Quality control slides and inspection method for an automated immunohistochemical staining system using them

Abstract

To provide a quality control slide which enables estimation of the presence or absence of abnormality in an automatic immunostaining device and causes thereof if abnormality is present.SOLUTION: A quality control slide is provided, having a spot (antigen(-)) containing no antigen, a spot (fixation(-) / antigen(+)) not histologically fixed and containing an antigen, and a spot (fixation(+) / antigen (+)) histologically fixed and containing the antigen.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to the configuration of a slide for accuracy management for managing the accuracy of an inspection device and an inspection method using the same, and particularly relates to a technique effective for the accuracy management of an automatic immunostaining device for pathological examination.

Background Art

[0002] Pathological examination is a method of diagnosing the presence or type of a lesion by creating a specimen sample in which a specimen such as cells or tissues collected from a patient is attached to a slide glass and observing it at the microscopic level. In pathological examination, a staining technique for the components of cells and tissues is essential for identifying lesions from colorless specimens. One of the main staining techniques used in pathological examination is immunostaining. This staining technique detects a specific antigen expressed in a specimen using an antibody reagent. It is a very important technique in pathological examination because it visualizes the localization of the antigen to be examined.

[0003] The number of immunostaining cases has been increasing year by year, and the demand for an automatic immunostaining device that realizes the automation of immunostaining is increasing. Current automatic immunostaining devices realize the automation of activation, washing, and staining. However, while automating multiple processes, it becomes difficult to determine in which process an abnormality has occurred when an abnormality occurs. To prevent abnormalities, daily inspections by users and regular inspections by device manufacturer engineers are required, which imposes a workload on users and engineers.

[0004] In this regard, a slide for accuracy management that can confirm whether all processes of an automatic immunostaining device have proceeded normally or an abnormality has occurred in any of the processes is already known. For example, in Patent Document 1, a slide glass having two spots each containing a binding substance and a non-binding substance is disclosed. If the spot containing the binding substance develops color and the spot not containing the binding substance does not develop color, it can be determined that the automatic immunostaining device is operating normally, and if any other color development pattern is confirmed, it can be determined that an abnormality has occurred in the automatic immunostaining device.

Prior Art Documents

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-98097 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, with the technology described in Patent Document 1, it is difficult to determine the type of abnormality when one occurs.

[0007] Therefore, the object of the present invention is to provide a quality control slide and an inspection method using the same that can determine whether or not there is a malfunction in an automated immunohistochemical staining device, and, if a malfunction occurs, can estimate the cause thereof. [Means for solving the problem]

[0008] To solve the above problems, the present invention is characterized by having spots that do not contain an antigen (antigen(-)), spots that do not undergo histological fixation and contain an antigen (fixed(-) / antigen(+)), and spots that undergo histological fixation and contain an antigen (fixed(+) / antigen(+)).

[0009] Furthermore, the present invention provides a method for inspecting an automated immunohistochemical staining apparatus using a quality control slide having the above-described features, characterized in that it determines whether or not there is an abnormality in the automated immunohistochemical staining apparatus, and if an abnormality occurs, estimates the cause thereof, based on the color development pattern of each spot. [Effects of the Invention]

[0010] According to the present invention, it is possible to realize a quality control slide and an inspection method using the same that can determine whether or not there is a malfunction in an automated immunohistochemical staining device, and if a malfunction occurs, can estimate the cause thereof.

[0011] Thereby, when using the automatic immunostaining apparatus, it is possible to confirm whether all steps have proceeded normally or an abnormality has occurred, and if an abnormality has occurred, the cause thereof can be estimated.

[0012] Also, by tracking changes over time, it is possible to detect signs of abnormality in the automatic immunostaining apparatus.

[0013] Problems, configurations, and effects other than those described above will be clarified by the description of the following embodiments.

Brief Description of the Drawings

[0014] [Figure 1] It is a diagram schematically showing the minimum configuration of a slide for accuracy management according to an embodiment of the present invention. [Figure 2] It is a diagram schematically showing the result of immunostaining the slide for accuracy management of FIG. 1. [Figure 3] It is a diagram schematically showing a slide for accuracy management of FIG. 1 with further spots added thereto. [Figure 4] It is a diagram schematically showing the result of immunostaining the slide for accuracy management of FIG. 3. [Figure 5] It is a diagram schematically showing a slide for accuracy management in which four levels are given to the antigen concentration and the fixation strength of spots, respectively. [Figure 6] It is a diagram schematically showing the result of immunostaining the slide for accuracy management of FIG. 5. [Figure 7] It is a diagram schematically showing the result of immunostaining the slide for accuracy management of FIG. 1 over time. [Figure 8] It is a diagram schematically showing the result of immunostaining the slide for accuracy management of FIG. 5 over time.

