Body preservation fixative based on in situ clearance of chromogens, preparation and preservation method

Abstract

This invention discloses a fixative solution for embalming and preservation of cadavers based on in-situ pigment removal, its preparation, and preservation methods, relating to the field of biological tissue preservation technology. The fixative solution, by weight / volume percentage, comprises the following components: formaldehyde, hexamethylenetetraacetic acid disodium salt, dipotassium glycyrrhizate, benzalkonium chloride, glycerol, sorbitol, and the balance being phosphate-buffered saline solution with a concentration of [value missing]. The value of the fixative solution is [value missing]. This invention utilizes a combination of low-concentration formaldehyde and a neutral environment, employing chelating agents and antioxidants to transfer and stabilize pigment precursors within the tissue during fixation in the liquid phase. It aims to solve the problems of tissue blackening and hardening caused by traditional high-concentration formaldehyde fixation, allowing specimens to maintain their natural morphology and structure for extended periods at room temperature.

Description

Technical Field

[0001] This invention relates to the field of biological tissue preservation technology, and in particular to a mortuary embalming fixative based on in situ pigment removal, its preparation and preservation method. Background Technology

[0002] In the field of anatomical specimen preparation, formaldehyde fixation has long been the gold standard. However, traditional formalin fixatives typically have high concentrations and a highly acidic environment, which leads to two irreversible and destructive processes.

[0003] First, there is the problem of rapid overfixation. High concentrations of formaldehyde cause proteins on the tissue surface to denature and coagulate in a very short time, forming a dense cross-linked layer and a permeability barrier, making it difficult for the fixative to penetrate evenly into the deeper tissue layers. Second, there is the problem of chemical pigment contamination. Under acidic conditions, formaldehyde reacts with blood components in the tissue to produce brownish-black formalin pigment, which deposits inside the tissue, resulting in a dull macroscopic color and blurred microscopic structure. Existing improvements, such as using buffered formalin, can alleviate the acidic environment, but they still cannot completely eliminate pigment deposition and often face the problem of solution instability due to poor component compatibility. In addition, traditional methods often rely on low temperatures to delay specimen deterioration, which significantly increases the energy consumption and operational complexity of preservation.

[0004] Therefore, how to achieve gentle fixation of remains or biological tissues and block pigment deposition at the source has become an urgent technical challenge. Summary of the Invention

[0005] The main objective of this invention is to provide a mortuary embalming fixation solution, its preparation and preservation method based on in-situ pigment removal, which aims to achieve gentle fixation of mortuaries or biological tissues and block pigment deposition from the source.

[0006] To achieve the above objectives, this invention proposes a mortuary preservation and fixation solution based on in-situ pigment removal, at a concentration of... In total, the stationary liquid consists of the following components: mass-volume percentage concentration Formaldehyde, and urotropine as a slow-release agent and odor inhibitor. Disodium ethylenediaminetetraacetate as a pigment precursor chelating agent Dipotassium glycyrrhizate as an antioxidant Benzalkonium chloride as a penetration enhancer First polyol moisturizer glycerol Sorbitol, a second polyol moisturizer and the remaining concentration is Phosphate-buffered saline; wherein, the fixative... Value .

[0007] Preferred, according to each In total, the formaldehyde is added at a mass percentage concentration of [missing information]. The formaldehyde solution is provided, and the amount added is... .

[0008] Preferably, the fixative further comprises an alkaline regulator, sodium hydroxide, for use in finalizing the fixative. Value adjusted to .

[0009] Preferably, the fixative is a clear liquid filtered through three layers of gauze, and the fixative is in Save in environment The diva maintained a clear statement and her The value remains at Within the range.

