TW202115373A - Process record slide for staining and method of using the same - Google Patents

Abstract

Described herein is a process record slide for staining. The process record slide includes a detection area for mounting a sample and a control area including one or more control targets. Also described herein is a method of using the process record slide in an immunohistochemical (IHC) or an immunochemistry (ICC) staining process.

Description

Glass slide for staining processing operation record and its use method

The present invention relates to a processing operation recording glass slide and a method of using the same, in particular to a processing operation recording glass slide for dyeing and a method of using the same.

Like other immunochemical methods, immunohistochemistry is a multi-step procedure. The above method includes a series of steps such as reagent exchange, culturing and washing. These procedures must be performed by skilled personnel and usually require the use of automated dyeing machines. The test results of different laboratories or institutions may vary greatly. The reason is that the quality control standards of the processing operations are different, or the quality control standards are lacking. The processed slides must be diagnosed and interpreted by a pathologist by subjective analysis, but in most cases, the pathologist is not involved in the staining operation.

For immunohistochemistry and other immunochemical methods, both automatic and manual procedures include steps that should be paid special attention to. For example, between two successive reagent application steps, on the one hand, the sample must be cleanly washed to remove unbound antibodies to prevent the residue from being amplified, and on the other hand, excess liquid must be removed to prevent the previous reagent from being carried in and / Or improperly dilute the next reagent, but do not dry out the sample. All the above procedures must be performed carefully to prevent the sample on the slide from being lost or damaged. In addition, although a sufficient amount of antibody reagent must be applied to cover a specific area of the slide, the reagent is expensive, so waste should be avoided as much as possible.

Furthermore, the reagents used in immunohistochemistry and other immunochemical methods (such as enzyme solutions and peroxidase color reagents) are not stable at working temperature or room temperature, so they must be prepared repeatedly. In addition, the phenomenon of non-specific antibody binding that leads to erroneous results remains to be resolved.

Generally speaking, immunohistochemistry (IHC) staining methods are used to assess whether there are specific antigenic sites in patient tissue sections. The IHC method requires subjective interpretation of the staining density in the tissue section to determine the diagnostic level of abnormal or cancerous conditions. In this process, it is usually assumed that the IHC processing operation can function correctly, and it is assumed that the tissue section will be marked by the chromogen markers that are used to distinguish abnormal or cancer conditions and are visible, but often because of the failure of antigen retrieval, Unrecognizable dyed products are produced due to errors of laboratory technicians or errors in the dyeing process. If the above situation occurs, the effect of the IHC method is the same as that of the hematoxylin-eosin (H&E) staining method. In addition, ineffective staining or staining errors may also lead to misdiagnosis, thus affecting subsequent treatment.

Methods and reagents that can improve staining results and reduce the frequency of reagent preparation may be beneficial to manual and automated immunohistochemistry. Many improved methods may be immediately applicable to related immunochemical methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence analysis and in situ hybridization.

From the above description, we can see that it is really difficult to eliminate the errors in immunohistochemistry and other immunochemistry methods. Therefore, the industry has developed a variety of methods to reveal the existence and/or degree of errors. Some of these methods and their limitations will be explained below.

Please refer to "Use of cultured cells as a control for quantitative immunocytochemical analysis of estrogen receptor in breast cancer: the Quicgel method" )” (the full text of which is incorporated into this article by reference). According to this reference, changes in tissue fixation, processing, and staining are the main reasons why the results of estrogen receptor (ER) immunohistochemical analysis are difficult to reproduce. The MCF-7 cell pellets with known ER content, frozen and suspended in Agar, were added to 55 Invasive Breast Tumor (IBC) samples as a control group. Use image analysis to determine the percentage of positive areas of MCF-7 cells and IBC (the percentage of positive nuclei in all analyzed nuclei) and the positive staining ratio (the sum of the optical density of the positive nucleus area divided by the sum of the optical density of all the analyzed nuclei ). According to the results of dextran-coated carbon analysis, the average ER in MCF-7 cells is 150 fmol/mg. The MCF-7 cell image analysis results of the 55 samples showed that the average percentage of positive areas was 70.81%. The positive staining ratio of IBC samples is between 0 and 98.5%. Then, using the known ER content, the percentage of positive area of MCF-7 cells and a conversion factor, the percentage of positive area of the clinical sample is converted into femtomole (10 ^-15 mol) equivalent. The femtomolar equivalent of the 55 IBC samples is between 0 and 1,790 (the average is 187). The control group with known ER femtomole equivalent provides internal standards for quality control and ER quantification.

Please refer to Chinese Patent No. 102435728 (the full text of which is incorporated herein by reference). This reference describes a method for preparing and using a positive control substance used to control the quality of immunohistochemistry operations. According to Chinese Patent No. 102435728, the manufacturing method of the control target is to make the glass slide adsorb peptides or proteins with different concentrations that can specifically react with antibodies. After that, the sample tissue is placed on the glass slide, and the conventional immunohistochemistry operation is performed on the glass slide. Since the sample tissue and the control target are subjected to the same staining operation, the results of the immunohistochemistry operation on the tissue can be seen from the staining results of the peptide/protein. Chinese Patent No. 102435728 also explains how to arrange positive control proteins or peptides on the immunohistochemistry slide.

Those with ordinary skills in the field can know that the density of the protein/peptide in the control target is different after evaluating the method of Chinese Patent No. 102435728. The reason for this is that the combination of peptide fragments and dextran polymer depends on the viscosity of the mixed solution, the washing operation used to remove excess peptides on the dextran, and the temperature. In addition, the size of the polymer precipitate varies with the concentration of the water bath, the reaction temperature and the amount of NaOH injected.

Those with ordinary skills in the field will know after evaluating the method of Chinese Patent No. 102435728 that the target with known reactivity (dyeing density) is not easy to manufacture. The reason for this is that the concentration of available peptides in the polymer precipitate cannot be easily obtained. Therefore, the obtained control substance can only be used to determine whether the primary antibody is present.

Those with ordinary skills in the field can know after evaluating the method of Chinese Patent No. 102435728 that although dextran can capture protein as the target of antibody, the captured protein can only provide the determination result of its existence, so , And it is impossible to establish a reference ruler suitable for detection such as digital imaging.

Those with ordinary skills in the field can know after evaluating the method of Chinese Patent No. 102435728 that the proteins/peptides adsorbed on the glass slide often leak out during the antigen retrieval operation. Due to the relatively small size of the slide, the control target is very close to the sample area, and the leaked protein/peptide may migrate into the tissue section, and then combine with the coexisting tissue section and the slide adhesive, resulting in non-specificity Staining results.

Please refer to the literature of Horizon Diagnostics. The structure of the slide provided by this institution is different from that described in Chinese Patent No. 102435728. A cell line cultured from the control target of Horizon Diagnostics slides. These cell lines are genetically engineered so that their DNA includes coding sequences for specific antigen peptides. These genetically modified cell lines are fixed in a tissue block on a glass slide with formalin and embedded in paraffin to form a loose cell slurry. In addition, the glass slide also includes a control target containing non-reactive cells as a negative control group. The method of manufacturing the control target array is to make a plurality of cell groups form a cylindrical core aligned with each other, and then cut a single section of the entire array and place it on a glass slide. The above-mentioned cell lines can be replicated as needed, so they can be regarded as reproducible.

A person with ordinary skills in the field will know after evaluating the slides manufactured by Horizon Diagnostics that since the density change of the reactive antigen in the control target cannot be controlled, these control targets can only be used to determine the presence of antibodies . The reason for this is that it is not easy to control the number of cells or the amount of antigen produced by the cells. The amount of antigen in each control target is therefore different. In addition, the antigen retrieval task received by the control target adds another variable to the amount of antigen in the control target.

After evaluating the slides manufactured by Horizon Diagnostics, those with ordinary skills in the field will know that only mixing the antigen-producing cell line and the control cell line in a specific ratio cannot control the amount of antigen in each cell well. The reason for this is that different cell lines usually have different electrostatic charges. Therefore, different cell lines tend to separate from each other during the cell culture process and grow in different regions. Therefore, it is impossible or impossible to establish a ruler of antigen density.

After evaluating the slides manufactured by Horizon Diagnostics, a person with ordinary skills in the field will know that due to the limited replication period of cell reproduction, the performance of the cell line is not consistent, and the antigen density of the new cell line cannot be guaranteed to be the same as the previous one. The antigen density of a cell line is the same.

After evaluating the slides manufactured by Horizon Diagnostics, a person with ordinary skills in the field will know that the method of manufacturing the Horizon Diagnostics control target is not cost-effective, because the tissue block is constructed, the tissue block is sliced, and the slice is sliced. Steps such as placing on a glass slide are very labor intensive.

Please refer to US Patent Application No. 2016/0274006A1 (the entire text of which is incorporated herein by reference), which describes a method and device that can be used as a control and calibrator when analyzing cells and tissues on microscope slides . The device of US Patent Application No. 2016/0274006A1 includes a moiety (such as a peptide epitope) connected to a particulate object (such as a transparent spherical bead, the size of which is preferably about the same as that of a cell). The quality control part (moiety) is designed to play a role similar to the analyte in the analysis process and produce a positive analysis reaction. The beads are placed on the microscope slide by a novel liquid matrix during the dyeing process, wherein the liquid matrix solidifies when dried, so that the beads are adhered to the microscope slide.

However, the control group and calibrator solution of US Patent Application No. 2016/0274006A1 is not very practical. Since the control target substance is coated on the beads and is sparsely distributed, the stability of the target substance is not good, and the data of the target substance is not easy to capture. In addition, when imaging a single bead, the color resulting from dyeing will gradually change from the top center to the edge, which adds additional variables.

Please refer to US Patent No. 7271008B2 (the entirety of which is incorporated herein by reference), which describes a device and method for determining the quality of reagents used in analytical operations (especially multi-step immunohistochemical analysis tasks). In detail, multiple compounds are attached to the substrate of the device, and each compound can react with one of the reagents used in the analysis operation.

The device of US Patent No. 7271008B2 provides a quality control method for evaluating the staining effect with secondary antibodies. However, the device of US Patent No. 7271008B2 did not undergo the same IHC staining step with the sample. Therefore, the representative of the IHC dyeing operation with this device is very limited. In addition, the base material of the device uses aminosilane, but the aminosilane cannot form a covalent bond that can survive the antigen retrieval operation. Furthermore, the alkaline phosphatase target may be decomposed at the temperature of the antigen retrieval operation. Therefore, although we know that the antigen retrieval operation will produce unknown variables in the IHC staining operation, the device of US Patent No. 7271008B2 cannot be used to monitor the antigen retrieval operation.

In view of the above-mentioned problems of the prior art, this specification uses several embodiments to describe a method for processing work recording slides and staining that can solve these problems. In detail, this manual describes a processing operation documentary slide that allows the patient sample and the control target to coexist and receive staining together. Therefore, when processing the sample, the control target will also be dyed to show whether there is an error in the processing operation and the degree of error (that is, the degree of deviation from the benchmark of the known target). Those skilled in the art should know that changes in the concentration of the staining reagent applied to the sample will affect the subjective analysis. If there are too many primary antibodies, the staining of tissue sections is often not specific. If the antigen retrieval operation is neglected, the effectiveness of the primary antibody, secondary antibody and chromogen reagent will affect the staining density (this density shows the antigen density on the tissue section). The aging of the antibody protein causes the hinge region between the antigen-binding region (Fab region) and the crystallizable region (Fc region) to break, and the Fc region is then separated. Only antibodies with Fab and Fc at the same time can play a role. Many chromogen reagents (which contain precipitable coloring agents) have a very short service life, and some chromogen reagents have a service life of only a few hours. Therefore, chromogen reagents of different ages have different effects, which in turn changes the dyeing density. It can be seen that the effectiveness of the reagent can affect and will definitely affect the staining density of the observed sample. Since the sample and the control target use the same staining reagent, the control target can reflect the changes in the age and dosage of the reagent, thereby providing more objective information for analysis. The processing operation document glass slides in these embodiments can perform effective, correct and accurate quality control for processing operations within a cost-sensitive price range, and therefore are suitable for various IHC and ICC slide glass slides.

