A method of preparing ffpe quality control articles with cell lines, reference articles, and kits

Abstract

The application provides a method for preparing FFPE quality control products by using cell lines, a reference product and a kit, and belongs to the fields of medicine, clinical laboratory science and biotechnology, and comprises the following steps: culturing cell lines by using microcarriers; mixing different cell lines based on requirements; centrifuging the mixed cell microcarrier suspension in a sealed malt pipe at one end; removing supernatant after the treatment; packaging the two ends of the malt pipe by using a semi-permeable membrane; removing the semi-permeable membrane after fixing, dehydrating and transparentizing the malt pipe by using a formaldehyde solution; separating the cell microcarriers after the wax immersion treatment from the malt pipe; and embedding the cell microcarriers into a paraffin block by using wax. The quality control product can be used in subsequent quality control processes, the mutation frequency of the quality control product can be kept in good uniformity, and batch production and large-scale production of the quality control product can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, clinical laboratory science and biotechnology, in particular to a method for preparing FFPE quality control products with cell lines, reference products and kits. BACKGROUND

[0002] In order to ensure the accuracy of test results, medical laboratories need to select appropriate quality control products when establishing a quality control system. Generally, certified reference materials / standard samples (CRM) are produced by a determined reference material / standard sample (RM) producer and are available on the market; laboratories can prepare quality control samples (QCM) for their own use to meet internal laboratory needs. Quality control samples (QCM) are sometimes also referred to as quality control samples or quality control products.

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

[0004] Clinical genetic testing is a special test item in the field of clinical medical testing, with rapid technological development. Currently, there are very few certified reference materials / standard samples (CRM) on the market, which cannot meet the quality control needs of different test items; the technical level of laboratories preparing quality control samples (QCM) themselves is uneven, and the uniformity and stability are difficult to guarantee.

[0005] Formalin-fixed paraffin-embedded (FFPE) tissues are an important sample type for clinical molecular pathology testing. FFPE quality control products include paraffin block quality control products, paraffin section quality control products, and paraffin roll quality control products, according to their forms. FFPE quality control products prepared from patient tissue samples are difficult to mass-produce due to heterogeneity between or within tissues, and are limited by ethical issues, making it impossible to mass-produce and use them. Currently, the quality control products commonly used in molecular pathology laboratories are prepared from cultured cell lines and embedded after dehydration to form paraffin blocks. However, due to the batch production process, it is difficult to control the uniformity and stability, which leads to the inability to be widely used. Most laboratories directly use cell lines to extract nucleic acids, and the quality control of the extraction process cannot be controlled.

[0006] The technical bottlenecks of the current cell line preparation FFPE quality control product include: 1) the space steric hindrance effect of different sizes of cells leads to uneven mixing (the number of cells in continuous sections is uneven, and the density of single cells is uneven); 2) the cell density in the wax block is insufficient (the nucleic acid yield extracted from single sections is small and uneven). Due to the conventional technical defects of 1) and 2), the uniformity of the mutation frequency of the quality control product in the subsequent quality control process is poor, and the batch and large-scale production of the quality control product still needs to be studied and solved. SUMMARY

[0007] To solve the above problems, the present application provides a method for preparing FFPE quality control products with cell lines, reference products and kits.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The present application provides a method for preparing FFPE quality control products with cell lines, comprising: culturing cell lines with microcarriers; mixing different cell lines based on demand; centrifuging the mixed cell microcarrier suspension in a sealed macaroni tube at one end, removing the supernatant after the treatment is completed, then packaging the macaroni tube with a semi-permeable membrane at both ends, fixing with formaldehyde solution, dehydrating and transparentizing, removing the semi-permeable membrane and immersing in wax; separating the cell microcarriers after wax immersion treatment from the macaroni tube, then embedding in wax to form a paraffin block.

[0010] Further, the microcarrier includes one or more of dextran matrix microcarrier, cellulose matrix microcarrier, gelatin matrix microcarrier, polystyrene matrix microcarrier, polyethylene matrix microcarrier, polygalacturonic acid matrix microcarrier, polypropylene matrix microcarrier, polyester fiber matrix microcarrier, chitosan matrix microcarrier, chitin matrix microcarrier, alginate gel microcarrier.

[0011] Further, the microcarrier is a dextran matrix microcarrier.

[0012] Further, the cell line is selected from NA12878, HEK293T, A549, BEAS-2B, all human adherent cell lines.

[0013] Further, the diameter of the macaroni tube is 0.3-2.0 cm, and the length of the macaroni tube is 5-15 cm.

