Multiplexed immunohistochemistry staining and digital pathology analysis dual-process quality control

Abstract

Provided herein is a multiplexed immunohistochemistry staining and digital pathology analysis dual-process quality control product, comprising a formalin-fixed paraffin-embedded (FFPE) tonsil sample, and a cell dispersion zone located in the center of the FFPE tonsil sample, the cell dispersion zone comprising dispersed PBMCs embedded with agarose gel and embedded with paraffin. Also provided herein are methods of preparing and using the quality control product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of life science and medical research, in particular, to a pathological tissue section multiplex immunohistochemical staining and digital pathology analysis double-process quality control product, and a preparation method and application thereof. BACKGROUND

[0002] Immunotherapy plays an increasingly important role in anti-tumor. Tumor microenvironment (TME) is closely related to the efficacy and prognosis of tumor immunotherapy. TME is composed of tumor cells, stromal cells and a large number of complex immune cells. The characterization of TME can be used to screen immunotherapy response patients, predict the efficacy and prognosis of immunotherapy, and develop clinical treatment programs, etc. For example, the ImmunoScore method calculated by the density of CD3 + and CD8 + T cells in the invasive margin and tumor center can be used to predict the risk of postoperative recurrence of patients, and assist in the postoperative chemotherapy decision-making process of early-stage colorectal cancer patients.

[0003] The characterization of TME not only includes the number and distribution of immune cells, but also involves the spatial distance between cells. For example, the closer the distance between CD8 + T cells and tumor cells, the better the prognosis of liver cancer patients; the distance between B cells and CD8+ T cells and M2 type macrophages is related to the response rate of liver cancer to cabozantinib and nivolumab neoadjuvant therapy. Multi-target positive and cell spatial distance need to be assisted by multiple immunohistochemistry (mIHC) technology. Studies have shown that compared with PD-L1 IHC, TMB, GEP methods, mIHC multi-target detection can more accurately predict the response of patients to anti-PD-1 / PD-L1 therapy (JAMA Oncol, 2019). The application of mIHC in tumor clinical diagnosis is attracting more and more attention.

[0004] Digital pathology analysis applies digital image processing and computer technology to analyze and diagnose based on tissue sections and cytological images. In tumor clinical diagnosis, digital pathology analysis plays an important role, enabling pathologists to more accurately, efficiently identify, classify and evaluate tumor tissues. After mIHC staining, digital images are generated by a scanner, and digital pathology analysis methods are used to analyze the number and spatial distribution of immune cells, which can more quickly, accurately and richly characterize TME, greatly promoting the application of mIHC in scientific research and clinical practice.

[0005] mIHC staining results are influenced by many factors, such as the conditions of antigen retrieval, the selection and use of antibodies, the conditions of color development reaction, and the consistency of operation, etc. Even minor adjustments can lead to differences in results. In addition, some tissue materials that can be suitable as mIHC staining process quality control products do not meet the subsequent needs as quality control products for digital pathology analysis due to their heterogeneity and irreproducibility.

[0006] Therefore, there is an urgent need to develop a dual-process quality control product suitable for quality control of mIHC staining and subsequent digital pathology analysis. SUMMARY

[0007] In one aspect, provided herein is a multiplexed immunohistochemistry staining and digital pathology analysis dual-process quality control product, comprising a formalin-fixed paraffin-embedded (FFPE) tonsil sample, and a cell dispersion zone located in the center of the FFPE tonsil sample, the cell dispersion zone comprising dispersed peripheral blood mononuclear cells (PBMCs) embedded with agarose gel and embedded with paraffin.

[0008] In another aspect, provided herein is a method for preparing a multiplexed immunohistochemistry staining and digital pathology analysis dual-process quality control product, comprising:

[0009] (a) subjecting peripheral blood mononuclear cells (PBMCs) isolated in vitro to a cell fixation treatment;

[0010] (b) preparing a PBMC suspension from the cell fixation-treated PBMCs;

[0011] (c) adding melted agarose gel to the PBMC suspension to prepare a PBMC-containing agarose gel, and performing a dehydration treatment after the gel is solidified;

[0012] (d) providing a formalin-fixed paraffin-embedded (FFPE) tonsil sample, removing a tissue block at the center position thereof to form a cavity;

[0013] (e) placing the dehydrated PBMC agarose gel into the cavity, and performing paraffin embedding thereon;

[0014] (f) sectioning the resulting sample to form the quality control product.