The content disclosed below provides a variety of embodiments or examples that implement different features of the subject matter of this case. To simplify the content of the disclosure, specific examples of each component and its configuration will be described below. Of course, these examples are for illustrative purposes only and are not restrictive. For example, in the following, forming the first feature on or above the second feature may include an embodiment in which the first and second features are in direct contact, or may include the first and second features because there are other features in between. Examples without direct contact. In addition, the content of this disclosure may reuse the same reference numerals and/or letters in different examples, and the repeated use is only for brevity, and does not represent the relevance between different embodiments and/or configurations.

Handling of operation record slides

Certain embodiments in this specification are directed to a processing operation record glass slide. In this manual, "process record slide" can also be called "slide", "PRS" (process record slide) or "PRS-IHC". The "IHC" in the term "PRS-IHC" is not used to limit the structure or function of such slides to immunohistochemical (IHC) staining operations. On the contrary, those with ordinary skills in the field should understand that the processing slides described herein can also be used for other staining operations, such as immunocytochemistry (ICC) staining operations.

Some examples in this specification are directed to a processing operation document slide that allows us to easily determine the efficacy of common steps in immunohistochemistry (IHC) or immunocytochemistry (ICC) analysis. These steps include removal of paraffin, antigen retrieval, primary staining, secondary staining, and so on.

Please refer to FIG. 1, in some embodiments, the processing operation recording slide 100 includes a substrate 101, an adhesive layer 103 and one or more control targets 141.

In some embodiments, the substrate 101 includes a glass substrate, a plastic substrate, or a polymer substrate.

In some embodiments, the adhesive layer 103 includes an adhesive. In some embodiments, the adhesive is covalently bonded to the substrate 101, such as a glass substrate.

In some embodiments, the adhesive provides a slightly hydrophilic surface on the glass slide. In some embodiments, the raw material of the adhesive includes end groups exposed to biological materials, and the surface wettability can be adjusted during manufacture. In certain embodiments, the end group includes one or more groups selected from the group consisting of: -ROH, -R(C=O)OH, -RNH ₃ , -R(C=O ) NH ₂ and -RNH ₂ . In some embodiments, the adhesive layer 103 includes an adhesive that is the same as or similar to the adhesive in the coating layer of the Thermo-Fisher SuperFrost glass slide GL4951P.

In some embodiments, the one or more control targets 141 are disposed on the substrate layer through the adhesive layer. In some embodiments, the one or more control targets are deposited in the control area 140 of the slide glass 100.

In some embodiments, the control target 141 includes a compound that can react with one or more reagents in the IHC or ICC operation and produce a readable indication (for example, a readable color). The concentration of the compound in each control target 141 is different. The change in the concentration of the compound can be identifiable after the control target 141 reacts with one or more of the reagents in the IHC or ICC operation, and the difference between the control target 141 can be distinguished.

In some embodiments, the processing operation recording glass slide 100 further includes a protective coating layer 105 covering the whole or a part of the glass slide 100. In some embodiments, the protective coating 105 can seal the control target 141 to isolate it from the external environment. The control target 141 sometimes includes proteins, peptides or other biologically derived targets (see the description below for details). Therefore, the protective coating 105 can protect the control target 141 from being oxidized or attacked by microorganisms.

In some embodiments, the protective coating layer 105 is a paraffin coating layer. In these embodiments, the paraffin coating layer and the paraffin used to embed the tissue/cell sample to be analyzed in the IHC or ICC analysis operation are removed in the same deparaffinization step.

Please refer to Figures 1 to 3C. In some embodiments, the processing operation record slide 100 includes a detection area 120 for holding samples in addition to the control area 140.

In some embodiments, the configuration of the detection area 120 is designed to hold patient samples such as tissue sections or loose cells.

In some embodiments, one or more control targets 141 held in the control area 140 receive staining together with the sample held in the detection area.

In some embodiments, the detection area 120 and the control area 140 have a clear boundary. In some embodiments, the detection area 120 and the control area 140 do not have a clear boundary, but are classified according to their individual functions.

In some embodiments, the control target 141 is arranged into one or more control target bearing points 1411. In some embodiments, the bearing points are arranged in a regular or irregular shape. In some embodiments, the regular shape includes a circle, an oval, a square, or a diamond. In some embodiments, the control target 141 is arranged in a two-dimensional (2D) or three-dimensional (3D) configuration.

In some embodiments, the control targets 141 are arranged in one or more control target arrays 1415, for example, one or more arrays formed by the control target bearing points 1411. In some embodiments, each control target array 1415 includes a control target with the same or similar chemical and biochemical properties. In some embodiments, each control target array 1415 includes a control target that can produce contrast optical properties.

In the above embodiment, since the detection area 120 and the control area 140 are located on the same glass slide, the sample held in the detection area 120 and the control target 141 in the control area 140 will undergo the same staining steps in the IHC or ICC operation. (See Figure 9). Therefore, the degree of staining of the control target 141 can reflect whether there are processing errors and the degree of errors in the IHC or ICC operation, and the control target 141 enables the processing errors in the IHC or ICC operation to deviate from the known target standard. Analyze the degree. Therefore, the processing operation document slides in these embodiments can complete effective, correct and accurate quality control at a relatively low cost, reduce subjective analysis, and provide a simple threshold for qualification determination. In addition, through the subsequent digital image capture and processing steps, the stained control target 141 can still be used as a ruler of antigen density. This ruler contains two scales: one is the value of antigen density, the other is color density, and both It can be applied to coexisting samples to assist diagnosis and interpretation (for example).

In some embodiments, the manner in which the control target 141 is deposited on the glass slide can prevent the control target 141 from being severely affected by the pretreatment steps commonly used in IHC or ICC staining operations. In some embodiments, the aforementioned pretreatment step includes deparaffinization or antigen retrieval. These pre-processing steps will be described in detail below.

In some embodiments, the slide glass 100 further includes a slide information area 160. In some embodiments, the slide information area 160 includes information about the type of slide, such as batch number, manufacturing date, type of control target, and so on. Since the slide information such as the type information of the comparison target is very extensive, the slide information area 160 in some embodiments includes a barcode for recording the slide information. In some embodiments, the barcode includes a website link that can be linked to a detailed description of the slide.

Please refer to FIG. 3. In some embodiments, the slide glass 100 further includes a protruding structure 107. In these embodiments, the raised structure 107 can ensure that the paraffin coating layer and the control target are not damaged by the adjacent glass slides when the glass slides are stacked on each other. In some embodiments, the protruding structure 107 includes a pair of strip-shaped structures extending outside the contrast area 140 or a plurality of dot-shaped structures extending outside the contrast area 140. These buffer structures help us to use direct-print label inkjet printers, because these printers usually feed unmarked slides from the bottom of the cassette. During the process of supplying the slides to the cassette, the buffer structures can ensure that the lower paraffin coating and the target will not be damaged by another slide passing from above.

Protective coating

In some embodiments, the protective coating layer 105 is a paraffin coating layer.

Please refer to Fig. 4, in some embodiments, the paraffin coating layer is a layer of paraffin wax on the control target and seals the control target.

Generally speaking, paraffin is a white or colorless soft solid derived from petroleum, coal or oil shale, and is composed of a mixture of hydrocarbon molecules containing 20 to 40 carbon atoms. It is a solid at room temperature, begins to melt when the temperature is higher than about 37°C (99°F), and has a boiling point higher than 370°C (698°F). The general applications of paraffin wax include lubrication, electrical insulation and candles. After dyeing, it can be made into crayons. Paraffin wax is clearly different from kerosene and other petroleum products sometimes called paraffin wax.

In the pathology laboratory, non-aqueous paraffin is used to impregnate tissues fixed with formaldehyde in order to cut tissue sample slices after immersion. In this procedure, the water content in the tissue is removed by increasing the concentration of ethanol (from 75% to anhydrous ethanol), and then organics such as xylene or its aliphatic substitutes (such as xylene isomers) are used. The solvent cleans the tissue. Then in a vacuum oven, the tissue is placed in liquid paraffin for a period of time to ensure that all air has been expelled. Then put it into the module frame and wait for the liquid wax to cool and solidify. Then cut the slices with a slicer.

Embedding tissue sections in paraffin is a method of long-term preservation of sectioned tissues. However, as far as the inventor knows, there is no relevant report about using paraffin as a coating layer on selected areas of microscope slides.

The control target slide contains peptides or proteins (see below for details), so it is a rich food source for bacteria or fungi. Peptides and proteins (and their antigenic sites, such as epitopes) are easily oxidized, and their ability to bind antibodies is sometimes reduced due to oxidation. In addition, if the hydroxyl groups involved in subsequent reactions react with acids and bases in the air, they may also be damaged. Therefore, glass slides containing protein deposits must usually be stored at a temperature where microorganisms cannot grow, and must be vacuum-sealed and packaged. Unprotected slides (such as slides exposed to the environment) have a storage life of only 2 to 5 days, depending on the ambient temperature, humidity and the concentration of pollutants in the air. All of the above are the limiting conditions for the effective use of restricted sediments.

Paraffin wax has antibacterial and antimicrobial properties, and can prevent oxidation of the material it seals. The inventors found that the paraffin coating layer can prolong the survival life of biological materials from 3 to 5 days to 1 to 2 years, thereby greatly extending the storage life of the processing glass slides.

In IHC or ICC staining operations, the paraffin used to embed the sample is generally removed (this step is called "deparaffinization"). Therefore, in some embodiments, the formula of the paraffin contained in the protective layer 105 is the same or similar to the formula of the paraffin used in the embedding operation, so that the paraffin coating layer is removed in the deparaffinization operation, thereby simplifying the dyeing operation.

In some embodiments, the paraffin wax is first liquefied, and then applied to the glass slide, and the paraffin wax coating layer is formed after it is solidified.

In some embodiments, the paraffin wax is mixed with a solvent to change its state from a solid to a liquid at room temperature. In some embodiments, the solvent includes xylene or an aliphatic solvent, such as xylene isomers. Mixing with this solvent can reduce viscosity and slow down the curing speed after deposition. In some embodiments, the solvent includes toluene, paint thinner or a 50:50 mixture of acetone and kerosene. Paraplast X-tra also includes dibutyl hydroxytoluene and phenolic antioxidants to further prevent degradation of proteins, peptides or inorganic targets due to oxidation.

In some embodiments, the temperature used to melt the solid paraffin is only 75°C higher than the melting point of the paraffin. After the paraffin is melted, slowly add an aliphatic solvent until the saturation point (that is, solid formation) can be observed. The resulting mixture was allowed to stand and cool to about 45°C. Then add more aliphatic solvent until the mixture is completely clear.

In some embodiments, the paraffin coating layer is applied to the biological material pre-deposited on the microscope slide and the special deposit that can react to staining. The biological materials include proteins, peptides, composite proteins, protein-coated beads, peptide-coated beads, conjugated coated beads, special end groups that can react to staining and capture specific staining substances, etc., see below for details The description.

In some embodiments, the paraffin wax is applied to the glass slide by spraying method, inkjet deposition method, transfer method (such as pad printing method), screen printing method or vapor deposition method. In some embodiments, the paraffin coating layer is applied to the glass slide by heating the paraffin coating layer to melt and/or mix the paraffin particles so that it can simultaneously seal the control target and the surrounding glass A single surface coating on the surface of the sheet.

In some embodiments, the glass slide is heated after the application of the paraffin wax coating layer to remove the solvent in the paraffin wax to ensure that the paraffin wax returns to a hardened state. One of the methods to remove the solvent is to irradiate the paraffin surface of the glass slide with infrared light. This method acts on the solvent instead of the paraffin or the biological material under the paraffin, so the required heat energy can be reduced. After the solvent evaporates, it can leave the paraffin freely without hindrance.