[0014] Further, before use, the macaroni tube is connected to a gas mixed with particulate matter for 30-60 minutes, then washed with an organic solvent, and the flow rate of the gas is wherein α is a correction factor, D is the diameter of the macaroni tube; the thermal conductivity of the particulate matter is at least 1.5 times that of the macaroni tube, the size of the particulate matter is not more than 0.2 mm, and the D5 particle size of the particulate matter is not more than 10 .

[0015] Further, the malt pipe separation process is that the outer wall of the malt pipe is heated while air pressure is applied to one end.

[0016] Further, the outer wall of the malt pipe is heated to T±5℃, wherein T is the wax dipping drop point; and the air pressure is 0.15-0.20 Mpa.

[0017] The application further provides a paraffin-embedded reference product prepared by the method.

[0018] The application further provides a genetic detection kit, which comprises the paraffin-embedded reference product.

[0019] The beneficial effects brought by the technical scheme provided by the embodiments of the application include:

[0020] The application adopts a microcarrier culture cell line, which is suitable for all adherent culture cells. The microcarrier plays a role of cell skeleton, and the arrangement mode of the cultured cells is more similar to tissues and closer to the arrangement characteristics of human tissues. The requirement that the quality control product should be close to a clinical sample is met. Secondly, the raw material cell line of the FFPE quality control product in the application does not need to be digested by trypsin or trypsin substitutes, and the cell microcarrier suspensions are directly mixed in proportion. Since the microcarriers are uniform in size, the distribution of the cells grown in unit time is relatively uniform, and the direct mixing of the microcarriers of the two cells can more easily mix the cells uniformly. The microcarrier plays a role of cell skeleton, and effectively overcomes the steric hindrance effect in the mixing process of different sizes of cells, which leads to uneven mixing. Finally, the microcarrier in the application plays a role of cell skeleton, and fully protects the relative positions of the cells in the subsequent cell centrifugation, dehydration, transparentization and embedding process, so that the cell distribution is more uniform. The traditional centrifugation, dehydration and transparentization process before cell embedding can cause cell loss, and the cell plasticization process before embedding uses a certain proportion of gel or similar substances, which causes the cell density in unit volume to be diluted. The application adopts a malt pipe to perform the centrifugation, dehydration, transparentization and plasticity of the FFPE quality control product raw material cell line, reduces the cell loss, does not need to add gel or similar materials, and increases the cell density in unit volume. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0022] Figure 1 The cell HE staining picture in the FFPE quality control product slice prepared in Embodiment 2 of the application;

[0023] Figure 2 Here are HE-stained images of cells in a section of FFPE quality control material prepared in Comparative Example 1 of this invention;

[0024] Figure 3 This is an image of cells stained with HE in a slice of FFPE quality control material prepared in Comparative Example 2 of this invention. Detailed Implementation

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Unless otherwise specified, all reagents used in this invention are commercially available products and do not affect the inventive points not claimed.

[0030] This invention provides a method for preparing FFPE quality control products using cell lines, comprising:

[0031] S1. Cell lines are cultured using microcarriers;

[0032] S2. Mix different cell lines based on requirements;

[0033] S3, the mixed cell microcarrier suspension is perfused into a sealed macaroni tube for centrifugal treatment, after the treatment, the supernatant is removed, then the two ends of the macaroni tube are packaged with a semi-permeable membrane, and after being fixed by a formaldehyde solution, dehydrated and transparentized, the semi-permeable membrane is removed for wax immersion treatment;

[0034] S4, the cell microcarrier after the wax immersion treatment is separated from the macaroni tube, and then embedded into a paraffin block by wax.

[0035] The microcarrier culture cell line adopted in the application is suitable for all adherent cells, the microcarrier plays a role of cell skeleton, the arrangement mode of the cultured cells is more similar to tissues, and is closer to the arrangement characteristics of human tissues. The quality control product meets the requirement that the quality control product should be close to a clinical sample; secondly, the raw material cell line of the FFPE quality control product in the application does not need to be digested by trypsin or trypsin substitutes, and the cell microcarrier suspension is directly mixed in proportion. Since the microcarriers are uniform in size, the distribution of the cells grown in unit time is relatively uniform, and the direct mixing of the microcarriers of two kinds of cells can more easily mix the cells uniformly. The microcarrier plays a role of cell skeleton, and effectively overcomes the space steric hindrance effect in the mixing process of different sizes of cells, which leads to uneven mixing. Finally, the microcarrier in the application plays a role of cell skeleton, and fully protects the relative positions of the cells in the subsequent cell centrifugation, dehydration, transparentization and embedding process, so that the cell distribution is more uniform. The traditional centrifugation, dehydration and transparentization process before cell embedding can cause cell loss, and the cell plasticization process before embedding uses a certain proportion of gel or similar substances, which causes the cell concentration in unit volume to be diluted. The application adopts the macaroni tube to perform the centrifugation, dehydration, transparentization and plasticity of the raw material cell line of the FFPE quality control product, reduces the cell loss, does not need to add gel or similar materials, and increases the cell density in unit volume.

