Mixed decalcification liquid, preparation method thereof and application of mixed decalcification liquid in pathological detection

Abstract

The invention relates to the technical field of medical detection, and particularly discloses a mixed decalcification solution, a preparation method thereof and application of the mixed decalcification solution in pathological detection. The invention relates to a mixed decalcification solution and a preparation method thereof. The mixed decalcification solution comprises the following raw materials in percentage: 3-5% of formaldehyde, 8-12% of formic acid, 3-5% of acetic acid, 0.8-1.2% of sodium gluconate, 0.3-5% of hydrochloric acid, 0.05-2% of trehalose and the balance of a phosphate buffer solution. The formic acid and the acetic acid are compounded to form a mixed acid system, so that the decalcification liquid has a higher decalcification speed and a certain tissue expansion protection effect, the decalcification process is milder, the damage of hydrochloric acid to antigens in bone tissues is reduced, and the dyeing effect in subsequent pathological detection is ensured. The chelation of sodium gluconate, trehalose and formaldehyde are utilized to protect the integrity of the tissue structure and reduce the damage to the antigen.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection, in particular to a mixed decalcification solution, a preparation method thereof and application in pathological detection. BACKGROUND

[0002] In the fields of biomedical science and material science, decalcification technology has important applications. With the continuous deepening of research, the requirements for decalcification effect and sample protection are also getting higher and higher. Decalcification technology plays a key role in bone tissue research, archaeology, forensic science and other aspects, which can help researchers observe the tissue structure more clearly, provide important basis for disease diagnosis, pathological detection and other aspects, and promote the development of related fields.

[0003] In pathological detection, hard tissues such as bones and teeth are often encountered. These tissues are rich in calcium and have high density and hard texture, so they cannot be directly made into wax blocks for sectioning. Therefore, the tissues need to be decalcified first. Traditional decalcification solutions mainly include strong acid decalcification solution, weak acid decalcification solution and neutral decalcification solution. The main component of strong acid decalcification solution is strong acid such as nitric acid, which has strong decalcification ability and rapid decalcification, but it can damage antigens and needs to be strictly controlled. The typical neutral decalcification solution is a chelating decalcification solution with ethylenediaminetetraacetic acid as the main component, which has the characteristics of mild decalcification effect and small damage to antigens, but the decalcification speed is extremely slow. The main component of weak acid decalcification solution is weak acid such as formic acid, but the decalcification ability and effect are between strong acid decalcification solution and neutral decalcification solution. SUMMARY

[0004] In order to improve the decalcification speed and effect of the existing decalcification solution, the present application provides a mixed decalcification solution, a preparation method thereof and application in pathological detection. In the first aspect, the present application provides a mixed decalcification solution, which adopts the following technical solution: A mixed decalcification solution, comprising the following percentage of raw materials: formaldehyde 3-5%, formic acid 8-12%, acetic acid 3-5%, sodium gluconate 0.8-1.2%, hydrochloric acid 0.3-5%, trehalose 0.05-2%, and the balance is phosphate buffer solution.

[0005] The mixed acid of formic acid and acetic acid can make calcium in the tissue precipitate rapidly, formic acid has a good decalcification speed, and acetic acid has a good swelling effect, the two are compounded to form a mixed acid system, which can make the decalcification solution have a faster decalcification speed and a certain tissue swelling protection effect, so that the decalcification process is more gentle, the damage of hydrochloric acid to the antigen in the bone tissue is reduced, and the staining effect in the subsequent pathological detection is ensured. Hydrochloric acid can maintain the acidic environment of the decalcification solution, quickly open the decalcification channel, and improve the decalcification speed. Sodium gluconate can effectively chelate calcium in the tissue, reduce the calcium salt precipitation caused by a large amount of Ca²⁺ released in the decalcification process, reduce the penetration of the decalcification solution to the bone tissue blocked by the calcium salt precipitation, and reduce the interference of the calcium salt precipitation on the subsequent pathological detection staining, thereby improving the decalcification speed. Trehalose can form a protective glassy matrix in extreme environments to prevent denaturation or degradation of proteins, lipids, cell membranes and DNA, protect the lipid bilayer structure of the cell membrane, reduce membrane damage and cell rupture caused by an acid environment, and protect the integrity of the tissue structure. Formaldehyde can cross-link and solidify biological macromolecules such as proteins and nucleic acids, maintain the structure of cells and tissues, and reduce the damage to the antigen; the phosphate buffer solution can keep the osmotic pressure and pH value of the decalcification solution relatively stable.