The paraffin used in some embodiments is a mixture of purified paraffin, synthetic polymer and other substances. In some embodiments, experiments are conducted on the proportion of paraffin wax components to obtain ideal paraffin wax melting point, hardness and viscosity. However, in order to protect biological materials, the paraffin used should be purified paraffin and contain no water.

In some embodiments, the thickness of the paraffin coating layer is less than 5 microns.

In some embodiments, the melting point of the paraffin contained in the protective coating layer 105 is lower than 60°C, such as lower than 56°C.

The paraffin used in some embodiments is soluble in xylene, xylene isomers or aliphatic substitutes.

The paraffin formulations used in some embodiments include but are not limited to TissuePrep and TissuePrep 2 (manufactured by Thermo Fisher, melting point 56°C), Paraplast and Paraplast Plus (manufactured by Leica, melting point 56°C), and Paraplast X- tra (manufactured by Leica, melting point 50~54°C). In some embodiments, the paraffin wax formulation is selected from all existing formulations containing dibutyl hydroxytoluene (if the dibutyl hydroxytoluene is not originally contained), which can make the paraffin wax coating layer reach the maximum hardness at ambient temperature.

Main target and main target array

In some embodiments, the control target 141 includes one or more primary targets for testing primary antibody reagents.

In this article, the term "primary target" refers to a composition containing reactive and non-reactive substances, wherein the reactive and non-reactive substances are exclusively bound to by only one of the multiple FcyR1 sites of the primary antibody The primary antibody. In this article, the term "major protein" refers to a protein that can bind to the FcyRI site of the primary antibody. In this article, the term "major dummy protein" refers to a protein that does not react with primary or secondary antibodies. In this article, the term "maximum target density" refers to the maximum monolayer density of adjacent proteins. In this article, the term "surrogate antigen" refers to a protein that is not the target of the primary antibody (derived from mouse or rabbit) used in IHC or ICC analysis, but binds to the primary antibody and only passes through the primary antibody One of the two FcyRI regions binds to the primary antibody. In this article, the term "mouse antibody-specific substitute antigen" refers to a protein or antigen that only specifically reacts with FcyRI derived from mouse primary antibodies. In this article, the term "rabbit antibody-specific substitute antigen" refers to a peptide or protein that only specifically reacts with FcyR1 derived from rabbit primary antibodies.

The weight of placental mammalian protein is between 50 and 65 kDa. The immunoglobulin G (IgG) of the primary antibody is composed of two Fab regions and an Fc region, and is usually represented by a Y-shaped model. The Fc region is connected to the two Fab regions through the hinge region. All unconjugated antibodies are cultured in mammalian hosts (mostly mice or rabbits) by inoculating the host with antagonistic antigen peptides. The host produces antibodies to resist the invasion of antagonistic antigen peptides, and the way to produce antibodies is to assemble the binding amino acid sequence in the antibody to capture the N-terminal side of the Fab region of the corresponding antigen. The Fc region of an antibody must be the Fc region of a mammalian host. The Fc region of the host antibody mostly has three binding sites for the secondary antibody to bind, and its binding affinity is from high to low: FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16).

The main dummy protein is a protein that does not react with mice, rabbits, goats, cows and sheep, because the proteins of these animals usually bind to secondary antibodies. In some embodiments, the main dummy protein is taken from any equine animal: horse, donkey, zebra, tapir or rhino. Bovine serum albumin (BSA) is not a suitable protein, because BSA must generate heat when it is covalently bound to tissues or most of the glass slide adhesive chemicals, and the binding process takes a long time. Proteins from cows, sheep and goats are also not suitable dummy proteins, because in some cases, the above-mentioned proteins may be confused with those of mice or rabbits, depending on the specificity of the primary antibody.

The main protein is the host's anti-mouse antibody or the host's anti-rabbit antibody. The host is limited to goats or any species of equine.

The main peptide-based protein can also be produced by reacting with mouse or rabbit FcyRI and the C-terminal cysteine residue is covalently bound to the carrier protein (such as the keyhole hemocyanin (KLH) subunit) Peptide composition. The free amine site of the KLH subunit is activated by Sulfo-SMCC, and then binds to the cysteine residue of the peptide by conjugation. The unactivated KLH subunit alone is sufficient to constitute the main dummy protein.

Please note that KLH in the state of whole protein weighs about 8000 kDa. In the state of whole protein, the pH or temperature of KLH is not stable. This instability may decompose the protein into the following subunits: KLH1 (weight 390 kDa) and KLH2 (weight 350 kDa). Any of the above units can be used as a carrier protein for peptides with cysteine residues (this residue is usually located at the C-terminus of the peptide) after being activated by Sulfo-SMCC.

Please refer to FIG. 5A. In some embodiments, the main target systems are arranged in a main target array 1415. In some embodiments, the main target array 1415 is a density gradient array of the main target. Each target in the array must have the same maximum target density (protein/surface area). However, the ratio of the main protein to the main dummy protein is changed in a gradual manner to ensure that the primary antibody dilutions within a wide concentration range can be distinguished by this. Generally speaking, two main surrogate antigen targets can be used to detect the concentration corresponding to the detection transfer curve. In some embodiments, the density gradient array of the main target includes a plurality of main target bearing points, one of the arrays is a target derived from mice, and the other is a target derived from rabbits.

In some embodiments, the density of the main target increases or decreases linearly, such as 100%, 80%, 60%, 40%, and so on. In some embodiments, the density of the main target is increased or decreased logarithmically, such as 100%, 33%, 10%, 3%, and so on.

In some embodiments, the main target density gradient array has a bearing point with the largest main target density, and the density of the main target may cause the main target to fail to saturate the bearing point during the dyeing process.

In some embodiments, the universal substitute antigen is protein A or protein G. Protein A binds to the FcyRI site of placental mammals and part of the Fab region. Protein G only binds to the FcyRI site of most placental mammals.

In certain embodiments, the surrogate antigen must be specific to mice or rabbits only. To have this specificity, the surrogate antigen can be composed of anti-mouse and anti-rabbit proteins, or composed of anti-mouse and anti-rabbit peptides on the carrier protein, or composed of anti-mouse and anti-rabbit VHH protein regions.

In certain embodiments, the dummy surrogate antigen includes antibodies from equine or hoofed placental mammals other than goats. The equine family includes animals such as horses, donkeys, tapirs, rhinos, and mules. The evolution and genes of equines are obviously different from other animals, so it can reduce the incidence of non-specific staining. Goats were excluded because anti-goat proteins were often used in the sequencing process of the secondary dyeing operation, and anti-goat proteins would bind to any unreacted main parts of the goat.

Although the interaction between the substitute antigen and the primary antibody used in the staining operation is different from the interaction between the real antigen and the primary antibody, the substitute antigen target can be used to evaluate the efficacy of the primary antibody. The primary antibody is accompanied by a concentration analysis report in mg/ml. However, this analysis report does not indicate that it can represent the percentage of intact immunoglobulin G (IgG). Since the density concentration of the substitute antigen in the target substance is known, the trapped amount of the primary antibody concentration used can be calculated based on this. The extrapolation method can be used to obtain the antigen density of coexisting tissue sections. The possibility of solving the antigen density in the tissue is limited by the cumulative replacement amount of the primary antibody (single strain or multiple strains) and the secondary antibody, and the enzyme amplification rate of the secondary staining operation. The concentration of the primary antibody used depends on the type of tissue and must have a certain margin. It is assumed here that the primary antibody will bind to all antigen sites available for binding within its replacement limit.

In some embodiments, the density gradient array of the main target is a universal surrogate antigen bearing point array. In certain embodiments, the load points in the array include increasing or decreasing surrogate antigen density. In some embodiments, the surrogate antigen system is mixed with one or more dummy proteins (for example, dummy surrogate antibodies), and the increase or decrease of the surrogate antigen concentration is achieved by changing the surrogate antigen and dummy protein under the condition that the maximum target density remains unchanged. The ratio of protein is achieved.

In certain embodiments, the control zone 140 includes at least two surrogate antigen target arrays. In some embodiments, the control area 140 includes a first-generation antigen target array and a second-generation antigen target array. The two arrays respectively include anti-mouse antibodies and anti-rabbit antibodies, and both are mixed with a dummy substitute antigen. , In order to form the required reaction density under the condition of the maximum target density unchanged.

Therefore, according to the above embodiment, the primary target of the substitute antigen can be used in the IHC or ICC step in combination with the enzyme amplification rate obtained through the secondary target and the antigen retrieval factor to evaluate the concentration of the primary antibody.

Antigen retrieval target

In certain embodiments, the control zone 140 includes an antigen retrieval target.

IHC and ICC staining analysis usually includes antigen retrieval (AR) operations. AR operation can use heat-induced epitope retrieval (HIER) method, also known as warm water antigen retrieval method. The results of antigen retrieval vary greatly. Different operators and even different slides may produce different results, depending on the method used and the implementation of the specific method. The results of direct measurement of AR buffer and buffer temperature are often inaccurate. However, if there is no appropriate control group, the operator usually has to blindly assume the temperature and state of the AR buffer. Therefore, the lack of a control group often causes errors in the AR operation to be inadvertently carried into the final evaluation operation, thereby causing misjudgments.

The most common problems in AR operations are insufficient repair and over repair. Too low AR temperature and insufficient exposure time may lead to insufficient repair. The AR temperature is too high, the time of exposure to the AR state is too long, or the state of the AR buffer is too severe (for example, pH>9.5 or pH<5.5), which may lead to excessive repair.

Therefore, some embodiments use antigen retrieval targets as indicators of insufficient, normal, and over-repair in AR operations.

In some embodiments, the control area 140 includes a 3D AR target (ARM3D) and a 2D AR target (ARM2D). The 2D and 3D targets will be explained in detail in the next section.

In some embodiments, the ARM2D target includes a 50:50 mixture of mouse and rabbit proteins (or proteins of other species) at a concentration of 100% and fixed with a minimum amount of formaldehyde. In certain embodiments, the protein is immunoglobulin G (IgG). Since the signal generated by the 2D target is less than the corresponding 3D target and the ARM2D target is minimally fixed, the staining of the ARM2D target represents insufficient antigen retrieval.

In some embodiments, the ARM3D target includes a 50:50 mixture of mouse and rabbit proteins (or proteins of other species) at a concentration of 100% and deposited on a 3D scaffold over-fixed with formaldehyde. Since the 3D target produces more signals than the corresponding 2D target and the ARM3D target is over-fixed, the ARM3D target is not dyed, which means that the AR operation is over repaired.

In some embodiments, the antigen retrieval target includes a primary target gradient array or a secondary target gradient array in addition to the ARM2D or ARM3D target. In some embodiments, the primary target array or the secondary target array is the same as or similar to those described above.

In some embodiments, the secondary target gradient array is a density gradient array of mice or rabbits. According to the above-mentioned embodiment, when 10% to 30% of the target bearing points in the gradient density array of mice and rabbits do not show a visually visible staining effect, it represents a normal repair state. Then, the repairing degree of AR operation can be evaluated according to the quantity of undyed low-concentration secondary targets.

2D/3D target

In some embodiments, the control target (eg, primary target, secondary target, or antigen retrieval target) includes a 2D target.

In this article, the term "2D target" refers to peptides, proteins, antigens, antibodies or any other control target substances deposited on the 2D surface of the glass slide.

The deposition of 2D targets is easier to implement, so the manufacturing cost is lower. Therefore, if there is a cost consideration, a 2D target can be used.

In some embodiments, the control target (eg, primary target, secondary target, or antigen retrieval target) includes a 3D target.

In this article, the term "3D target" means that at least one of peptides, proteins, antigens, antibodies or any other control target substances is applied to a support structure so that the resulting complex is located on the glass slide. In 3D space.

In one or more embodiments, the method of constructing a 3D target is to form a 3D support on a glass slide, and then deposit the control target substance on the support. In certain embodiments, the 3D scaffold is a polysaccharide cluster. The polysaccharide forms a covalent bond with the hydroxyl or amine group on the surface of the peptide or protein, thereby linking the protein into one body and fixing the target on the adhesive layer of the glass slide. In some embodiments, the control target is fixed on the 3D stent through formaldehyde fixation. Therefore, the target has both 2D and 3D components.

