A pathological sample slide for long-term preservation and its preparation method and application

Abstract

The present invention discloses a pathological sample slide for long-term storage, and a preparation method and application thereof, which belong to the technical field of immunohistochemistry quality control products. The pathological sample slide includes a dried sample spot prepared using a pathological sample, and the sample spot is evenly covered with a layer of protective agent, and the protective agent includes at least one selected from a carbohydrate protective agent and / or a polyhydroxy compound protective agent. The preparation method includes first preparing a sample spot on a slide using a pathological sample, and then evenly covering the sample spot with a layer of the protective agent. The pathological sample slide of the present invention has stable performance of the target target on the sample spot, and the staining performance is not affected after being stored at 2-8°C for 6 months, and will not affect subsequent detection, and has very important application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunohistochemistry quality control products, and in particular, relates to a pathological sample slide for long-term preservation, a preparation method thereof and an application thereof. Background Art

[0002] Cytopathology is a pathological examination and diagnosis based on cells from the tumor site. It is simple, easy to carry out, economical and practical, and has a high diagnostic positive rate. It is currently the main method for diagnosing esophageal cancer and cervical cancer. Clinically, cytopathology diagnosis is a method of direct scraping or smear examination of superficial tumors or natural secretions that have cavities that communicate with the outside world.

[0003] In order to ensure the accuracy of the test results, cytopathology testing usually requires liquid cell quality control products. Generally, liquid cell quality control products are directly dripped onto the glass slide containing the cell sample to be tested, and after drying, cytopathology testing can be completed quickly. However, liquid cell quality control products have a short shelf life and can weaken staining in 3 weeks. They need to be prepared frequently, making it difficult to ensure the consistency of quality control. Summary of the invention

[0004] In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:

[0005] A first aspect of the present invention provides a pathological sample slide for long-term preservation, wherein the pathological sample slide comprises a dried sample spot prepared using a pathological sample, and the sample spot is evenly covered with a layer of protective agent, wherein the protective agent comprises at least one selected from a carbohydrate protective agent and / or a polyhydroxy compound protective agent.

[0006] In immunoassays, quality control products are standardized materials used to calibrate and verify experimental results. They usually contain antigens, antibodies or other immune reaction components of known quantity and characteristics to ensure the accuracy and repeatability of immunoassays. Such quality control products are widely used in immunological tests such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, Western blot, flow cytometry, etc. In this field, quality control products, especially cell quality control products, are generally liquid quality control products. The present invention innovatively proposes a quality control slide form including a sample spot to extend the storage time without affecting the performance.

[0007] In the present invention, the inventor unexpectedly discovered that after forming a sample spot on a glass slide, evenly covering it with a layer of protective agent can effectively prolong the storage time of the sample spot. After being stored at 2-8°C for up to 6 months, the staining performance of the target point has not changed significantly, and the quality control consistency of detection at different times can be guaranteed. It should be particularly noted that the protective agent cannot be mixed with the pathological sample before forming the sample spot. Instead, the pathological sample needs to be formed into a sample spot on a glass slide first, and then a layer of protective agent is evenly covered on the sample spot. This sequence is one of the cores of the present technical solution. The formation of the pathological sample spot and the covering operation of the protective agent are further defined in the subsequent preparation method, but the present invention does not limit the preparation method of the pathological sample spot and the covering method of the protective agent, especially the covering method of the protective agent. As long as the technical solution can evenly cover the formed pathological sample spot with a layer of protective agent, it should fall within the protection scope of the present invention.

[0008] In the present invention, pathological samples include cytological samples and histological samples. Histological samples are generally obtained through open surgery, endoscopic examination or percutaneous puncture biopsy, while cytological samples are generally obtained through body fluids, netting, fine needle puncture, exfoliated cells, etc. Of course, in the present invention, the pathological samples can further include cell suspensions (including liquid and semi-solid) containing target cells or cell fragments, cell paraffin sections, and cell fragment suspensions prepared from cell paraffin sections. For different pathological samples, the method of preparing sample spots is different. Optionally, the sample spots are prepared by one of the following methods:

[0009] (1) preparing the histological sample into a tissue paraffin section or a tissue frozen section, attaching it to a glass slide, and drying it to obtain the sample spot;

[0010] (2) preparing the histological sample into a tissue suspension, dropping it onto a glass slide, and drying it to obtain the sample spot;

[0011] (3) using the Pap smear method or the liquid-based thin-layer cytology preparation method to obtain the sample spot on a glass slide;

[0012] (4) preparing a cytological sample or a cell suspension containing target cells into a cell paraffin section, attaching it to a glass slide, and drying it to obtain the sample spot;

[0013] (5) Dropping a cell suspension containing target cells and / or cell fragments onto a glass slide and drying the suspension to obtain the sample spot.