[0006] The strong acid hydrochloric acid and the high-efficiency acid formic acid are used to make the decalcification solution have a faster decalcification speed, formaldehyde is used to provide core protein cross-linking and fixation, trehalose is used to provide additional stability protection and resist acid damage at the molecular and cellular levels, and the swelling effect of acetic acid is used to maintain the tissue morphology and flexibility, so that the three work together to protect the tissue morphology and structure, and ensure that the tissue after decalcification can be made into a higher-quality section; through the combined action of various components, the decalcification solution can have a faster decalcification speed while better protecting the antigen in the tissue.

[0007] Preferably, the mixed decalcification solution comprises the following percentage of raw materials: 3-5% of formaldehyde, 8-12% of formic acid, 3-5% of acetic acid, 0.8-1.2% of sodium gluconate, 0.38-3.8% of hydrochloric acid, 0.1-1% of trehalose, and the rest is a phosphate buffer solution.

[0008] By controlling the use amount of hydrochloric acid and trehalose within a suitable range, the decalcification speed of various bone tissues can be accelerated, and the decalcification time can be shortened, and a reasonable amount of trehalose can better protect the antigen and nucleic acid in the tissue, thereby ensuring the staining results of subsequent molecular pathology.

[0009] Preferably, the pH of the phosphate buffer solution is 7-7.8.

[0010] Preferably, the concentration of phosphate in the phosphate buffer solution is 0.005-0.015 mol / L.

[0011] In a second aspect, the application provides a preparation method of mixed decalcification solution, which adopts the following technical scheme: A preparation method of mixed decalcification solution, comprising the following specific steps: Mixing formaldehyde, formic acid, acetic acid, sodium gluconate, hydrochloric acid, trehalose and phosphate buffer solution, stirring uniformly to obtain the mixed decalcification solution.

[0012] By adopting the above technical scheme, through the synergistic effect of various components, the decalcification speed of various bone tissues can be accelerated, the decalcification time is shortened, and the antigens and nucleic acids in the tissues are protected, so that the staining results in subsequent pathological detection are ensured.

[0013] In a third aspect, the application provides an application of the mixed decalcification solution in pathological detection, which adopts the following technical scheme: An application of the mixed decalcification solution in pathological detection, comprising the following specific steps: Putting the bone tissue into the mixed decalcification solution for decalcification, wherein the mixed decalcification solution is prepared by the preparation method of the mixed decalcification solution, placing the decalcified bone tissue in running water for cleaning, sequentially immersing the cleaned bone tissue in ethanol, xylene and melted wax solution, preparing a wax block and slicing, taking the slices for HE, IHC and molecular pathological staining, and observing the staining effect diagram of the bone tissue, so as to complete the application of the mixed decalcification solution in pathological detection.

[0014] By adopting the above technical scheme, the bone tissue treated by the decalcification solution prepared by the application can maintain complete cell morphology, clear outline and good staining effect.

[0015] Preferably, the bone tissue is put into the mixed decalcification solution for decalcification, and the decalcification is completed when the bone tissue floats up.

[0016] Preferably, the cleaned bone tissue is immersed in an ethanol solution with a mass fraction of 60%-100%, and then sequentially immersed in xylene and melted wax solution.

[0017] In summary, the application has the following beneficial effects: 1. Since the application adopts the mixed acid system formed by the combination of formic acid and acetic acid, the decalcification solution has both fast decalcification speed and certain tissue swelling protection effect, the decalcification process is more gentle, the damage of hydrochloric acid to antigens in the bone tissue is reduced, and the staining effect in subsequent pathological detection is ensured. The chelation of sodium gluconate reduces the calcium salt precipitation caused by the release of a large amount of Ca²⁺ in the decalcification process, and improves the decalcification speed. Trehalose and formaldehyde are used to protect the integrity of the tissue structure and reduce the damage to the antigens.