In one or more embodiments, the 3D target is constructed by submicron beads covalently bonded to the primary or secondary target material, and then the primary or secondary target material is fixed by formaldehyde fixation , So that it can withstand normal antigen retrieval operations. The target deposited in this way includes both 2D and 3D components.

Compared to 2D targets, 3D targets behave more like stained samples. IHC or ICC stained samples (such as tissue sections and cells) have a high degree. The sample height is usually between 4 microns and 10 microns. The target antigen site in the staining operation can be located at any position on the exposed surface structure of the tissue section. Therefore, the antigen site in the sample can be flat at any position on the top surface, bottom surface or the side wall of the tissue, and the amount of chromogen that can be deposited by the antigen on the side wall of the sample is significantly more than that of the 2D target. The amount that can be deposited. The 2D component and the 3D component that exist at the same time form a first-order position difference, which can be applied to other 2D targets to form a virtual 3D array. Experiments have found that when any 3D target is larger than 0.5 microns, the color depth of its staining is the same as that of tissue sections.

In addition, 3,3'-diaminobenzidine (DAB; one of the commonly used reagents in dyeing operations) will age in a relatively short period of time, resulting in changes in color and color density. The inventor found that the color and color density of a 3D target dyed with DAB are similar to those of the sample.

Special dye

In certain embodiments, the control target includes a special dye.

In some embodiments, the special dye includes Elson Blue, Aniline Blue-Orange G Solution, Azan Dye, Bielschowsky Silver Dye, Brown and Bren & Brenn)-Glan's stain, Cresol Violet, DAB, Fontana-Masson (Fontana-Masson) stain, Gordon-Sweet (Gordon-Sweet) silver stain, Grocott ( Grocott hexamethylenetetramine silver stain, Hall bilirubin stain, Jones hexamethylenetetramine silver stain, Luxol fast blue, Luxol fast blue-cresol purple, mucus carmine (Mel Method), Muller-Mowry Colloidal Iron, Orange G, Nuclear Fast Red, Periodic Acid-Schiff-Amylase (PAS-D) Dye, Periodic Acid-Schiff (PAS) dye, phosphotungstic acid, hematoxylin, Sirius red dye, acidified toluidine blue, Gomori single-step three-color dye, Mason three-color dye, Victoria blue, Von Kossa dye, Weigert (Weigert) resorcinol fuchsin, Weigert iron hematoxylin, Gini's stain or a combination of the above.

Imaging reference target

In some embodiments, the control area 140 further includes an imaging reference target. In some embodiments, the imaging reference target includes one or more black targets, one or more white targets, or transparent targets.

The method of digital imaging of microscope slides containing stained biological materials has been continuously evolving, and it is hoped that pre-screening of stained substances and even complete diagnostic interpretation can be made. In some embodiments, the imaging operation of the glass slide is performed immediately after the staining operation of the glass slide is completed, and during the imaging process, the illuminance or exposure can be adjusted to prevent the digital image from being compressed at the black/white boundary. In another method, the black and white targets are located at predetermined positions of the label. The basic assumption is that the black and white targets can represent the ultimate strength of the dyeing signal. However, when implementing this method, since the black target is much darker than the staining effect that the tissue section can produce, and the white target is also far whiter than the staining effect that the tissue section can produce, the digital scale will be compressed.

In some embodiments, the imaging reference target is printed on a glass slide. In some embodiments, the imaging reference target includes a pair of black and white targets. In some embodiments, the imaging reference target is a printed paint deposit and does not react with the reagents used in the dyeing operation.

Some imaging systems only use transmitted light (illuminated from the bottom of the slide). Some imaging systems use both transmitted light and reflected light (illuminated from the top surface of the slide). The third type of transparent reference target is suitable for reflected light illumination. Therefore, when illuminated by transmitted light, the black and white reference objects are a black pigment target and a transparent target, respectively, and when illuminated by reflected light, the black and white reference objects are a transparent target and a white target, respectively. Please refer to the example of a single antibody slide in Figure 12 and the example of a double antibody slide in Figure 13. The black and white targets in the picture are each a series of dots, but they can also be changed to a strip structure to function as a buffer structure at the same time to prevent the target bearing points from being damaged when the inkjet printer supplies the slides. . The inkjet printer supplies slides from the bottom of the cassette. The above-mentioned buffer structure can ensure that the paraffin coating layer and the target on the glass slide are not worn by the remaining glass slides in the stack. These buffer structures can also ensure that the paraffin layer will not adhere to the bottom of adjacent glass slides when multiple slides are shipped in a packaged form.

If imaging with transmitted light, the antigen density on the tissue will not be displayed correctly. The reason for this is that not all antigen sites of the tissue are located on the same plane as a single sheet after staining. The antigenic site of the tissue may be located at any position in its thickness. The coloring agent particles in the secondary dyeing operation will continue to precipitate until the particles cover the enzyme sites and block subsequent precipitation. If an antigen site is located on the sidewall of the tissue section, the enzyme site will never be covered, causing the precipitation to over-represent the presence of antigen. When using transmitted light, the deposited colorant will produce "dark areas" due to the increase in thickness. On the contrary, the reflected light only "sees" the top surface of the pile of colorants, so the antigen density can be displayed more accurately.

In some embodiments, the color of the white target is close to pure white. Since pure white is not easy to obtain, the color of the white target in some embodiments differs from pure white by 5%-10%. White that differs from pure white by less than 5% is not easy to manufacture, and colors with a difference of more than 10% from pure white may not be accurate enough as a control of the intensity of the dyeing signal.

In some embodiments, the white target includes a white pigment. In some embodiments, the white pigment is a metal oxide or metal sulfide pigment, and can remain stable unless it is exposed to strong light. In certain embodiments, the white pigment includes aluminum oxide, titanium oxide, or barium sulfate. In some embodiments, the metal oxide or metal sulfide is in the form of beads.

In some embodiments, the black target includes black pigment. In some embodiments, the black pigment includes a carbon-based pigment. In some embodiments, the black pigment includes carbon powder. In some embodiments, the diameter of the carbon powder is less than 2 microns.

In some embodiments, the imaging reference target includes an anhydride-based epoxy resin. In some embodiments, the method of manufacturing the imaging reference target is to mix the pigment into an anhydride-based epoxy resin, and then cure the anhydride-based epoxy resin. There is no special restriction on the matching method of the epoxy resin adhesive and the pigment, as long as a good wetting effect can be produced between the used pigment and the epoxy resin adhesive.

In some embodiments, the anhydride-based epoxy resin includes an anhydride catalyst, and the anhydride catalyst can eliminate unreacted amine groups of the aminosilane-based catalyst. Free amine groups can also capture biological materials and part of the staining reagents, thus producing non-specific staining results in IHC and ICC operations. Eliminate unreacted amine groups to avoid non-specific dyeing results.

In some embodiments, the anhydride-based epoxy resin coating includes an anhydride catalyst, such as methyltetrahydrophthalic anhydride or diphenyliodonium hexafluoroarsenate.

In some embodiments, curing the anhydride-based epoxy resin includes irradiating the anhydride-based epoxy resin with ultraviolet light (UV) (for example, ultraviolet light with a wavelength of 365 nm). In some embodiments, curing the anhydride-based epoxy resin coating further includes heating the anhydride-based epoxy resin coating to cause the epoxy resin to undergo a cross-linking reaction. There are no special restrictions on the anhydride-based epoxy resin and its associated reagents that can be cured by ultraviolet light. Those with ordinary skills in the field only need to search for the products of anhydride manufacturers to find ideal epoxy resins based on anhydrides.

In some embodiments, an imaging reference target including an anhydride-based epoxy resin is first deposited on a glass slide, and then a primary target, a secondary target, or an antigen retrieval target is deposited. The reason for this is that the heat treatment step used to cure the anhydride-based epoxy resin may destroy the primary target, the secondary target, or the biological material in the antigen retrieval target. In some embodiments, the anhydride-based epoxy resin is cured by ultraviolet light. Therefore, in order to prevent ultraviolet light from damaging the peptides and proteins in the primary target, secondary target, or antigen retrieval target, the reference target is imaged. The deposition step of the object is later than the deposition step of the main target, the secondary target or the antigen retrieval target.

In some embodiments, the formulation of the imaging reference target is completely free of surfactants, so as to prevent the ink/paint used from reacting with the various dyes and reagents that may be accepted by the glass slide.

In some embodiments, the imaging reference target is printed on a glass slide using a printer. In other embodiments, the imaging reference target is printed with a syringe because the syringe can control the deposition size of the target.

The use of imaging reference targets facilitates the optimization of digital analysis, because the entire range of digitization has been defined by the black/white (not pure black and pure white references) defined by the glass slide. Digital analysis helps to determine the color of the control target by interpolation, thereby more accurately determining the amount of antigen in the stained sample. The use of imaging reference targets is also helpful for the correction of digital analysis, so as to provide a reference point for measuring the reaction volume of the control target.

Method of depositing control target

Certain embodiments in this specification are directed to a method for depositing control targets on glass slides for processing operation records. In some embodiments, the slide glass and the control targets are the same as or similar to those described above.

In some embodiments, the method for depositing a control target includes depositing a primary control target or a secondary control target on a glass slide.

In some embodiments, depositing the primary control target or the secondary control target includes: preparing a solution of the control target at a predetermined concentration; fixing the control target; and printing the solution on a glass slide.

In some embodiments, preparing the solution of the control target includes preparing a solution including the control target and a polysaccharide, wherein the polysaccharide acts as a linking agent between the protein and the glass slide adhesive.

In some embodiments, fixing the control target includes: fixing the control target with formaldehyde. Formaldehyde can cause the control target to produce a cross-linking reaction, so that the control target can withstand normal antigen retrieval operations.

In certain embodiments, the control target has been cross-linked. The protein in the tissue is bound to other parts of the tissue that will prevent the protein from spreading and denaturing. The control target in the solution includes loose protein, so it is more sensitive to heat and pH, and easily spreads and denatures on the glass slide, especially in the AR operation of IHC or ICC analysis.

In some embodiments, the main target system is prepared in the following manner: Prepare a set of main protein dilutions, each of which has a volume of 1 ml and a concentration of 45 μg/ml. In detail, donkey anti-mouse protein, donkey anti-rabbit protein, and donkey immunoglobulin G (IgG) (H&L) are each _{diluted with distilled water (dH 2} O) as necessary to make the concentration 45 μg/ml. The concentration of the above-mentioned protein at the time of purchase is mostly between 1 and 10 mg/ml. Add 10 μl thimerosal as a fungal growth inhibitor. At a temperature of 40~60°C, each main dilution is fixed with 10 μl of 0.2% formaldehyde for 1~4 hours. Add 30 μl of amylose at a concentration of 0.45% as a linear polymer (thimerosal is added as a fungal growth inhibitor), and mix for 30 minutes. Add 20 μl of 0.1 M ammonium bicarbonate to quench any unreacted formaldehyde. Prepare donkey anti-mouse and donkey anti-rabbit protein target mixture from the obtained protein master solution, so that each target contains 700 μl of protein master solution.

In some embodiments, the secondary target system is prepared in the following manner: Prepare a set of primary dilutions of secondary protein, each primary dilution has a volume of 1 ml and a concentration of 45 μg/ml. In detail, mouse, rabbit, and donkey immunoglobulin G (IgG) (H&L) are each _{diluted with distilled water (dH 2} O) as needed to make the concentration 45 μg/ml. The concentration of the above-mentioned protein at the time of purchase is mostly between 10 and 60 mg/ml. Add 10 μl thimerosal as a fungal growth inhibitor. At a temperature of 40~50°C, each main dilution is fixed with 10 μl of 0.2% formaldehyde for 1~4 hours. Add 30 μl of amylose at a concentration of 0.45% as a linear polymer (thimerosal is added as a fungal growth inhibitor), and mix for 30 minutes. Add 20 μl of 0.1 M ammonium bicarbonate to quench any unreacted formaldehyde. Prepare donkey anti-mouse and donkey anti-rabbit protein target mixture from the obtained protein master solution, so that each target contains 700 μl of protein master solution.

