution is added to the aqueous phase solution and sheared continuously for 10-15 min to form a uniform milky white microemulsion. S4. Finally, after cooling to room temperature, add benzyl alcohol and stir well to obtain an aqueous dewaxing reagent.

[0030] Example 2 This embodiment provides an aqueous dewaxing reagent comprising the following components by weight percentage: 0.8% polyoxyethylene-8-octylanisole, 0.4% C10 alkyl glucoside, 0.4% sodium C10-C12 alcohol polyoxyethylene ether sulfate, 0.3% sodium C10 alkylbenzene sulfonate, 0.8% D-limonene, 0.2% α-terpineol, 0.1% phytic acid, 0.03% vitamin E, 0.1% pentanediol, 0.08% phosphate buffer, and the balance being distilled water. The preparation method of the aqueous dewaxing reagent is the same as in Example 1.

[0031] Example 3 This embodiment provides an aqueous dewaxing reagent comprising the following components by weight percentage: 0.8% polyoxyethylene-8-octylanisole, 0.4% C10 alkyl glucoside, 0.4% sodium C10-C12 alcohol polyoxyethylene ether sulfate, 0.3% sodium C10 alkylbenzene sulfonate, 0.12% D-limonene, 0.4% α-terpineol, 0.1% phytic acid, 0.03% vitamin E, 0.1% pentanediol, 0.08% phosphate buffer, and the balance being distilled water. The preparation method of the aqueous dewaxing reagent is the same as in Example 1.

[0032] Example 4 This embodiment provides an aqueous dewaxing reagent comprising the following components by weight percentage: 0.8% polyoxyethylene-8-octylanisole, 0.4% C10 alkyl glucoside, 0.4% sodium C10-C12 alcohol polyoxyethylene ether sulfate, 0.3% sodium C10 alkylbenzene sulfonate, 0.10% D-limonene, 0.5% α-terpineol, 0.1% phytic acid, 0.03% vitamin E, 0.1% pentanediol, 0.08% phosphate buffer, and the balance being distilled water. The preparation method of the aqueous dewaxing reagent is the same as in Example 1.

[0033] Example 5 This embodiment provides an aqueous dewaxing reagent comprising the following components by weight percentage: 0.8% polyoxyethylene-8-octylanisole, 0.4% C10 alkyl glucoside, 0.4% sodium C10-C12 alcohol polyoxyethylene ether sulfate, 0.3% sodium C10 alkylbenzene sulfonate, 0.4% D-limonene, 0.4% α-terpineol, 0.1% phytic acid, 0.03% vitamin E, 0.1% pentanediol, 0.08% phosphate buffer, and the balance being distilled water. The preparation method of the aqueous dewaxing reagent is the same as in Example 1.

[0034] Example 6 This embodiment provides an aqueous dewaxing reagent comprising the following components by weight percentage: 2.0% polyoxyethylene-8-octylanisole, 0.8% C14 alkyl glucoside, 1.0% sodium C14-C16 alcohol polyoxyethylene ether sulfate, 0.6% sodium C14 alkylbenzene sulfonate, 2.0% D-limonene, 0.4% α-terpineol, 0.2% phytic acid, 0.08% vitamin E, 0.2% benzyl alcohol, 0.15% Trip-HCl buffer, and the balance being distilled water. The preparation method of the aqueous dewaxing reagent is the same as in Example 1.

[0035] Example 7 The preparation method of this embodiment is the same as that of Example 1, except that only 1.5% of D-limonene is used in the components and α-terpineol is not added.

[0036] Example 8 The preparation method of this embodiment is the same as that of Example 1, except that only 0.3% D-limonene is used in the components, and no D-limonene is added.

[0037] Comparative Example 1 The comparative example was prepared using the same method as Example 1, except that D-limonene and α-terpineol were not added.

[0038] Comparative Example 2 The comparative example was prepared using the same method as in Example 1, except that the amount of D-limonene was 3.5% and the amount of α-terpineol was 0.8%.

[0039] Performance testing Take paraffin sections of human tonsils, bake the sections, add the dewaxing reagents used in Examples 1-8 and Comparative Examples 1-2 respectively, and heat to 65°C for immersion. Take out the paraffin sections of tissue, wash with water to complete the dewaxing, and then stain for observation.