[0018] 2, The use amount of hydrochloric acid and trehalose in the application is controlled and verified within the range, which can accelerate the decalcification speed of various bone tissues and shorten the decalcification time. Meanwhile, the reasonable use amount of trehalose can better protect the antigens and nucleic acids in the tissues, and ensure the subsequent molecular pathological staining results. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the HE staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the examples 1-4 and the comparative example 1 of the application; Figure 1 is the HE staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 1 of the application; Figure 1 is the HE staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 2 of the application; Figure 1 is the HE staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 3 of the application; Figure 1 is the HE staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 4 of the application; Figure 1 is the HE staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the comparative example 1 of the application; Figure 2 is the IHC staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the examples 1-4 and the comparative example 1 of the application; Figure 2 is the IHC staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 1 of the application; Figure 2 is the IHC staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 2 of the application; Figure 2 is the IHC staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 3 of the application; Figure 2 is the IHC staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 4 of the application; Figure 2 is the IHC staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the comparative example 1 of the application; Figure 3 is the molecular pathological staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the examples 1-4 and the comparative example 1 of the application.

[0020] Figure 3 is the molecular pathological staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in the example 1 of the application; Figure 3Fig. 2 is a molecular pathology staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in Example 2; Figure 3 Fig. 3 is a molecular pathology staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in Example 3; Figure 3 Fig. 4 is a molecular pathology staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in Example 4; Figure 3 Fig. 5 is a molecular pathology staining result diagram of the bone tissue treated by the mixed decalcification solution prepared in Comparative Example 1. DETAILED DESCRIPTION

[0021] The application will be further described in detail below in combination with examples.

[0022] All raw materials in the examples can be obtained by market purchase.

[0023] Formic acid is selected from China Reagent Group 80065618.

[0024] Acetic acid is selected from China Reagent Group C124040025.

[0025] Hydrochloric acid is selected from China Reagent Group 10011018.

[0026] Sodium gluconate is selected from Sigma-S2054.

[0027] Trehalose is selected from Shanghai McLean Biochemical Technology Co., Ltd. L823931.

[0028] Formaldehyde is selected from China Reagent Group XW015000003. EXAMPLE Example 1

[0029] The present embodiment provides a mixed decalcification solution, which comprises the following percentage of raw materials: formaldehyde 3.8%, formic acid 10%, acetic acid 4%, sodium gluconate 1%, hydrochloric acid 0.38%, trehalose 1%, and the balance is phosphate buffer solution. The phosphate buffer solution has a pH of 7.4, and the concentration of phosphate in the phosphate buffer solution is 0.01 mol / L, which is selected from Beijing Zhongshanjinqiao Biotechnology Co., Ltd. ZLI-9062.

[0030] The preparation method of the mixed decalcification solution comprises the following specific steps: Mixing formaldehyde, formic acid, acetic acid, sodium gluconate, hydrochloric acid, trehalose and phosphate buffer solution, and stirring uniformly to obtain the mixed decalcification solution.

[0031] The application of a mixed decalcification solution in pathological detection comprises the following specific steps: S1: Put the bone tissue into the mixed decalcification solution for decalcification, the mixed decalcification solution is prepared by the preparation method of the mixed decalcification solution, when the bone tissue floats, the decalcification is completed, and the decalcified bone tissue is placed in running water for cleaning for 30 min.

[0032] S2: The cleaned bone tissue is sequentially soaked in ethanol solutions with mass fractions of 60%, 70%, 80%, 90% and 100% for 30 min each time, the 100% ethanol solution is continuously soaked for three times, and then the tissue after soaking is soaked in dimethylbenzene for 30 min, continuously soaked for three times, and then soaked in molten paraffin solution for three times, to prepare a wax block and slice.

[0033] S3: The slice is taken for HE, IHC and molecular pathology staining respectively, the stained slice is sequentially cleaned by using ethanol solutions with mass fractions of 75%, 95% and 100%, the 100% ethanol solution is cleaned for three times, and then dried, and after being sealed by using a sealing machine, the staining effect diagram of the bone tissue is observed under a microscope, and the application of the mixed decalcification solution in pathological detection is completed. Example 2

[0034] Example 2 differs from Example 1 in that the mixed decalcification solution comprises the following percentage of raw materials: formaldehyde 3.8%, formic acid 10%, acetic acid 4%, sodium gluconate 1%, hydrochloric acid 0.38%, trehalose 0.1%, and the balance is a phosphate buffer solution. Example 3

[0035] Example 3 differs from Example 1 in that the mixed decalcification solution comprises the following percentage of raw materials: formaldehyde 3.8%, formic acid 10%, acetic acid 4%, sodium gluconate 1%, hydrochloric acid 3.8%, trehalose 1%, and the balance is a phosphate buffer solution. Example 4