[0010] This application also discloses a method for preparing a mortuary embalming and fixation solution based on in-situ pigment removal, characterized by comprising the following steps: (1) Measure the final volume The phosphate-buffered saline solution was then added sequentially with hexamethylenetetraacetic acid (HTA), disodium EDTA, sorbitol, and dipotassium glycyrrhizate, and stirred until dissolved. (2) Add the glycerol and benzalkonium chloride to the solution obtained in step (1) and stir until homogeneous; (3) Add to the solution obtained in step (2) The rate of addition of mass percentage concentration is Formaldehyde solution and stir; (4) After bringing the phosphate-buffered saline solution to the target volume, add sodium hydroxide solution dropwise to adjust the final volume. Value to .

[0011] This application also discloses a method for room temperature preservation of remains using a mortuary embalming and fixation solution, characterized in that the remains or biological tissues are completely immersed in the fixation solution, and... Save it in the specified environment.

[0012] The above technical solution has the following advantages: The embalming and fixation solution for remains based on in-situ pigment removal provided by this invention achieves excellent preservation and color retention effects through the synergistic effect of its components. This is achieved by controlling the formaldehyde concentration at a mass-volume percentage concentration of [missing information]. The lower level, combined with The phosphate-buffered saline solution creates a neutral-to-alkaline environment, enabling a gentle yet deep-penetrating fixation process that effectively prevents excessive hardening of the tissue surface. Urotropin, introduced into the system, acts as a slow-release agent, binding with free formaldehyde to significantly reduce irritating odor while maintaining fixation efficacy. Disodium EDTA and dipotassium glycyrrhizate, key components of the fixative, exert chelating and antioxidant effects, respectively, transferring and locking pigment precursors such as iron ions that have seeped from the tissue into the liquid phase under neutral conditions, thus blocking the formation and deposition of formalin pigments in the tissue at their source. Benzalkonium chloride, acting as a penetration enhancer, further reduces the surface tension of the liquid, while the dual moisturizing system of glycerol and sorbitol prevents tissue dehydration and hardening. This fixative exhibits excellent chemical stability at room temperature, allowing specimens to maintain their natural visual morphology and clear microstructure over a long period, significantly reducing preservation challenges. Attached Figure Description

[0013] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings, wherein: Figure 1 A schematic diagram of the initial fixed phenomenon provided in an embodiment of the present invention. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0015] This invention provides a mortuary preservation strategy based on gentle fixation and in-situ pigment removal. It aims to overcome the technical bottlenecks of traditional high-concentration formaldehyde fixation, such as tissue blackening, surface hardening, and uneven penetration, by constructing a novel chemical protection system. The core mechanism of this invention lies in using low-concentration formaldehyde in conjunction with a neutral to slightly alkaline buffer environment to achieve gentle fixation. Simultaneously, chelating agents and antioxidants are introduced to transfer and lock pigment precursors within the tissue into the liquid phase during fixation, thereby allowing the specimen to maintain its natural visual morphology and clear microstructure for a long period at room temperature.

[0016] Example 1 This embodiment provides a mortuary preservation and fixation solution based on in-situ pigment removal, at a concentration of per... The stationary phase, in total volume, consists of the following components: [Concentration percentage is missing from original text]. Formaldehyde, and urotropine as a slow-release agent and odor inhibitor. Disodium ethylenediaminetetraacetate (EDTA-2Na) serves as a chelating agent for pigment precursors. Dipotassium glycyrrhizate as an antioxidant Benzalkonium chloride as a penetration enhancer First polyol moisturizer glycerol Sorbitol, a second polyol moisturizer and the remaining concentration is Phosphate-buffered saline (PBS) is used. The final fixative solution... The value is set to .

[0017] The specific preparation method of the above-mentioned embalming and fixation solution based on in-situ pigment removal includes the following steps: First, measure the final volume. Phosphate-buffered saline solution, i.e., phosphate-buffered saline solution. concentration And initially Value Phosphate-buffered saline solution was injected into the preparation container. Under continuous stirring, urotropine was added sequentially to the container. Disodium ethylenediaminetetraacetate Sorbitol and dipotassium glycyrrhizate Stir until the solid particles are completely dissolved. Urotropin binds with free formaldehyde in the system to form a slow-release complex, thus significantly reducing the irritating odor of the liquid while maintaining the fixation effect. Disodium EDTA, as a strong chelating agent, captures iron ions that have seeped from the tissue, preventing them from participating in the formation of a black precipitate.

