Preparation method of easily-identified cytoplasm control point wax block

Abstract

The application discloses a preparation method of an easily-recognized cytoplasm control point wax block. The application provides a method for preparing an easily-recognized cytoplasm control point wax block. The method is used for obtaining a cell column by sampling a donor cell wax block, placing the cell column in colored receptor paraffin which is mixed by dye and melted paraffin, and further preparing the cell column into an identifiable quality control wax block containing a quality control point. In the application, the applicant proves that the performance of the wax block is different under different volume ratios of dye and paraffin, and the most suitable ratio of the two is screened. The method provided by the application not only facilitates the recognition of the cytoplasm control point, but also does not interfere with the IHC staining result in the later stage, and has a wide application prospect in clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical testing technology, and in particular to a method for preparing a wax block with easily identifiable cell quality control points. Background Technology

[0002] Immunohistochemical staining, or immunohistochemical assay for short, involves using a specific primary antibody that binds specifically to the antigen in the tissue sample. A universal secondary antibody then binds to the primary antibody, and a chromogenic molecule attached to the secondary antibody develops the color. The immunohistochemical assay (antigen-antibody reaction) is a lengthy and complex process, using a wide variety of reagents. It is also susceptible to changes caused by numerous factors (including temperature, humidity, concentration, batch-to-batch variation, and human error), which can easily lead to inaccurate results. Therefore, using quality control samples as negative and positive controls is an indispensable part of the assay to ensure accuracy.

[0003] Because of individual differences and unstable sources of diseased tissues in the human body, cytology control blocks have become an important quality control material. However, commercially available cytology control blocks are often white and semi-transparent, making it difficult to observe cytology control points and hindering clinical application.

[0004] Therefore, it is crucial to provide a paraffin block preparation technique that allows for easy identification of cell quality control points. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for preparing paraffin blocks that is easy to identify cell quality control points, while not generating background interference or affecting IHC staining results.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a method for preparing easily identifiable cell quality control point wax blocks.

[0007] Furthermore, the preparation method includes the following steps: The dye and molten paraffin are mixed evenly and then cooled and solidified to obtain the coloring acceptor paraffin. The recipient paraffin is perforated using a perforation device to form N cavities, where N≥1; Cell columns are obtained by using a perforation device to extract donor cell wax blocks. The cell columns are placed in the cavity and heated to fuse the cell columns with the staining receptor paraffin to obtain a wax block with easily identifiable cell quality control points.

[0008] Furthermore, the heating refers to the local heating of the interface between the cell column and the cavity, so that the paraffin at the interface melts slightly and then solidifies again to form a cell wax block with an integrated structure and easily identifiable cell quality control points.

[0009] Furthermore, the temperature of the local heating is 55°C to 70°C, preferably 65°C; the heating time is 5 to 10 minutes.

[0010] In this invention, the applicant, through extensive and in-depth research, provides a method for preparing paraffin blocks with easily identifiable cell quality control points. This method stains the paraffin with color, leaving the cell quality control points unstained, thus achieving easy identification of the cell quality control points without interfering with subsequent IHC staining.

[0011] In this invention, the cell column refers to a solid cell aggregate with a certain volume and shape formed by taking materials from donor cell wax blocks.

[0012] In this invention, the cell quality control point refers to the cellular region exposed for microscopic observation and interpretation after slicing the cross-section of the cell quality control paraffin block. This region corresponds to the cross-section of the cell column embedded inside the paraffin block.

[0013] In this invention, the cavity is a hole obtained by drilling holes in the pigmented receptor paraffin to accommodate the cell column.

[0014] Furthermore, when N>1, the N cavities are arranged in a regular matrix array or a concentric circle array on the acceptor wax block.

[0015] Furthermore, the number of cavities is the same as the number of cell columns obtained from the sampling.

[0016] Furthermore, the volume ratio of the dye to the melted paraffin is 1:2000 to 1:40.

[0017] Furthermore, the volume ratio of the dye to the melted paraffin is 1:2000 to 1:100.

[0018] Furthermore, the volume ratio of the dye to the melted paraffin is 1:2000.

[0019] Furthermore, the dye is an oil-soluble liquid candle dye.

[0020] In this invention, the oil-soluble candle liquid dye is a commercially available product commonly used in the field and can be obtained through commercial purchase.

[0021] Furthermore, the method for preparing the donor cell paraffin block includes the steps of target cell collection, fixation, dehydration, clearing, paraffin impregnation, and embedding.

[0022] In some implementations, the target cells refer to target protein-negative and / or target protein-positive cells.

