Colored frozen embedding agent, preparation method thereof, and frozen embedding agent kit

Abstract

A colored cryoembedding agent, its preparation method, and a cryoembedding agent kit relate to the field of frozen section technology and address the technical issues of ice crystals easily forming within tissue cells, poor continuity and integrity, and the inability to provide color identification. Polyvinyl alcohol and pure water are mixed and heated to a boil, followed by the addition of potassium bicarbonate, eosin Y, methylene blue, and alcian blue 8GX. After stirring and dissolving, the mixture is cooled at room temperature, followed by the addition of polyethylene glycol 400 and sodium benzoate. The solution is heated in a water bath until clear, and pure water is added to compensate for evaporation losses. Finally, sucrose and dimethyl sulfoxide are added and mixed uniformly to obtain the cryoembedding agent. The kit contains at least two different colored cryoembedding agents. The present invention can be applied to tissue embedding in frozen sections, and is particularly suitable for pathological testing of breast cancer, thyroid cancer, and other cancers to distinguish tissue sites within the same patient.

Description

Technical Field

[0001] The invention relates to the technical field of frozen sections, and in particular to a colored freezing embedding agent, a preparation method thereof, and a freezing embedding agent kit. Background Art

[0002] Intraoperative pathological analysis is a crucial step in the surgical process, providing clinicians with rapid and accurate histopathological diagnoses, which directly impacts surgical planning and patient outcomes. Frozen section technology, as a core tool for intraoperative pathological analysis, directly determines the accuracy and reliability of pathological diagnoses. However, the core challenge of frozen section technology lies in maximizing the morphological integrity and biological activity of tissue cells during the rapid freezing process, thereby providing high-quality tissue samples for pathological diagnosis.

[0003] Currently, clinical application of frozen sectioning technology still faces numerous technical bottlenecks. Existing cryoembedding agents suffer from poor moisture control and insufficient permeability, leading to inconsistent temperature gradients inside and outside the tissue during freezing. This makes it difficult to effectively control ice crystal formation within tissue cells, resulting in structural damage, which in turn affects section quality and diagnostic accuracy. Furthermore, while inorganic thickeners such as aluminum silicate can form thixotropic gels, they lack the necessary adhesion and, after freezing, cannot meet sectioning requirements. Organic thickeners such as sodium carboxymethylcellulose, while effective at thickening, require adjustment to a pH of 8–9 and are difficult to fade after dye binding, making them unsuitable for preparing colored cryoembedding agents. Cellulose thickeners such as methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose can increase viscosity through hydrogen bonding, but their increased toughness and elasticity can hinder the binding of the thickener solution to the frozen tissue, thus compromising the quality of frozen sections. Consequently, conventional cryoembedding agents struggle to ensure continuity and integrity during tissue sectioning, often resulting in wrinkling and fragmentation of tissue sections. Thirdly, existing embedding agents still lack effective support and positioning capabilities for small tissue samples, increasing the operational risks for diagnosticians. These issues not only limit the further development of frozen section technology but also pose a serious challenge to the reliability of intraoperative pathological diagnosis.

[0004] In summary, there is an urgent need to develop a new type of embedding agent for intraoperative frozen tissue sample processing to solve the following problems of the freezing embedding agent in the existing technology: First, ice crystals are easily formed in tissue cells during rapid freezing, causing tissue cell damage; second, the continuity and integrity of tissue sections are poor, and wrinkles and fragmentation are prone to occur; third, it is impossible to provide a color-coded support base for small tissue samples, which poses an operational risk; fourth, the freezing embedding agent is toxic, affecting the accuracy of subsequent immunohistochemistry testing. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a colored freezing embedding agent, a preparation method thereof, and a freezing embedding agent kit.

[0006] The technical solutions of the present invention are as follows:

[0007] A colored freezing embedding medium comprising the following components in parts by weight:

[0008] 20-200 parts of polyvinyl alcohol, 4-40 parts of sucrose, 19-25 parts of potassium bicarbonate, 5-20 parts of sodium benzoate, 11-77 parts of dimethyl sulfoxide, 0.5-2 parts of eosin Y or 0.25-20 parts of methylene blue or 0.25-20 parts of Alcian blue 8GX, 22-102 parts of polyethylene glycol 400, and 2000 parts of purified water.

[0009] The present invention also provides a method for preparing the colored frozen embedding medium, which is characterized by comprising the following steps:

[0010] Mix polyvinyl alcohol and pure water, heat and boil, then add potassium bicarbonate, eosin Y / methylene blue / Alcian blue 8GX, stir to dissolve, cool at room temperature, add polyethylene glycol 400 and sodium benzoate, heat in a water bath until the solution is clear, add the pure water lost by evaporation, and finally add sucrose and dimethyl sulfoxide, mix well to obtain a frozen embedding medium.