[0036] In step S1, the microcarrier includes one or more of a dextran matrix microcarrier, a cellulose matrix microcarrier, a gelatin matrix microcarrier, a polystyrene matrix microcarrier, a polyethylene matrix microcarrier, a polygalacturonic acid matrix microcarrier, a polypropylene matrix microcarrier, a polyester fiber matrix microcarrier, a chitosan matrix microcarrier, a chitin matrix microcarrier, and an alginate gel microcarrier. Preferably, one kind of microcarrier is used, and at this time, the size of the microcarrier is more uniform, so that when the microcarrier is used as a cell skeleton, the uniformity of the cell distribution is more improved. The above-mentioned microcarriers are all commercially available products.

[0037] Preferably, the microcarrier is a dextran matrix microcarrier. Such a microcarrier is based on a cross-linked dextran framework, has a large specific surface area (>4000 cm 2 / g), and the surface of the microcarrier is covered with a part of positively charged DEAE groups by special process treatment, so as to meet the efficient growth of cells.

[0038] Preferably, in the embodiments of the present application, 5 mg of dextran matrix microcarriers (such as commercially available Cytodex 1 microcarriers) are used for 1 mL of culture medium.

[0039] The cell line in the present application is an in vitro adherent culture cell line, and the culture medium is the culture medium recommended by the cell line preservation agency; as a preferred, the raw material cell line of the quality control product can be selected from all human adherent culture cell lines such as NA12878, HEK293T, A549, and BEAS-2B.

[0040] The cell fusion degree of the unit mass microcarrier in the present application reaches 70%-90% (such as 5 mg of commercially available Cytodex 1 microcarriers is equivalent to 30 cm 2 surface) can be passaged (can be passaged for 5-10 times) or collected; as a preferred, the cell growth fusion degree is about 85% when collected, and can be continuously passaged for 5 times.

[0041] In the embodiments of the present application, in order to measure the cell density, after the cell line is cultured, it is mixed thoroughly, 5-10 mL of microcarrier suspension is taken, digested with trypsin or trypsin substitute, and the digested cells are collected for counting, and the cell density in unit volume of culture medium of each cell line (more than 1 × 10 7 cells / mL) can be calculated.

[0042] In step S2, the cell line microcarrier does not need to be digested with trypsin or trypsin substitute, and is directly mixed in proportion. The mixing ratio is mixed according to the target mutation frequency of the quality control product and the density of two or more cell lines, and if a single cell line quality control product is prepared, this step is omitted.

[0043] After the microcarriers with growing cells in the present application are mixed, centrifugal treatment is performed, the centrifugal treatment adopts 1000-3000g, and centrifugal treatment is performed for at least 5 min, preferably 2000g centrifugal treatment is performed for 5 min-15 min, the supernatant is discarded and the cell microcarrier precipitate is retained. The cell microcarrier precipitate is resuspended with 1×PBS, 2000g centrifugation is performed, the supernatant is discarded and the cell microcarrier precipitate is retained, and the process is repeated twice. As a preferred, 1×PBS can be used in an amount of 30 mL-200 mL, which is more than 20 times the volume of the microcarrier precipitate. After the cell microcarrier precipitate (one or more than one cell line) after three 1×PBS washes in the present application is resuspended with 1×PBS solution in an amount of 1 times the volume, the cells are fixed.

[0044] In the step S3, the mixed cell microcarrier suspension is perfused into a sealed end of a malt pipe for centrifugal treatment. The sealing method is not specifically limited, and the purpose is to ensure that the liquid does not flow out through the sealed end during subsequent centrifugal treatment. The centrifugal treatment is performed at a centrifugal force of 1500g-2500g for 8min-15min. After centrifugation, the supernatant is removed. It should be noted that the centrifugal treatment process is very critical. Along the longitudinal direction of the malt pipe, the density of the cell microcarriers away from the rotation center of the centrifuge is larger, and the density of the cell microcarriers close to the rotation center of the centrifuge is smaller, thereby causing the density of the prepared cell microcarriers to differ along the longitudinal direction of the malt pipe. In order to solve the above problem, the centrifugal force and the centrifugal time of the centrifugal treatment are limited, and the size of the malt pipe is combined to ensure that the liquid can be effectively removed while the cell microcarriers can quickly recover to a state of uniform distribution along the longitudinal direction of the malt pipe after the centrifugal treatment is completed.