[0023] In this invention, the target protein is not limited and includes any one of known diagnostic biomarkers, therapeutic target proteins, and disease-related mutant proteins. It encompasses any one or more combinations of tumor-associated antigens, hormone receptors, cell proliferation markers, cell cycle regulatory proteins, tyrosine kinase receptors, cell lineage-specific markers, transcription factors, and infectious disease-related antigens. In a specific embodiment of this invention, paraffin blocks with easily identifiable cellular quality control points are prepared using P40-positive and negative target protein cells as examples.

[0024] Those skilled in the art can determine the number of cells collected in this invention based on the specific intended use. Preferably, 1 10×10 7 (1×10 7 2×10 7 3×10 7 4×10 7 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 10×10 7 The cell number is appropriate. In a specific embodiment of the invention, the cell number is 9 × 10⁻⁶. 7 .

[0025] Furthermore, the fixation step includes fixation using a fixative and adding cell gel to the fixed cells.

[0026] Furthermore, the fixatives include, but are not limited to, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, and FAA fixative.

[0027] Furthermore, the fixative is selected from 10% neutral formalin.

[0028] Furthermore, the fixed time is 24~48 hours.

[0029] Furthermore, the cell gel is a polysaccharide.

[0030] Furthermore, the cell gel is agarose.

[0031] Furthermore, the dehydration, clearing, and paraffin impregnation steps include sequentially treating the prepared cell blocks with the following: 70% ethanol for 2 hours, 85% ethanol for 2 hours, 95% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol for 1 hour, anhydrous ethanol for 1 hour, anhydrous ethanol in a 1:1 ratio for 30 minutes, xylene for 30 minutes, xylene for 30 minutes, paraffin at high temperature for 1 hour, paraffin at high temperature for 1 hour, and paraffin at high temperature for 1 hour. The prepared cell wax blocks are used as donors, and by inserting colored paraffin block acceptors, they are further integrated into identifiable quality control wax blocks.

[0032] Furthermore, the cell column includes one or more of the target protein negative control cell column and the positive control cell column.

[0033] Furthermore, the target protein negative control cell column and the positive control cell column were derived from different donor cell blocks.

[0034] Furthermore, the perforation device is a tissue punch or a tissue chip fabrication instrument.

[0035] A second aspect of the present invention provides a cell wax block with easily identifiable cell quality control points.

[0036] Furthermore, the cell wax block is prepared by the method described in the first aspect of the present invention.

[0037] A third aspect of the present invention provides a cell slice.

[0038] Furthermore, the cell slices are obtained from the cell wax block slices described in the second aspect of the present invention.

[0039] Furthermore, the thickness of the cell slices is 3-5 μm.

[0040] The fourth aspect of the present invention provides a product.

[0041] Furthermore, the product includes dyes and paraffin.

[0042] Furthermore, the dye is an oil-soluble liquid dye.

[0043] Furthermore, the dye is a liquid-dye-for-candle dye.

[0044] Furthermore, the paraffin wax is in solid, granular, or molten form.

[0045] In some embodiments, the product comprises a component consisting of a premixed mixture of melted paraffin and a dye, wherein the dye is uniformly dispersed in the paraffin.

[0046] In some implementations, the paraffin and dye are packaged separately in a physically separated state and mixed during use.

[0047] The fifth aspect of the present invention provides a cell wax block embedding mold.

[0048] Furthermore, the embedding mold includes a mold body, and the cavity of the mold body is pre-filled with the colored paraffin wax described in the first aspect of the present invention.

[0049] The sixth aspect of the present invention provides any of the following applications: 1) The application of the cell paraffin blocks described in the second aspect of the present invention or the cell sections described in the third aspect of the present invention in internal quality control and external quality assessment of immunohistochemical detection; 2) The use of the cell blocks described in the second aspect of the present invention or the cell sections described in the third aspect of the present invention in immunohistochemical detection or in the preparation of products for immunohistochemical detection; 3) The application of the product described in the fourth aspect of the present invention or the embedding mold described in the fifth aspect of the present invention in the preparation of cell wax blocks.

[0050] The seventh aspect of the present invention provides a system for preparing easily identifiable cell quality control point wax blocks.

[0051] Furthermore, the system includes: Coloring receptor paraffin preparation module: used to mix the dye and molten paraffin evenly, and obtain coloring receptor paraffin after cooling and solidification; Preparation of cavity modules: used to drill holes in the recipient paraffin using a drilling device to form N cavities, where N≥1; Cell column extraction module: used to extract cell columns from donor cell wax blocks using a perforation device; Cell column implantation module: used to place the cell column in the cavity; Fusion module: Heating fuses the cell column with the staining receptor paraffin to obtain a wax block with easily identifiable cell quality control points.