[0011] Preferably, the stirring and dissolving time is 50 min to 120 min.

[0012] Preferably, the temperature after cooling at room temperature is 50°C.

[0013] Preferably, the water bath heating temperature is 90°C.

[0014] The present invention also provides a freezing embedding agent kit, which contains at least two of the above colored freezing embedding agent reagents of different colors.

[0015] Compared with the prior art, the present invention has the following specific beneficial effects:

[0016] 1. In the prior art, dyes are mostly used for tissue staining rather than embedding and marking. The embedding agent provided in this application embeds specific dyes (eosin Y / methylene blue / Alcian Blue 8GX) during a boiling stage. The synergistic effect of the specific dyes and potassium bicarbonate allows them to bind to polyvinyl alcohol chains, achieving nanoscale dispersion. This uniform dispersion of the dyes within the embedding matrix creates a reversible bond, preventing the dyes from penetrating into the tissue. Firstly, the dyes within the embedding agent develop distinct colors and high transparency in a potassium bicarbonate buffered environment. By using different colored embedding agents, the kit can significantly support color differentiation between left and right tissues, making it particularly suitable for surgical procedures such as breast and thyroid cancers, where precise differentiation between different tissue sites within the same patient is required. This color differentiation design not only facilitates rapid intraoperative location and identification of tissue samples, but also effectively reduces operational risks and improves the efficiency and accuracy of intraoperative pathological diagnosis. Secondly, after treatment with 75% alcohol, the dyes completely fade without residue, allowing for secondary staining of faded tissue samples, which exhibits significantly improved color and transparency compared to samples treated with conventional embedding agents. The background staining rate is significantly reduced, ensuring the clarity of microscopic observation of tissue sections.

[0017] 2. The present application forms a gradient permeation protective layer in a low-temperature environment through a specific ratio of dimethyl sulfoxide and sucrose. Dimethyl sulfoxide has good tissue cell permeability, which helps to penetrate into the intercellular spaces and cells of the tissues. The dye combines with water molecules, thereby reducing the formation of ice crystals inside and outside the tissue cells, and does not affect the freezing effect of the tissue. At the same time, the use of sucrose can not only form a good embedding matrix to provide a soft support base for the tissue block to be frozen, but also effectively absorb moisture from the surface and section of the tissue block. Experiments show that the combined use of dimethyl sulfoxide and sucrose can play a synergistic role, jointly reduce the formation of ice crystals in the tissue cells, improve the continuity and integrity of the tissue sections, reduce the wrinkling and fragmentation during the slicing process, and reduce tissue cell damage, thereby effectively improving the quality of the frozen sections and overcoming the technical bottleneck of the traditional antifreeze agent that the penetration efficiency decreases at a high cooling rate.

[0018] 3. The coexistence of high transparency and immunohistochemical compatibility proves that the embedding agent of the present invention has no invasive damage to tissues, is safe and non-toxic, and does not affect the accuracy of subsequent immunohistochemical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a photo of the sample after embedding;

[0020] Figure 2 HE staining observation of lung tissue;

[0021] Figure 3 This is the HE staining observation picture of thyroid tissue;

[0022] Figure 4 HE staining observation picture of breast tissue;

[0023] Figure 5 This is the HE staining observation picture of adenomyosis tissue;

[0024] Figure 6 The immunohistochemical staining observations of TTF-1, AE and P40. DETAILED DESCRIPTION

[0025] In order to make the technical solution of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the specification of the present invention. It should be noted that the following embodiments are only used to better understand the technical solution of the present invention and should not be understood as limiting the present invention.

[0026] Example 1.

[0027] Mix 150 g of polyvinyl alcohol and 2000 mL of pure water, heat and boil, then add 20 g of potassium bicarbonate and 0.5 g of eosin Y. Stir continuously with a stirrer for 100 minutes, cool to 50°C at room temperature, add 40 mL of polyethylene glycol 400 and 5 g of sodium benzoate, and heat in a water bath until the precipitate is completely dissolved. Then add the amount of pure water lost by evaporation, and finally add 4 g of sucrose and 10 mL of dimethyl sulfoxide. Mix well to obtain an orange frozen embedding medium.

[0028] Example 2.