[0014] In the present invention, the drying refers to natural drying (room temperature) or low-temperature heating drying at about 45°C.

[0015] In the present invention, no matter in which form the sample spot is prepared, if it is used as a quality control, it is also called a quality control point or a quality control spot.

[0016] In the first method, the histological sample is obtained by open biopsy (through surgery), endoscopic biopsy (using biopsy forceps) or percutaneous puncture biopsy (using biopsy needle). The obtained histological sample is fixed, dehydrated, transparent, wax-impregnated, embedded and sliced ​​to obtain the tissue paraffin section, or rapidly frozen, fixed and sliced ​​to obtain the tissue frozen section.

[0017] In the second method, fresh tissue or dewaxed and hydrated tissue paraffin sections are broken by ultrasound to prepare a tissue suspension. The fresh tissue or dewaxed and hydrated tissue includes transplanted tumor tissue.

[0018] In the third method, the cytological samples include body fluids (such as pleural effusion, ascites, etc.), esophageal and gastric reticular cells, endometrial and cervical exfoliated cells, etc. The Pap smear method uses a scraper to scrape exfoliated cells from the cervix and then directly smears them on a glass slide; the liquid-based thin layer cytological preparation method can be mainly divided into two categories: membrane preparation (Thin Prep Cytologic Test, TCT) and sedimentation preparation (Liquid-Based Cytologic Test / Liquid-Based Cytologic Preparation, LCT / LBP). TCT collects cells, mixes them, and filters them under negative pressure, and then takes the filtered cells and makes a thin layer on a glass slide. LCT / LBP uses two centrifugations and independent staining, which has great advantages in automation and standardization, and can achieve good control of the quality of pathological diagnosis. It has become the mainstream cytological preparation method. In addition to being mainly used in cervical cytology testing, it has more advantages in the detection of non-gynecological samples such as sputum, pleural effusion, urine, and bronchoalveolar lavage fluid.

[0019] In the fourth method, the cytological sample or the cell suspension containing the target cells can be centrifuged, and the precipitate can be further fixed, dehydrated, transparent, wax-impregnated, embedded and sliced ​​to prepare cell paraffin sections by referring to the method of histological samples. Among them, the cell suspension containing the target cells is usually a liquid cell quality control product.

[0020] In the fifth method, the cell suspension containing target cells and / or cell fragments is a suspension obtained by cell paraffin sectioning. In this case, the suspension contains broken cells, i.e., cell fragments, due to the physical sectioning, and also contains detectable antigens, which can be used for detection.

[0021] In some embodiments of the present invention, the method for preparing the cell suspension containing target cells and / or cell fragments is as follows: dewaxing and hydrating the cell paraffin sections, and adding a semisolid buffer to prepare a semisolid cell suspension. In some specific embodiments of the present invention, the preparation method of the cell suspension containing target cells and / or cell fragments is as follows: placing cell paraffin sections in a centrifuge tube; pouring about 25 mL of dewaxing solution into the centrifuge tube, and oscillating to dissolve the paraffin in the sections; centrifuging the mixture at 2500 rpm for 5 min, pouring off the dewaxing solution, adding 25 mL of isopropanol, and then oscillating the cell pellet with ethanol; centrifuging the mixture at 2500 rpm for 5 min, pouring off the isopropanol, and then adding about 1 mL of anhydrous ethanol to the centrifuge tube, and blowing evenly with a pipette tip; placing the mixture in a 1.5 mL centrifuge tube, centrifuging at 2500 rpm for 5 min, absorbing the ethanol with a pipette tip, adding 25% v / v ethanol, the added volume of 25% v / v ethanol being 30 times the volume of the sections, and oscillating to form a cell suspension, which includes cell fragments and intact cells.

[0022] Further, 1 to 3 μL of the cell suspension containing target cells and / or cell fragments is added dropwise to the quality control area of ​​the slide, and dried in a manner including but not limited to natural drying at room temperature or oven drying, and a sample spot containing cells or cell fragments is formed at the position of the cell suspension. Taking 2 μL of sample volume as an example, the diameter of the formed sample spot is 3 to 5 mm. It should be noted that the quality control area described here is for the convenience of explaining the designated or delineated area, which is usually located at about 1 / 3 of both ends of the slide.