[0015] In another aspect, provided herein is a method for using the quality control product in a multiplexed immunohistochemistry staining process combined with a digital pathology analysis process, comprising:

[0016] subjecting the quality control product to multiplexed immunohistochemistry staining together with a test piece;

[0017] The multiplex immunohistochemistry staining process is evaluated according to the tonsil staining results of the quality control product. If the distribution of positive cells in the tonsil tissue of the quality control product meets the expectation, the multiplex immunohistochemistry staining process is evaluated as qualified.

[0018] The density or percentage of each positive cell in the cell dispersion area is analyzed. If the result is within the specified range of the reference value of the quality control product (e.g. within ± 30% of the reference value), the digital pathology analysis process is evaluated as qualified.

[0019] In another aspect, the present disclosure provides use of the quality control product in a multiplex immunohistochemistry staining combined with digital pathology analysis process. BRIEF DESCRIPTION OF DRAWINGS

[0020] The present disclosure will be further described with reference to the drawings, which are presented herein for illustration purposes only and are not intended to limit the scope of the present disclosure.

[0021] Figure 1 Photographs showing the agarose gel embedded with PBMC cells (left) and the tonsil / PBMC quality control wax block (right) prepared according to one of the embodiments described herein.

[0022] Figure 2 Photograph showing a quality control slide stained by mIHC CD3 / CD4 / CD8 / PD-L1 target combination according to one of the embodiments described herein.

[0023] Figure 3 Photograph showing the quality control results of the mIHC staining process according to one of the embodiments described herein. DETAILED DESCRIPTION

[0024] In the present disclosure, the meanings of technical terms are consistent with the common understanding of those skilled in the art, unless otherwise specified. In the present disclosure, “one” or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In the present disclosure, when multiple numerical values, numerical value ranges, or combinations thereof are given to describe the same parameter or variable, it is equivalent to specifically disclosing these numerical values, range end values, and numerical value ranges formed by any combination thereof. In the present disclosure, any numerical value, whether or not modified by words such as “about”, always covers the approximate range that can be understood by those skilled in the art, for example, plus or minus 10%, 5%, etc. In the present disclosure, each “embodiment” equally refers to and covers the embodiments of the methods and systems disclosed in the present disclosure. In the present disclosure, one or more technical features in any embodiment can be freely combined with one or more technical features in any one or more other embodiments, and the embodiments thus obtained also belong to the disclosure.

[0025] Multiple immunohistochemistry (mIHC) is a tissue detection technique developed from standard IHC for detecting single target protein, which can achieve simultaneous detection of multiple targets in situ on one slice. The mIHC technology based on tyramide signal amplification (TSA) has become a powerful tool for studying the biology of complex diseases. The basic working principle is that the primary antibody is first combined with the antigen, and then the secondary antibody with horseradish peroxidase (HRP) is combined with the primary antibody; HRP catalyzes the addition of tyramide fluorescein substrate to the reaction system to produce activated fluorescent substrate; the activated substrate can be covalently combined with tyrosine residues on the antigen, so that the tyramide fluorescein is stably covalently combined on the sample; then the non-covalently combined primary antibody-secondary antibody-HRP complex is washed away by heat repair method; the second round of incubation is repeated with another kind of primary antibody-secondary antibody-HRP, and another tyramide fluorescein substrate is used to label the protein, and so on, to achieve multiple labeling. The TSA method can currently achieve simultaneous labeling of up to 8 targets on the same tissue section. This method has stable fluorescence labeling, high detection sensitivity, and no antibody species limitation.

