CN111521460A - FFPE reference product, preparation method and application thereof - Google Patents
FFPE reference product, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an FFPE reference product, and a preparation method and application thereof. The preparation method of the FFPE reference substance comprises the following steps: selecting a cell line of interest; culturing cells of a target cell line to a preset concentration, collecting the cells, fixing the cells by formalin, and preparing paraffin blocks; and slicing the paraffin block to obtain the FFPE reference product. By selecting a proper cell line and then culturing the cell line to prepare the FFPE sample which is the same as the clinical sample, the reference comparison function from nucleic acid extraction to detection result can be provided for the clinical FFPE sample, the quality control function is played for the whole process of FFPE sample detection, and the accuracy of the detection result is improved. The method is suitable for preparing the FFPE reference product of the tumor cell strain with a specific gene mutation ratio in a large scale, so that the problem that the internal quality control of the FFPE reference product has no whole process when an enterprise researches and develops products such as a preparation kit and the like and clinically inspects is solved.
Description
Technical Field
The invention relates to the field of FFPE sample detection, in particular to an FFPE reference product, and a preparation method and application thereof.
Background
In recent years, with the continuous development of molecular biology technology, especially the wide application of PCR technology and high throughput sequencing technology, gene detection technology gradually moves from laboratory to clinical application, especially in cancer treatment, most of targeted drugs are related to specific gene mutation types, and therefore, in the process of developing and verifying analysis performance of gene detection products, enterprise reference products containing different mutation frequencies of specific genes are needed to evaluate the sensitivity, accuracy, specificity and repeatability of products. Meanwhile, in the gene detection process of the third-party clinical examination, some reference products with specific mutation frequencies of genes are also needed to check the accuracy of the detection result from nucleic acid extraction.
Tissue samples commonly used in clinical studies are formalin-fixed and paraffin-embedded (FFPE) tissue samples, however, currently common corporate reference preparations are made in the form of: (1) mixing the mutant plasmid DNA and the wild plasmid DNA; (2) mixing the mutant plasmid DNA with wild cell line DNA; (3) mixing the mutant cell line DNA with the wild cell line DNA; (4) mutant clinical sample DNA was mixed with wild-type cell line DNA. Most of the reference products are prepared by mixing DNA, and the condition of FFPE real samples cannot be simulated well, so that the reference products are difficult to provide accurate reference.
Disclosure of Invention
The invention mainly aims to provide an FFPE reference product, a preparation method and application thereof, and aims to solve the problem that the FFPE reference product in the prior art cannot play an accurate reference role.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method of preparing an FFPE reference, the method comprising: selecting a cell line of interest; culturing cells of a target cell line to a preset concentration, collecting the cells, fixing the cells by formalin, and preparing paraffin blocks; and slicing the paraffin block to obtain the FFPE reference product.
Further, the FFPE reference substance is an FFPE negative reference substance, the target cell line is a wild-type cell line, and preferably, the wild-type cell line is any one or more selected from BEAS-2B cells, GM12878 cells and Hela cells.
Further, the predetermined concentration is 4 × 106～5×106cells/mL.
Further, the FFPE reference substance is an FFPE positive reference substance, the target cell line is a wild type cell line and a mutant cell line, and preferably the wild type cell line is selected from any one or more of BEAS-2B cells, GM12878 cells and Hela cells; preferably the mutant cell line is selected from any one or more of SW48, H1650, KYSE450 and H1975.
Further, after culturing the cells of the target cell line to a predetermined concentration, collecting the cells and formalin-fixing them to make paraffin blocks, comprising: respectively diluting cells of the wild type cell line and the mutant cell line to obtain diluted wild type cells and diluted mutant cells; culturing the diluted wild type cells and the diluted mutant type cells to a predetermined concentration respectively; mixing the wild type cells and the mutant type cells reaching the preset concentration to obtain mixed cells; the mixed cells were collected and formalin-fixed, thereby preparing paraffin blocks.
Further, the concentration of the diluted wild-type cells and the diluted mutant cells were each independently 0.5 × 103～5×103cells/mL, preferably, the predetermined concentration is 1.5 × 106～5×106cells/mL, more preferably 2 × 106～3×106cells/mL; preferably, the wild-type cells and the mutant cells are mixed at a set mutation frequency.
Further, before selecting the target cell line, the method further comprises a step of verifying the genetic variation of the target cell line, preferably by adopting a NGS or ddPCR method.
Further, the paraffin block is sliced to obtain a paraffin roll with the thickness of 8-12 mu m, and the paraffin roll is the FFPE reference product.