[0036] Example 4 differs from Example 1 in that the mixed decalcification solution comprises the following percentage of raw materials: formaldehyde 3.8%, formic acid 10%, acetic acid 4%, sodium gluconate 1%, hydrochloric acid 3.8%, trehalose 0.1%, and the balance is a phosphate buffer solution. Example 5

[0037] Example 5 differs from Example 1 in that the mixed decalcification solution comprises the following percentage of raw materials: formaldehyde 3%, formic acid 8%, acetic acid 5%, sodium gluconate 0.8%, hydrochloric acid 5%, trehalose 0.05%, and the balance is a phosphate buffer solution. Example 6

[0038] Example 6 differs from Example 1 in that the mixed decalcification solution comprises the following percentages of raw materials: formaldehyde 5%, formic acid 12%, acetic acid 3%, sodium gluconate 1.2%, hydrochloric acid 0.3%, trehalose 2%, and the balance is a phosphate buffer solution.

[0039] Comparative Example 1 Comparative Example 1 differs from Example 1 in that the decalcification solution prepared in Comparative Example 1 is a common commercially available EDTA decalcification solution, which is selected from E1117, pH 7.2, of Beijing Solaybao Technology Co., Ltd.

[0040] The decalcification solutions provided in Examples 1-6 and Comparative Example 1 were subjected to the following performance tests, and the specific test results are shown in the following table.

[0041] I. Decalcification speed test Take 50 ml of the mixed decalcification solution prepared in the application, and take bone tissue with a size of about 1.0 cm long x 0.5 cm wide x 0.3 cm thick, and respectively put it into the seven decalcification solutions prepared in Examples 1-6 and Comparative Example 1, and observe the phenomenon and record it.

[0042] Table 1: Performance test result data table of decalcification speed of decalcification solution Example Decalcification phenomenon Time required for completion of decalcification Example 1 Bubbles appeared on the surface of the bone tissue after 6 h and the bone tissue floated after 12 h 12h Example 2 Bubbles appeared on the surface of the bone tissue after 4 h and the bone tissue floated after 8 h 8h Example 3 Bubbles appeared on the surface of the bone tissue after 2 h and the bone tissue floated after 6 h 6h Example 4 Bubbles appeared on the surface of the bone tissue after 2 h and the bone tissue floated after 4 h 4h Example 5 Bubbles appeared on the surface of the bone tissue after 6 h and the bone tissue floated after 12 h 12h Example 6 Bubbles appeared on the surface of the bone tissue after 6 h and the bone tissue floated after 12 h 12h Comparative Example 1 No bubbles appeared on the surface of the bone tissue and the bone tissue floated after 24 h 24h As can be seen from the performance test results in Table 1, the mixed decalcification solution prepared in the application can significantly accelerate the decalcification speed of bone tissue and shorten the decalcification time. As can be seen by comparing Examples 1-4 and Examples 5-6, the decalcification time in Examples 2-4 is shorter, which further illustrates that the use of hydrochloric acid and sodium gluconate at a certain concentration has an important influence on the decalcification speed and effectively shortens the decalcification time. As can be seen by comparing Comparative Example 1 and Example 1, the decalcification speed of the decalcification solution used in Comparative Example 1 is significantly slower, which further illustrates that the conventional ethylenediaminetetraacetic acid decalcification solution, although has a relatively mild decalcification effect, has a very slow decalcification speed.

[0043] II. Application of pathological detection Using a microscope, the HE, IHC, and molecular pathology staining effect diagrams of the bone tissue treated by the decalcification solutions prepared in Examples 1-4 and Comparative Example 1 were observed.

[0044] As shown in FIG. a, the bone tissue treated by the mixed decalcification solution prepared in Example 1 has a good bone tissue structure, the bone trabeculae are pink, the bone marrow nuclei are blue-violet, and the cytoplasm and red blood cells are red, and the cell morphology is complete and the outline is clear. Figure 1 As shown in FIG. c, the bone tissue treated by the mixed decalcification solution prepared in Example 3 has a loose bone tissue structure, the bone trabeculae are pink, the bone marrow nuclei are blue-violet, and the cytoplasm and red blood cells are less common; and as shown in FIG. d, the bone tissue treated by the mixed decalcification solution prepared in Example 4 has a loose bone tissue structure, the bone trabeculae are pink, the bone marrow nuclei are blue-violet, and the cytoplasm and red blood cells are less common. Figure 2As shown in the IHC staining results, the bone tissue treated with the mixed decalcification solution prepared in Example 1 (Figure a) and Example 2 (Figure b) showed intact cell morphology, clear outlines, and obvious nuclear staining, but the former had a heavier staining background. The bone tissue treated with the mixed decalcification solution prepared in Example 3 (Figure c) and Example 4 (Figure d) showed incomplete cell morphology, unclear outlines, and few nuclear staining. The bone tissue treated with the decalcification solution prepared in Comparative Example 1 (Figure e) showed unclear cell outlines and blurred nuclear staining.

