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

Abstract

The invention discloses a colored freezing embedding agent, a preparation method thereof and a freezing embedding agent kit, relates to the technical field of frozen sections, and solves the technical problems that ice crystals are easy to form in tissue cells, the continuity and the integrity are poor and color identification cannot be provided. The preparation method comprises the following steps: mixing polyvinyl alcohol and pure water, heating and boiling, adding potassium bicarbonate, eosin Y / methylene blue / Alzheimer blue 8GX, stirring and dissolving, cooling at room temperature, adding polyethylene glycol 400 and sodium benzoate, heating in water bath until the solution is clear, supplementing pure water lost by evaporation, finally adding cane sugar and dimethyl sulfoxide, and uniformly mixing to obtain the frozen embedding medium. The kit comprises at least two colored freezing embedding agent reagents with different colors. The method can be applied to tissue embedding in frozen sections, and is particularly suitable for pathological detection of breast cancer, thyroid cancer and the like so as to distinguish tissue parts of 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 and a preparation method thereof and a freezing embedding agent kit. Background Art

[0002] Intraoperative pathological analysis is a crucial part of the surgical process. It can provide clinicians with rapid and accurate histopathological diagnosis, which directly affects the formulation of surgical plans and the treatment effect of patients. Among them, frozen section technology is the core means of intraoperative pathological analysis, and its quality directly determines the accuracy and reliability of pathological diagnosis. However, the core challenge of frozen section technology is how to maximize the morphological integrity and biological activity of tissue cells during the rapid freezing process, so as to provide high-quality tissue samples for pathological diagnosis.

[0003] At present, frozen section technology still faces many technical bottlenecks in clinical applications. Due to the poor water control ability and insufficient permeability of the frozen embedding agents used in the prior art, the temperature gradient inside and outside the tissue is inconsistent during the freezing process. It is difficult to effectively control the formation of ice crystals in tissue cells during the freezing process, resulting in cell structure damage, which in turn affects the quality of the slice and the accuracy of the diagnosis. In addition, in the selection of thickeners, although inorganic thickeners such as aluminum silicate can form thixotropic gels, they lack the necessary adhesion and cannot meet the requirements of the slice after freezing; although organic thickeners such as sodium carboxymethyl cellulose have a thickening effect, they need to adjust the pH value to 8~9, and are difficult to fade after combining with dyes, and are not suitable for preparing colored frozen embedding agents; although cellulose thickeners such as methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose can increase the viscosity of the system through hydrogen bonding, their high toughness and elasticity will affect the combination of thickener aqueous solution and frozen tissue, thereby affecting the frozen section effect. Therefore, it is difficult for traditional frozen embedding agents to ensure the continuity and integrity of the tissue section process, and wrinkles and fragmentation of tissue sections often occur. Thirdly, the existing embedding agents still lack effective support and positioning functions for small tissue samples, which increases the operating risks of diagnostic workers. The existence of these problems not only limits the further development of frozen section technology, but also poses a severe 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 prior art: 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 immunohistochemical 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 and a preparation method thereof and a freezing embedding agent kit.

[0006] The technical solution of the present invention is as follows: A colored freezing embedding medium, comprising the following components in parts by weight: 20 to 200 parts of polyvinyl alcohol, 4 to 40 parts of sucrose, 19 to 25 parts of potassium bicarbonate, 5 to 20 parts of sodium benzoate, 11 to 77 parts of dimethyl sulfoxide, 0.5 to 2 parts of eosin Y or 0.25 to 20 parts of methylene blue or 0.25 to 20 parts of Alcian blue 8GX, 22 to 102 parts of polyethylene glycol 400, and 2000 parts of pure water.

[0007] The present invention also provides a method for preparing the colored cryoembedding agent, which is characterized by comprising the following steps: Mix polyvinyl alcohol and pure water, heat to 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 agent.

[0008] Preferably, the stirring and dissolving time is 50 min to 120 min.

[0009] Preferably, the temperature after cooling at room temperature is 50°C.

[0010] Preferably, the water bath heating temperature is 90°C.

[0011] The present invention also provides a freezing embedding agent kit, wherein the kit comprises at least two of the above colored freezing embedding agent reagents of different colors.