[0026] The combined application of mIHC and digital pathology analysis will have unique technical advantages in pathological analysis, which can achieve multiple sample fluorescence imaging and obtain high signal-to-noise ratio images to assist accurate multiple quantification, location and morphology analysis. However, the combined application is still limited by the lack of a universal quality control product suitable for the double-process quality control of mIHC combined with digital pathology analysis. The development and application of such a quality control product can provide guidance for accurate pathological analysis interpretation, which is of great significance for accurate diagnosis and treatment of diseases.

[0027] After long-term exploration and research, the inventors have developed a quality control product suitable for the above double-process.

[0028] In one aspect, provided herein is a multiple immunohistochemistry staining and digital pathology analysis double-process quality control product, which comprises a formalin-fixed paraffin-embedded (FFPE) tonsil sample, and a cell dispersion zone located in the center of the FFPE tonsil sample, the cell dispersion zone comprising dispersed peripheral blood mononuclear cells (PBMCs) embedded in agarose gel and embedded in paraffin.

[0029] In some embodiments, the quality control product is a section. In some embodiments, the quality control product is a section with a thickness of 2-8 μm (e.g., 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, or a range formed by any two of the above values as endpoints).

[0030] In some specific embodiments, the tonsil sample is a FFPE tonsil sample with normal detection results after mIHC antibody combination staining experiment.

[0031] As used herein, "FFPE tonsil sample" and "FFPE sample" are used interchangeably to refer to a sample of a tonsil that has been processed using formalin fixation paraffin embedding (FFPE).

[0032] In some embodiments, the tonsil is a tonsil having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) intact tissue periphery.

[0033] The cell dispersion zone comprising dispersed PBMCs embedded in agarose gel and embedded in paraffin is centrally located in the FFPE tonsil sample.

[0034] As used herein, "centrally located in the FFPE tonsil sample" refers to being located in the FFPE tonsil sample in a transverse plane that is perpendicular to the line of sight from which the FFPE sample is expected to be viewed (e.g., a plane parallel to the plane of the FFPE sample section), and in a cross-section taken in that transverse plane.

[0035] In some embodiments, the PBMCs used to prepare the cell dispersion zone have a nucleated cell rate of > 90%.

[0036] In some embodiments, the PBMCs used to prepare the cell dispersion zone have a viability of > 90%.

[0037] In some embodiments, the PBMCs are human PBMCs.

[0038] In some embodiments, the cell dispersion zone can optionally include one or more exogenous positive cells incorporated therein.

[0039] In some embodiments, the plurality of PBMCs has a concentration of 1 x 10 7 cells / mL to 1 x 10 8 cells / mL, e.g., 1 x 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 , 1 x 10 8 cells / mL, or a range formed by any two of these values as endpoints.

[0040] In some embodiments, a cell dispersion zone in the center of the FFPE sample does not contain tonsil tissue in the FFPE sample in the longitudinal direction, with the longitudinal direction being the direction perpendicular to the line of sight intended for viewing the FFPE sample (e.g., the direction perpendicular to the FFPE sample section). In some embodiments, the cell dispersion zone does not overlap with tonsil tissue in the FFPE sample in the longitudinal direction. In some embodiments, the cell dispersion zone is present in a manner that replaces an equivalent size region in the center of the FFPE sample. In one specific embodiment, the cell dispersion zone is formed by removing (e.g., removing or scooping out) the tissue in the center of the FFPE sample, forming a cavity, and filling the cavity with the agarose gel having PBMCs dispersed therein.

[0041] In some embodiments, the cell dispersion zone is surrounded by tonsil tissue in the FFPE sample. In some embodiments, at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the perimeter of the cell dispersion zone is surrounded by tonsil tissue in the FFPE sample.

[0042] In some embodiments, in a cross-sectional plane that is perpendicular to the line of sight intended for viewing the FFPE sample (e.g., a plane parallel to the FFPE sample section), the area ratio of the cell dispersion zone to the surrounding tonsil tissue is 1:4 to 1:10 (e.g., 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or a range formed by any two of these values as endpoints).