Modes for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In each drawing, the same components are denoted by the same reference numerals, and detailed descriptions of overlapping parts are omitted.

[0016] The slide for accuracy control of the present invention can be used for accuracy control of immunostaining. As the most suitable application, it is to be installed and used in an automatic immunostaining apparatus when using the automatic immunostaining apparatus. The slide for accuracy control of the present invention is used when performing immunostaining of a specimen.

[0017] Immunostaining is a technique for detecting a specific antigen using an antibody. The immunostaining referred to in the present invention includes steps of activating a specimen, adding an antibody reagent to prepare a reaction system, performing an antigen-antibody reaction, washing unreacted antibody reagent, performing a color development reaction, and washing unreacted color development reagent.

[0018] By mounting a specimen on the specimen carrier part of the slide for accuracy control of the present invention, the specimen and the spot can be immunostained on the same slide, and the specimen and the spot can be processed under the same conditions. At this time, by confirming the color development pattern of the spot, the reliability of the staining state of the specimen can be evaluated. In addition, the slide for accuracy control of the present invention can confirm whether all steps of immunostaining are proceeding normally or whether an abnormality has occurred, and if an abnormality has occurred, the cause can be estimated.

[0019] FIG. 1 schematically shows the minimum configuration of the slide for accuracy control of the present invention.

[0020] As shown in FIG. 1, the slide 9 for accuracy control of the present invention is composed of a spot (antigen (-)) 1 that does not contain an antigen, a spot (fixed (-) / antigen (+)) 2 that has not been subjected to histological immobilization and contains an antigen, a spot (fixed (+) / antigen (+)) 3 that has been subjected to histological immobilization and contains an antigen, and a specimen carrier part 8. The slide 9 for accuracy control of the present invention is characterized by having at least three or more types of spots among spots 1 to 3.

[0021] In this invention, histological fixation refers to a chemical treatment to protect specimens of biological origin from deterioration due to autodegradation or decomposition. Generally, specimens used in immunohistochemistry are histologically fixed. One representative histological fixation method is the use of aldehyde compounds. By crosslinking the antigen in the specimen with the surrounding tissue components using aldehydes, it becomes possible to retain the antigen in a state close to that of the living organism for a long period of time. On the other hand, because histological fixation shields the reaction sites of the antigen, immunohistochemistry will not proceed normally unless the antigen is activated by heat treatment or the like immediately before immunohistochemistry.

[0022] If spot (antigen(-))1 does not develop color, it is presumed that the immunohistochemical staining is not progressing normally, that the staining reagent is not concentrated or active enough, that the reaction system has not been prepared correctly, or that the activation is incomplete. If spot (antigen(-))1 develops strong color, it is presumed that there is residual staining reagent.

[0023] If spot (fixed(-) / antigen(+))2 does not develop color, a failure in reaction system preparation is suspected. If spot (fixed(-) / antigen(+))2 develops weak color, insufficient concentration or insufficient activity of the staining reagent is suspected. If spot (fixed(-) / antigen(+))2 develops color, normal progression of immunohistochemistry or incomplete activation is suspected. If spot (fixed(-) / antigen(+))2 develops strong color, residual staining reagent is suspected.

[0024] If spot (fixed(+) / antigen(+))3 does not develop color, a failure in reaction system preparation is suspected. If spot (fixed(+) / antigen(+))3 develops weak color, insufficient concentration or activity of the staining reagent, or incomplete activation is suspected. If spot (fixed(+) / antigen(+))3 develops color, normal progress of immunohistochemical staining is suspected. If spot (fixed(+) / antigen(+))3 develops strong color, residual staining reagent is suspected.

[0025] By using these spots in combination, it is possible to correlate the color development pattern with the type of abnormality in immunohistochemistry and estimate the abnormality on the automated immunohistochemistry system.

[0026] Figure 2 schematically shows the results of immunostaining of slide 9 used for quality control in Figure 1.