[0012] Compared with the prior art, the specific beneficial effects of the present invention are: 1. In the prior art, dyes are mostly used for tissue staining rather than embedding identification. The embedding agent provided in this application embeds a specific dye (eosin Y / methylene blue / alcian blue 8GX) during the boiling stage. Through the synergistic effect of the specific dye and potassium bicarbonate, it is combined with the polyvinyl alcohol chain to achieve nano-level dispersion, so that the dye is evenly dispersed in the embedding matrix to form a reversible bond, and the dye in the embedding agent will not penetrate into the tissue. On the one hand, the dye in the embedding agent has a bright color and high transparency under the potassium bicarbonate buffer environment. By using embedding agents of different colors, the kit can significantly support the color distinction of left and right tissues, which is particularly suitable for surgical scenarios such as breast cancer and thyroid cancer that require accurate distinction between different tissue parts of the same patient. This color distinction design not only facilitates the rapid positioning and identification of tissue samples during surgery, but also effectively reduces operational risks and improves the efficiency and accuracy of intraoperative pathological diagnosis. On the other hand, after being treated with 75% alcohol, the dye can be completely faded without residue. The faded tissue sample can be secondary stained, and the color transparency after secondary staining is significantly improved compared with the sample treated with conventional embedding agents. The background staining rate is significantly reduced, ensuring the clarity of microscopic observation of tissue sections.

[0013] 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 gaps between tissue cells and cells. The dye combines with water molecules to reduce 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 wrinkles and fragmentation during the slicing process, and reduce tissue cell damage, thereby effectively improving the quality of frozen sections, overcoming the technical bottleneck of the decline in the penetration efficiency of traditional antifreeze agents at high cooling rates.

[0014] 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

[0015] Figure 1 This is a photo of the sample after embedding; Figure 2 HE staining observation of lung tissue; Figure 3 This is the HE staining observation picture of thyroid tissue; Figure 4 HE staining observation picture of breast tissue; Figure 5 This is the HE staining observation picture of adenomyosis tissue; Figure 6 These are the immunohistochemical staining observations of TTF-1, AE and P40. DETAILED DESCRIPTION

[0016] 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 a limitation to the present invention.

[0017] Example 1. Mix 150g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 20g of potassium bicarbonate and 0.5g of eosin Y, stir continuously with a stirrer for 100min, 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, add the pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well, and obtain an orange frozen embedding agent.

[0018] Example 2. Mix 50g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 20g of potassium bicarbonate and 0.5g of eosin Y, stir continuously with a stirrer for 80min, 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, add the pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well to obtain an orange frozen embedding agent.

[0019] Example 3. Mix 200g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 25g of potassium bicarbonate and 1.5g of eosin Y, stir continuously with a stirrer for 120min, 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, add the pure water lost by evaporation, and finally add 30g of sucrose and 50mL of dimethyl sulfoxide, mix well, and obtain an orange frozen embedding agent.

[0020] Example 4. 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 100min, 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, add the pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well to obtain a blue frozen embedding agent.

[0021] Example 5. 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 80min, 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, add the pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well to obtain a blue frozen embedding agent.

[0022] Example 6. 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 120min, 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, add the pure water lost by evaporation, and finally add 30g of sucrose and 50mL of dimethyl sulfoxide, mix well, and obtain a blue frozen embedding agent.

[0023] Example 7. 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 100min, 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, add the pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well, and obtain a green frozen embedding agent.

[0024] Example 8. Mix 50g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 20g of potassium bicarbonate and 0.5g of Alcian Blue 8GX, stir continuously with a stirrer for 80min, 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, add the pure water lost by evaporation, and finally add 4g of sucrose and 10mL of dimethyl sulfoxide, mix well to obtain a green frozen embedding agent.

[0025] Example 9. Mix 200g of polyvinyl alcohol and 2000mL of pure water, heat and boil, then add 25g of potassium bicarbonate and 12g of Alcian Blue 8GX, stir continuously with a stirrer for 120min, 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, add the pure water lost by evaporation, and finally add 30g of sucrose and 50mL of dimethyl sulfoxide, mix well, and obtain a green frozen embedding agent.

[0026] Comparative Example 1. The difference from Example 1 is that potassium bicarbonate is replaced with pure water, and the remaining operations are the same as Example 1.

[0027] Comparative Example 2. The difference from Example 1 is that dimethyl sulfoxide is replaced by pure water, and the other operations are the same as Example 1.

[0028] Comparative Example 3. The difference from Example 1 is that sucrose is replaced by pure water, and the rest of the operations are the same as in Example 1.

[0029] Effect example 1. Standardize the sampling of quick frozen pathological specimens during surgery. Under the premise of ensuring that it does not affect the pathological diagnosis, cut several pieces of tissue of roughly the same size and thickness. The length and width of the tissue block are less than 1.5cm, and the thickness is less than 0.3cm. Standardize the sampling, dehydration, and paraffin embedding to make paraffin wax blocks.