[0043] In some embodiments, in a cross-sectional plane that is perpendicular to the line of sight intended for viewing the FFPE sample (e.g., a plane parallel to the FFPE sample section), the area of the cell dispersion zone does not exceed at least 90% of the outer perimeter of the surrounding tonsil tissue, e.g., does not exceed the outer perimeter of the surrounding tonsil tissue.

[0044] In another aspect, provided herein is a method of preparing a multiplexed immunohistochemical staining and digital pathology analysis dual-process quality control article, comprising:

[0045] (a) subjecting an in-vitro isolated peripheral blood mononuclear cell (PBMC) to a cell fixation treatment;

[0046] (b) preparing a PBMC suspension from the cell fixation treated PBMC;

[0047] (c) adding a melted agarose gel to the PBMC suspension to prepare a PBMC-containing agarose gel, and subjecting the gel to a dehydration treatment after the gel solidifies;

[0048] (d) providing a formalin-fixed paraffin-embedded (FFPE) tonsil sample, removing a tissue block from a central location thereof, forming a cavity;

[0049] (e) placing the dehydrated PBMC agarose gel into the cavity and paraffin-embedding it;

[0050] (f) sectioning the resulting sample to form the quality control.

[0051] In one embodiment, the PBMCs are isolated in vitro from collected blood, e.g., human blood. In some embodiments, the PBMCs are isolated using lymphoprep.

[0052] In some embodiments, the PBMCs have a nucleated cell rate of > 90%.

[0053] In some embodiments, the PBMCs have a cell viability of > 90%.

[0054] In some embodiments, the method further comprises, prior to step (a), detecting the nucleated cell rate and viability of the PBMCs. In one particular embodiment, the method comprises detecting the viability and nucleated cell rate of the plurality of peripheral blood mononuclear cells (PBMCs) isolated in vitro using trypan blue and AO / PI staining solution prior to subjecting the cells to cell fixation.

[0055] In some embodiments, the PBMCs are human PBMCs.

[0056] In some embodiments, the PBMCs optionally have one or more exogenous positive cells incorporated therein.

[0057] In some embodiments, the PBMCs are fixed using a cell fixative. Examples of cell fixatives include, but are not limited to, 4% paraformaldehyde.

[0058] In some embodiments, the fixation of the PBMCs is performed at room temperature, e.g., 20 to 25 °C.

[0059] In some embodiments, the concentration of the PBMCs is 1 x 10 7 cells / mL to 1 x 10 8 cells / mL, e.g., 1 x 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 , 1 x 10 8about 1 x 106cells / mL, or a range formed with any two of the listed values as end points.

[0060] In some embodiments, step (b) comprises suspending the cell-fixed PBMCs in phosphate buffered saline (PBS).

[0061] In some embodiments, step (c) comprises adding the melted agarose gel to the PBMC suspension at a volume ratio of 1 :0.8 to 1 : 1.2 (e.g., 1 : 1). In some embodiments, the melted agarose gel is obtained by heating an agarose gel. In some embodiments, the concentration of agarose in the melted agarose gel is 2-4% (e.g., 2%, 3%, or 4%). In some embodiments, the agarose is low-melting agarose. "Low-melting agarose" generally refers to agarose that has been modified to have a lower gelation temperature and a lower melting point. For example, low-melting agarose has hydroxyethyl, methoxy, or the like groups introduced into its polysaccharide chains. The melting point of low-melting agarose is about 65°C (e.g., a melting point of no higher than 70°C, no higher than 68°C, or no higher than 65°C), and can gel at about 30°C.

[0062] In some embodiments, step (c) further comprises pouring the mixture of agarose and PBMC suspension into a container (e.g., a plastic tubular container) having a shape, and allowing it to cool to prepare a PBMC-containing agarose gel. The shape of the container can be determined based on the volume of the mixture, and the shape of the tissue block to be removed from the FFPE sample. In some embodiments, the PBMC-containing agarose gel formed is removed from the container after dehydrating the PBMC-containing agarose gel.

