Method for preparing FFPE quality control product by using cell line, reference product and kit

Abstract

The invention provides a method for preparing an FFPE quality control product by using a cell line, a reference product and a kit, and belongs to the technical field of medicine, clinical laboratory science and biologication.The method comprises the steps that the cell line is cultured by adopting a microcarrier; mixing different cell lines based on requirements; pouring the mixed cell microcarrier suspension into a straw with one sealed end, carrying out centrifugal treatment, removing the supernatant after the treatment is finished, then packaging the two ends of the straw by adopting semi-permeable membranes, carrying out formaldehyde solution fixation, dehydration and transparent treatment, removing the semi-permeable membranes, and carrying out wax dipping treatment; and separating the cell microcarrier subjected to wax dipping treatment from the straw, and then embedding the cell microcarrier with wax to form a paraffin block. The mutation frequency of the quality control product can keep good uniformity in the subsequent quality control process, and batch and large-scale production of the quality control product is achieved.

Description

Technical Field

[0001] This invention relates to the fields of medicine, clinical laboratory science, and biotechnology, and specifically to a method for preparing FFPE quality control products using cell lines, reference products, and kits. Background Technology

[0002] To ensure the accuracy of test results, medical testing laboratories need to select appropriate quality control materials when establishing a quality control system. Certified reference materials (CRMs) are typically produced by designated CRM manufacturers and are commercially available. Laboratories can prepare their own quality control samples (QCMs) for internal use. QCMs are sometimes simply referred to as quality control samples or quality control materials.

[0003] The uses of quality control materials include (but are not limited to): validation of laboratory-developed tests (LDTs), verification of commercial reagent kits, internal quality control (IQC), external quality assessment (EQA) or proficiency testing (PT), and verification of the comparability of test results. They can also be used in the development and analytical performance validation process of commercial reagent kits.

[0004] Clinical genetic testing is a special testing item in the field of clinical medical testing. The technology is developing rapidly, but there are still very few certified reference materials / standard samples (CRM) on the market, which cannot meet the quality control needs of different testing items. The technical level of laboratories that can prepare their own quality control samples (QCM) is still uneven, and the homogeneity and stability are difficult to guarantee.

[0005] Formalin-fixed paraffin-embedded (FFPE) tissue is an important sample type for clinical molecular pathology testing. FFPE quality control materials, according to their form, include: paraffin block quality control materials, paraffin section quality control materials, and paraffin roll quality control materials. FFPE quality control materials prepared from patient tissue samples are difficult to obtain in large quantities due to inter-tissue or intra-tissue heterogeneity, and are also restricted by ethical issues, preventing mass production and use. Currently, the commonly used quality control materials in molecular pathology laboratories are typically prepared from cultured cell lines, dehydrated, embedded, and then into paraffin blocks. However, the batch processing, homogeneity, and stability of this preparation process are difficult to control, limiting its widespread use. Most laboratories directly extract nucleic acids from cell lines, making it impossible to perform quality control on the extraction process itself.

[0006] Current technical bottlenecks in the preparation of FFPE quality control products from cell lines include: 1) steric hindrance effects of cells of different sizes leading to uneven mixing (uneven cell count in continuous sections and uneven cell density in single sheets); 2) insufficient cell density in the paraffin block (low and uneven nucleic acid yield from a single section). Due to the conventional technical deficiencies in 1) and 2), the uniformity of mutation frequency in subsequent quality control processes is poor, and the batch and large-scale production of quality control products still requires further research and solutions. Summary of the Invention

[0007] To address the above problems, this invention provides a method for preparing FFPE quality control products using cell lines, a reference product, and a kit.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing FFPE quality control products using cell lines, comprising: culturing cell lines using microcarriers; mixing different cell lines according to requirements; perfusing the mixed cell microcarrier suspension into a straw sealed at one end for centrifugation; removing the supernatant after centrifugation; then encapsulating both ends of the straw with a semi-permeable membrane; fixing, dehydrating, and clearing the straw with formaldehyde solution; removing the semi-permeable membrane and performing a wax impregnation process; separating the wax-impregnated cell microcarriers from the straw and then embedding them in paraffin wax to form paraffin blocks.