According to a second aspect of the present application, there is provided an FFPE reference, the FFPE reference being a paraffin-embedded section.
Further, the paraffin embedded section is a cell paraffin embedded section; preferably, the thickness of the paraffin-embedded section is 8-12 μm; preferably, the FFPE reference is an FFPE negative reference, and the paraffin-embedded section is a paraffin-embedded section of the wild-type cell; preferably, the FFPE reference is an FFPE positive reference and the paraffin-embedded section is a paraffin-embedded section of a mixture of wild-type cells and mutant cells.
In order to achieve the above object, according to one aspect of the present invention, there is provided an FFPE sample detection kit, which includes any one of the FFPE reference products described above, or an FFPE reference product prepared by any one of the preparation methods described above.
According to another aspect of the present invention, there is provided an FFPE reference product prepared by any one of the above-mentioned preparation methods, or an application of any one of the above-mentioned FFPE reference products in development of an FFPE sample variation detection product.
By applying the technical scheme of the invention, the FFPE sample which is the same as the clinical sample is prepared by selecting the appropriate cell line and culturing, so that a reference comparison function from nucleic acid extraction to a detection result can be provided for the clinical FFPE sample, a quality control function is played for the whole process of FFPE sample detection, and the accuracy of the detection result is improved. The method is suitable for preparing the FFPE reference product of the tumor cell strain with a specific gene mutation ratio in a large scale, so that the problem that the internal quality control of the FFPE reference product has no whole process when an enterprise researches and develops products such as a preparation kit and the like and clinically inspects is solved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIGS. 1 to 4 are scattergrams for examination and verification of gene mutations carried by different cell lines by the digital PCR method according to example 1 of the present invention, in which FIG. 1 shows the results of examination of the p.G719S mutation carried by SW48 cell line, FIG. 2 shows the results of examination of the E746_ A750del mutation carried by H1650 cell line, FIG. 3 shows the results of examination of the p.S768I mutation carried by KYSE450 cell line, and FIG. 4 shows the results of examination of the p.L858R mutation carried by H1975 cell line, respectively.
FIGS. 5 to 8 are scatter plots showing the results of detection of the positive reference for 5% mutation frequency in example 1 according to the present invention, wherein FIG. 5 shows the results of detection of the p.G719S mutation carried by the SW48 cell line, FIG. 6 shows the results of detection of the E746_ A750del mutation carried by the H1650 cell line, FIG. 7 shows the results of detection of the p.S768I mutation carried by the KYSE450 cell line, and FIG. 8 shows the results of detection of the p.L858R mutation carried by the H1975 cell line, respectively.
Figure 9 shows a graph of the results of testing a clinical sample using the positive and negative references of the present application in example 3 according to the present invention, where a represents the clinical sample, B represents the FFPE positive reference, and C represents the FFPE negative reference.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background art, the tissue sample that is clinically common at present is an FFPE sample, and when such a sample is detected, the reference substances used are DNA mixtures of different forms, so that the reference effect cannot be accurately provided for the clinical tissue sample from DNA extraction to library construction, and then the detection result. To this end, the applicant proposes an improved reference to make the reference closer to the real clinical FFPE sample, thereby serving as a more accurate reference for the clinical FFPE sample.
In an exemplary embodiment of the present application, there is provided a method of making an FFPE reference, the method comprising: selecting a cell line of interest; culturing cells of a target cell line to a preset concentration, collecting the cells, fixing the cells by formalin, and preparing paraffin blocks; and slicing the paraffin block to obtain the FFPE reference product.
According to the preparation method of the FFPE reference product, the appropriate cell line is selected, and then the FFPE sample which is the same as the clinical sample is prepared after culture, so that a reference comparison effect from nucleic acid extraction to a detection result can be provided for the clinical FFPE sample, a quality control effect is achieved for the whole process of detection of the FFPE sample, and the accuracy of the detection result is improved. The method is suitable for preparing the FFPE reference product of the tumor cell strain with a specific gene mutation ratio in a large scale, so that the problem that the internal quality control of the FFPE reference product has no whole process when an enterprise researches and develops products such as a preparation kit and the like and clinically inspects is solved.
The FFPE reference substance can be used for specifically preparing a negative reference substance and a positive reference substance according to different effects. When the FFPE reference product is an FFPE negative reference product, the target cell line is a wild-type cell line, preferably the wild-type cell line is selected from any one or more of BEAS-2B cells, GM12878 cells and Hela cells.