[0052] Furthermore, the system also includes a donor cell paraffin block preparation module; the donor cell paraffin block preparation module includes a target cell collection unit, a fixation unit, a dehydration and transparent paraffin impregnation unit, and an embedding unit.

[0053] Furthermore, the cell collection unit is used to collect cell deposits.

[0054] Furthermore, the fixation unit is used to fix cells with a fixative and to add cell gel to the fixed cells.

[0055] Furthermore, the dehydrated transparent wax-impregnation unit is used to sequentially treat the prepared cell clumps as follows: 70% ethanol treatment for 2 h, 85% ethanol treatment for 2 h, 95% ethanol treatment for 1 h, 95% ethanol treatment for 1 h, anhydrous ethanol treatment for 1 h, anhydrous ethanol treatment for 1 h, anhydrous ethanol:xylene treatment in a 1:1 ratio for 30 min, xylene treatment for 30 min, xylene treatment for 30 min, paraffin high-temperature treatment for 1 h, paraffin high-temperature treatment for 1 h, paraffin high-temperature treatment for 1 h.

[0056] In some implementations, the system may be considered as being implemented in the form of a computer-readable storage medium configured with computer programs, wherein such a storage medium enables a computer to operate in a particular and predefined manner to perform the functions of this application.

[0057] The eighth aspect of the present invention provides a computer-readable storage medium.

[0058] Furthermore, the computer-readable storage medium includes a computer program that, when executed, implements the functions of the modules in the system described in the seventh aspect of the present invention.

[0059] Advantages and beneficial effects of the present invention: This invention provides a method for preparing easily identifiable cell quality control point paraffin blocks. The method involves obtaining cell columns from donor cell paraffin blocks, placing the cell columns in a staining acceptor paraffin mixture of dye and melted paraffin, and further preparing an identifiable quality control paraffin block containing quality control points. Furthermore, the applicant has demonstrated that the performance of the paraffin blocks varies with different dye-to-paraffin volume ratios and has screened out the optimal ratio. The method provided by this invention not only facilitates the identification of cell quality control points but also does not interfere with subsequent IHC staining, showing broad application prospects in clinical diagnosis. Attached Figure Description

[0060] Figure 1 Images showing the appearance of different concentrations of dyes after paraffin embedding; Figure 2 This is a slide image after paraffin sections have been cut and unfolded. Figure 3 IHC results of paraffin sections with different concentrations of dye. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0062] Example I. Experimental Methods This invention uses P40 positive and negative cells as an example to prepare cell paraffin blocks with easily identifiable cell quality control points. The specific steps are as follows: 1. Preparation of donor wax blocks (1) Collect 9×10 7 P40 negative and P40 positive cell pellets were fixed with 1 ml of 10% neutral formalin fixative for 24-48 h. (2) Centrifuge to remove fixative, add 1 ml of 2.5% agarose cell gel to the fixed cells, and prepare cell clumps; (3) The prepared cell clumps were subjected to the following treatments in sequence: 70% ethanol for 2 h, 85% ethanol for 2 h, 95% ethanol for 1 h, 95% ethanol for 1 h, anhydrous ethanol for 1 h, anhydrous ethanol for 1 h, anhydrous ethanol for 30 min, anhydrous ethanol: xylene in a ratio of 1:1, xylene for 30 min, xylene for 30 min, paraffin high temperature treatment for 1 h, paraffin high temperature treatment for 1 h, paraffin high temperature treatment for 1 h; (4) Cell blocks were prepared by paraffin embedding and used as donors. Cell columns were obtained by extracting material from the donor paraffin blocks using a tissue punch with a diameter of 2 mm.

[0063] 2. Prepare cell paraffin blocks with easily identifiable cell quality control points. (1) After melting the paraffin at 65°C, add the dye (oil-soluble candle liquid dye (peach red), liquid-dye-for-candle, purchased from Taobao and Candle Handmade Shop, product specification 10mL / bottle). Mix the dye and paraffin at a ratio of 1:20000, 1:2000, 1:100, 1:40, 1:20 to make colored paraffin (colored paraffin). (Note: Do not heat the paraffin and dye together after mixing. The dye will deteriorate and clump after heating, and will not be able to mix evenly with the paraffin. It will exist in the wax block as an impurity and will not dye the paraffin into a uniform color.) (2) Prepare wax blocks using the colored paraffin prepared in step (1) above, and punch holes to form array cavities, which serve as the wax blocks for embedding cell columns; (3) Place the P40 negative and P40 positive cell columns into the corresponding array cavity of the pre-prepared stained receptor paraffin block, ensuring that the upper edge of the cell column is flush with the surface of the receptor paraffin block, and finally heat and fuse them to form an integrated structure of the cell column and the receptor paraffin block.