[0023] In some embodiments of the present invention, the protective agent includes at least one selected from a carbohydrate protective agent and / or a polyhydroxy compound protective agent. The inventors have verified that the same protective effect can be obtained by covering the sample spot with a plurality of single carbohydrate or polyhydroxy compound protective agents or a mixture of at least two of them.

[0024] In some specific embodiments of the present invention, the carbohydrate protective agent is selected from at least one of glucose, aD-pyranose mannoside, sucrose, lactose, trehalose, cellobiose, mannose, maltose, inositol, soft sugar, inulin, anhydroglucose, maltodextrin, maltopolysaccharide, sucrose octasulfate, heparin, and 2-hydroxypropyl-B cyclodextrin.

[0025] In some specific embodiments of the present invention, the polyhydroxy compound protective agent is selected from at least one of glycerol, mannitol, sorbitol, inositol, thiol and polyethylene glycol.

[0026] The second aspect of the present invention provides a method for preparing a pathological sample slide for long-term storage according to the first aspect of the present invention, comprising the following steps:

[0027] obtaining a slide including the sample spot;

[0028] The sample spot is evenly covered with a layer of the protective agent to obtain the pathological sample slide.

[0029] As described above, the present invention is not limited to a specific implementation method of uniformly covering the sample spot with a layer of the protective agent, and those skilled in the art may implement it in any manner as long as the order of forming the sample spot first and then covering it with the protective agent is ensured.

[0030] In the present invention, as an example, a specific implementation scheme of uniformly covering a layer of the protective agent is provided, that is, spraying a first long-term protective liquid containing the protective agent onto the sample spot and drying the solution.

[0031] In the present invention, as an example, another specific implementation scheme for uniformly covering a layer of the protective agent is provided, that is, a second long-term protective liquid containing the protective agent is dripped onto the sample spot and dried, the solvent of the second long-term protective liquid is an aqueous solution of a volatile organic solvent, and the mass volume fraction (m / v) or volume fraction (v / v) of the protective agent in the second long-term protective liquid is 2~10%.

[0032] Among them, the selection of the volatile organic solvent needs to comprehensively consider its surface tension and volatility, so that the second long-term protective solution can form full coverage after being dripped onto the sample spot, so that the protective agent is evenly covered on the sample spot; and the volatile organic solvent should be able to evaporate as quickly as possible to avoid affecting the distribution or performance of cells in the sample spot. In some embodiments of the present invention, the volatile organic solvent is selected from at least one of ethanol, isopropanol, butanol, ethyl acetate and butyl acetate. In some specific embodiments of the present invention, the volatile organic solvent is ethanol. In some preferred embodiments of the present invention, the solvent of the second long-term protective solution is an ethanol aqueous solution with a volume fraction of 20-50%.

[0033] It is necessary to reiterate that the main purpose of using volatile organic solvents in the present invention is to make the protective agent evenly and completely cover the sample spot. Those skilled in the art may also use spraying and other forms to perform the operation. In this case, there is no limitation on the solvent, such as water.

[0034] Furthermore, the first long-term protection solution or the second long-term protection solution further comprises 0.5-2% by weight volume fraction of a protein protective agent and 0.0005-1% by weight volume fraction of a surfactant.

[0035] In some specific embodiments of the present invention, the protein protective agent is selected from at least one of BSA, skimmed milk powder, HSA, OVA, and fetal bovine serum.

[0036] In some specific embodiments of the present invention, the surfactant is selected from at least one of Tween-20, Tween-80, Triton-100, Span20, sucrose fatty acid ester, hydroxypropyl-β-cyclodextrin, sodium lauryl sulfate and fatty alcohol polyoxyethylene ether.

[0037] In some embodiments of the present invention, a protein protective agent is used in combination with a surfactant, and the type of the surfactant can be arbitrarily selected.

[0038] In some embodiments of the present invention, in order to further extend the storage time, the first long-term protection solution or the second long-term protection solution may also include a preservative or the like.

[0039] In the present invention, the glass slide refers to a glass slide commonly used in the art, but those skilled in the art may also use other planar supports to replace the glass slide.

[0040] The third aspect of the present invention provides a pathology detection kit, comprising the pathology sample slide described in the first aspect of the present invention. The pathology detection kit includes an immunohistochemistry detection kit, an immunofluorescence detection kit, and the like.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The method of the present invention is used to prepare a pathological sample slide. A slide containing a sample spot prepared from a pathological sample is used as a raw material. A long-term protective liquid containing a protective agent is dripped onto the sample spot. After drying, a protective agent film is formed to isolate the slide from air oxidation, thereby extending the shelf life of the pathological sample slide. When the slide is tested again, the protective agent can be easily removed by rinsing with water without affecting subsequent testing.