[0063] In some embodiments, step (d) comprises removing a volume and shape of tissue block from a central location of the FFPE tonsil sample. The volume and shape of the tissue block or cavity can match the volume and shape of the dehydrated PBMC-containing agarose gel.

[0064] In some embodiments, in a cross-sectional plane that is perpendicular to the line of sight direction in which the FFPE sample is intended to be viewed (e.g., a plane parallel to the plane of the FFPE sample section), the ratio of the area of the removed tonsil tissue block to the surrounding tonsil tissue is 1 :4 to 1 : 10 (e.g., 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, or a range formed with any two of the listed values as end points). In some embodiments, in a cross-sectional plane that is perpendicular to the line of sight direction in which the FFPE sample is intended to be viewed (e.g., a plane parallel to the plane of the FFPE sample section), the removed tonsil tissue block does not exceed at least 70% of the outer perimeter of the tonsil tissue, e.g., does not exceed the outer edge of the tonsil tissue.

[0065] In some embodiments, the thickness of the quality control section obtained in step (f) is 2-8 pm (e.g., 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, or a range formed by any two of these values as endpoints).

[0066] The resulting quality control section comprises a FFPE tonsil sample, and a cell dispersion zone located in the center thereof.

[0067] In some embodiments, with the line of sight direction in which the FFPE sample is expected to be observed (e.g., the direction perpendicular to the FFPE sample section) as the longitudinal direction, the cell dispersion zone located in the center of the FFPE sample does not contain tonsil tissue in the FFPE sample in the longitudinal direction. In some embodiments, the cell dispersion zone does not overlap with tonsil tissue in the FFPE sample in the longitudinal direction. In some embodiments, the cell dispersion zone is present in a manner to replace a same size area in the center of the FFPE sample. In one specific embodiment, the cell dispersion zone is formed by removing the tissue in the center of the FFPE sample, forming a cavity, and filling the cavity with dispersed PBMCs (e.g., dispersed PBMCs embedded with agarose gel and embedded with paraffin).

[0068] In some embodiments, the cell dispersion zone is surrounded by tonsil tissue in the FFPE sample. In some embodiments, at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the perimeter of the cell dispersion zone is surrounded by tonsil tissue in the FFPE sample.

[0069] In some embodiments, the method further comprises determining a reference value for the density or percentage of positive cells of the quality control. The reference value for the density or percentage can be determined based on the average of multiple sections of the quality control obtained by serial sectioning.

[0070] In some embodiments, the method further optionally comprises step (g): detection of the quality control.

[0071] In one specific exemplary embodiment, the detection of the quality control can comprise:

[0072] selecting multiple (e.g., 9) sections of the quality control by serial sectioning, dividing the sections into at least 3 groups (e.g., 3 sections / group), and performing mIHC by at least two detectors;

[0073] determining the distribution of one or more positive cells on the tonsil in the mIHC staining results;

[0074] determining the density or percentage of one or more positive cells in the cell dispersion zone;

[0075] Selecting a quality control sample with correct distribution of one or more positive cells in tonsil tissue area and one or more positive cells in cell dispersion area, and calculating the reference value of the density or percentage of positive cells in the quality control sample.

[0076] In another aspect, the present disclosure provides a method of using the quality control sample in a multiplexed immunohistochemistry staining process combined with a digital pathology analysis process, comprising:

[0077] Performing multiplexed immunohistochemistry staining on the quality control sample together with the test slides;

[0078] Evaluating the multiplexed immunohistochemistry staining process according to the staining results of the tonsil tissue in the quality control sample, wherein if the distribution of positive cells in the tonsil tissue in the quality control sample is as expected, the multiplexed immunohistochemistry staining process is evaluated as qualified.

[0079] Analyzing the density or percentage of each positive cell in the cell dispersion area, wherein if the result is within a specified range (e.g., ±30% of the reference value) of the reference value of the quality control sample, the digital pathology analysis process is evaluated as qualified.

[0080] In another aspect, the present disclosure provides the use of the quality control sample in a multiplexed immunohistochemistry staining process combined with a digital pathology analysis process.