[0009] Furthermore, the microcarriers include one or more of the following: dextran matrix microcarriers, cellulose matrix microcarriers, gelatin matrix microcarriers, polystyrene matrix microcarriers, polyethylene matrix microcarriers, polygalacturonic acid matrix microcarriers, polypropylene matrix microcarriers, polyester fiber matrix microcarriers, chitosan matrix microcarriers, chitin matrix microcarriers, and alginate gel microcarriers.

[0010] Furthermore, the microcarrier is a dextran-based microcarrier.

[0011] Furthermore, the cell lines selected are all human-derived adherent cell lines, including NA12878, HEK293T, A549, and BEAS-2B.

[0012] Furthermore, the diameter of the straw is 0.3cm-2.0cm, and the length of the straw is 5cm-15cm.

[0013] Furthermore, before use, a gas mixed with particulate matter is introduced into one end of the straw for 30-60 minutes, followed by cleaning with an organic solvent. The flow rate of the gas is [missing information]. Where α is a correction factor and D is the diameter of the straw; the thermal conductivity of the particles is at least 1.5 times that of the straw, the size of the particles does not exceed 0.2 mm, and the D5 particle size of the particles does not exceed 10 mm. .

[0014] Furthermore, the wheat straw separation process involves heating the outer wall of the wheat straw while applying air pressure to one end.

[0015] Furthermore, the outer wall of the straw is heated to T±5℃, where T is the dropping point of the impregnated wax; the air pressure is 0.15-0.20 MPa.

[0016] The present invention also provides a paraffin-embedded reference prepared by the above method.

[0017] The present invention also provides a gene detection kit, the kit comprising the above-mentioned paraffin-embedded reference material.

[0018] The beneficial effects of the technical solutions provided by the embodiments of the present invention include: This invention utilizes microcarriers to culture cell lines, suitable for all adherent cell cultures. The microcarriers act as a cytoskeleton, resulting in a cell arrangement more similar to tissue, closely resembling the distribution characteristics of human tissue. This meets the requirement that quality control products should closely approximate clinical samples. Secondly, the raw material cell lines for the FFPE quality control products in this invention do not require digestion with trypsin or trypsin substitutes; the cell microcarrier suspension is directly mixed in a specific ratio. Due to the uniform size of the microcarriers, the distribution of cells growing per unit time is relatively uniform. Directly mixing two types of cell microcarriers makes it easier to homogenize the cells. The microcarriers, acting as a cytoskeleton, effectively overcome the spatial steric hindrance effect during the mixing of cells of different sizes, which can lead to uneven mixing. Finally, the microcarriers in this invention, acting as a cytoskeleton, fully protect the relative positions of cells during subsequent cell centrifugation, dehydration, clearing, and embedding processes, resulting in a more uniform cell distribution. Traditional cell centrifugation, dehydration, and clearing processes before embedding can lead to cell loss, and the use of a certain proportion of gel or similar substances during the pre-embedding cell shaping process dilutes the cell concentration per unit volume. This invention uses wheat tubes to centrifuge, dehydrate, clear, and plasticize the cell lines of FFPE quality control raw materials, reducing cell loss, eliminating the need to add gels or similar materials, and increasing the cell density per unit volume. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 HE-stained cell images from FFPE quality control sections prepared in Example 2 of this invention; Figure 2Here are HE-stained images of cells in a section of FFPE quality control material prepared in Comparative Example 1 of this invention; Figure 3 This is an image of cells stained with HE in a section of FFPE quality control material prepared in Comparative Example 2 of this invention. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0022] As used herein, the terms “comprising,” “including,” “having,” or any other variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus that includes listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Unless the context clearly specifies otherwise, the singular forms “an” and “the” include a plural of objects under discussion.

[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, this invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described in the embodiments of this invention, based on the knowledge of those skilled in the art and the description of this invention.