In some embodiments, the primary or secondary target or the imaging reference target prepared in the above manner is printed on the glass slide through a single printing cycle in the following manner: Print the target solution on a microscope slide covered with adhesive. Air-dry at 60°C until the moisture is completely evaporated, then let it stand to cool. Spray the paraffin-solvent mixture on the printed target array. Reflow the paraffin to seal the target array and remove the solvent, so that the paraffin restores its hardened solid state.

IHC or ICC staining method

Certain examples in this specification are for an immunohistochemistry (IHC) or immunocytochemistry (ICC) staining method. Some examples in this specification are directed to an immunohistochemistry (IHC) or immunocytochemistry (ICC) staining method using the aforementioned processing slides.

In some embodiments, the IHC or ICC staining method includes embedding the fixed sample in paraffin and removing the paraffin to expose the antigenic sites in the cell structure of the sample.

In some embodiments, removing the paraffin wax includes heating the paraffin wax at a temperature of 65 to 75°C for 3 to 10 minutes to make it semi-liquid, and then adding an aliphatic solvent (such as xylene or its isomers) to The semi-liquid paraffin is liquefied, and then rehydrated with absolute ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and a salt-based buffer solution in sequence.

In some embodiments, the IHC or ICC staining method further includes removing the formaldehyde used for fixation to expose the antigen sites in the sample. In some embodiments, removing the formaldehyde used for fixation includes performing a heat-induced epitope retrieval (HIER) operation, that is, performing multiple warm water antigen retrieval cycles.

In some embodiments, the HIER operation includes heating the sample in the presence of water to break the Schiff base bond between the formaldehyde and the tissue. In some embodiments, heating the sample includes subjecting the sample to a temperature of 89~95°C. In some embodiments, the sample is exposed to a buffer reagent when heated. In some embodiments, the pH of the buffer reagent is between 6 and 10, such as between 6 and 9. The choice of the pH value of the reagent depends on the type of sample, such as the type of tissue.

In some embodiments, the water-based antigen retrieval operation includes subjecting the sample to a melting point that is higher than the melting point of the embedding paraffin (which is between about 60°C and about 65°C) by about 10°C.的温度。 The temperature. In some embodiments, the water-based antigen retrieval operation includes washing the sample with soap and continuous washing to dissolve and remove the paraffin.

Please note that during the process of removing paraffin and unfixing, the operator's mistakes or work defects may hinder the subsequent dyeing, resulting in false negative results. Although inspection of the staining results of the control target in the processing operation record glass can be used to identify such false negative results, the operator's error or work defect will still cause waste of samples, time and other resources and should be avoided.

At this point, the antigen site has been exposed, so staining reagents can be applied to generate a signal that can show the presence and amount of the target antigen.

In some embodiments, the IHC or ICC staining method further includes the administration of one or more primary antibodies. If more than one primary antibody needs to be administered, the primary antibodies must come from different animal species to avoid cross-reaction with the secondary antibodies. For example, if two primary antibodies are used, the first of which is a mouse antibody, the second primary antibody must be a non-mouse antibody, such as a rabbit antibody. According to the above embodiment, the one or more primary antibodies will bind to the matched antigen site in the sample and the main control target (such as protein/peptide antigen or surrogate antigen). In some embodiments, primary antibodies include antibodies targeting ER, PR, Her2, or Ki67.

Please refer to Figure 11. In some embodiments, the primary antibody system is combined with a reporter substance (such as a chromogen reporter substance (such as an enzyme chromogen reporter substance)). These examples do not require the use of secondary antibodies.

In some embodiments, the primary antibody is not conjugated to the reporter substance. These examples additionally administer secondary antibodies that target the primary antibody. In some embodiments, the secondary antibody is conjugated to the reporter substance.

As shown in Fig. 11, some embodiments perform a signal amplification operation including multiple steps to further amplify the signal of the antigen. Examples of such multi-step signal amplification operations include: reacting the enzyme-labeled tertiary antibody with the enzyme-labeled secondary antibody (three steps, indirect) to provide 2x amplification; and making alkaline phosphatase resistant to alkaline phosphatase (APAAP) ) The immune complex reacts with the secondary antibody to provide 2x magnification. In some embodiments, the primary antibody and the antibody system in the immune complex are produced from the same species. The above-mentioned signal amplification tasks include: labeling avidin-biotin (LAB) method and labeling streptavidin-biotin (LSAB) method, even if the enzyme labeling (streptavidin) and biotin Catalytic signal amplification (CSA) technology, such as the biotinylated secondary antibody is located on the primary antibody, and the streptavidin-enzyme complex is located on the biotinylated secondary antibody. On the antibody, to provide >10x magnification; and to bind a polymer containing 10 secondary antibodies and 70 enzyme sites to a single primary antibody to provide >10x magnification.

In some embodiments, the reporter substance is an enzyme that can precipitate the chromogen in the presence of the dyeing substrate (hereinafter referred to as the reporter enzyme). All reported enzymes can achieve the same final state of chromogen precipitation.

In certain embodiments, the reporter enzyme includes horseradish peroxidase (HRP), alkaline phosphatase (AP), or glucose oxidase.

In some embodiments, one of the three common secondary dye groups, multiple counterstains, or two of the above is used, namely: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and Nuclear counterstain.

In certain embodiments, the aforementioned chromogens include 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), DAB+nickel enhancer, fast red, 3, 3'5,5'-Tetramethylbenzidine (TMB), StayYellow (StayYellow), 5-Bromo-4-chloro-3-indolyl phosphate (BCIP)/Nitro blue tetrazole (NBT), BCIP/Tetranitro Blue Tetrazolium (TNBT), Naphthol AS-MX Phosphate + Fast Blue BB, Naphthol AS-MX Phosphate + Fast Red TR, Naphthol AS-MX Phosphate + New Fuchsin, Long-lasting Green ( StayGreen), NBT or a combination of the above.

In some embodiments, the combination of the above-mentioned dyeing substrate and the reporter enzyme and the colors produced are as follows: HRP (Wasabi peroxidase) DAB (3,3'-diaminobenzidine) ＞＞ Brown to reddish brown AEC (3-amino-9-ethylcarbazole) ＞＞ red DAB+nickel enhancer ＞＞ black TNB (3,3'5,5'-Tetramethylbenzidine) ＞＞ blue StayYellow ＞＞ yellow AP (Alkaline Phosphatase) BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazole) ＞＞ blue Naphthol AS-MX Phosphate + Fast Blue ＞＞ blue Naphthol AS-MX Phosphate + Fast Red ＞＞ red Naphthol AS-MX Phosphate + New Fuchsin ＞＞ red StayGreen ＞＞ green GO (Glucose Oxidase) NBT (nitro blue tetrazolium) ＞＞ Blue to purple

In some embodiments, the IHC or ICC staining method further includes counterstaining, such as nuclear counterstaining. In some embodiments, the nuclear counterstain uses one or more nuclear stains, such as hematoxylin that can produce blue.

The choice of the above-mentioned dyeing substrate or compound depends on the required color, the required stability and local regulations and standards.

For example, DAB is a dyeing substrate commonly used in the United States and China, while AEC is a dyeing substrate commonly used in other countries. Since the brown-red produced by DAB is more saturated than the red of AEC, DAB is generally used instead of AEC. However, AEC staining is more stable than DAB staining. Experimental results show that the early DAB will age rapidly in a short period of time, causing its color saturation to drop significantly within 4 hours. Newer DAB formulations add stabilizers to extend the life of DAB from several hours to several days. In addition, DAB may be washed away in subsequent buffer washing cycles, while AEC can remain stable for weeks or even months.

The regulations and standards of various countries in the world are trying to require or mandate that any reagents, methods and instruments used to treat tissue sections and loose cells should be included in their inspection operations once they are feasible and available for use. This kind of legal control group has long existed in hematology and clinical chemistry. On the one hand, it is used to verify the treatment result and on the other hand to ensure the quality of the treatment. The test results of this control group can be plotted as a Levey-Jennings chart (Westgard et al., 1981). Please refer to "A multi-rule Shewhart chart for quality control in clinical chemistry" by Westgard J, Barry P, Hunt M, Groth T (Clinical Chemistry Journal No. Pages 493-501, Issue 27, 1981).

Extrapolation calculation of antigen imaging ruler

In some embodiments, the IHC or ICC staining method further includes performing extrapolation calculations for the staining results of the primary and secondary targets to estimate the concentration of the antigen in the patient sample coexisting with it.

In some embodiments, the IHC or ICC staining method further includes performing extrapolation calculations for the staining results of the primary and secondary targets to evaluate the processing quality of the IHC or ICC staining step.

Since the deposition amount of the control target on the glass slide is predetermined, each target can be calculated by knowing the molecular weight and quantity of the various protein control target molecules in the sediment, the area of each target, and the porosity of the glass slide coating. The active surface protein density of the substance.

The application concentration, application volume, and surface area of the primary antibody that can be contacted with the reagent on the slide are all known. We can reasonably assume that most of the suspended antibodies have fallen and were captured by antigenic sites that can accept these antibodies during the period of time when the reagents can be contacted. In detail, only the antibody that directly falls on the antigen site will be captured, and the antibody that has not been captured will be washed away in the buffer washing step. Therefore, the concentration of deposited antibody can be obtained under appropriate conditions (for example, when the concentration is higher than the cut-off concentration by more than 25% and the saturation concentration is less than 25%). In this article, "cut-off" is defined as the density of the target site is not sufficient to capture the administered concentration of protein, and "saturation" is defined as the antigen concentration cannot capture all the administered protein.

As long as the dilution ratio of the main target is known, the correct density target of the main target can be selected and the concentration of the main target can be verified.

In an embodiment of the present invention, each secondary and main target is a mixture of [(mouse or rabbit IgG) + (donkey IgG + cross-linking agent + fungal inhibitor)], or [(with antigen A) KLH or KLH with antigen B)+(unconjugated KLH+crosslinker+fungal inhibitor)], or alternative antigen [(donkey anti-mouse IgG or donkey anti-rabbit IgG)+crosslinker+donkey IgG+ Fungus inhibitor)] mixture. Each target carrying point has the same total protein volume, but the mixing ratio is slightly adjusted, so that the protein in each target has a different atomic mass.

The molecular weights of some examples of proteins are as follows: Mouse IgG = 155 kDa Rabbit IgG = 150 kDa Donkey IgG, donkey anti-mouse and donkey anti-rabbit = 160 kDa Protein A = 42 kDa Protein G = 58 or 65 kDa Protein A/G = 50 kDa Protein L = 76 kDa Chicken Immunoglobulin Y (IgY) = 180 kDa KLH subunit: KLH1 = 350 kDa, KLH2 = 390 kDa

In another embodiment of the present invention, the gradient of the 2D secondary target includes a gradual dilution ratio from 1 to 1000:1. In some embodiments, the dilution ratio follows a -20 log (dilution) curve, where the gradient of the dilution ratio is -3 dBd. "-20 log (dilution) = dBd" describes the dilution ratio in a semi-logarithmic manner, and its purpose is to linearize the data so that the modification items can be added. The term "(dilution)" refers to the dilution rate X, where X is [1..1000], that is, 1:1 to 1,000:1. The term "dBd" is defined as the number of decibels of dilution or the intensity of dilution. The above-mentioned modification items include the loss caused by antigen retrieval, the amplification rate of the enzyme, or the dilution ratio of the primary antibody reagent.

The secondary staining operation involves an enzyme magnification function whose value (enzyme magnification) is between 1 and 20×. This function is a function of the structure of the dyeing reagent. When the magnification increases, the lower concentration of the secondary target will become saturated, and when the magnification drops to 1, only the high concentration of the secondary target can produce visually visible dyeing results.