[0045] The malt pipe in the embodiment of the application is a commercially available product, such as a frozen sperm tube commonly used in the field of reproduction, which has biocompatibility.

[0046] Specifically, the diameter of the malt pipe is 0.3cm-2.0cm, and the length of the malt pipe is 5cm-15cm.

[0047] After the centrifugal treatment, the length of the malt pipe is cut based on the length of the microcarrier sediment, and the two ends are packaged with a semi-permeable membrane. Preferably, when the length of the malt pipe is cut, the reserved length of the malt pipe is L+(1-3)cm, and L is the length of the sediment, that is, the distance between the interface of the supernatant and the microcarrier sediment after the centrifugal treatment and the sealed end.

[0048] Subsequently, the two ends of the malt pipe are packaged with a semi-permeable membrane, and then fixed with a formaldehyde solution, dehydrated and transparently treated, and then immersed in wax after removing the semi-permeable membrane. Specifically, the semi-permeable membrane is a commercially available product, which can allow formaldehyde solution, ethanol and xylene to pass through but not allow cell microcarriers to pass through.

[0049] The macaroni (cell microcarrier) packaged by the semi-permeable membrane in the application is placed in a 20%-40% neutral formaldehyde solution for fixation, and after the dehydration treatment by ethanol gradient and the xylene transparency step, the semi-permeable membrane is removed, the semi-permeable membrane at one end of the macaroni can be removed, or the semi-permeable membrane at both ends of the macaroni can be removed, and then the cells are fully immersed in wax. Specifically, neutral formalin is added to the macaroni, shaken and then inclined in a refrigerator at 5-10°C for 30-60 minutes, then centrifuged at 1000-2000g for 5-10 minutes, and the supernatant is discarded; then washed once with PBS, and then centrifuged at 1000-2000g for 5-10 minutes. According to the gradient from low to high concentration of ethanol, when each dehydration step is completed, centrifuge at 1000-2000g for 5-10 minutes, and discard the supernatant; replace the anhydrous ethanol with xylene gradually in a fume hood, and the specific operation is as follows: first treat with 2 / 3 anhydrous ethanol and 1 / 3 xylene for 10-20 minutes, and then suck the supernatant; then treat with 1 / 3 anhydrous ethanol and 2 / 3 xylene for 10-20 minutes, and then suck the supernatant; then replace with pure xylene for 20-30 minutes, and then suck the xylene to proceed to the paraffin embedding step of the cells.

[0050] The macaroni (wax-processed cell microcarrier) in the application is placed in a freezer at -15°C to -25°C for more than 1 hour. If embedding is not performed on the same day, it can be temporarily stored at -15°C to -25°C until embedding.

[0051] The macaroni in the application can be sprayed with an international one-dimensional code (128 code) in the middle of the macaroni by using an automatic code spraying device. The sprayed ink is low-temperature resistant (-196°C), and the code serves as the unique identification of the cell sample, which can better realize traceability management in large-scale production processes.

[0052] The wax-processed cell microcarrier is separated from the macaroni, and then embedded in a paraffin block. The macaroni separation process is: heating the outer wall of the macaroni while applying air pressure to one end of the macaroni. Specifically, before use, the one end of the macaroni is connected to a gas mixed with particles for 30-60 minutes, and then cleaned with an organic solvent. The flow rate of the gas is , where a is a correction factor, and D is the diameter of the macaroni; the thermal conductivity of the particles is at least 1.5 times that of the macaroni, the size of the particles is not more than 0.2 mm, and the D5 particle size of the particles is not more than 10 . The above treatment is to treat the macaroni wall. On the one hand, the larger particles rub the surface of the macaroni to form a certain surface roughness on the inner surface of the macaroni, but the size of the groove on the inner surface is small enough, and the paraffin will not enter the groove to increase the resistance of the wax-processed cell microcarrier to separate from the macaroni under the action of surface tension. On the other hand, the D5 particle size of the particles is not more than 10 The size is small enough, and part of the size can be embedded in the inner surface of the tube, so as to increase the heat conduction efficiency. The cell microcarrier immersed in wax is quickly melted at the contact position between the inner wall of the tube and the cell microcarrier. Since the tube is made of a high polymer material, the heat conductivity coefficient is low, and the heat conductivity coefficient of paraffin is also low. Therefore, during the heating process, the cell microcarrier immersed in wax is melted to a high degree, and under the action of air pressure, the cell microcarrier immersed in wax cannot be effectively formed, resulting in poor uniformity of mutation frequency of the quality control product in the subsequent quality control process, and the quality control product cannot be mass produced. The small particles embedded in the tube wall can quickly melt the cell microcarrier immersed in wax at the contact position between the inner wall of the tube and the cell microcarrier, avoid heat transfer to the core of the cell microcarrier, and ensure that the cell microcarrier immersed in wax is formed well and does not cause problems such as adhesion.