[0064] II. Experimental Results Images of the appearance of different proportions of colored paraffin embedded in the sample are shown below. Figure 1As shown, when the staining solution to paraffin ratio is 1:20000, the color difference between the cell spots and the paraffin is small, making it difficult to observe the cell spots. When the staining solution to paraffin ratio is 1:20, the paraffin color covering the cell spots will obscure some of the cell spot locations, and the slides are easily broken. Upon contact with water, the paraffin rapidly dissolves and shatters, causing the spots to separate and making it impossible to retrieve the slides for subsequent baking experiments. Figure 2 Therefore, a solution ratio of 1:2000 to 1:40 for staining and paraffin is preferred, as cell spots can be clearly observed within this concentration range.

[0065] Furthermore, the prepared cell paraffin sections were sectioned and stained with IHC, and the results were as follows: Figure 3 As shown, the paraffin blocks prepared using this method have no effect on the subsequent immunohistochemical results.

[0066] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a paraffin block with easily identifiable cell quality control points, characterized in that, The preparation method includes the following steps: The dye and molten paraffin are mixed evenly and then cooled and solidified to obtain the coloring acceptor paraffin. The recipient paraffin is perforated using a perforation device to form N cavities, where N≥1; Cell columns are obtained by using a perforation device to extract donor cell wax blocks. The cell columns are placed in the cavity and heated to fuse the cell columns with the staining receptor paraffin to obtain a wax block with easily identifiable cell quality control points.

2. The preparation method according to claim 1, characterized in that, The volume ratio of the dye to the melted paraffin is 1:2000 to 1:40; Preferably, the volume ratio of the dye to the melted paraffin is 1:2000 to 1:100; Preferably, the volume ratio of the dye to the melted paraffin is 1:2000; Preferably, the dye is an oil-soluble liquid candle dye.

3. The preparation method according to claim 1, characterized in that, The method for preparing the donor cell paraffin block includes the steps of target cell collection, fixation, dehydration, clearing, paraffin impregnation, and embedding. Preferably, the cell column includes one or more of the target protein negative control cell column and the positive control cell column; Preferably, the punching device is a tissue punch or a tissue chip fabrication instrument.

4. A cell paraffin block for easily identifying cell quality control points, characterized in that, The cell wax block is prepared by the method according to any one of claims 1-3.

5. A cell slice, characterized in that, The cell slices were obtained from the cell paraffin block slices as described in claim 4.

6. A product characterized in that, The products include dyes and paraffin wax; Preferably, the dye is an oil-soluble liquid dye; Preferably, the dye is a liquid-dye-for-candle dye; Preferably, the paraffin wax is solid, granular, or molten.

7. A cell wax block embedding mold, characterized in that, The embedding mold includes a mold body, and the cavity of the mold body is pre-filled with the coloring receptor paraffin as described in claim 1 or 2.

8. Any of the following applications: 1) The application of the cell paraffin block as described in claim 4 or the cell section as described in claim 5 in internal quality control and external quality assessment of immunohistochemical detection; 2) The use of the cell block of claim 4 or the cell slice of claim 5 in immunohistochemical detection or in the preparation of products for immunohistochemical detection; 3) The application of the product of claim 6 or the embedding mold of claim 7 in the preparation of cell wax blocks.

9. A system for preparing easily identifiable cell quality control point paraffin blocks, characterized in that, The system includes: Coloring receptor paraffin preparation module: used to mix the dye and molten paraffin evenly, and obtain coloring receptor paraffin after cooling and solidification; Preparation of cavity modules: used to drill holes in the recipient paraffin using a drilling device to form N cavities, where N≥1; Cell column extraction module: used to extract cell columns from donor cell wax blocks using a perforation device; Cell column implantation module: used to place the cell column in the cavity; Fusion module: Heating fuses cell columns with staining receptor paraffin to obtain a wax block with easily identifiable cell quality control points; Preferably, the system further includes a donor cell paraffin block preparation module; the donor cell paraffin block preparation module includes a target cell collection unit, a fixation unit, a dehydration and transparent paraffin impregnation unit, and an embedding unit.

10. A computer-readable storage medium, characterized in that, The computer-readable storage medium includes a computer program that, when executed, performs the functions of the modules in the system of claim 9.

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