[0018] Subsequently, glycerol was added to the solution of the dissolved solid components. And benzalkonium chloride stock solution. In this embodiment, a mass percentage concentration of [missing information] is used. The amount of benzalkonium chloride stock solution added is calculated based on its concentration to ensure that the effective component concentration in the final fixative is within the specified range. Between. The specific calculation method uses the following formula: The target final concentration is set as follows: The specific concentration of the stock solution is Precisely add via micropipette The benzalkonium chloride stock solution allows benzalkonium chloride to... The final concentration of the active ingredient in the system reached Benzalkonium chloride, as a surfactant, can effectively reduce the surface tension of liquids and promote the penetration of immobilized components into deeper tissues. Because its concentration is controlled within... The extremely low level of [acid] completely eliminates the risk of it reacting with other anionic components in the system and producing precipitation, thus ensuring the dynamic stability of the system.

[0019] In a fume hood environment, with continuous stirring, slowly add dropwise the solution of the above mixture at a mass percentage concentration of [missing value]. The formaldehyde solution is a formaldehyde solution with a mass percentage concentration of 37%. Control the dropping speed to By controlling the dropping rate and stirring intensity, excessive protein denaturation caused by sudden spikes in localized formaldehyde concentration can be avoided. After the dropping is complete, utilize... Adjust the volume of phosphate-buffered saline to the target volume. .

[0020] Finally, using calibrated precision... The system was monitored, and dilute sodium hydroxide solution was added dropwise to the final volumetric solution. Value precisely adjusted to The prepared fixative was filtered through three layers of gauze to remove any trace impurities, resulting in a clear, transparent finished fixative with a faint aromatic odor. This fixative should be... Store in a cool, dark, and sealed environment.

[0021] The fixative provided in this embodiment demonstrates excellent in-situ pigment removal performance in practical applications. Fresh biological tissue specimens, such as pig heart or pig liver tissue, are completely immersed in the fixative of this embodiment and placed... Store at room temperature. During the initial fixation phase... Within hours, a key technical phenomenon can be observed: the fixative gradually changes from its initial colorless and transparent state to a clear, slightly brownish color, while the specimen itself retains a relatively reddish hue.

[0022] The scientific mechanism of this phenomenon lies in the fact that this embodiment... Low concentrations of formaldehyde and The combination of phosphate-buffered saline and physiological saline creates a mild, stable environment. This environment is unlike traditional... Like formalin at certain concentrations, it rapidly coagulates surface proteins into a dense, cross-linked barrier. Therefore, components such as hemoglobin within the tissue can be gently exchanged and diffused into the liquid phase under a neutral to slightly alkaline environment. The exudated hemoglobin, protected by the chelation of disodium EDTA and the antioxidant effect of dipotassium glycyrrhizate, avoids drastic denaturation under acidic conditions, preventing the formation of the dark brown formalin pigment. These pigment precursors are successfully "transferred" and "locked" in the fixative, resulting in a clear, slightly brown liquid.

[0023] After 30 days of observation at room temperature, specimens preserved using the fixative in this embodiment exhibited a uniform, natural pale yellow to pale brown color, without the mottled browning commonly seen in traditional methods. Histological H&E staining of sections showed an extremely clean tissue background with no pigment granule deposition, and clear cell nuclei and cytoplasmic structures, demonstrating that this formulation achieves reliable preservation while simultaneously providing high-quality pigment removal and structural preservation. Furthermore, the fixative remained clear throughout the 30-day observation period. The value stabilizes at The above demonstrates excellent chemical stability and buffering capacity.