[0024] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, immunology, laboratory medicine, gene sequencing technology, bioinformatics technology, and related fields.

[0025] Unless otherwise specified, all reagents used in this invention are commercially available products and do not affect the inventive points not applied for.

[0026] This invention provides a method for preparing FFPE quality control products using cell lines, comprising: S1. Cell lines are cultured using microcarriers; S2. Mix different cell lines based on requirements; S3. The mixed cell microcarrier suspension is perfused into a wheat tube sealed at one end and centrifuged. After the treatment, the supernatant is removed. Then, the two ends of the wheat tube are sealed with a semi-permeable membrane. After fixation with formaldehyde solution, dehydration and transparency treatment are performed. The semi-permeable membrane is removed and the tube is then impregnated with wax. S4. The wax-treated cell microcarriers are separated from the wheat tubes and then embedded in wax to form paraffin blocks.

[0027] This invention utilizes microcarriers to culture cell lines, suitable for all adherent cell cultures. The microcarriers act as a cytoskeleton, resulting in a cell arrangement more similar to tissue, closely resembling the distribution characteristics of human tissue. This meets the requirement that quality control products should closely approximate clinical samples. Secondly, the raw material cell lines for the FFPE quality control products in this invention do not require digestion with trypsin or trypsin substitutes; the cell microcarrier suspension is directly mixed in a specific ratio. Due to the uniform size of the microcarriers, the distribution of cells growing per unit time is relatively uniform. Directly mixing two types of cell microcarriers makes it easier to homogenize the cells. The microcarriers, acting as a cytoskeleton, effectively overcome the spatial steric hindrance effect during the mixing of cells of different sizes, which can lead to uneven mixing. Finally, the microcarriers in this invention, acting as a cytoskeleton, fully protect the relative positions of cells during subsequent cell centrifugation, dehydration, clearing, and embedding processes, resulting in a more uniform cell distribution. Traditional cell centrifugation, dehydration, and clearing processes before embedding can lead to cell loss, and the use of a certain proportion of gel or similar substances during the pre-embedding cell shaping process dilutes the cell concentration per unit volume. This invention uses wheat tubes to centrifuge, dehydrate, clear, and plasticize the cell lines of FFPE quality control raw materials, reducing cell loss, eliminating the need to add gels or similar materials, and increasing the cell density per unit volume.

[0028] In step S1, the microcarriers include one or more of the following: dextran-based microcarriers, cellulose-based microcarriers, gelatin-based microcarriers, polystyrene-based microcarriers, polyethylene-based microcarriers, polygalacturonic acid-based microcarriers, polypropylene-based microcarriers, polyester fiber-based microcarriers, chitosan-based microcarriers, chitin-based microcarriers, and alginate gel microcarriers. Preferably, only one type of microcarrier is used, as this results in more uniform microcarrier size, which is more conducive to improving the uniformity of cell distribution when used as a cytoskeleton. All of the above microcarriers are commercially available products.

[0029] Preferably, the microcarrier is a dextran-based microcarrier. Such microcarriers, based on a cross-linked dextran framework, possess a very large specific surface area (>4000 cm²). 2 / g), the surface of the microcarrier is treated with a special process to cover part of the positively charged DEAE groups, which can meet the requirements of efficient cell growth.

[0030] Preferably, in this embodiment of the invention, 5 mg of dextran matrix microcarriers (e.g., commercially available Cytodex1 microcarriers) are used per 1 mL of culture medium. The cell lines used in this invention are in vitro adherent cell lines, and the culture medium is the culture medium recommended by the cell line preservation institution. Preferably, the cell lines used as raw materials for quality control products can be any human-derived adherent cell lines such as NA12878, HEK293T, A549, and BEAS-2B.

[0031] The cell confluence achieved by the microcarriers in this invention is 70%-90% (e.g., 5 mg of commercially available Cytodex1 microcarriers is equivalent to 30 cm³ of cells). 2 After assessing the surface area, cells can be passaged (5-10 times) or collected; preferably, cells are collected when the confluence of growth is around 85%, and can be passaged 5 times consecutively.