[0040] The dewaxing time, tissue integrity, antigen preservation, and background cleanliness of each of Examples 1-8 and Comparative Examples 1-2 were tested respectively, and the test results are shown in Table 1.

[0041] Table 1 Detection indicators for each sample According to the comparison of data from Example 1 and Examples 4-5 in Table 1, D-limonene and α-terpineol have a synergistic effect, which can significantly shorten the dewaxing time and have excellent tissue integrity and antigen preservation, and a clean background.

[0042] As can be seen from the comparison of Examples 1 to 5, when the mass ratio of D-limonene to α-terpineol is in the range of 2 to 4:1, the dewaxing time is shorter and the tissue integrity and antigen preservation are excellent.

[0043] Compared to Comparative Example 1, without the addition of a penetration enhancer, the dewaxing time significantly increased. The prolonged dewaxing process led to decreased tissue integrity, reduced antigen preservation, and the appearance of trace sediment in the background. A comparison between Example 1 and Comparative Example 2 shows that while adding excessive penetration enhancer can shorten the dewaxing time, it causes localized tissue damage, poor antigen preservation, and oily residue in the background.

[0044] In summary, this application, by using an appropriate amount of penetration enhancer, can achieve rapid penetration and dewaxing of paraffin sections. It has excellent dewaxing effect on thick sections and dense tissues with high fat content. At the same time, the integrity of the tissue and the preservation of antigens are improved after dewaxing, and the background is clean.

[0045] Those skilled in the art should understand that the above embodiments are merely examples and do not limit the invention. The objectives of the invention have been fully and effectively achieved. The functions and principles of the invention have been shown and explained in the embodiments, and any variations or modifications can be made to the implementation of the invention without departing from the stated principles.

Claims

1. An aqueous dewaxing agent, characterized in that, It comprises the following components by weight percentage: 0.3-6% surfactant and 0.5-3% composite penetration enhancer, with the balance being water. The composite penetration enhancer is composed of limonene and terpineol, wherein the mass of terpineol is not less than 10% of the total mass of the composite penetration enhancer.

2. The aqueous dewaxing agent as described in claim 1, characterized in that, The mass ratio of limonene to terpineol is 2:1 to 4:

1.

3. The aqueous dewaxing agent as described in claim 1, characterized in that, The limonene is selected from D-limonene or L-limonene, and the terpineol is selected from α-terpineol, β-terpineol or γ-terpineol.

4. The aqueous dewaxing agent as described in claim 1, characterized in that, The surfactant is composed of a nonionic surfactant and anionic surfactant, with the amounts of the nonionic surfactant being 0.15-3.5% and the anionic surfactant being 0.15-2.5%, respectively.

5. The aqueous dewaxing agent as described in claim 1, characterized in that, The nonionic surfactant is composed of polyoxyethylene-8-octylphenyl ether and at least one C8-C16 alkyl glucoside; the anionic surfactant is composed of at least one C10-C16 sodium alcohol polyoxyethylene ether sulfate and at least one C10-C16 sodium alkylbenzene sulfonate.

6. The aqueous dewaxing agent as described in claim 5, characterized in that, The nonionic surfactant contains 0.1-2.5% polyoxyethylene-8-octylphenyl ether and 0.05-1% C8-C16 alkyl glucoside by weight, respectively; the anionic surfactant contains 0.1-2.0% sodium C10-C16 alcohol polyoxyethylene ether sulfate and 0.05-0.5% sodium C10-C16 alkylbenzene sulfonate by weight, respectively.

7. The aqueous dewaxing agent according to any one of claims 1-6, characterized in that, It also includes the following components by weight percentage: 0.05-0.4% antioxidant, 0.02-0.3% preservative, and 0.05-0.2% pH buffer.

8. The aqueous dewaxing agent as described in claim 7, characterized in that, The antioxidant is a composition of phytic acid and vitamin E, wherein the weight amounts of phytic acid are 0.04-0.3% and vitamin E are 0.01-0.1%, respectively.

9. The method for preparing the aqueous dewaxing agent according to claims 1-6, characterized in that, It is composed of surfactants, composite penetration enhancers and water.

10. A method for preparing the aqueous dewaxing agent according to claim 7 or 8, characterized in that, The process includes the following steps: emulsifying the surfactant, composite penetration enhancer, antioxidant, pH buffer and water, then adding the preservative and stirring until homogeneous to obtain the water-based dewaxing reagent.

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