[0029] Mix 50 g of polyvinyl alcohol and 2000 mL of pure water, heat to a boil, then add 20 g of potassium bicarbonate and 0.5 g of eosin Y. Stir continuously with a stirrer for 80 min, cool to 50°C at room temperature, add 40 mL of polyethylene glycol 400 and 5 g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, then add the amount of pure water lost by evaporation, and finally add 4 g of sucrose and 10 mL of dimethyl sulfoxide. Mix well to obtain an orange frozen embedding medium.

[0030] Example 3.

[0031] Mix 200 g of polyvinyl alcohol and 2000 mL of pure water, heat to a boil, then add 25 g of potassium bicarbonate and 1.5 g of eosin Y. Stir continuously with a stirrer for 120 min, cool to 50°C at room temperature, add 80 mL of polyethylene glycol 400 and 20 g of sodium benzoate, and heat in a water bath until the precipitate is completely dissolved. Replace the amount of pure water lost by evaporation, and finally add 30 g of sucrose and 50 mL of dimethyl sulfoxide. Mix well to obtain an orange frozen embedding medium.

[0032] Example 4.

[0033] Mix 150g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 20g of potassium bicarbonate and 0.25g of methylene blue, stir continuously with a stirrer for 100 minutes, cool to 50°C at room temperature, add 40mL of polyethylene glycol 400 and 5g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, then add the amount of pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well, and obtain a blue frozen embedding medium.

[0034] Example 5.

[0035] Mix 50g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 20g of potassium bicarbonate and 0.5g of methylene blue, stir continuously with a stirrer for 80 minutes, cool to 50°C at room temperature, add 40mL of polyethylene glycol 400 and 5g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, then add the amount of pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well, and obtain a blue frozen embedding medium.

[0036] Example 6.

[0037] Mix 200g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 25g of potassium bicarbonate and 12g of methylene blue, stir continuously with a stirrer for 120 minutes, cool to 50°C at room temperature, add 80mL of polyethylene glycol 400 and 20g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, then add the amount of pure water lost by evaporation, and finally add 30g of sucrose and 50mL of dimethyl sulfoxide, mix well, and obtain a blue frozen embedding medium.

[0038] Example 7.

[0039] Mix 150g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 20g of potassium bicarbonate and 0.25g of Alcian Blue 8GX, stir continuously with a stirrer for 100 minutes, cool to 50°C at room temperature, add 40mL of polyethylene glycol 400 and 5g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, then add the amount of pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well, and obtain a green frozen embedding medium.

[0040] Example 8.

[0041] Mix 50 g of polyvinyl alcohol and 2000 mL of pure water, heat and boil, then add 20 g of potassium bicarbonate and 0.5 g of Alcian Blue 8GX, stir continuously with a stirrer for 80 minutes, cool to 50°C at room temperature, add 40 mL of polyethylene glycol 400 and 5 g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, add the amount of pure water lost by evaporation, and finally add 4 g of sucrose and 10 mL of dimethyl sulfoxide, mix well, and obtain a green frozen embedding medium.

[0042] Example 9.

[0043] Mix 200 g of polyvinyl alcohol and 2000 mL of pure water, heat and boil, then add 25 g of potassium bicarbonate and 12 g of Alcian Blue 8GX, stir continuously with a stirrer for 120 minutes, cool to 50°C at room temperature, add 80 mL of polyethylene glycol 400 and 20 g of sodium benzoate, heat in a water bath until the precipitate is completely dissolved, add the amount of pure water lost by evaporation, and finally add 30 g of sucrose and 50 mL of dimethyl sulfoxide, mix well, and obtain a green frozen embedding medium.

[0044] Comparative Example 1.

[0045] The difference from Example 1 is that potassium bicarbonate is replaced with pure water, and the other operations are the same as in Example 1.

[0046] Comparative Example 2.

[0047] The difference from Example 1 is that dimethyl sulfoxide is replaced by pure water, and the other operations are the same as those in Example 1.

[0048] Comparative Example 3.

[0049] The difference from Example 1 is that sucrose is replaced by pure water, and the other operations are the same as those in Example 1.

[0050] Effect example 1.

[0051] Standardize the sampling of rapidly frozen pathological specimens during surgery. Ensure that this does not affect the pathological diagnosis by cutting several tissue blocks of approximately equal size and thickness. The length and width of the tissue blocks should be less than 1.5 cm, and the thickness should be less than 0.3 cm. Standardize the sampling, dehydration, and paraffin embedding to create paraffin blocks.

[0052] 2-3 mL of cryoembedding agent was dripped onto each frozen sample holder. Excised tissue blocks were then placed onto the holders. After the tissue blocks settled naturally, the cryoembedding agents described in Examples 1-9, Comparative Examples 1-3, and commercially available transparent OCT were dripped onto the surfaces of the tissue blocks until they were completely covered. The frozen sample holders and the tissue blocks embedded with the cryoembedding agents were then placed on the freezing table of a cryostat for rapid freezing. Conventional rapid frozen sections were then performed using the cryostat.