[0045] like Figure 3 As shown, based on the molecular pathological staining results, the bone tissue treated with the mixed decalcification solution prepared in Example 1 (Figure a) and Example 2 (Figure b) showed intact cell morphology, clear outlines, and obvious cytoplasmic and membrane staining, but the former had a heavier staining background. The bone tissue treated with the mixed decalcification solution prepared in Example 3 (Figure c) and Example 4 (Figure d) showed incomplete cell morphology, unclear outlines, and little cytoplasmic and membrane staining. The bone tissue treated with the decalcification solution prepared in Comparative Example 1 (Figure e) showed unclear cell outlines and blurred cytoplasmic and membrane staining.

[0046] The staining performance tests show that the staining effects of Examples 1-2 in this application are better. Considering the decalcification rate, the decalcification solution prepared in Example 2 has better overall performance. The mixed decalcification solution adjusts the ratio of the components used in the raw materials, which effectively balances the decalcification rate and antigen protection. A comparison between Comparative Example 1 and Example 1 shows that the bone tissue treated with the decalcification solution prepared in Comparative Example 1 (Figure e) has unclear bone tissue and cell outlines, and the staining localization is blurred.

[0047] To further explain, the addition of formic acid and acetic acid in this application makes the decalcification process gentler. By controlling the ratio of hydrochloric acid and sodium gluconate within an appropriate range, the decalcification speed of various bone tissues can be significantly accelerated and the decalcification time shortened. By utilizing trehalose, antigens and nucleic acids in the tissues are better protected, ensuring the subsequent staining results.

[0048] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A mixed decalcification solution, characterized in that, The raw materials include the following percentages: formaldehyde 3-5%, formic acid 8-12%, acetic acid 3-5%, sodium gluconate 0.8-1.2%, hydrochloric acid 0.3-5%, trehalose 0.05-2%, and the balance is phosphate buffer solution.

2. The mixed decalcification solution according to claim 1, characterized in that, The mixed decalcification solution comprises the following percentages of raw materials: formaldehyde 3-5%, formic acid 8-12%, acetic acid 3-5%, sodium gluconate 0.8-1.2%, hydrochloric acid 0.38-3.8%, trehalose 0.1-1%, and the balance being a phosphate buffer solution.

3. The mixed decalcification solution according to claim 1, characterized in that, The pH of the phosphate buffer solution is 7-7.

8.

4. The mixed decalcification solution according to claim 3, characterized in that, The concentration of phosphate in the phosphate buffer solution is 0.005-0.015 mol / L.

5. A method for preparing the mixed decalcification solution as described in any one of claims 1-4, characterized in that, The specific steps include the following: Formaldehyde, formic acid, acetic acid, sodium gluconate, hydrochloric acid, trehalose, and phosphate buffer solution are mixed and stirred until homogeneous to obtain a mixed decalcification solution.

6. The application of the mixed decalcification solution according to any one of claims 1-5 in pathological testing, characterized in that, The specific steps include the following: Bone tissue is immersed in a mixed decalcification solution for decalcification. The mixed decalcification solution is prepared by the method described in claims 1-5. The decalcified bone tissue is washed in running water. The washed bone tissue is then soaked in ethanol, xylene, and melted wax in sequence to form wax blocks, which are then sectioned. The sections are stained with hematoxylin and eosin (HE), iodine chromatograph (IHC), and molecular pathology, respectively. The staining effect of the bone tissue is observed, thus completing the application of the mixed decalcification solution in pathological testing.

7. The application of the mixed decalcification solution according to claim 6 in pathological testing, characterized in that, Bone tissue is immersed in a mixed decalcification solution for decalcification. Decalcification is complete when the bone tissue floats to the surface.

8. The application of the mixed decalcification solution according to claim 6 in pathological testing, characterized in that, The cleaned bone tissue was successively immersed in ethanol solutions with mass fractions of 60%, 70%, 80%, 90%, and 100%, and then successively immersed in xylene and melted wax.

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