When the cells of the above wild-type cell line are selected and cultured for preparing a paraffin-embedded sample, the predetermined concentration to be attained by the cell culture can be determined according to actual needs, and in the present application, the predetermined concentration is preferably 4 × 106～5×106cells/mL. Setting the predetermined concentration of cells within the above range enables keeping the cells in good condition and rarely causing cell aging and death.
When the FFPE reference product is an FFPE positive reference product, the target cell line is a wild-type cell line and a mutant cell line, preferably the wild-type cell line is selected from any one or more of BEAS-2B cells, GM12878 cells and Hela cells; preferably the mutant cell line is selected from any one or more of SW48, H1650, KYSE450 and H1975.
The above cell lines are all commercial cell lines. Can be purchased from the market according to actual needs. It should be noted that the cell lines of the present application are not limited to the above mentioned ones, and in practical development or application scenarios, the cell lines carrying the target mutation sites can be reasonably selected according to practical needs.
When preparing the FFPE positive reference substance, respectively culturing a wild cell line and a mutant cell line, then mixing cell lines of different sources according to the target mutation frequency, and finally preparing the mixed cells into paraffin embedded sections. In a preferred embodiment of the present application, after culturing the cells of the target cell line to a predetermined concentration, collecting the cells and formalin-fixing the cells to make paraffin blocks comprises: respectively diluting cells of the wild type cell line and the mutant cell line to obtain diluted wild type cells and diluted mutant cells; culturing the diluted wild type cells and the diluted mutant type cells to a predetermined concentration respectively; mixing the wild type cells and the mutant type cells reaching the preset concentration to obtain mixed cells; the mixed cells were collected and formalin-fixed, thereby preparing paraffin blocks.
Depending on the original concentration of each cell line, the cells may be diluted to the appropriate starting culture concentration prior to culturing, hi a preferred embodiment, the diluted wild type cells and the diluted mutant cells are each independently at a concentration of 0.5 × 103～5×103cells/mL。
In a preferred embodiment, the cells of the two cell lines are separately cultured to a predetermined concentration of 1.5 × 106～5×106cells/mL, more preferably 2 × 106～3×106cells/mL. In a preferred embodiment, the mixing ratio of the wild-type cells and the mutant cells is calculated according to the actual variation frequency.
In order to further improve the accuracy of the reference, in a preferred embodiment of the present application, before selecting the target cell line, a step of verifying the genetic variation of the target cell line is further included, preferably by NGS or ddPCR. The accuracy of the selected reference substance is ensured by verifying that the selected target cell line does not have target mutation or mutation exists on a target gene locus, and a reliable basis is provided for the variation detection of a subsequent FFPE sample to be detected.
The slice thickness of the prepared FFPE reference product can be reasonably set according to the slice thickness of the clinical FFPE reference product. In a preferred embodiment of the present application, the paraffin block is sliced to obtain a paraffin roll with a thickness of 8-12 μm, and the paraffin roll is an FFPE reference. I.e., whether a negative reference or a positive reference, the thickness can be within the above ranges.
In a second exemplary embodiment of the present application, an FFPE reference is provided, which is a paraffin-embedded section. By providing the paraffin-embedded section sample which is the same as the clinical sample, the reference quality control function of the whole process of nucleic acid extraction to detection results can be provided for the clinical FFPE sample, so that the steps and reasons which possibly cause problems can be conveniently detected.
The FFPE reference substance, preferably the paraffin-embedded section is a cell paraffin-embedded section. The embedded section of cells is formed by selecting positive cells that are verified to carry the variation of the target gene and negative cells that are verified to have no variation of the target gene.
The paraffin-embedded section thickness of the FFPE reference was determined from the thickness of the clinical FFPE sample as described above. In a preferred embodiment, the thickness of the paraffin-embedded section is 8-12 μm; more preferably 10 μm.
The specific embedded cells varied according to the control type of the FFPE reference. When the FFPE reference is an FFPE negative reference, the paraffin-embedded section is a paraffin-embedded section of the wild-type cell. When the FFPE reference substance is an FFPE positive reference substance, the paraffin-embedded section is a paraffin-embedded section formed by mixing wild-type cells and mutant cells.
In a third exemplary embodiment of the present application, there is provided an FFPE sample variation detection kit, which comprises any one of the FFPE references described above, or an FFPE reference prepared by any one of the preparation methods described above.
In a fourth exemplary embodiment of the present application, there is further provided an application of any one of the FFPE references described above, or an FFPE reference prepared by any one of the preparation methods described above, in development of a variation detection product for an FFPE sample.