[0053] The sample photos after embedding are as follows Figure 1 As shown, the leftmost sample is the one with a colorless embedding medium, the middle sample is orange, and the backmost sample is the one with a blue embedding medium. It can be seen that the dye in the embedding medium develops brightly in a potassium bicarbonate buffered environment, enabling significant color differentiation between the left and right sides. This allows for the identification of breast and thyroid cancer tissue sites within the same patient, facilitating rapid intraoperative localization and reducing operational risks.

[0054] The sliced samples were stained with HE and immunohistochemistry, and the frozen slice tissues were observed with a fluorescence microscope. The HE staining observation images of lung tissue, thyroid tissue, breast tissue and adenomyosis tissue were as follows: Figures 2 to 5 The immunohistochemical staining results of TTF-1, AE and P40 are shown in Figure 6 The above observation results prove that the addition of dye to the embedding medium provided by the present application does not affect the results of HE and immunohistochemical staining. At the same time, it improves the diagnostic efficiency of frozen tissue.

[0055] Effect example 2.

[0056] The tissue sections treated with the embedding agents of Examples 1, 4, 7, and Comparative Example 1 were immersed in 75% alcohol for 5 minutes to observe the fading effect. The faded samples were then subjected to a second conventional HE staining, and the background cleanliness and staining transparency were evaluated under a microscope. The evaluation results are as follows:

[0057]

[0058] The above results demonstrate that the complexation of potassium bicarbonate with the dye ensures complete fading (Comparative Example 1 shows a low fading rate and high residual color). The dye nanodispersion (Example Group) prevents penetration into the tissue, and secondary staining is free of background interference.

[0059] Effect example 3.

[0060] Transmission electron microscopy was used to observe the morphology and distribution of ice crystals within frozen tissue cells after sectioning, and the wrinkling rate (fragmentation / wrinkling ratio per 100 sections) was calculated. The average ice crystal diameter in the Example group was observed to be less than 5 μm, and the intracellular ice crystal density was reduced by over 30%. In contrast, the ice crystal density corresponding to Comparative Example 2 increased significantly, with a wrinkling rate greater than 50%. The sections corresponding to Comparative Example 3 had insufficient surface water adsorption and a high rate of edge fragmentation. The number of complete sections that could be cut consecutively from the same tissue block was recorded, and laser confocal microscopy was used to observe the integrity of the cytoskeleton within the sections. The Example group had ≥50 consecutive sections, while the Comparative Example 3 group had an increased rate of cytoskeleton fragmentation. The commercially available OCT embedding medium resulted in approximately 30 sections. This suggests that the lack of dimethyl sulfoxide leads to insufficient penetration.

[0061] It will be understood that the present invention is described by way of some embodiments, and it will be appreciated by those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are intended to be protected by the present invention.

Claims

1. A method for preparing a colored frozen embedding medium, characterized in that: The colored freezing embedding medium comprises the following components in parts by weight: 20-200 parts of polyvinyl alcohol, 4-40 parts of sucrose, 19-25 parts of potassium bicarbonate, 5-20 parts of sodium benzoate, 11-77 parts of dimethyl sulfoxide, 0.5-2 parts of eosin Y or 0.25-20 parts of methylene blue or 0.25-20 parts of Alcian Blue 8GX, 22-102 parts of polyethylene glycol 400, and 2000 parts of purified water; The preparation method comprises the following steps: Mix polyvinyl alcohol and pure water, heat and boil, then add potassium bicarbonate, eosin Y / methylene blue / Alcian blue 8GX, stir to dissolve, cool at room temperature, add polyethylene glycol 400 and sodium benzoate, heat in a water bath until the solution is clear, add the pure water lost by evaporation, and finally add sucrose and dimethyl sulfoxide, mix well to obtain a frozen embedding medium.

2. The method for preparing the colored frozen embedding medium according to claim 1, wherein: The stirring and dissolving time is 50 min to 120 min.

3. The method for preparing the colored frozen embedding medium according to claim 1, characterized in that: The temperature after cooling at room temperature is 50°C.

4. The method for preparing the colored frozen embedding medium according to claim 1, wherein: The water bath heating temperature is 90°C.

5. A freezing embedding agent kit, characterized in that: The kit comprises at least two colored frozen embedding agents of different colors prepared by the preparation method according to any one of claims 1 to 4.

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