[0024] Example 2 This embodiment provides another embalming and fixation solution based on in-situ pigment removal, aiming to verify the applicability and stability of the formulation at different component ratios. (Per...) In total, the stationary phase consists of the following components: a mass-volume percentage concentration of... Formaldehyde, and urotropine as a slow-release agent and odor inhibitor. Disodium ethylenediaminetetraacetate as a pigment precursor chelating agent Dipotassium glycyrrhizate as an antioxidant Benzalkonium chloride as a penetration enhancer First polyol moisturizer glycerol Sorbitol, a second polyol moisturizer and the remaining concentration is Phosphate-buffered saline solution. The final fixative solution... Value adjusted to .

[0025] In this embodiment, formaldehyde is added at a mass percentage concentration of... The formaldehyde solution is provided, and the specific amount added is... This concentration of formaldehyde provides moderate fixation strength, avoiding the problem of instantaneous denaturation and coagulation of surface proteins and the formation of a permeability barrier caused by traditional high-concentration formaldehyde.

[0026] The preparation process of the fixative strictly follows a specific process sequence to ensure the physicochemical compatibility of each component: First step, measure concentration Phosphate-buffered saline solution is placed in a container, the volume of which accounts for a certain percentage of the final volume. Subsequently, solid components, namely hexamethylenetetramine, were added to the brine in sequence. Disodium ethylenediaminetetraacetate Sorbitol and dipotassium glycyrrhizate Turn on the agitator and stir thoroughly until all solid solutes are completely dissolved in the buffer brine.

[0027] The second step involves adding the liquid component, namely glycerol, to the solution after the solid has completely dissolved. And benzalkonium chloride. The use of benzalkonium chloride can effectively reduce the surface tension of the solution, thereby promoting the penetration of the liquid into the deeper layers of the remains or biological tissues during subsequent fixation.

[0028] The third step is to add the mixture obtained in the second step to... The concentration was added slowly at a rate of [missing information - likely a concentration percentage]. The formaldehyde solution was prepared and stirred continuously. Strict control of the dropping rate was necessary to prevent excessive local formaldehyde concentration, which could lead to excessive protein cross-linking.

[0029] The fourth step is to use a concentration of The solution was brought to a final volume using phosphate-buffered saline until the total volume reached the target value. After adjusting the volume, use a precision... Monitoring was performed, and an appropriate amount of alkaline regulator, namely sodium hydroxide solution, was added dropwise to bring the final solution of the stationary phase to a suitable level. Value adjusted to The scope of this invention is limited. This invention provides a powerful dynamic buffer for physiological saline using phosphate-buffered saline. Buffering capacity, combined with initial Precise calibration of values ​​can effectively resist the impact of acidic substances released by tissues in the early stages of fixation.

[0030] The prepared fixative solution needs to be physically filtered through three layers of gauze to ensure that the final product is a clear and transparent liquid. Experimental results show that this fixative solution... After being stored at room temperature for 30 days, it still maintains a clear state, and its The value remained stable at Within the specified range, it exhibits excellent physicochemical stability.

[0031] The specific method for room temperature preservation involved in this embodiment is as follows: the remains or biological tissues requiring preservation are completely immersed in the prepared fixative solution, and then... Store in a static environment.

[0032] To further verify the technical advantages of this invention in in-situ pigment removal, a mechanism verification experiment was conducted in this embodiment. A control group, namely the CF group, was set up in the experiment, using traditional... Neutral buffered formalin fixative, and the experimental group (EF group), used the fixative described in this embodiment. It was stored at room temperature. to Hours later, it was observed that although the fixative in the CF group remained clear, the tissue specimens rapidly turned gray and white. This is because the high concentration of formaldehyde caused severe denaturation of the surface proteins of the tissue, resulting in the instantaneous disintegration of red blood cells and the in-situ denaturation and precipitation of hemoglobin within the tissue.