[0030] 2-3 mL of freezing embedding agent was dripped onto the frozen sample tray, and then the cut tissue blocks were placed onto the frozen sample tray. After the tissue blocks were naturally settled, the transparent OCT freezing embedding agents in Examples 1-9, Comparative Examples 1-3, and commercially available were dripped onto the surface of the tissue blocks until the tissue blocks were completely covered. Then, the frozen sample tray and the tissue blocks embedded with the freezing embedding agent were placed on the freezing table of the freezing microtome for quick freezing, and then the freezing microtome was used for conventional quick frozen sectioning.

[0031] The sample photos after embedding are as follows Figure 1 As shown, the leftmost side is the sample corresponding to the colorless embedding medium, the middle side is orange, and the last side is the sample corresponding to the blue embedding medium. It can be seen that the dye in the embedding medium is brightly colored under the potassium bicarbonate buffer environment, which supports significant color distinction between the left and right sides, and distinguishes the tissue sites of the same patient for breast cancer and thyroid cancer, facilitating rapid positioning during surgery and reducing operational risks.

[0032] HE and immunohistochemical staining were performed on the sliced ​​samples, and the frozen slice tissues were observed under a fluorescence microscope. The HE staining observation images of lung tissue, thyroid tissue, breast tissue and adenomyosis tissue were obtained as shown in the following figure. Figures 2~5 The immunohistochemical staining results of TTF-1, AE and P40 are shown in Figure 6 The above observation results can prove that the addition of dyes to the embedding agent 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 tissues.

[0033] Effect example 2. The tissue sections treated with the embedding agents of Example 1, Example 4, Example 7 and Comparative Example 1 were immersed in 75% alcohol for 5 minutes to observe the fading effect. The faded samples were subjected to secondary conventional HE staining, and the background cleanliness and staining transparency were observed and evaluated under a microscope. The evaluation results are as follows:

[0034] The above results can prove that the complexation of potassium bicarbonate with the dye ensures complete fading (the fading rate of comparative example 1 is low and the residual is high). The dye nanodispersion (example group) avoids infiltration into the tissue, and the secondary dyeing has no background interference.

[0035] Effect example 3. The morphology and distribution of ice crystals in tissue cells after frozen sectioning were observed by transmission electron microscopy, and the wrinkle rate of the slices (fragmentation / wrinkle ratio in every 100 slices) was calculated. It was observed that the average diameter of ice crystals in the embodiment group was less than 5 μm, and the density of ice crystals in cells was reduced by more than 30%; while the ice crystal density corresponding to comparative example 2 increased significantly, the wrinkle rate was greater than 50%, and the surface water adsorption of the slices corresponding to comparative example 3 was insufficient, and the fragmentation rate of the slice edges was high. The number of complete slices that can be cut out continuously from the same tissue block was recorded, and the integrity of the cytoskeleton in the slices was observed by laser confocal microscopy. It was observed that the number of continuous slices in the embodiment group was ≥50, the cytoskeleton fracture rate in comparative example 3 group increased, and the number of slices corresponding to the commercially available OCT embedding agent was about 30. This shows that the lack of dimethyl sulfoxide leads to insufficient penetration.

[0036] It is to be understood that the present invention is described by some embodiments, and it is known to 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 by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A colored freezing embedding medium, characterized in that: The composition includes the following components in parts by weight: 20 to 200 parts of polyvinyl alcohol, 4 to 40 parts of sucrose, 19 to 25 parts of potassium bicarbonate, 5 to 20 parts of sodium benzoate, 11 to 77 parts of dimethyl sulfoxide, 0.5 to 2 parts of eosin Y or 0.25 to 20 parts of methylene blue or 0.25 to 20 parts of Alcian blue 8GX, 22 to 102 parts of polyethylene glycol 400, and 2000 parts of pure water.

2. A method for preparing the colored frozen embedding agent according to claim 1, characterized in that: The steps include: Mix polyvinyl alcohol and pure water, heat to 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 agent.

3. The method for preparing the colored frozen embedding agent according to claim 2, characterized in that: The stirring and dissolving time is 50 min to 120 min.

4. The method for preparing the colored cryoembedding according to claim 2, characterized in that: The temperature after cooling at room temperature is 50°C.

5. The method for preparing the colored cryoembedding according to claim 2, characterized in that: The water bath heating temperature is 90°C.

6. A freezing embedding agent kit, characterized in that: The kit comprises at least two different colored frozen embedding media as claimed in claim 1.

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