[0081] The tissues and cells used in the quality control sample described herein are derived from humans, and the target protein expression is similar to that of the test sample, thus being more suitable than cell lines as a quality control sample for a pathology analysis process. Some cell lines are cancerous cells themselves, and the protein expression is different from that of normal cells of the same type in vivo; and even if normal cells are used, their genes will change due to long-term culture.

[0082] The quality control sample described herein includes a tonsil tissue area and a PBMC cell area, which matches most immune cells found in TME, and can be used as a quality control sample for mIHC combined staining of multiple target immune cells. If a cell line is used as a reference, different expression levels of cells or cell lines need to be screened for each target, and it is difficult to quickly adjust to meet the needs of changes in mIHC target combinations.

[0083] The specific distribution of immune cells in tonsil tissue makes the mIHC staining results suitable for quality control experimental procedures, but the number of positive cells will vary between each section of the tonsil sample, and due to the close distribution of tissue cells, cell overlap leads to inaccurate cell counting, thus being unsuitable for quality control of digital pathology analysis processes. The dispersed distribution of cells in the cell dispersion area is stable in terms of cell composition between sections, thus meeting the need for accurate cell counting, and being suitable as a quality control sample for digital pathology analysis.

[0084] Because the cell mass is small, and the surrounding cells in the staining process will be due to flushing and other factors caused by the unwanted edge effect, further lead to uneven staining, or lead to cell mass damage and other impact on the results of subsequent digital pathology analysis. And the cell dispersion zone embedded in the tonsil tissue, greatly reduces this edge effect.

[0085] Embodiments

[0086] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not to limit the scope of the application. Those skilled in the art can make appropriate modifications and changes to the application, and these modifications and changes are within the scope of the application.

[0087] 1. Quality control preparation process

[0088] 1.1 PBMC separation and fixation: collect 10-20ml of human blood collected with EDTA collection tube, separate PBMC cells with lymph separation liquid;

[0089] 1.2 PBMC is washed with PBS, then stained with trypan blue and AO / PI staining solution (ThermoFisher, A49905) to detect cell activity and cell nuclear rate, both of which should be ≥90%;

[0090] 1.3 After centrifugation of PBMC at 800g for 5 minutes, remove the supernatant, and fix with 1mL cell fixation solution (4% paraformaldehyde) at room temperature for 1 hour;

[0091] 1.4 Resuspend with appropriate amount of PBS, determine the cell concentration, and the cell concentration is about 5×10 6 / mL.

[0092] 2. FFPE tonsil samples

[0093] The FFPE tonsil sample which has passed the mIHC antibody combination staining experiment is used.

[0094] 3. PBMC embedding

[0095] 3.1 Resuspend PBMC in PBS at a certain concentration (8×10 7 / mL);

[0096] 3.2 Add an equal volume of melted 2% low-melting-point agarose gel, and mix gently;

[0097] 3.3 Pour the mixed solution into a plastic tube and cool and solidify;

[0098] 3.4 After removing the agarose gel strip, dehydrate;

[0099] 3.5 A cylindrical hole was drilled in the center of the FFPE tonsil sample using a tissue puncher. The wax block was melted and the tissue was removed. The dehydrated cell agarose block was placed into the hole of the tonsil and re-embedded in paraffin;

[0100] 3.6 Sections were cut at 3-5um thickness and made into quality control slides.

[0101] Figure 1 Photographs of the prepared agarose gel embedded with PBMC cells (left panel) and tonsil / PBMC quality control wax block (right panel) are shown.

[0102] 4. Quality control slide detection

[0103] 4.1 Nine sections were cut from the quality control slide and divided into 3 groups for mIHC by operators A and B, respectively. Operator A did 1 group and operator B did 1 group in two time periods.

[0104] 4.2 The positive cells in the mIHC staining results were distributed correctly on the tonsil.