[0043] After the pathological sample slide of the present invention is stored for a long time under refrigerated conditions (2-8° C.), the dispersion of cells or tissue fragments is still good and the staining performance is not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The figures show the covering effects of different treatment methods on cells on quality control points in Example 2 of the present invention, A: control group, liquid cells were added and dried without adding any protective solution; B: experimental group 1, liquid cells were added and dried and then long-term protective solution 1 was added; C: experimental group 2, liquid cells were added and dried and then long-term protective solution 2 was added; D: experimental group 3, liquid cells were added and dried and then 25% v / v ethanol was added; E: experimental group 4, protective agent was added to the liquid cells in advance and then added to the glass slide and dried without adding any protective solution.

[0045] Figure 2The effects of different treatment methods on the cell staining performance at the quality control point in Example 2 of the present invention are shown, A: control group, liquid cells were added and dried without adding any protective solution; B: experimental group 1, liquid cells were added and dried, and long-term protective solution 1 was added; C: experimental group 2, liquid cells were added and dried, and long-term protective solution 2 was added; D: experimental group 3, liquid cells were added and dried, and 25% v / v ethanol was added; E: experimental group 4, protective agent was added to the liquid cells in advance, then added to the glass slide and dried, and no protective solution was added; F: staining results of qualified quality control products.

[0046] Figure 3 The figures show the covering effect of the long-term protective solution containing different protective agents on the cells on the quality control points in Example 3 of the present invention, A: long-term protective solution 1-1; B: long-term protective solution 1-2; C: long-term protective solution 1-3; D: long-term protective solution 1-4.

[0047] Figure 4 The effects of the long-term protection solutions containing different protective agents in Example 3 of the present invention on the cell staining performance at the quality control point are shown, A: long-term protection solution 1-1; B: long-term protection solution 1-2; C: long-term protection solution 1-3; D: long-term protection solution 1-4.

[0048] Figure 5 The effect of the long-term protection solution containing different protein + surfactant combinations in Example 4 of the present invention on the cell staining performance at the quality control point is shown, A: control; B: long-term protection solution 2-1; C: long-term protection solution 2-2; D: long-term protection solution 2-3; E: long-term protection solution 2-4; F: long-term protection solution 2-5; G: long-term protection solution 2-6; H: long-term protection solution 2-7; I: long-term protection solution 2-8.

[0049] Figure 6 The effects of different long-term protection solutions on TCT staining performance in Example 5 of the present invention are shown, A: control; B: long-term protection solution 1; C: long-term protection solution 2-1.

[0050] Figure 7 The effects of different storage methods on the staining performance of cell quality control points in Example 6 of the present invention are shown: A: conventional liquid cell group stored for 3 months; B: liquid cell group with added protective agent stored for 3 months; C: quality control slide group stored for 3 months; D: liquid cell group with added protective agent stored for 6 months; E: quality control slide group stored for 6 months.

[0051] Figure 8The effects of different storage methods on the staining performance of cell quality control points in Example 7 of the present invention are shown, A: staining of tissue sections immediately after dewaxing; B: staining of tissue sections after being dewaxed and stored for 1 month without adding long-term protective solution; C: staining of tissue sections after being dewaxed and covered with long-term protective solution and stored for 1 month; D: staining of undewaxed tissue sections after being stored for 1 month; E: staining of tissue sections after being dewaxed and covered with long-term protective solution and stored for 3 months; F: staining of undewaxed tissue sections after being stored for 3 months. DETAILED DESCRIPTION

[0052] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0053] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally regarded as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0054] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0055] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0056] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0058] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0059] In the following examples, the immunohistochemistry method was performed using the LYNX480 fully automatic immunohistochemistry staining machine of Toulin (Hangzhou) Biopharmaceutical Co., Ltd. The target primary antibody, secondary antibody, auxiliary reagents, etc. used in conjunction were purchased from the commercial kit of Toulin (Hangzhou) Biopharmaceutical Co., Ltd. The experiment adopted the conventional immunohistochemistry experimental process and was performed according to the instructions of the kit.

[0060] The preparation method of liquid cell quality control is as follows: place a thin layer of wax-embedded cell sample slices into a centrifuge tube; pour about 25 mL of dewaxing solution into the centrifuge tube and oscillate to dissolve the paraffin in the slices; centrifuge the mixture at 2500 rpm for 5 minutes, pour off the dewaxing solution, add 25 mL of isopropanol, and then oscillate the cell pellet and ethanol; centrifuge the mixture at 2500 rpm for 5 minutes, pour off the isopropanol, and then add about 1 mL of anhydrous ethanol to the centrifuge tube and blow evenly with a pipette; put the mixture into a 1.5 mL centrifuge tube, centrifuge at 2500 rpm for 5 minutes, remove the ethanol with a pipette, add 25% v / v ethanol, the added volume of 25% v / v ethanol is 30 times the volume of the slice, and oscillate to form a cell suspension, which is the liquid cell quality control.