[0033] In stark contrast, the fixative in the EF group gradually turned a clear, slightly brownish color, while the tissue specimens initially remained reddish, subsequently transforming slowly and uniformly into a near-natural pale yellow hue. This key phenomenon of the fixative turning slightly brown directly demonstrates the role of the in-situ pigment removal mechanism: the exudated hemoglobin, under the neutral environment and the chelating protection of disodium EDTA, avoids rapid denaturation within the tissue, instead dissolving and locking in the fixative phase in a stable state.

[0034] Long-term preservation tests showed that after 30 days of storage at room temperature, the CF group specimens exhibited significant mottled browning, due to formalin pigment deposition generated under acidic conditions. In contrast, the EF group specimens showed a uniform and natural color. Subsequent histological evidence indicated that the H&E-stained sections of the EF group had a clean background, completely free of brown-black pigment granules, and the cells and tissue structures were clearly visible, fully demonstrating the significant technical effect of the synergistic effect of gentle fixation and in-situ pigment removal in this invention.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Those skilled in the art should understand that, within the scope of the present invention, various reasonable adjustments and optimizations can be made to the above component ratios, preparation steps, and application methods. For example, adjustments can be made based on specific environmental temperatures or biological tissue types. to Fine-tune the formaldehyde concentration within the range, or within to The dosage of urotropine, acting as a sustained-release agent and odor inhibitor, was adjusted within a certain range to achieve optimal fixation and deodorization effects for different specimen types. The pigment-based in-situ removal mechanism disclosed in this invention is not only suitable for the long-term preservation of teaching specimens but also possesses excellent potential for industrial production. Any modifications, equivalent substitutions, and improvements made within the technical spirit and protection principles of this invention should be included within the scope of protection of this invention.

Claims

1. A embalming and fixation solution for remains based on in-situ pigment removal, characterized in that, According to each In total, the stationary liquid consists of the following components: mass-volume percentage concentration Formaldehyde, and urotropine as a slow-release agent and odor inhibitor. Disodium ethylenediaminetetraacetate as a pigment precursor chelating agent Dipotassium glycyrrhizate as an antioxidant Benzalkonium chloride as a penetration enhancer First polyol moisturizer glycerol Sorbitol, a second polyol moisturizer and the remaining concentration is Phosphate-buffered saline; wherein, the fixative... Value .

2. The embalming and fixation solution for remains based on in-situ pigment removal according to claim 1, characterized in that, According to each In total, the formaldehyde is added at a mass percentage concentration of [missing information]. The formaldehyde solution is provided, and the amount added is... .

3. The embalming and fixation solution for remains based on in-situ pigment removal according to claim 1, characterized in that, The fixative also contains sodium hydroxide, an alkaline regulator, for finalizing the fixative solution. Value adjusted to .

4. The embalming and fixation solution for remains based on in-situ pigment removal according to claim 1, characterized in that, The fixative is a clear liquid filtered through three layers of gauze, and the fixative is in Save in environment The diva maintained a clear statement and her The value remains at Within the range.

5. The method for preparing a embalming and fixation solution based on in-situ pigment removal according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Measure the final volume The phosphate-buffered saline solution was then added sequentially with hexamethylenetetraacetic acid (HTA), disodium EDTA, sorbitol, and dipotassium glycyrrhizate, and stirred until dissolved. (2) Add the glycerol and benzalkonium chloride to the solution obtained in step (1) and stir until homogeneous; (3) Add to the solution obtained in step (2) The rate of addition of mass percentage concentration is Formaldehyde solution and stir; (4) After bringing the phosphate-buffered saline solution to the target volume, add sodium hydroxide solution dropwise to adjust the final volume. Value to .

6. A method for preserving remains at room temperature using the embalming and fixative solution according to any one of claims 1 to 4, characterized in that, The remains or biological tissues are completely immersed in the fixative solution, and Save it in the specified environment.

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