[0105] 4.3 The density of each positive cell in the PBMC cell cluster area of the tonsil was analyzed using the analysis module of the HighPlex IHC-FL of the Indica Labs HALO analysis software. Intra-batch precision analysis was performed within each group. Inter-person and inter-day precision analysis was performed for the results of operators A and B. The CV values of intra-batch, inter-person and inter-day precision should be less than a specified value (e.g. < 30%).

[0106] 4.4 The average value of the positive cell density values of the 9 quality control slides was set as the reference value of the positive cell density of the quality control.

[0107] The quality control slide stained by mIHC CD3 / CD4 / CD8 / PD-L1 combination is shown in Figure 2 The tonsil / PBMC quality control slide was scanned after mIHC CD3 / CD4 / CD8 / PD-L1 combination staining to show the staining effect of the tonsil and the 4-color target staining results of the cell cluster in the tissue.

[0108] The analysis results are shown in Table 1.

[0109] Table 1. Precision analysis of CD3 / CD4 / CD8 / PD-L1 target staining.

[0110]

[0111]

[0112] The analysis was performed as described in 4.3 above, and the intra-batch, inter-person and inter-day precision were all < 30%.

[0113] The average value of the density of the positive cells in the 9 quality control slices was taken as the reference value of the positive cell density of the quality control. The reference value of the positive cell density of the quality control is shown in Table 2.

[0114] Table 2. Reference value of positive cell density of quality control

[0115]

[0116] 5. Application of quality control slides in clinical sample mIHC detection

[0117] 5.1 The quality control slices were subjected to mIHC staining together with the test slices.

[0118] 5.2 After the staining, the mIHC staining process was evaluated according to the staining results of the tonsil in the quality control slices.

[0119] 5.3 The density values of the positive cells in the cell clusters in the tonsil were analyzed.

[0120] 5.4 If the results were within the reference value of the quality control, such as ± 30%, the digital pathology analysis process was determined to be qualified.

[0121] The quality control results of the mIHC staining process are shown in Figure 3 The quality control slices were subjected to mIHC staining together with the test samples, and the distribution of the positive cells on the tonsil was observed after scanning the mIHC staining: (1) PD-L1 had weak staining in the germinal center, and showed strong positive staining in the stratified epithelial cells; (2) CD3 positive cells were mainly distributed in the T cell area and lymphoid follicles; (3) CD4 positive cells were mainly distributed in the T cell area and lymphoid follicles; (4) CD8 positive cells were mainly present in the T cell area, around the lymphoid follicles and in the marginal zone. The distribution of the positive cells was consistent with the expectation, and the experimental process was qualified.

[0122] The quality control results of the digital pathology analysis process are shown in Table 3.

[0123] Table 3. Quality control results of the analysis process

[0124]

[0125] The same digital pathology analysis process Indica Labs HighPlex IHC-FL was used for the quality control and the test samples. The positive fluorescence threshold was determined by an experienced pathologist, and the other analysis parameters were consistent. The density values of the positive cells in the cell clusters in the quality control were analyzed. The CV value was ≤ 30%, which met the expected requirements, and the digital pathology analysis process was qualified.

[0126] The tissues and cells used to prepare the double-process quality control product are derived from humans, and the target protein expression is similar to that of the detection sample, thus being more suitable than cell lines as a process quality control product for pathological analysis. Some cell lines are cancerous cells themselves, and the protein expression is different from that of normal cells of the same type in vivo; and even if normal cells are used, the genes will change due to long-term culture.

[0127] The prepared double-process quality control product includes a tonsil tissue area and a PBMC cell dispersion area, which matches most of the immune cells found in the TME, and thus can be used as a quality control product for the combined staining of a variety of mIHC target immune cells. If a cell line is used as a reference, different expression levels of cells or cell lines need to be screened for each target, and it is difficult to quickly adjust to meet the needs of changes in the mIHC target combination.

[0128] The prepared double-process quality control product, which includes a cell dispersion area with stable composition and meets the needs of accurate cell counting, overcomes the problem of differences in the number of positive cells between each section of the tonsil sample, and the problem of inaccurate cell counting due to the close and overlapping distribution of tissue cells, and meets the needs of process quality control for digital pathological analysis.