[0053] Specifically, alpha is a correction coefficient, and the unit is L / min.m 2 / 3 The value range is 450-550, and D is the diameter of the tube, the unit is m.

[0054] In the embodiment of the application, the particulate matter can be iron sand or fine sand.

[0055] The tube separation process is that the outer wall of the tube is heated while air pressure is applied to one end of the tube. The outer wall of the tube is heated to T±5℃, wherein T is the drop point of the immersed wax; and the air pressure is 0.15-0.20 Mpa.

[0056] Specifically, the -15℃~-25℃ frozen tube (cell microcarrier immersed in wax) in the application is placed in a 65℃ water bath / metal bath, and at the same time, the cell microcarrier immersed in wax is quickly pushed out by using a matching air pressure device.

[0057] The cell microcarrier immersed in wax in the application is placed on a tissue embedding table, and quickly embedded into a paraffin block by using wax. Each paraffin block embedding can embed a cell microcarrier immersed in wax with a length of about 5mm, and a long cell microcarrier immersed in wax can be segmented and embedded into a paraffin block quality control product.

[0058] The wax block quality control product in the application is cut into a 4-7μm thick paraffin section quality control product or a paraffin roll piece quality control product.

[0059] The embodiment of the application also provides a paraffin embedding reference product prepared by using the above method.

[0060] The embodiment of the application also provides a gene detection kit, and the kit comprises the above paraffin embedding reference product.

[0061] In order to better illustrate the embodiments of the application, the application will be further described in detail through specific examples.

[0062] Example 1

[0063] FFPE quality control preparation

[0064] The main instruments and equipment are shown in Table 1.

[0065] Table 1 Equipment used in the examples

[0066]

[0067] The main reagent consumables are shown in Table 2.

[0068] Table 2 Reagent consumables used in the examples

[0069]

[0070] The cell line quality control raw materials are shown in Table 3.

[0071] Table 3 Quality control source materials used in the examples

[0072]

[0073] Preparation method:

[0074] The cells provided by the preservation center are in a growing state, and the cells are placed in T-25 plastic culture bottles, with a quantity of about 1-2 × 10 6 The cells are eluted from the bottle wall with trypsin, and the cells are transferred to a Virya sterile independent packaging 500 mL triangular cell culture conical flask, 20 mL of the corresponding culture medium and an equal proportion of Cytodex1 microcarrier (5 mg of dextran matrix microcarrier is used per mL of culture medium) are added.

[0075] The two cells are subjected to microscopic examination, digestion and subculture according to the subculture method recommended by the China Typical Culture Collection Center. After subculture to 5 generations (the cell fusion degree reaches more than 85%), the subculture is stopped, and there is no need for trypsin digestion, and all the cell microcarriers in the triangular cell culture conical flask are directly collected. (Batch industrial production can use bioreactors for large-scale culture)

[0076] Take 5-10 mL of cell microcarrier suspension and digest with trypsin or trypsin substitute, collect the digested cells and count them. Calculate the cell density in each cell line per unit volume of culture medium (per unit mass of microcarrier). A small amount of microcarrier is digested and counted for subsequent calculation of the mixing ratio of the cells.

[0077] Mix two cell lines microcarriers (BEAS-2B and A549) in the ratio of 1:1 by cell number. Centrifuge the mixed cell microcarriers at 2000g, discard the supernatant and keep the cell microcarrier pellet. Resuspend with 20 times volume of 1x PBS, centrifuge at 2000g, discard the supernatant and keep the cell microcarrier pellet, repeat the process for 2 times. Resuspend the cell microcarriers with 1 times volume of 1x PBS solution.