[0061] The spotting and drying of liquid cell quality control products were performed using the fully automatic slide processing system Plus60 of Tuling (Hangzhou) Biotechnology Co., Ltd. (the sample volume was set to 2μL, with a heating plate drying function).

[0062] For other experimental methods, unless otherwise specified, they are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0063] Example 1 Exploration of long-term storage of liquid cell quality control products

[0064] First, the inventors tried to drip the liquid cell quality control product onto a glass slide and then preserve it using a resin sealant. However, when testing was needed, they found that the resin sealant was difficult to remove and could not be used as a quality control product to complete the testing of the cell sample.

[0065] Next, the inventors tried to drip the liquid cell quality control onto the glass slide and then coat it with a layer of wax for preservation, but found that the wax removal was not effective because the amount of coating was difficult to control. In addition, the liquid cells would be damaged if they were exposed to 60°C wax liquid again, which would eventually lead to poor staining results or even no staining.

[0066] Example 2 Selection of base solvent for preparing long-term protective solution for liquid cell quality control slides

[0067] Based on the exploration in Example 1, the inventor tried to drip the liquid cell quality control product on the glass slide, then drip the protective liquid and dry it for long-term storage. At this time, the inventor believes that the solvent is a very critical component, so the solvent is selected first. The inventor used the liquid cell quality control product CD20 (Ramos cells) as the test object and performed the following treatments respectively:

[0068] Control group: Take 2 µL of the mixed liquid cell quality control product CD20 suspension and drop it on the glass slide. After drying, it forms a quality control point (about 3-5 mm). No protective solution is added.

[0069] Experimental group 1: The liquid cell quality control product CD20 was placed on the glass slide to form a quality control spot in the same way as the control group, and about 8 μL of long-term protection solution 1 (5% m / v glucose, 25% v / v ethanol solvent) was added and then dried;

[0070] Experimental Group 2: The liquid cell quality control product CD20 was placed on the glass slide to form a quality control spot in the same manner as the control group, and about 8 μL of long-term protection solution 2 (5% m / v glucose, purified water solvent) was added and then dried;

[0071] Experimental group 3: The liquid cell quality control product CD20 was placed on the glass slide to form a quality control spot in the same way as the control group, and about 8 μL of 25% v / v ethanol was added and then dried;

[0072] Experimental Group 4: 5% m / v glucose was added to the liquid cell quality control product in advance, and then the liquid cell quality control product CD20 was formed on the glass slide to form a quality control point in the same way as the control group, and no protective solution was added.

[0073] The protective coverage effect of the protective liquid on the quality control point is as follows Figure 1 As shown. Figure 1 As shown in Figure A, the control group did not receive any treatment, so there was no coverage effect. Figure 1 From B, we can see that after adding a drop of long-term protection solution 1 to the experimental group, the quality control point is evenly and completely covered; Figure 1 From C, we can see that after adding long-term protection solution 2 to the experimental group 2, the quality control points are not covered evenly and completely. Figure 1 As shown in Figure D, the addition of 25% v / v ethanol alone to the third drop of the experimental group also had no significant covering effect on the quality control point; Figure 1 As shown in Figure E, in Experimental Group 4, a single sugar component (5% m / v glucose) was added to the liquid cell quality control product before it was dropped onto the glass slide. There was also no obvious covering effect, and local crystallization was found on the quality control point under a microscope.

[0074] The detection target corresponding to the liquid cell quality control product CD20 is CD20. After the quality control slides prepared by the above method were placed at 4°C for 3 months, the performance of the target at each quality control point was verified by immunohistochemical staining. The results are as follows Figure 2 shown.

[0075] Depend on Figure 2 As shown in Figure A, after the quality control slides were placed at 4°C for 3 months, the quality control of the control group basically showed negative staining. Figure 2 As shown in Figure D, the staining of the experimental group with three drops of 25% v / v ethanol was similar, that is, a negative result. Figure 2 As shown in Figure B, the staining performance of the quality control point covered by the long-term protection solution 1 in the experimental group is comparable to that of the qualified quality control product (such as Figure 2 F) is consistent with strong staining and high positive rate; Figure 2 As shown in Figure C, the staining intensity and positive rate of the quality control point covered by the long-term protection solution 2 in the experimental group 2 were lower than those of the qualified quality control products, indicating that the long-term protection solution 2 did not have a protective effect on the quality control products during long-term storage at 4°C, and that cell debris was prone to diffusion during the experiment, which would contaminate adjacent samples and affect the interpretation of the subsequent results. Figure 2 As shown in Figure E, the staining intensity and positive rate of the quality control points in experimental group 4, where a single sugar component was pre-added, were weaker than those in experimental group 2 with protective solution, which obviously did not meet the requirements.