[0032] In this embodiment of the invention, to measure cell density, after the cell lines are cultured and thoroughly mixed, 5-10 mL of the microcarrier suspension is taken and digested with trypsin or a trypsin substitute. The digested cells are collected and counted, and the cell density per unit volume of culture medium for each cell line is calculated (greater than 1 × 10⁻⁶). 7 (cells / mL can be used for the next mixing step).

[0033] In step S2, the cell line microcarriers do not require digestion with trypsin or trypsin substitutes; they are directly mixed in the specified proportions. The mixing ratio is determined according to the target mutation frequency of the quality control sample and the density of two or more cell lines. This step is omitted if a single cell line quality control sample is being prepared.

[0034] After the microcarriers containing growing cells in this invention are mixed, they are centrifuged at 1000-3000g for at least 5 minutes, preferably 2000g for 5-15 minutes. The supernatant is discarded, and the cell microcarrier precipitate is retained. The cell microcarrier precipitate is resuspended in 1×PBS, centrifuged at 2000g, and the supernatant is discarded, retaining the cell microcarrier precipitate. This process is repeated twice. Preferably, 30-200mL of 1×PBS can be used, which is more than 20 times the volume of the microcarrier precipitate. The cell microcarrier precipitate (of one or more cell lines) after three washes with 1×PBS in this invention is resuspended in one volume of 1×PBS solution and then allowed to fix the cells.

[0035] In step S3, the mixed cell microcarrier suspension is infused into a wheat tube sealed at one end for centrifugation. The specific sealing method is not limited, the purpose being to ensure that the liquid does not flow out through the sealed end during subsequent centrifugation. Centrifugation is performed at a centrifugal force of 1500g-2500g for 8-15 minutes. After centrifugation, the supernatant is removed. It is important to note that this centrifugation process is crucial. Along the longitudinal direction of the wheat tube, during centrifugation, the density of cell microcarriers further away from the centrifuge's rotation center is higher, while the density of cell microcarriers closer to the centrifuge's rotation center is lower, resulting in a difference in the density of the prepared cell microcarriers along the longitudinal direction of the wheat tube. To solve the above problem, this application limits the centrifugal force and centrifugation time, and combines this with the size of the wheat tube, ensuring that the liquid can be effectively removed while the cell microcarriers can quickly return to a uniform distribution along the longitudinal direction of the wheat tube after centrifugation.

[0036] The "wheat tube" in this embodiment of the invention is a commercially available product, such as the frozen sperm tube commonly used in the field of reproduction, which is biocompatible.

[0037] Specifically, the diameter of the straw is 0.3cm-2.0cm, and the length of the straw is 5cm-15cm.

[0038] After centrifugation, the length of the wheat tube is cut based on the length of the microcarrier precipitate, and both ends are sealed with a semi-permeable membrane. Preferably, when cutting the wheat tube, the reserved length of the wheat tube is L + (1-3) cm, where L is the precipitate length, that is, the distance between the interface between the supernatant and the microcarrier precipitate and the sealed end after centrifugation.

[0039] The wheat tubes were then sealed at both ends with semi-permeable membranes, followed by fixation with formaldehyde solution, dehydration, and transparency treatment. The semi-permeable membranes were then removed, and the tubes underwent wax impregnation. Specifically, commercially available semi-permeable membranes were used, allowing formaldehyde melt, ethanol, and xylene to pass through while preventing the passage of cellular microcarriers.