Below isThe advantageous effects of the present application will be further explained with reference to specific examples. It should be noted that the cell lines in the following examples were purchased from Nanjing Kebai Biotech Co., Ltd; the cell DNA extraction kit is a blood/cell/tissue genome DNA extraction kit (DP304) of Tiangen Biochemical technology (Beijing) Co., Ltd; the FFPE tissue DNA extraction kit is a paraffin-embedded tissue DNA rapid extraction kit (DP330) of Tiangen Biochemical technology (Beijing) Ltd; the digital PCR reagent is selected from QuantStaudio of Thermo Fisher ScientificTM3D Digital PCR 20KChip Kit v2 and Master Mix(A26317)。
Example 1
Aiming at an EGFR gene, preparing FFPE positive reference products of 4 mutation sites of EGFR genes p.G719S, Exon19Del, p.S768I and p.L858R, and comprising the following steps:
(1) cell line purchase, detailed information as follows:
table 1:
(2) and (4) culturing the cells, and extracting DNA from the cultured cell line.
(3) Using QuantStudio for the extracted cell linesTM3D Digital PCR was performed for validation to determine the mutation frequency of the site.
As shown in the following table:
table 2:
| serial number | Cell line name | Name of mutation | Digital PCR detection frequency | Digital PCR scatter plot |
| 1 | SW48 | p.G719S | 27.43％ | FIG. 1 shows a schematic view of a |
| 2 | H1650 | E746_A750del | 60.07％ | FIG. 2 |
| 3 | KYSE450 | p.S768I | 49.70％ | FIG. 3 |
| 4 | H1975 | p.L858R | 67.85％ | FIG. 4 |
(4) And (3) diluting the cells by adopting a limiting dilution method, selecting monoclonal cells containing target gene mutation, culturing, counting at 12h, 24h, 36h, 48h and 60h respectively, and calculating the proliferation rate.
(5) The final concentration of each cell was selected to be 2 × 10 according to the proliferation rate of each cell6～3×106cells/mL was used as the number of mixed cells, and different volumes of cells were mixed according to the mutation frequency of 5% of the reference, as shown in the following table.
Table 3:
(6) cell embedding and Paraffin coil preparation
And (3) carrying out vortex oscillation on the tumor cell lines mixed according to a specific proportion, fully and uniformly mixing, centrifuging, then removing supernatant, and collecting cells. After 3 washes with PBS, the cell pellet was fixed with 10% formalin for 2 hours and processed according to a conventional dehydration procedure. After the dehydration procedure was completed, the tissue blocks were removed from the automated dehydrator and embedded in normal paraffin. And (3) freezing the embedded cell line wax blocks on a freezing table, and cutting each wax block into a plurality of paraffin coils with the thickness of 10 mu m after the wax blocks are solidified and cooled.
(7) Extracting genome DNA of a paraffin roll newly prepared according to a specific proportion, using QuantStaudio (TM) 3DDigital PCR to verify mutation frequency, and determining a prepared mutation frequency result.
(8) The results of the detection of the positive reference at 5% mutation frequency were as follows:
table 4:
| serial number | Name of positive reference | Mutation name (frequency) | Digital PCR detection frequency | Digital PCR scatter plot |
| 1 | EGFR-P1-5 | p.G719S(5％) | 4.20％ | FIG. 5 |
| 2 | EGFR-P2-5 | p.Ex19Del(5％) | 4.70％ | FIG. 6 |
| 3 | EGFR-P3-5 | p.S768I(5％) | 4.41％ | FIG. 7 |
| 4 | EGFR-P4-5 | p.L858R(5％) | 6.00％ | FIG. 8 |
Example 2
Preparation of a wild-type FFPE cell line reference:
(1) cell line purchase, detailed information as follows:
table 5:
(2) and (3) cell culture, wherein DNA extraction is carried out on the cultured cell line, and Sanger sequencing is carried out on the extracted genomic DNA to verify the gene mutation condition.
(3) After the verification is passed, the wild type cell line is amplified and cultured, and the cell number reaches 4 × 106～5×106At cells/mL, the adherent cells were digested and collected by centrifugation.
(4) Cell embedding and Paraffin coil preparation
The collected cells were washed 3 times with PBS and then cell pellets were fixed with 10% formalin for 2 hours and processed according to a conventional dehydration procedure. After the dehydration procedure was completed, the tissue blocks were removed from the automated dehydrator and embedded in normal paraffin. And (3) freezing the embedded cell line wax blocks on a freezing table, and cutting each wax block into a plurality of paraffin coils with the thickness of 10 mu m after the wax blocks are solidified and cooled.