[0076] From the comparison of the results between the control group and experimental groups 1, 2 and 3, it can be seen that without any protection, the quality control points on the quality control slides are difficult to be preserved for a long time at 4°C. Furthermore, by comparing experimental groups 1 and 2, it was found that the solvent of the long-term protection solution has a greater impact on the protection effect. Using 25% v / v ethanol as a solvent can cover the sample spots more evenly and completely than using water as a solvent, and thus the protection effect is better. However, if only 25% v / v ethanol is added (experimental group 3), there is no protective effect, indicating that the protective agent is also crucial. Furthermore, by comparing experimental groups 1 and 4, it can be seen that the order of adding the protective agent is also very critical to the stable preservation of the quality control product. If the protective agent is added in advance and then dried, the protection effect is not as good as adding the solvent containing the protective agent after the quality control point is formed.

[0077] When the long-term protection solution composition was further optimized in the subsequent examples, 25% v / v ethanol was used as the solvent.

[0078] Example 3 Selection of different protective agents in long-term protective solution

[0079] In order to further improve the performance of the long-term protection solution, the inventors tried to replace the glucose in the long-term protection solution 1 in Example 2 with different protection types to explore the protection effect on the quality control points, specifically:

[0080] As in Example 2, 2 μL of the mixed liquid cell quality control product CD20 suspension was dropped on a glass slide, and after drying, quality control points were formed. About 8 μL of one of the long-term protection solutions shown in Table 1 was dropped on different quality control points and then dried.

[0081] Table 1 Long-term protection solution of different polysaccharide protective agents

[0082]

[0083] The protection coverage effect of quality control points is as follows Figure 3 As shown. Figure 3 It can be seen that the long-term protective solution containing different protective agents has a uniform and complete coverage effect on the quality control points, which meets the requirements.

[0084] After different long-term protection solutions were added to the quality control points and dried, the quality control slides were placed at 4°C for 3 months, and the immunohistochemical staining performance of the quality control points was verified. The control was a control without adding any protection solution (the same as the control group in Example 2). The results are as follows: Figure 4 shown.

[0085] Depend on Figure 4 It can be seen that after the slides were placed at 4°C for 3 months after the long-term protection solution containing different types of protective agents was added and dried, the immunohistochemical staining performance of the quality control points was still good, indicating that different types of protective agents have a protective effect on the long-term placement of quality control slides.

[0086] Example 4 Further adding components to the long-term protection solution

[0087] In order to further improve the performance of the long-term protection solution, the inventors tried to add different components (protein protective agents and surfactants) to the long-term protection solution 1 in Example 2 to explore its protective effect on the quality control points, specifically:

[0088] As in Example 2, 2 μL of the mixed liquid cell quality control product CD20 suspension was dropped on a glass slide, and quality control points were formed after drying. About 8 μL of one of the long-term protection solutions shown in Table 2 was added to different quality control points and then dried. Long-term protection solution 1 was used as a control.

[0089] Table 2 Long-term protection solution with different components added

[0090]

[0091] After the liquid cell quality control products with different long-term protection solutions were placed at 4°C for 3 months, the immunohistochemical staining performance was verified, and the control without any protection solution (the same as the control group in Example 2) was used as the control. The results are as follows Figure 5 shown.

[0092] Depend on Figure 5 It can be seen that after adding long-term protective solution containing different protein protective agents and surfactants and placing it at 4°C for 6 months after drying, the immunohistochemical staining performance of the quality control points was better than that of the control. Among them, the staining performance of the quality control product with added skimmed milk powder protein protective agent was weaker, but still stronger than the control.

[0093] The above results show that different types of protein protective agents and surfactants have a further protective effect on the staining performance of the quality control film after long-term storage.

[0094] Example 5 Application in cervical tissue cell smear detection

[0095] In order to verify that the above long-term protection solution is also applicable to pathological tissue cell smears, such as cervical tissue cell smears (TCT), the inventors used the same operation as the liquid cell quality control to protect the tissue cell smears. Specifically, long-term protection solution 1, long-term protection solution 1-1~1-4 and long-term protection solution 2-1~2-8 were dripped onto the cell smears and dried, and then placed at 4°C for 6 months before performance testing.