[0040] In this invention, wheat tubes (containing cell microcarriers) encapsulated with a semi-permeable membrane are fixed in a 20%-40% neutral formaldehyde solution. After ethanol gradient dehydration and xylene clearing, the semi-permeable membrane is removed. This can be done by removing the semi-permeable membrane from one end or both ends of the wheat tube. The cells are then thoroughly immersed in paraffin wax. Specifically, neutral formalin is added to the wheat tubes, shaken well, and then fixed at 5℃-10℃ for 30-60 minutes. The tubes are then centrifuged at 1000g-2000g for 5-10 minutes, and the supernatant is discarded. The tubes are washed once with PBS, and then centrifuged at 1000g-2000g for 5-10 minutes. Following a gradient of ethanol concentration from low to high, centrifuge at 1000g-2000g for 5-10 minutes after each dehydration step, discarding the supernatant. Gradually replace anhydrous ethanol with xylene in a fume hood, as follows: first treat with 2 / 3 anhydrous ethanol and 1 / 3 xylene for 10-20 minutes, then aspirate the supernatant; next treat with 1 / 3 anhydrous ethanol and 2 / 3 xylene for 10-20 minutes, then aspirate the supernatant; finally, treat with pure xylene for 20-30 minutes, then aspirate the xylene before proceeding with the paraffin embedding step.

[0041] The wheat tubes (cell microcarriers after wax impregnation) in this invention are frozen at -15℃ to -25℃ for more than 1 hour. If embedding is not performed on the same day, they can be temporarily stored at -15℃ to -25℃ until embedding.

[0042] The wheat tubes in this invention utilize an automatic inkjet printing device to spray an internationally recognized one-dimensional barcode (code 128) onto the middle of the wheat tube. The ink used for spraying is resistant to low temperatures (-196°C). The code serves as a unique identifier for cell samples, enabling better traceability management for large-scale production processes.

[0043] The paraffin-impregnated cell microcarriers were separated from the wheat tubes and then embedded in paraffin to form paraffin blocks. The wheat tube separation process involved heating the outer wall of the wheat tubes while applying gas pressure to one end. Specifically, before use, a gas containing particulate matter was introduced into one end of the wheat tube for 30-60 minutes, followed by cleaning with an organic solvent. The gas flow rate was [not specified]. Where α is a correction factor and D is the diameter of the straw; the thermal conductivity of the particles is at least 1.5 times that of the straw, the size of the particles does not exceed 0.2 mm, and the D5 particle size of the particles does not exceed 10 mm. The purpose of the above treatment is to improve the appearance of the wheat tube walls. On one hand, the larger particles rub against the surface of the wheat tubes, creating a certain surface roughness on the inner surface. However, the grooves on the inner surface are small enough that paraffin wax, under surface tension, will not penetrate the grooves and increase the resistance to the separation of the wax-treated cell microcarriers from the wheat tubes. On the other hand, the D5 particle size of the particles does not exceed 10 mm. The small size allows for partial embedding into the inner surface of the straw, increasing thermal conductivity and rapidly melting the contact area between the wax-impregnated cell microcarriers and the inner wall of the straw. Because the straw is made of polymer materials, its thermal conductivity is low, and paraffin wax also has a low thermal conductivity. Therefore, during heating, the wax-impregnated cell microcarriers melt to a high degree. Under pressure, the ejected wax-impregnated cell microcarriers cannot be effectively shaped, resulting in poor uniformity of mutation frequency in subsequent quality control processes, hindering mass production. The small particles embedded in the straw wall rapidly melt the contact area between the wax-impregnated cell microcarriers and the inner wall of the straw, preventing heat transfer to the core of the cell microcarriers. This ensures good shaping of the ejected wax-impregnated cell microcarriers and prevents adhesion problems.

[0044] Specifically, α is the correction factor, with units of L / min·m. 2 / 3 Its value ranges from 450 to 550, where D is the diameter of the straw in meters.

[0045] In this embodiment of the invention, the particulate matter may be iron sand or fine sand.

[0046] The wheat straw separation process is as follows: while heating the outer wall of the wheat straw, air pressure is applied to one end of the wheat straw. The outer wall of the wheat straw is heated to T±5℃, where T is the dropping point of the impregnated wax; the air pressure is 0.15-0.20 MPa.

[0047] Specifically, in this invention, the wheat tubes frozen at -15℃ to -25℃ (cell microcarriers after wax impregnation) are placed in a 65℃ water bath / metal bath, and at the same time, the wax-impregnated cell microcarriers are quickly pushed out using a matching pneumatic device.