Example 3
The prepared FFPE positive reference substance, the prepared FFPE negative reference substance and a clinical FFPE sample are adopted, the FFPE extraction kit of Tiangen is adopted to extract genome DNA, the extracted DNA is subjected to fluorescent quantitative PCR detection by using a primer and a probe for detecting EGFR p.L858R, and the result of an amplification curve is shown in figure 9.
As shown in fig. 9, a is the clinical FFPE sample, B is the FFPE positive reference, and C is the FFPE negative reference. As can be seen from FIG. 9, the reference substance of the present application can serve as a reference control for the whole process of nucleic acid extraction and PCR detection for clinical samples, thereby playing a good quality control role in the development of related detection reagent products and the clinical detection process.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the FFPE reference substance is prepared according to the specific mutation frequency, and the accuracy of the mutation frequency is verified through digital PCR detection, so that the FFPE reference substance prepared according to the method can simulate a clinical real sample, and is suitable for preparing the FFPE reference substance of the tumor cell strain containing the specific gene mutation ratio in a large scale, and the problem that an enterprise has no FFPE reference substance in internal quality control during research and development of products such as a preparation kit and clinical examination is solved.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A preparation method of an FFPE reference product is characterized by comprising the following steps:
selecting a cell line of interest;
culturing the cells of the target cell line to a preset concentration, collecting the cells, fixing the cells by formalin, and preparing paraffin blocks;
and slicing the paraffin block to obtain the FFPE reference product.
2. The method according to claim 1, wherein the FFPE reference product is an FFPE negative reference product, and the target cell line is a wild-type cell line, preferably the wild-type cell line is selected from any one or more of BEAS-2B cells, GM12878 cells and Hela cells.
3. The method of claim 1 or 2, wherein the predetermined concentration is 4 × 106～5×106cells/mL.
4. The method of claim 1, wherein the FFPE reference is an FFPE positive reference, the target cell line is a wild-type cell line and a mutant cell line,
preferably, the wild type cell line is selected from any one or more of BEAS-2B cells, GM12878 cells and Hela cells;
preferably the mutant cell line is selected from any one or more of SW48, H1650, KYSE450 and H1975.
5. The method of claim 4, wherein the culturing the cells of the target cell line to a predetermined concentration, and then collecting the cells and formalin-fixing the cells to make paraffin blocks comprises:
diluting cells of the wild type cell line and the mutant cell line respectively to obtain diluted wild type cells and diluted mutant cells;
culturing the diluted wild-type cells and the diluted mutant cells to the predetermined concentrations, respectively;
mixing the wild type cells and the mutant type cells reaching the predetermined concentration to obtain mixed cells;
the mixed cells were collected and formalin-fixed, thereby preparing the paraffin block.
6. The method according to claim 5, wherein the concentration of the diluted wild-type cell and the diluted mutant-type cell is 0.5 × 103～5×103cells/mL；
Preferably, the predetermined concentration is 1.5 × 106～5×106cells/mL, more preferably 2 × 106～3×106cells/mL；
Preferably, the wild-type cell and the mutant cell are mixed at a set mutation frequency.
7. The method of claim 1, wherein the method further comprises a step of verifying the genetic variation of the target cell line, preferably NGS or ddPCR, before selecting the target cell line.
8. The preparation method according to claim 1, wherein the paraffin block is sliced to obtain a paraffin roll with a thickness of 8-12 μm, and the paraffin roll is the FFPE reference product.
9. An FFPE reference substance is characterized in that the FFPE reference substance is a paraffin embedded section.
10. The FFPE reference product of claim 9, wherein the paraffin-embedded section is a cell paraffin-embedded section;
preferably, the thickness of the paraffin-embedded section is 8-12 μm;
preferably, the FFPE reference is an FFPE negative reference, and the paraffin-embedded section is a paraffin-embedded section of a wild-type cell;
preferably, the FFPE reference is an FFPE positive reference, and the paraffin-embedded section is a paraffin-embedded section in which wild-type cells and mutant cells are mixed.
11. An FFPE sample detection kit comprising the FFPE reference of claim 9 or 10, or the FFPE reference prepared by the preparation method of any one of claims 1 to 8.
12. The FFPE reference product prepared by the preparation method of any one of claims 1 to 8, or the FFPE reference product of claim 9 or 10, for use in development of an FFPE sample mutation detection product.
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