[0096] TCT corresponds to the target p16 & Ki-67. After long-term placement, double-staining cytological detection method (reagents purchased from Tuling, primary antibody number I12622, secondary antibody number I20092) was used for staining verification.

[0097] The results showed that for tissue cell smears, the long-term protection solution suitable for liquid cell quality control products also has a protective effect on cell smears. After long-term storage, the staining performance of tissue cell smears dripped with long-term protection solution 1, long-term protection solution 1-1~1-4 and long-term protection solution 2-1~2-8 is significantly better than that of conventionally preserved cell smears, and the protection effect of long-term protection solution 2-1~2-8 is significantly better than that of long-term protection solution 1 and long-term protection solution 1-1~1-4. Among them, the staining results of the control (conventionally preserved cell smears) are as follows Figure 6 As shown in A; the staining results of the tissue cell smear with long-term protection solution 1 are as follows Figure 6 As shown in B; the staining results of the tissue cell smear with the long-term protection solution 2-1 added are as follows Figure 6 As shown in C. This indicates that the long-term protective solution also has a protective effect on tissue cell smears, which can prolong the effective use time, and the addition of protein protective agents and surfactants can further improve the protective effect.

[0098] Example 6 Verification of other liquid cell quality control products

[0099] In order to verify the preservation performance of the prepared quality control slides, the inventors used liquid cell quality control product CD3 (Molt-4 cells, 25% v / v ethanol as solvent) as the test object and performed the following operations:

[0100] Conventional liquid cell group: Place the liquid cell quality control product (Molt-4 cells) at 4°C for a long period of time (3 months or 6 months) and mix it. Take 2 µL of the mixed liquid cell quality control product (Molt-4 cells) and drop it on the glass slide. After drying, it forms a quality control point, and then perform immunohistochemical staining.

[0101] Liquid cell group with added protective agent: The liquid cell quality control product (Molt-4 cells) was centrifuged and re-dissolved with long-term protective solution 2-1 as the liquid cell quality control product. After being placed at 4°C for a long time (3 months or 6 months), it was mixed. 2µL of the mixed liquid cell quality control product (Molt-4 cells) was dropped on the glass slide. After drying, quality control spots were formed, and then immunohistochemical staining was performed.

[0102] Quality control slide group: Take 2µL of the mixed liquid cell quality control product (Molt-4 cells) and drop it on the slide. After drying, a quality control point is formed. 10μL of long-term protection solution 2-1 is added to the quality control point. After drying, place it at 4℃ for a long time (3 months or 6 months) and then perform immunohistochemical staining.

[0103] The target of the liquid cell quality control (Molt-4 cells) is CD3. After being stored at 4°C (normally stored at -20°C) for 3 and 6 months, the target was tested by immunohistochemical staining for performance verification. The results are as follows: Figure 7 shown.

[0104] Depend on Figure 7 It can be seen that after 3 months, the immunohistochemical staining intensity and positive rate of the quality control points of the conventional liquid cell group decreased significantly; the staining results of the quality control points of the liquid cell group with added protective agent and the quality control points of the quality control slide group met the requirements, but the immunohistochemical staining performance of the liquid cell group with added protective agent was slightly weaker than that of the quality control points of the quality control slide group. After continued storage for 6 months, the immunohistochemical staining intensity and positive rate of the quality control points of the liquid cell group with added protective agent decreased significantly, while the staining intensity of the quality control points of the quality control slide group still met the requirements.

[0105] The above results show that the storage time of quality control cells in long-term protective solution (in the form of liquid cell quality control products) can be extended to a certain extent, but the staining performance is obviously not as good as that of quality control slides.

[0106] Example 7 Application in detection of seminoma tissue sections

[0107] In order to verify that the above-mentioned long-term protection solution is also suitable for pathological tissue sections, such as seminoma tissue sections, the inventors dewaxed the sections in conventional immunohistochemistry experiments and attached them to glass slides. The tissue sections were protected using the same operation as the liquid cell quality control product. Specifically, long-term protection solution 2-1 was added dropwise to the dewaxed sections to completely cover them and dry them. A blank control was used without adding protection solution. The sections were placed at 4°C for 1 and 3 months before performance testing, and compared with the performance of undewaxed tissue sections stored for the same time.

[0108] The target site PLAP for seminoma was placed for a long time and then stained and verified using immunohistochemistry (reagents purchased from Tuling (Hangzhou) Biopharmaceutical Co., Ltd.). The results are as follows Figure 8 shown.