[0048] In this invention, the paraffin-impregnated cell microcarriers are placed on a tissue embedding stage and quickly embedded in paraffin to form paraffin blocks. Each paraffin block can embed approximately 5 mm of the paraffin-impregnated cell microcarriers, and longer paraffin-impregnated cell microcarriers can be segmented and embedded into paraffin blocks for quality control.

[0049] The quality control products of the wax blocks in this invention are cut into paraffin slices or rolls with a thickness of 4-7 μm.

[0050] This invention also provides a paraffin-embedded reference material prepared using the above method.

[0051] This invention also provides a gene detection kit, which includes the above-mentioned paraffin-embedded reference material.

[0052] To better illustrate the embodiments of the present invention, the present invention will be further described in detail below through specific examples.

[0053] Example 1 FFPE quality control product preparation The main instruments and equipment are shown in Table 1.

[0054] Table 1. Equipment used in the embodiments

[0055] The main reagents and consumables are shown in Table 2.

[0056] Table 2. Reagents and consumables used in the examples.

[0057] The raw materials for cell line quality control products are shown in Table 3.

[0058] Table 3 shows the quality control materials used in the examples.

[0059] Preparation method: The cells provided by the preservation center are in a growing state and are placed in T-25 plastic culture flasks, with a quantity of approximately 1~2 × 10⁶ cells / year. 6 Cells / bottle. Use trypsin to detach cells from the bottle wall, transfer cells to a 500mL individually packaged Virya triangular cell culture flask, add 20mL of the corresponding culture medium and an equal proportion of Cytodex1 microcarriers (1mL of culture medium uses 5mg of dextran matrix microcarriers).

[0060] Both cell types were examined under a microscope, digested, and passaged according to the passage method recommended by the China Center for Type Culture Collection. After five passages (when cell confluence reached 85% or higher), passage was stopped, and all cell microcarriers in the triangular cell culture flasks were collected directly without trypsin digestion. (For large-scale industrial production, a bioreactor can be used for culture.) Take 5-10 mL of cell microcarrier suspension, digest with trypsin or a trypsin substitute, and collect and count the digested cells. Calculate the cell density per unit volume of culture medium (per unit mass of microcarrier) for each cell line. Take a small amount of microcarriers for digestion and counting, which will be used for subsequent calculation of cell mixing ratios.

[0061] Mix the two cell line microcarriers (BEAS-2B and A549) at a 1:1 cell ratio. Centrifuge the mixed cell microcarriers at 2000g, discard the supernatant, and retain the cell microcarrier pellet. Resuspend the pellet in 20 volumes of 1×PBS, centrifuge at 2000g, discard the supernatant, and repeat this process twice. Resuspend the cell microcarriers in 1 volume of 1×PBS solution.

[0062] After mixing the cell microcarriers, they are filled into straws (CBS™ High Security straws) with one end blocked (polyamine fiber fabric) and a diameter of 0.3 cm. Before use, the straw is filled with gas mixed with particulate matter for 30 minutes, and then washed with absolute ethanol. The flow rate of the gas is , where α is a correction factor with a value of 500 L / min.m 2 / 3 . The diameter of the straw is 0.3 cm. Through calculation, the flow rate of the gas is 10 L / min.

[0063] The thermal conductivity of the particulate matter is 1.5 times that of the straw. The size of the particulate matter does not exceed 0.2 mm, and the D5 particle size of the particulate matter is 10 μm.

[0064] Centrifuge at 2000 g for 10 minutes. After centrifugation, discard the supernatant; cut the straw according to the length of the cell microcarrier precipitate, leaving 1 cm at each end, and encapsulate both ends with a semi-permeable membrane.

[0065] Place the straw containing the cell microcarriers sealed with a semi-permeable membrane into a 40% neutral formaldehyde solution for fixation. After ethanol gradient dehydration treatment and xylene clearing steps, the cells are fully infiltrated with wax. After spraying the code on the straw containing the infiltrated cell microcarriers, place it at -15°C to -25°C and freeze overnight.