The KLH subunit and peptide-based secondary and the conjugated main target protein are very different in size. Therefore, the dummy diluent must be an unactivated KLH unit. The reason is that the mass of the KLH subunit will indeed be greatly affected by the peptide. change.

The estimated maximum binding capacity of KLH subunits to protein A, G and A/G is four to six protein molecules. Appropriate KLH dummy dilution should be combined with chicken IgY (this protein will not react with protein A, G, A/G or L at all). Although the size of chicken IgY is about four times the size of protein A/G, the final result is about the same due to the smaller amount of binding to KLH. No matter which method is selected, the mass of the KLH subunit will be between 650 and 700 kDa. The mass value of the KLH subunit can be used for subsequent calculations. The following will take mouse IgG and rabbit IgG as examples for detailed explanation.

In another embodiment of the present invention, the main target can be made of protein A, G or A/G. Primary antibodies are mainly based on the host Fc region of mouse IgG or rabbit IgG. However, the FcyRI site is located on mouse IgG1 and rabbit IgG. Protein A can bind strongly to rabbit IgG, but its binding ability to mouse IgG1 is weak. Protein G can bind strongly to rabbit IgG, and the binding force between it and mouse IgG1 is moderate. Protein A/G can bind strongly to rabbit IgG, and the binding force between it and mouse IgG1 is also moderate. Protein L can bind strongly to mouse IgG1, but the binding force to rabbit IgG is weak (its binding position is the FcyRI site of the Fab light chain instead of other chains). Certain embodiments use protein G or A/G. The two proteins both appear to have dipolar structures and can only bind to the primary antibody in a single connection.

Proteins A, G, A/G and L can bind to most placental mammalian IgG. BSA (Bovine Serum Albumin) does not bind to the above-mentioned proteins. Therefore, if BSA is used, the blocking step performed before the use of the secondary staining kit will be invalid. In detail, the blocking step is to prevent goat protein from binding to protein A, G, A/G or L. This design is different from other methods because most of the secondary staining kits use goat-derived host protein in a certain step. Therefore, it is strongly recommended to use blockers derived from equines, such as immunoglobulin G (IgG) from horses, donkeys, tapirs or rhinoceros. Equine genes are different from other placental mammals at the beginning of evolution, and the difference between the two is sufficient to make most equine proteins not react with secondary staining reagents, but they can fully bind to proteins A, G and A/G to block the unreacted part of the main target.

The bound primary antibody must have an unbound FcyRI site in its two FcyRI sites that can still react with the secondary antibody. Therefore, after the primary antibody is captured, it must have at least one FcyRI site for secondary staining. Therefore, the function is not affected. Therefore, the captured and stained primary antibody can represent the actual concentration of the primary antibody, rather than the analysis/dilution value of the primary antibody.

The average atomic mass of the primary antibody is 150 kDa. Therefore, the molecular weight of a single antibody molecule is 150 kDa (1.6605×10 ¹² ), and its weight is equal to 249×10 ^-12 ng. Assuming that the slide has only one exposed area, the dosage of the primary antibody reagent can be calculated accordingly. For example, if a closed capillary gap with an internal size of 20.3 mm×20.3 mm×0.14 mm is used, the volume of the gap is 57.2 µl. Therefore, the application amount of the primary antibody reagent is 2.832 per 1 micron width of the target area. nl.

The primary antibody reagent is diluted from the concentrate to an intermediate dilution with a concentration of 10 μg/ml. Then the intermediate dilution is diluted in a ratio of 1:1 to 1000:1, and the final dilution is applied to the glass slide. When the dilution ratio is between 1:1 and 25.1:1, the number of antibodies deposited on 1 square micron area is between 31.5 and 7.08.

In order to ensure 100% catching power, the main target should have a safety factor between 100× and 1000×. When the selected safety factor is 1000×, the main target must contain 4×10 ⁶ antigenic sites. Although the KLH subunit is larger than the antibody administered, this size difference is still insufficient to cause a greater than 1:1 change in the number of antibodies captured. The average atomic mass of each KLH subunit is 370 kDa, and its weight is equal to 614.4×10 ^-12 ng.

The volume of protein molecules can be estimated based on the molecular weight and average partial specific volume of the protein (partial specific volume = volume/molecular weight). The average partial specific volume of soluble globular protein obtained through experiments is ~0.73 cm ³ /g. This value will vary with protein, but the range of variation is very small. The calculation formula can be simplified as: protein volume = ~(1.212×10 ^-3 ×MW) nm ³ . Therefore, the volume of an individual KLH subunit is 448.44 nm ³ . Assuming that the protein molecule is spherical, the diameter of the spherical is 0.132×MW ^1/3 nm. Calculated in this way, the diameter of a single KLH subunit is about 9.436 nm.

If a control target bearing point has a diameter of 1 mm and includes a single layer of KLH subunits, a total of 11.237×10 ²⁷ protein molecules are required. If the density of the active target is 4×10 ⁶ protein molecules, the minimum dilution ratio is 1:2.8×10 ²¹ . In practical applications, as long as the determination method of the primary antibody mainly depends on the concentration of its active protein, any dilution ratio close to 1:1000 can be used. Therefore, the density of the target is only limited by its lowest concentration value.

In one embodiment, the secondary target array is a dilution that is gradually diluted at a ratio of 1 to 1000:1. The linear slope of the dilution ratio can be expressed in dBd =-20 log (dilution). When the dilution ratio is 1 to 1,000:1, the semi-logarithmic range is 0 dB to -60 dBd. In some embodiments, a series of dilution ratios of -0, -3, -6, -9, -12, -15,-can be obtained by gradually diluting the secondary target with a gradual dilution of -3 dB. 18 and -21 dBd dilution.

Both the secondary and main target arrays are fixed in a semi-reversible manner, so the degradation degree in AR operations is lower than that of the sample or the AR target. The above degradation comes from the protein fragments that have been shed rather than the intact protein. In some embodiments, when the protein target and the sample slice are continuously subjected to the AR operation, the damage caused by the AR causes the pattern of the gradient scale to shift toward the position of 100%. On the other hand, the enzyme magnification of the secondary staining operation shifts the gradient array to 10%. In some embodiments, the enzyme magnification is 1, 2, 4, 5, 8, 10, 15 or 20 times, and the magnitudes that cause the secondary target array to shift toward 10% of the target are: 1. 20x All targets offset -26 dBd 2. 15x All targets offset-23.52 3. 10x All targets offset -20 4. 5x All targets offset -13.98 5. 4x All targets offset-12.04 6. 2x All targets offset-6.02 7. 1x Only 2D 100% load point is close to black

Generally speaking, if the damage caused by AR causes the secondary target array to shift to 100% by more than three bearing points, the damage can be regarded as excessive damage. In this case, the enzyme magnification should be higher. Re-stain with a secondary staining kit or higher antibody concentration.

Therefore, the color density of the main antigen target is the sum of the antibody concentration multiplied by the enzyme magnification of the secondary staining kit. The density of the secondary target is simply the enzyme amplification rate multiplied by the protein concentration of the secondary target.

Changes in the illumination intensity of the digital imaging system will shift the dynamic range of the image toward saturation (gradually darker colors) or cut-off (gradually lighter colors), depending on the digital imaging system used. The above changes will cause the antigen color scale to shift, but will not cause the antigen density value scale to shift. Therefore, the numerical scale has nothing to do with the illumination intensity, and the color scale depends on the illumination intensity.

Please refer to Figure 13. In an embodiment of the present invention, the aforementioned secondary protein target array in the control area 140 has two rows: one row is mouse IgG, and the other row is rabbit IgG mixed with dummy IgG serum protein to form Contain at least five steps and a gradient density series with a linear slope of -20 log (dilution) from the maximum density to the minimum density. The initial dilution ratio is 1000:1, and then the dilution ratio is 1:1 to 1,000:1. .

In another embodiment of the present invention, the aforementioned secondary target array contains a mixture of mouse IgG or rabbit IgG-donkey IgG at three densities of 33%, 16.5%, and 4%.

In another embodiment, the antigen site found in the final processing step is colored due to chromogen precipitation. Therefore, the mouse and rabbit target arrays reflect the -20 log (dilution) linear slope of the chromogen precipitation of the secondary staining kit, or the target arrays reflect the reactivity and enzyme amplification of the secondary stain.

In another embodiment, whether the main target density gradient array can be successfully formed depends on whether the target mixture can be successfully prepared, whether the mixture can be successfully deposited on a glass slide with an adhesive coating layer, and whether it can be The adhesive forms a covalent bond with the target substance.

In another embodiment, if it can be inferred that the target array has been deposited successfully and the primary and secondary staining reagents have performed properly, a computer algorithm can be used to perform curve matching between the data sets. In another embodiment, the primary stain is selected from any one of IHC or ICC approved antibodies, including mouse or rabbit host proteins that will not bind to fluorescent markers or bind to enzyme sites (such as HRP or AP) . In another embodiment, the secondary stain includes a plurality of secondary stains with a magnification ratio of between 1× and 25×, which are not related to mice and rabbits, and use different chromogens.

It is worth mentioning that in another embodiment of the present invention, the absolute detection result of one slide may not be exactly the same as the detection result of another slide at another time point. The reason is that the effect of the secondary staining kit varies from batch to batch, and the same is true for the effectiveness of the primary stain and the primary antibody. However, the test results of the glass slides processed in this case can be verified by the control target, and are equivalent to the test results of another glass slide using different staining reagents.

In some embodiments, the concentration scale of the main antigen is applied to coexisting tissue sections, so as to find cell defects in the tissue sections, such as cancer cells.

Various embodiments will be described below as examples. Those who are familiar with the art should know that not only can these embodiments be modified in a variety of ways, but other embodiments can also be used, all without departing from the broader scope of the present invention. The above and other changes made to the exemplary embodiments are all covered by the scope of the present invention.

example

The following examples are only provided to illustrate the present invention, and should not be construed as limiting the scope of the present invention.

Example 1 Formation of paraffin shielding coating by spraying method

The surface of the glass slide is sprayed with a low-speed airflow. Some embodiments use a low velocity liquid-air mixture. The mixture was sprayed on the glass slide through the mask to cover the control target. Usually one or two passes are sprayed to form a layer with a thickness of less than 5 microns, during which there is no need to reheat the paraffin seal to make it flow. The paraffin mixture storage tank and nozzle are heated to slightly higher than 56°C to ensure that the paraffin is sprayed in a liquid state and is still liquid during the process of spraying onto the glass slide. The nominal spray coverage of the nozzle is 0.375 inches wide. In order to reflux the deposited paraffin mixture, the ambient temperature is increased to 60°C and maintained for one minute. This step removes the solvent and restores the paraffin to a hardened state after cooling.

Example 2 Formation of paraffin shielding coating by screen printing

The current is passed through the wire between the two parallel sides of the stainless steel printed screen to heat the screen. The temperature of the screen is slightly lower than the melting point of the paraffin wax to prevent the paraffin paste from oozing out from the bottom of the screen. At this temperature, paraffin wax behaves like a paste rather than a liquid. In order to reflow the deposited paraffin, the ambient temperature is increased to 60°C and maintained for one minute. This step can remove any solvent in the applied paraffin mixture and restore the paraffin to a hardened state.

Example 3 Formation of paraffin shielding coating by inkjet method

The inkjet head used is combined with a heater to keep the paraffin in a liquid state. The way to convert paraffin wax from solid to liquid is to use aliphatic solvents (such as any isomer of xylene). In order to reflux the deposited paraffin mixture, the ambient temperature is increased to 60°C and maintained for one minute. This step can remove the solvent in the applied paraffin mixture and restore the paraffin to a hardened state.

Example 4 Formation of paraffin shielding coating by roller transfer method

The heated roller pulls a layer of paraffin film from the heated storage tank, and then transfers the paraffin film to a glass slide. The transfer method is similar to painting the wall with a raising roller. In order to reflow the deposited paraffin, the ambient temperature is increased to 60°C and maintained for one minute. This step can remove the solvent in the applied paraffin mixture and restore the paraffin to a hardened state.