[0109] Depend on Figure 8 It can be seen that compared with the staining immediately after dewaxing (such as Figure 8 Middle A), staining of tissue sections became significantly weaker 1 month after dewaxing (e.g. Figure 8 B), while the sections covered with long-term protection solution 2-1 after dewaxing (such as Figure 8 C) and undewaxed sections ( Figure 8 D) and the staining intensity was comparable to that before storage ( Figure 8 There is no obvious difference in A). After 3 months of storage, the staining intensity of the sections covered with long-term protection solution 2-1 after dewaxing (such as Figure 8 E) and the staining intensity of undewaxed sections ( Figure 8 (F) There was no significant decrease, and the staining performance did not deteriorate, and it still met the detection requirements.

[0110] In summary, for tissue sections, the long-term protection solution 2-1 suitable for liquid cell quality control products is also effective in covering and protecting tissue sections. After the tissue sections are dewaxed, covered with the long-term protection solution 2-1 and dried, the long-term staining performance is significantly better than that of the tissue sections conventionally stored after dewaxing. This shows that the long-term preservation solution 2-1 also has a protective effect on tissue sections, which can extend the effective use time, and the addition of protein protective agents and surfactants can further extend the shelf life.

[0111] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A pathological sample slide for long-term storage, characterized in that: The pathological sample slide includes a dried sample spot prepared using a pathological sample, and the sample spot is evenly covered with a layer of protective agent, wherein the protective agent includes a carbohydrate protective agent and / or a polyhydroxy compound protective agent, and the evenly covering with a layer of the protective agent is performed by dripping a second long-term protective solution containing the protective agent onto the sample spot and drying the solution, wherein the solvent of the second long-term protective solution is an aqueous solution of a volatile organic solvent, and the mass volume fraction of the protective agent in the second long-term protective solution is 2-10%.

2. A pathological sample slide for long-term storage according to claim 1, characterized in that: The sample plaque is prepared by one of the following methods: (1) preparing the histological sample into a tissue paraffin section or a tissue frozen section, attaching it to a glass slide, and drying it to obtain the sample spot; (2) preparing the histological sample into a tissue suspension, dropping it onto a glass slide, and drying it to obtain the sample spot; (3) preparing the sample spot on a glass slide using a Pap smear method or a liquid-based thin-layer cytology preparation method; (4) preparing a cytological sample or a cell suspension containing target cells into a cell paraffin section, attaching it to a glass slide, and drying it to obtain the sample spot; (5) Dropping a cell suspension containing target cells and / or cell fragments onto a glass slide and drying the suspension to obtain the sample spot.

3. A pathological sample slide for long-term storage according to claim 1 or 2, characterized in that: The carbohydrate protective agent includes at least one of glucose, aD-pyranose, sucrose, lactose, trehalose, cellobiose, mannose, maltose, inositol, soft sugar, inulin, anhydroglucose, maltodextrin, maltopolysaccharide, sucrose octasulfate, heparin and 2-hydroxypropyl-B cyclodextrin; the polyhydroxy compound protective agent includes at least one of glycerol, mannitol, sorbitol, inositol, thiol and polyethylene glycol.

4. A method for preparing a pathological sample slide for long-term storage according to any one of claims 1 to 3, characterized in that: The following steps are involved: obtaining a slide including the sample spot; The pathological sample slide is obtained by uniformly covering the sample spot with a layer of the protective agent, wherein the uniform covering includes dripping a second long-term protective solution containing the protective agent onto the sample spot and drying the solution, wherein the solvent of the second long-term protective solution is an aqueous solution of a volatile organic solvent, and the mass volume fraction of the protective agent in the second long-term protective solution is 2-10%.

5. The preparation method according to claim 4, characterized in that: The volatile organic solvent is selected from at least one of ethanol, isopropanol, butanol, ethyl acetate and butyl acetate.

6. The preparation method according to claim 4 or 5, characterized in that: The second long-term protection solution also includes a protein protective agent with a mass volume fraction of 0.5-2% and a surfactant with a mass volume fraction of 0.0005-1%.

7. The preparation method according to claim 6, characterized in that: The protein protective agent is selected from at least one of BSA, skimmed milk powder, HSA, OVA, and fetal bovine serum; the surfactant is selected from at least one of Tween-20, Tween-80, Triton-100, Span20, sucrose fatty acid ester, hydroxypropyl-β-cyclodextrin, sodium dodecyl sulfate, and fatty alcohol polyoxyethylene ether.

8. A pathology detection kit, characterized in that: The invention comprises the pathological sample slide according to any one of claims 1 to 3.

Images