[0129] The prepared double-process quality control product greatly reduces or avoids the occurrence of edge effects, and the staining is uniform, ensuring accurate results for subsequent digital pathological analysis.

Claims

1. A dual-process quality control for multiplex immunohistochemical staining and digital pathology analysis, comprising a formalin-fixed paraffin-embedded tonsil sample, and a cell dispersion zone located in the center of the formalin-fixed paraffin-embedded tonsil sample, the cell dispersion zone comprising dispersed peripheral blood mononuclear cells embedded with agarose gel and embedded with paraffin, wherein the formalin-fixed paraffin-embedded tonsil sample can be stained, wherein the tonsil sample is used for multiplex immunohistochemical staining process evaluation, and the cell dispersion zone is used for digital pathology analysis process evaluation.

2. The quality control according to claim 1, wherein, The quality control is a section.

3. The quality control according to claim 1, wherein, The quality control is a section with a thickness of 2-8 μm.

4. The quality control according to claim 1, wherein, The tonsil is a formalin-fixed paraffin-embedded tonsil with at least 70% intact tissue margin.

5. The quality control according to claim 1, wherein, The peripheral blood mononuclear cells used to prepare the cell dispersion zone have a nucleated cell rate of ≥ 90%, and the peripheral blood mononuclear cells used to prepare the cell dispersion zone have a viability of ≥ 90%.

6. The quality control according to claim 1, wherein, The concentration of the peripheral blood mononuclear cells is 1 x 10 7 cells / mL to 1 x 10 8 cells / mL.

7. A method for preparing the dual-process quality control for multiplex immunohistochemical staining and digital pathology analysis according to claim 1, comprising: (a) subjecting in vitro isolated peripheral blood mononuclear cells to cell fixation treatment; (b) preparing a peripheral blood mononuclear cell suspension from the cell fixation treated peripheral blood mononuclear cells; (c) adding melted agarose gel to the peripheral blood mononuclear cell suspension to prepare a peripheral blood mononuclear cell-containing agarose gel, and subjecting the gel to dehydration treatment after solidification; (d) providing a formalin-fixed paraffin-embedded tonsil sample, removing a tissue block in the center to form a cavity; (e) placing the dehydrated peripheral blood mononuclear cell-containing agarose gel into the cavity, and embedding it with paraffin; (f) sectioning the resulting sample to form the quality control.

8. The method of claim 7, wherein, Step (c) comprises adding melted agarose gel to the peripheral blood mononuclear cell suspension at a volume ratio of 1:0.8-1:1.

2.

9. The method of claim 7, wherein, Step (c) comprises adding melted agarose gel to the peripheral blood mononuclear cell suspension at a volume ratio of 1:

1.

10. The method of claim 8 or 9, wherein, The agarose concentration in the melted agarose gel is 2-4%.

11. The method of claim 8 or 9, wherein, The agarose gel is a low-melting-point agarose gel.

12. The method of claim 7, wherein, The ratio of the area of the removed tonsil tissue block to the surrounding tonsil tissue in the cross-section of the transverse plane, which is perpendicular to the line of sight direction for expected observation of the formalin-fixed paraffin-embedded tonsil sample, is 1:4-1:

10.

13. A method for using the quality control according to claim 1 in a multiplex immunohistochemical staining process combined with a digital pathology analysis process, comprising: subjecting the quality control to multiplex immunohistochemical staining together with a detection sheet; evaluating the multiplex immunohistochemical staining process according to the staining results of the tonsil in the quality control, wherein if the distribution of positive cells in the tonsil tissue in the quality control is as expected, the multiplex immunohistochemical staining process is evaluated as qualified; analyzing the density or percentage of each positive cell in the cell dispersion zone, wherein if the results are within the specified range of the reference value of the quality control, the digital pathology analysis process is evaluated as qualified.

14. The method of claim 13, wherein, The specified range of the reference value of the quality control is the reference value ± 30%.

15. Use of the quality control of claim 1 in a multiplexed immunohistochemical staining combined with digital pathology analysis process.

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