Example 5 The relationship between antigen retrieval exposure time and degradation of PRS secondary targets arranged in a simple logarithmic series

The test in this example is to verify that the change of AR exposure time will be reflected in the 2D secondary target and AR target.

The expected result is that the attenuation of the signal intensity varies with the antigen retrieval exposure time, and its rate of change is a linear slope. Sure enough, if the time required to increase the temperature of the AR buffer above 89°C is not considered, the slope in the relationship graph is linear. In order to achieve the best white balance and contrast settings, the PRS black/white target system accepts 8-bit digitization, and the resulting slope is 1.3 lsb/minute (+/- 0.2 lsb). The experimental results show that after the 89°C time scale, the signals of the AR target and the secondary target decay in a linear manner with the increase of the AR exposure time, for a period of 20 minutes. And after the end of this 20 minutes, because more than 50% of the control targets have been under extreme pressure, the effectiveness of the secondary targets is reduced.

Example 6 Consistency of secondary targets

The experiment in this example is to explore the following two factors:

I. Compare the same batch of glass slides made with the same batch number of secondary protein sources and dilutions in a point-to-point manner.

II. Point-to-point comparison of glass slides made with different batches of secondary protein sources and dilutions.

This experiment uses 100% and 40% target formulations. A total of 100 slides were printed, and all slides were processed with the avidin-biotin complex (ABC) mouse and rabbit secondary staining kit manufactured by Scytek. As antigen retrieval operations will add additional variables, antigen retrieval was not performed in this test. The distribution of the two slides is within 1.5%.

Example 7 Choice of dummy protein

Ten different secondary target arrays are made with two different batches of mouse, rabbit and bovine immunoglobulin G (IgG). In detail, these arrays are made with 100%, 40% and 20% dilutions of the aforementioned IgG. The distribution of 100% and 40% dilution groups are both within 1.5%. The dyeing intensity of the 20% dilution group increased unexpectedly. The inventors found that the unexpected increase was due to the unexpected interaction between bovine IgG and the biotinylated goat anti-polyvalent reagent in the above-mentioned ABC staining kit. The solution to this problem is to replace bovine IgG with donkey protein. After retesting, the distribution of the 20% dilution group was also within 1.5%.

Example 8 Perform quality control with PRS secondary targets arranged in a logarithmic series

When used for quality control, coexisting targets can provide feedback for IHC processing operations, as shown in Figure 6.

Please refer to Figure 6. A total of four secondary target arrays (50:50 mixtures of mice and rabbits) are subjected to different levels of antigen retrieval, that is, low, slightly over, over, and extreme over antigen retrieval. The protein loss rate was 5%, 10%, 30% and 40% respectively. The staining results of the four different slides in the figure represent the above four conditions respectively. The purpose of antigen retrieval is to destroy the Schiff base bond between formaldehyde and protein, so that the antigen site is no longer covered. The rate of antigen exposure depends on the reaction temperature. When the temperature rises, nucleate boiling may occur. Nucleate boiling can cause damage to tissues and protein deposits. Under ideal conditions, the antigen retrieval (AR) activity of the entire slide is uniform, but this is often not the case in practice: some areas have slightly more antigen retrieval activities, while others are slightly less. If the above-mentioned inconsistencies of antigen retrieval activities are ignored, the following methods can be used to determine whether slides can be used for diagnostic interpretation.

If AR is insufficient or excessive, the secondary target array may not be able to reflect this failure state. However, two AR targets can show the failure state of insufficient or excessive AR. a. The 2D/3D targets that are under-fixed and the 2D targets that are over-fixed appear black to represent low AR. At this time, the dyeing result of the secondary target array will appear to be perfect, and the target has not shifted to the left due to AR. In the IHC dyeing operation, if the following situations occur, the AR activity may be less: i. The AR heater does not work, or the temperature setting is far below 80°C ii. AR buffer is neutral, that is, the pH value is 7 instead of 6 or 9 iii. Exposure time is too short b. The 2D/3D target with insufficient fixation is extremely white and the 2D target with over fixation is less than 50% black, which means high AR. At this time, the secondary target array will also be roughly white. If the following conditions occur in IHC dyeing operations, it may lead to more AR activities: i. The working temperature of the heater is higher than 95°C ii. Exposure time is too long c. The precipitation of chromogen may be wrong in the following two situations: i. The dyeing intensity of high-concentration secondary targets suddenly drops and does not reach the maximum color depth. The staining intensity of the secondary target array should increase with the density of the reaction site without exception. If it is not incremental, the reagents in the secondary staining kit have been exhausted due to chromogen precipitation. The solution is to increase the dilution ratio of the primary antibody (that is, reduce the antibody concentration). ii. The chromogen reagent has begun to deteriorate after activation (this is a common condition of DAB). The solution is to use a new DAB mixture.

Saturation or cut-off may occur during the staining process, depending on the concentration of the primary antibody and the enzymatic amplification rate of the secondary staining kit. When the density of the enzyme sites exceeds the maximum amount that the enzyme sites can precipitate the coloring agent in the chromogen, it is saturated; at this time, the dyeing result will show the maximum color depth that can be achieved. When the concentration of the primary antibody and the enzymatic amplification rate of the secondary staining kit are too low, resulting in insufficient precipitation of the colorant, it is in the cut-off state. The above two factors cause the color depth of the secondary target array to shift toward saturation (100%) or cut-off (0%). In Figure 6, this offset phenomenon can be seen by the number of visually visible targets. When the enzymatic magnification of the secondary dyeing operation increases, the density of the 100% bearing point shifts to the 0% position. Common enzyme magnifications are 1, 2, 4, 5, 8, 10, 15 and 20 times. The above-mentioned enzyme magnification will shift the secondary target array to 0% by the following amplitudes: 20x All targets offset -26 dBd 15x All targets offset-23.52 10x All targets offset -20 5x All targets offset -13.98 4x All targets offset-12.04 2x All targets offset-6.02 1x Only 2D 100% load point is close to black

If the main target array exists, increasing the enzymatic magnification of the secondary dyeing operation will shift the dyeing density toward the low-concentration main target bearing point. The same phenomenon occurs when the concentration of the primary antibody is increased. Antigen retrieval operation degrades both the primary and secondary targets to a certain degree, thus causing a reverse shift toward the cut-off state. If more than three loading points disappear at the end of the IHC staining operation, it may mean that the antigen retrieval time of the slide is too long, the antigen retrieval temperature is too high, or both occur at the same time, causing the tissue to lose too many antigen sites; in this case , The diagnostic interpretation result of the slide will be meaningless. The above judgment has nothing to do with the effectiveness of the primary antibody, because the staining results have proved that the secondary staining operation is invalid. This level of destruction cannot be overcome by any action in the antibody step.

Example 9 Use the PRS antigen density ruler to track the illuminance

Observing a microscope slide through a conventional microscope is a subjective behavior, because the transmitted light illuminance is not fixed during each observation. When performing full slide imaging (WSI), the scanner uses pure white and pure black holes to establish white balance and contrast. But this is not the case with manual microscopes. Figure 10 shows the impact on the image when the illumination is too low (-5% difference from the best illuminance), best (+0), and too high (+10 or +15% different from the best illuminance). When the illuminance is lower than the optimal illuminance, the dyeing density is compressed. As far as cancer tumor stage is concerned, the compressed staining density may make the grade of diagnosis result higher than the actual grade by one grade. When the illuminance is higher than the optimal illuminance, the image becomes whitish. As far as the stage of cancer is concerned, the whiter image may make the diagnosis result one grade lower than the actual grade. The color density of the antigen and its numerical scale are derived from the primary and secondary targets, and can be superimposed on the WSI image. The numerical scale is an independent item, and the color density is a dependent item. When applying the antigen color density and numerical scale to WSI, even if the user adjusts the illuminance up or down, the numerical scale remains fixed, and the color density scale changes with the illuminance. The advantage is that the user can choose to increase/decrease the apparent illuminance to "see" the features in the tissue image, but the numerical relationship between illuminance and color density still exists. When the magnification is changed, the above effects remain unchanged.

Example 10 Constructing an antigen density scale

According to some embodiments, there are two types of antigen density scales.

1. The Type A ruler uses a secondary target array. The basic assumption is that more primary antibodies are administered than the tissue antigen sites, and the proportion of the former exceeding the latter must not be greater than 10%.

2. Type B rulers use the main target array (gradient density array of main antigens).

A Class: Antigen ruler based only on secondary targets

This type of ruler uses only secondary target arrays. The identification information contained in the 2D barcode includes (a) the data of the primary antibody: the host species of the antibody, and the dilution ratio expressed in -dBd (dBd represents the number of decibels (dB) of dilution); and (b) the enzyme for the secondary staining operation magnification.

The secondary target gradient density array is composed of the known concentration of the secondary target protein, and the concentration of the secondary target decreases by -3dBd. The choice of the maximum concentration depends on the minimum dilution ratio of the primary antibody. The user usually dilutes according to the concentration specifications provided by the antibody reagent manufacturer, first prepares it to a fixed intermediate concentration of 1 μg/ml, and then prepares all other dilutions to cater for different types of tissues. Generally speaking, the dilution ratio of the second set of primary antibodies is between 1:1 and 1,000:1.

In response to the range of enzyme magnification in the secondary staining operation, the secondary target array includes a larger range of dilution ratios. As far as the -3 dBd gradient is concerned, the minimum dilution ratio of the secondary target array is 1,000:1 or -60 dBd, and this dilution ratio is expressed in S dBd (the secondary target dB dilution ratio). Therefore, the highest dilution ratio for a series of eight load points is -0 dBd or 1:1. The degradation of the secondary target due to antigen retrieval is expressed in AR dBd (antigen retrieval dB dilution ratio). Any one of the eight bearing points of the secondary target array represents a -3 dBd gradual change. The loss of two targets due to antigen retrieval (disappeared) means that the loss caused by antigen retrieval is +6 dBd. In other words, for 2D targets, the secondary target array is (-S + AR) dBd or [+6 to -54 dBd]. Now consider the antibody concentration and the enzymatic amplification rate of the secondary staining operation. The antibody concentration is A dBd, and the enzyme amplification rate is E dBd. Therefore, the secondary target array is (-S +AR -E) dBd, and the organization is (+AR -E +A) dBd. Another factor that needs to be considered is the 100% difference between 2D and 3D. The dyeing difference between the 3D structure of the 100% 2D/3D target and the 100% 2D target represents the chromogen precipitation constant of the secondary dye. This constant is used to specify the color density of the numerical scale and expressed in D dBd. The difference in color density is applied to each 2D target in the array. Therefore, the color density of the 2D array can be expressed by (+AR -E +A +D) dBd.

If the enzyme amplification rate is 10×, then E = -20 dBd. Therefore, the 2D secondary target array is: -14, -17, -20, -23, -26, -29, blank and blank dBd. The two adjacent 0% load points are damaged by the antigen retrieval operation so that they cannot be recovered by staining, so they are blank. If the 2D/3D color density difference is 10×, then D = +20 dBd, so that the 3D secondary target array becomes -34, -37, -40, -43, -46, -49, blank and blank dBd . It is assumed here that the primary antibody reagent will find the appropriate antigen site in the main target, and then achieve a 100% yield. It is also assumed here that although each KLH protein molecule has more than two strands of antigen peptides, each KLH protein molecule can only effectively bind to one antibody molecule and stain the antibody molecule. If other antibody molecules find other suitable antigens on the same KLH protein molecule, these antibody molecules will not be able to bind to the KLH protein molecule because the secondary dye occupies the same position. Therefore, the number of detectable antigen sites on each major antigen protein is one. Since the main target contains the same number of protein molecules per microliter as the secondary target, the primary dilution diluted from 500 μg/ml antibody masterbatch is applied to the data of the secondary target array, so that the secondary target Adjust the color density of the target to the value of the antigen density. When monitoring secondary targets, you can select targets with intermediate color density. The intermediate color density is defined as the 50% point between the maximum black value and the maximum white value. This position is equivalent to the 1.5 dBd position in the 3 dBd range. This site is the anchor point established by the antigen density ruler. If the target range mentioned at the end of the previous article is used, the midpoint is -41.5 dBd.