[0066] Quickly place the straw containing the infiltrated cell microcarriers frozen at -15°C to -25°C into a 65°C water bath / metal bath (if using a metal bath, it should be a special slender inner diameter, matching the straw, and can be considered customized for mass production), and at the same time, use the pneumatic device supporting this invention to push out the infiltrated cell microcarriers.

[0067] Place the infiltrated cell microcarriers on a tissue embedding table and quickly embed them into paraffin blocks with paraffin. Each paraffin block can embed about 5 mm of the infiltrated cell microcarriers, and the long infiltrated cell microcarriers can be segmented and embedded into paraffin block quality control products.

[0068] Cut the paraffin block quality control products into paraffin section quality control products or paraffin strip quality control products with a thickness of 4 μm.

[0069] The comparison between the paraffin block quality control products prepared by the method of this invention and the commercially available paraffin block quality control products is shown in Table 4.

[0070] Table 4 Parameters of the commercially available products and the quality control products prepared by this invention

[0071] As shown in the table above, the paraffin block quality control prepared by the method of this invention has twice the cell content of commercially available paraffin block quality control; the slide quality control prepared by the method of this invention is thinner than commercially available slide quality control, which improves the efficiency of dewaxing, digestion, and decrosslinking of the quality control, and improves the quality of extracted nucleic acids; the paraffin block quality control prepared by the method of this invention can obtain nearly three times the number of slides compared to commercially available paraffin block quality control, and the DNA extraction yield meets the general molecular biology testing requirements of downstream applications. Therefore, the paraffin block quality control prepared by the method of this invention meets the needs of clinical applications, including but not limited to: internal quality control, external quality assessment, and comparability of test results.

[0072] Example 2 Unlike Example 1, in this embodiment of the invention, before use, one end of the straw is purged with gas containing particulate matter for 60 minutes, followed by cleaning with anhydrous ethanol. The flow rate of the gas is [missing information]. Where α is a correction factor, with a value of 500 L / min·m 2 / 3 The diameter of the straw is 1 cm, and the calculated gas flow rate is 23 L / min.

[0073] The thermal conductivity of the particles is 1.5 times that of the straw, the size of the particles does not exceed 0.2 mm, and the D5 particle size of the particles is 10 μm.

[0074] The paraffin-impregnated cell microcarriers were placed on a tissue embedding stage and quickly embedded in paraffin to form paraffin blocks. Several rolls of slides from the start, middle, and end positions of three paraffin blocks were randomly selected for quality control. One slide was extracted from each block, two blocks, or every three months to assess the concentration, purity, and fragment integrity of the nucleic acids, and to detect any mutations. Figure 1 As shown in Table 5, the cells in the paraffin block quality control slices prepared in the embodiments of the present invention are densely and evenly distributed. The extraction quality of the prepared quality control is shown in Table 6, and the mutation detection results of the quality control are shown in Table 6.

[0075] Table 5 Quality control sample extraction quality

[0076] Table 6 Results of Mutation Detection of Quality Control Products

[0077] Two rolls of paraffin section quality control were randomly selected from the beginning, middle, and end positions. For each paraffin section quality control, at least three positions were selected under the same light microscope parameters. The average cell count per field of view was calculated. The same procedure was repeated for paraffin section quality control at different positions to calculate the cell count for the entire section. The statistical results of calculating the cell count for the entire section based on the cell count per field of view are shown in Table 7.

[0078] Table 7. Statistical results of cell number at different locations

[0079] Example 3 Unlike Example 1, in this embodiment of the invention, before use, one end of the straw is purged with gas containing particulate matter for 30 minutes, followed by cleaning with anhydrous ethanol. The flow rate of the gas is [missing information]. Where α is a correction factor, with a value of 500 L / min·m 2 / 3 The diameter of the straw is 2cm, and the calculated gas flow rate is 37L / min.

[0080] The thermal conductivity of the particles is at least 1.5 times that of straw, the particle size does not exceed 0.2 mm, and the D5 particle size of the particles is 8 mm. .