[0078] After mixing the cell microcarriers, fill them into 0.3cm diameter one end blocked (polyamide fabric) straws (CBS™ High Security straws). Before use, pass the one end of the straw with the mixed particles into the gas for 30min, then clean with anhydrous ethanol, the flow rate of the gas is wherein a is the correction factor, the value is 500 L / min.m 2 / 3 The diameter of the straw is 0.3cm, by calculation, the flow rate of the gas is 10 L / min.

[0079] The thermal conductivity of the particles is 1.5 times of the straw, the size of the particles is no more than 0.2mm, the D5 particle size of the particles is 10μm.

[0080] Centrifuge at 2000g for 10min. After centrifugation, discard the supernatant; cut the straw according to the length of the cell microcarrier pellet, leave 1cm at both ends, and package the two ends with semi-permeable membrane.

[0081] Put the semi-permeable membrane closed cell microcarrier filled straw into 40% neutral formaldehyde solution for fixation. After ethanol gradient dehydration treatment and xylene transparency steps, the cells are fully immersed in wax. After wax immersion, the cell microcarrier straw is sprayed and placed at -15℃~-25℃ overnight.

[0082] Put the -15℃~-25℃ frozen wax immersed cell microcarrier straw into a 65℃ water bath / metal bath (if a metal bath is used, it should be a special long inner diameter, matched with the straw, and when mass production, customization can be considered) quickly, and at the same time, use the gas pressure device matched with the invention to push out the wax immersed cell microcarrier.

[0083] Put the wax immersed cell microcarrier on the tissue embedding table, and quickly embed it into a paraffin block with paraffin. Each paraffin block can embed about 5mm of wax immersed cell microcarrier, and long wax immersed cell microcarrier can be segmented and embedded into paraffin block quality control.

[0084] Cut the wax block quality control into 4μm thick paraffin section quality control or paraffin roll quality control.

[0085] The comparison of the paraffin block quality control prepared by the method of the invention and the commercially available paraffin block quality control is shown in Table 4.

[0086] Table 4. Parameters of commercially available products and the prepared quality control products of the application

[0087]

[0088] As shown in the above table, the cell content of the paraffin block quality control product prepared by the method of the application is twice that of the commercially available paraffin block quality control product; the section quality control product prepared by the method of the application is thinner than the commercially available section quality control product, which can improve the efficiency of dewaxing, digestion and decrosslinking of the quality control product and improve the quality of extracted nucleic acid; the paraffin block quality control product prepared by the method of the application can obtain nearly three times the number of sections compared with the commercially available paraffin block quality control product, and the DNA yield meets the downstream general molecular biology test requirements. Therefore, the paraffin block quality control product prepared by the method of the application meets the clinical application requirements, including but not limited to: internal quality control, interlaboratory quality evaluation, and comparability of test results.

[0089] Example 2

[0090] Different from example 1, in the embodiment of the application, before use, one end of the straw is connected to the gas mixed with particulate matter for 60 min, and then washed with anhydrous ethanol, and the flow rate of the gas is wherein a is a correction factor, and the value is 500 L / min.m 2 / 3 The diameter of the straw is 1 cm, and the flow rate of the gas is 23 L / min through calculation.

[0091] The thermal conductivity of the particulate matter is 1.5 times that of the straw, the size of the particulate matter is not more than 0.2 mm, and the D5 particle size of the particulate matter is 10 μm.

[0092] After the cells are immersed in wax, the cell microcarriers are placed on a tissue embedding table and quickly embedded into paraffin blocks. Randomly take several rolls from the start, middle and end positions of each of the three paraffin block quality control products, extract one piece every one / two / three months, and detect and evaluate the concentration, purity and fragment integrity of nucleic acid, and detect the mutation of nucleic acid. As shown in Figure 1 The cells in the paraffin block quality control product prepared by the embodiment of the application are tightly and uniformly distributed, and the extraction quality of the prepared quality control product is shown in Table 5, and the mutation detection result of the quality control product is shown in Table 6.

[0093] Table 5. Quality control product extraction quality

[0094]

[0095] Table 6. Mutation detection results of quality control products

[0096]

[0097] 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.

[0098] Table 7. Statistical results of cell number at different locations

[0099]

[0100] Example 3

[0101] 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.

[0102] 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. .

[0103] 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.

[0104] Comparative Example 1

[0105] 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.

[0106] Comparative Example 2

[0107] 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.

[0108] Comparative Example 3

[0109] 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.

[0110] 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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