The secondary target is diluted to the primary dilution of 10 μg/ml. Each array is a mixture of mouse (or rabbit) and donkey immunoglobulin G (IgG). The above-mentioned proteins have different atomic masses, but it is assumed here that their atomic masses are all 150 kDa, and it is assumed that the total number of protein molecules is the same despite the different mixing ratios of the target carrying points. Only the concentration of active protein is considered here. When the atomic mass is 150 kDa, the molecular weight MW of individual protein molecules = 249.07×10 ^-12 ng. The diameter of the standard target bearing point is 1 mm. If the printed sediment is 1 µm thick and the sediment concentration is 10 µg/ml, the number of protein molecules deposited is 31.5×10 ⁶ . Therefore, there will be 31.5 protein molecules in the 1 µm diameter range. If a protein molecule is equivalent to an antigen site, the antigen density can be further calculated. Each deposit in the secondary target array uses the same number of protein molecules, but the ratio of mice (or rabbits) to donkeys varies as the concentration of mice (or rabbits) decreases. 100% of the targets are all mice (or rabbits) and correspond to the 0 dBd point on the ruler.

The secondary staining kit will only produce tissue staining when the primary antibody binds to the antigen site on the tissue. There is no particular restriction on the concentration of the antibody to be administered, but the concentration of the antibody must be sufficient to bind to the antigen site available for binding. Therefore, the result of tissue antigen density measurement is a constant amount, but its value must be corrected according to the loss caused by antigen retrieval and the enzymatic amplification rate of the secondary staining operation. In this way, the color density can be harmonized with the measured value.

In the previous example, the enzyme magnification was 10×, and antigen retrieval caused the secondary target array to lose two load points. In other words, the enzyme amplification rate is -20 dBd, and the loss caused by antigen retrieval is +6 dBd. The result is -14 dBd. Each dilution can be represented by the following table: Density value dBd Color density dBd % Target (mouse/rabbit: donkey) Antigen density (mouse/rabbit antigen in ^{1 µm 2)} 0 -14 100 31.5 -3 -17 70.8 23.32 -6 -20 50.1 15.78 -9 -twenty three 35.5 11.18 -12 -26 25.1 7.90 -15 -29 17.78 5.60 -17 -32 12.59 3.96 -20 -35 9.9 3.12

B Class: Ruler based on the main antigen

This ruler uses both primary and secondary target arrays. The identification information contained in the 2D barcode includes (a) the data of the primary antibody: the host species of the antibody, and the dilution ratio expressed in dBd; and (b) the enzymatic amplification rate of the secondary staining operation. The lot number data includes information on the main target combination used.

If a series of main targets exist, the series can include three bearing points, the bearing point with the highest concentration has the same 100% concentration as the secondary target array, but the concentration gradient of the three bearing points is -6 dBd. In fact, the dilution ratios of the primary target array and the secondary target array have the same slope. The main targets are: -0, -6 and -12 dBd, and expressed in terms of P dBd (the main target dB dilution ratio). It can be reasonably expected here that the loss caused by antigen retrieval is almost the same as the loss suffered by the secondary target array. When the main target array reacts with the secondary dye, it is also affected by the same enzyme magnification function. Therefore, the main target array can be represented by (-A +AR -E) dBd, where the density of the main target is controlled by the dilution ratio of the primary antibody. The only necessary condition is: P is always greater than A. If the enzyme amplification rate is 10× (ie E = -20 dBd) and the loss caused by antigen retrieval is +6 dbd, the main target array is -20, -26, and -32 dBd. The loss of antigen retrieval to the main target is not enough to make the main target blank (it can be known from the influence of the secondary target array). Although the secondary target array is not sufficient to establish an antigen density scale, it is necessary to confirm that the primary dilution system is correctly applied so that the primary target can play its role.

The above description has summarized the features of various embodiments, so that those who are familiar with the art can further understand the state of the content of this disclosure. Those who are familiar with the art should know that they can easily design or modify other methods and/or structures based on this disclosure to achieve the same purpose and/or advantages as the embodiments described herein. Those who are familiar with this technique should also understand that the above-mentioned equivalent structure does not deviate from the scope of the content of this disclosure, and these people can change, replace and modify in a variety of ways without departing from the spirit and scope of the content of this disclosure. The details disclosed in this article.

100: Process slides for operating records

101: Substrate

103: Adhesive layer

105: Protective coating

107: raised structure

120: detection area

140: control area

141: Control target

160: Slide Information Area

1411: Control target carrying point

1415: Control target array

Reading the following detailed description in conjunction with the attached drawings can clearly understand the various aspects of the disclosure. Please note that many features are not drawn to scale, but this is standard practice in the industry. In fact, the size of many features may have been arbitrarily enlarged or reduced for clear discussion.

Figure 1 is a processing operation record glass slide in some embodiments. Fig. 2 is a processing operation record glass slide in some embodiments. FIG. 3A is a processing operation documentary glass slide that has not yet been manufactured in some embodiments. FIG. 3B is a processing operation documentary slide that has been manufactured in some embodiments. FIG. 3C is a processing operation record glass slide of the dyeing operation in some embodiments. Figure 4 is a processing operation documentary glass slide that includes a paraffin coating in some embodiments. Figures 5A and 5B are the primary target density gradient array and the secondary target density array of the slide in some embodiments. Figure 6 shows the antigen retrieval targets of the slides in some examples. Figure 7 shows the control area of the slide in some embodiments. Fig. 8A and Fig. 8B are the glass slides that have been stained in some embodiments. Figure 9 shows the staining operation with the slides in some embodiments. Figure 10 shows the imaging results of stained glass slides in some examples. Figure 11 shows the enzymatic amplification operation in some embodiments. Figure 12 is a single primary antibody control slide that can be used with any primary antibody derived from mouse or rabbit in certain embodiments. Figure 13 is a double primary antibody control slide that can use a primary antibody derived from a mouse and a primary antibody derived from a rabbit in some embodiments.

Claims (20)

A glass slide containing: The detection area, which can be used to hold samples containing tissue sections or loose cells; and Control area, which can be used for: In the immunohistochemistry or immunochemical detection operation, display the error of the intermediate step and the measurement result of the implementation effect, and Provide a reference for qualitatively or quantitatively determining the color and antigen density of stained tissues or cells. The slide glass according to claim 1, further comprising an adhesive coating layer that can be combined with moieties, wherein the adhesive coating layer includes at least one end group selected from the group consisting of:- ROH, -R(C=O)OH, -RNH ₃ , -R(C=O)NH ₂ and -RNH ₂ . The slide glass according to claim 1, wherein the control area includes a main target array, the main target array includes one or more main target bearing points, and each of the one or more main target bearing points The main target-bearing site includes a protein site, the protein site includes a protein that can react with the host FcyRI peptide on the primary antibody, and the protein can react with a single or multiple primary antibody hosts, and each of the protein sites can react with the host FcyRI peptide on the primary antibody. A single primary antibody protein is immobilized on the slide. The slide glass according to claim 3, wherein the FcyRI peptide is linked to a carrier protein, and the carrier protein comprises a non-reactive protein, and the non-reactive protein does not interact with the immunohistochemical or immunochemical detection operation The secondary staining reagent used in the reaction. The slide glass according to claim 4, wherein the main target array includes a plurality of the main target bearing points, and each of the plurality of main target bearing points includes the same reactive FcyRI Peptide, the concentration of the reactive FcyRI peptide possessed by each main target carrying point in the plurality of main target carrying points is different from the concentration of the reactive FcyRI peptide possessed by other main target carrying points in the plurality of main target carrying points Concentration of reactive FcyRI peptide. The slide glass according to claim 4, wherein the main target array includes a plurality of the main target bearing points, and each of the plurality of main target bearing points includes a different reactive peptide , The concentration of the reactive peptide contained in each main target bearing point of the plurality of main target bearing points is different from the reactivity possessed by other main target bearing points of the plurality of main target bearing points Concentration of peptide. The slide glass according to claim 1, wherein the control area includes a main target array, the main target array includes a main target bearing point, the main target bearing point includes a plurality of main targets, and the plurality of targets Each main target in the main target includes an antibody that can resist the host protein of the primary antibody used in the immunohistochemistry or immunochemical detection operation. The slide glass according to claim 1, wherein the control area includes a main target array, the main target array includes a main target bearing point, the main target bearing point includes a plurality of main targets, and the plurality of targets Each of the main targets in the main target includes protein A, protein G, protein A/G or protein L that can resist the host protein of the primary antibody used in the immunohistochemistry or immunochemical detection operation. The slide glass according to claim 1, wherein the control area includes a secondary target array, the secondary target array includes a secondary target bearing point, and the secondary target bearing point includes a plurality of secondary targets , And each secondary target of the plurality of secondary targets includes: The host protein of the primary antibody used in the immunohistochemistry or immunochemical detection operation; and Dummy protein. The slide glass according to claim 9, wherein the control area contains a plurality of the secondary target arrays, and each secondary target in the same secondary target array contains the same host protein and is different Each of the secondary targets in the secondary target array contains a different host protein. The slide glass according to claim 1, wherein the control area further includes an imaging reference object bearing point. The slide glass according to claim 11, wherein the imaging reference object bearing point includes at least one of the following: Black reference target, which contains toner; A white reference target, wherein the white reference target comprises a metal oxide or a metal sulfide; or A transparent reference target, wherein the transparent reference target contains a non-reactive protein that does not react with any immunohistochemical or immunochemical reagents. The slide glass according to claim 11, wherein the imaging reference object bearing point comprises: An anhydride-based epoxy resin, which is used for the white reference target or the black reference target; and Equine protein, which is used for the transparent reference target. The glass slide according to claim 1, wherein the control area further includes an antigen retrieval monitor carrying point, which can be used to monitor the degree of antigen retrieval in the immunohistochemistry or immunochemical detection operation. The slide glass according to claim 14, wherein the antigen retrieval monitor bearing point comprises a carrier protein or submicron beads, which comprises: Host protein, which is covalently attached to define three-dimensional (3D) particles; and The fixative is located below or above the host protein. The slide glass according to claim 1, wherein the control area is covered by a paraffin coating layer. The glass slide described in claim 1, further comprising at least one of the following: Marks used to identify the type of slides; The code used to identify the antigen contained in the control area; or batch number. A method for immunohistochemical staining using glass slides, the method comprising the following steps: Remove part of the fixation, where the part is sufficient to expose the antigen site in the tissue section and expose the control target; The tissue section and the control targets are stained, wherein the staining includes: One or more primary antibodies are applied to the slide, and one or more secondary antibodies that are attached to the moiety are applied to the slide; or one or more are attached to the moiety to the slide Primary antibody; and Applying dyeing reagents that can produce color in the presence of the moiety, so as to show the presence of the target antigen; and Evaluate the quality of the staining operation, wherein the evaluation includes evaluating the staining results of the control targets or the staining results of the antigen retrieval monitors in the control area, the control area being separated from the tissue section. The method according to claim 18, further comprising removing the paraffin protective layer of the control targets, wherein removing the paraffin protective layer comprises: Heat the slide to a temperature between 65°C and 75°C for 3 to 10 minutes to form semi-liquid paraffin on the control area and the tissue section; Liquefying the semi-liquid paraffin with an organic solvent; Sequentially, rehydrate the control area and the test area with absolute ethanol and ethanol of increasing concentration but not exceeding 60%; and The control targets are rehydrated in the buffer solution. The method according to claim 19, wherein the organic solvent comprises at least one selected from the group consisting of aliphatic solvents, xylene and xylene isomers, and the buffer solution is a salt The basic buffer solution.

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