[0081] The paraffin-impregnated cell microcarriers were placed on a tissue embedding stage and quickly embedded in paraffin to form paraffin blocks. The same characterization methods as in Example 2 were used, and the results were similar.

[0082] Comparative Example 1 Compared to Example 2, the inner surface of the wheat straw in this comparative example was not treated. The resulting paraffin-impregnated cell microcarriers exhibited high softening and, under the influence of surface tension, tended to aggregate, leading to changes in cell distribution. The prepared paraffin block quality control sections, after staining, showed... Figure 2 As shown, the cells are unevenly distributed and loosely arranged.

[0083] Comparative Example 2 Compared to Example 2, the gas flow rate in this comparative example was 28 L / min. The greater frictional force of the wheat tube on the cell microcarriers resulted in a longer ejection time, leading to a higher degree of softening of the paraffin-impregnated cell microcarriers. Under the influence of surface tension, these microcarriers tended to clump together, causing changes in cell distribution. The prepared paraffin block quality control sections, after staining, showed... Figure 3 As shown, the cells are unevenly distributed and loosely arranged.

[0084] Comparative Example 3 Compared to Example 2, the gas flow rate in this comparative example was 20 L / min. The particulate matter could not effectively heat the contact area between the wax-impregnated cell microcarriers and the surface of the wheat tube, resulting in a longer ejection time. This led to a higher degree of softening of the wax-impregnated cell microcarriers, which, under the influence of surface tension, tended to agglomerate, causing changes in cell distribution, resulting in uneven and loose cell distribution.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing FFPE quality control products using cell lines, characterized in that, include: Cell lines were cultured using microcarriers; Different cell lines are mixed based on requirements; The mixed cell microcarrier suspension was perfused into a wheat tube sealed at one end and centrifuged. After the treatment, the supernatant was removed. Then, the two ends of the wheat tube were sealed with a semi-permeable membrane. After fixation with formaldehyde solution, dehydration and transparency treatment were performed. The semi-permeable membrane was removed and the tube was then impregnated with wax. The wax-impregnated cell microcarriers were separated from the wheat tubes and then embedded in wax to form paraffin blocks.

2. The method according to claim 1, characterized in that, The microcarriers include one or more of the following: dextran matrix microcarriers, cellulose matrix microcarriers, gelatin matrix microcarriers, polystyrene matrix microcarriers, polyethylene matrix microcarriers, polygalacturonic acid matrix microcarriers, polypropylene matrix microcarriers, polyester fiber matrix microcarriers, chitosan matrix microcarriers, chitin matrix microcarriers, and alginate gel microcarriers.

3. The method according to claim 2, characterized in that, The microcarrier is a dextran-based microcarrier.

4. The method according to claim 1, characterized in that, The cell lines used were all native adherent cell lines, including NA12878, HEK293T, A549, and BEAS-2B.

5. The method according to claim 1, characterized in that, The diameter of the straw is 0.3cm-2.0cm, and the length of the straw is 5cm-15cm.

6. The method according to claim 5, characterized in that, Before use, a gas mixed with particulate matter is introduced into one end of the straw for 30-60 minutes, followed by cleaning with an organic solvent. The flow rate of the gas is [missing information]. , where α is the correction factor and D is the diameter of the straw; The thermal conductivity of the particles is at least 1.5 times that of the straw, the size of the particles does not exceed 0.2 mm, and the D5 particle size of the particles does not exceed 10 mm. .

7. The method according to claim 6, characterized in that, The wheat straw separation process involves heating the outer wall of the wheat straw while applying air pressure to one end.

8. The method according to claim 7, characterized in that, The outer wall of the straw is heated to T±5℃, where T is the dropping point of the impregnated wax; The air pressure is 0.15-0.20 MPa.

9. A paraffin-embedded reference prepared by the method according to any one of claims 1-8.

10. A gene detection kit, characterized in that, The kit includes the paraffin-embedded reference as described in claim 9.

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