[0027] For example, as shown in Figure 2a, if spot (antigen(-))1 does not develop color, but spot (fixed(-) / antigen(+))2 and spot (fixed(+) / antigen(+))3 develop color, normal progression of immunohistochemistry is presumed. No abnormalities are found, and the entire process is presumed to be normal.

[0028] As shown in Figure 2b, if none of the spots (antigen(-)), spot (fixed(-) / antigen(+)), and spot (fixed(+) / antigen(+)) 3 develop color, a failure in reaction system preparation is suspected. In other words, a reagent dispensing failure is suspected as the abnormality.

[0029] As shown in Figure 2c, if spot (antigen(-))1, spot (fixed(-) / antigen(+))2, and spot (fixed(+) / antigen(+))3 all show strong coloration, residual staining reagent is suspected. In other words, insufficient washing is suspected as the abnormality.

[0030] As shown in Figure 2d, if spot (antigen(-))1 does not develop color, spot (fixed(-) / antigen(+))2 develops color, and spot (fixed(+) / antigen(+))3 develops weak color, incomplete activation is suspected. In other words, insufficient activation is suspected as the abnormality.

[0031] As shown in Figure 2e, if spot (antigen(-))1 does not develop color, and spots (fixed(-) / antigen(+))2 and spot (fixed(+) / antigen(+))3 develop weak color, it is presumed that the staining reagent is either too concentrated or too active. In other words, the abnormality is presumed to be a staining reagent abnormality, including deterioration of the staining reagent.

[0032] By checking the color development patterns of at least three types of spots in this way, it is possible to confirm whether the entire immunohistochemical staining process is normal, or if an abnormality has occurred, and if an abnormality has occurred, to estimate its cause.

[0033] Figure 3 schematically shows the same slide 9 used for quality control in Figure 1, but with an additional spotlight added.

[0034] The precision control slide 9 of the present invention can also estimate the type of anomaly in more detail by adding further spots to the three types of spots described above.

[0035] Excessive histological fixation is applied, and by adding spot 4 containing antigen (fixed (+++) / antigen (+)), excessive retrieval can be estimated.

[0036] In this invention, excessive histological fixation refers to fixation such that the immunohistochemical properties of the antigen are not restored even after normal retrieval. Specifically, it is preferable that excessive histological fixation fixes 50% or more of the amino groups of the antigen. Furthermore, the immunoreactivity of the antigen is not lost even with excessive retrieval.

[0037] By adding Spot (Primary Antibody (+)) 5, which contains a primary antibody that binds to the secondary antibody of the antibody reagent used in the test, and Spot (HRP (+)) 6, which contains an enzyme that reacts with the chromogenic reagent used in the test, it is possible to estimate the details of staining reagent abnormalities such as staining reagent degradation, and also to estimate errors in the selection of the primary antibody.

[0038] If spot (immobilized (+++) / antigen (+)) 4 does not develop color, it is suspected that the immunohistochemical staining is not progressing normally, the reaction system preparation is incomplete, the activation is incomplete, the primary antibody concentration or activity is insufficient, the secondary antibody concentration or activity is insufficient, the chromogenic reagent concentration or activity is insufficient, or the primary antibody is unsuitable. If spot (immobilized (+++) / antigen (+)) 4 develops color, it is suspected that the activation is excessive. If spot (immobilized (+++) / antigen (+)) 4 develops color strongly, it is suspected that there is residual staining reagent.

[0039] If spot (primary antibody (+)) 5 does not develop color, a failure in reaction system preparation is suspected. If spot (primary antibody (+)) 5 develops weak color, insufficient concentration or activity of the secondary antibody or insufficient concentration or activity of the chromogenic reagent is suspected. If spot (primary antibody (+)) 5 develops color, normal progression of immunohistochemistry, incomplete activation, insufficient concentration or activity of the primary antibody, incompatible primary antibody, or excessive activation is suspected. If spot (primary antibody (+)) 5 develops strongly color, residual staining reagent is suspected.

[0040] If spot (HRP(+))6 does not develop color, a failure in reaction system preparation is suspected. If spot (HRP(+))6 develops weakly, incomplete activation, insufficient concentration or activity of the chromogenic reagent is suspected. If spot (HRP(+))6 develops color, normal progress of immunohistochemistry, insufficient concentration or activity of the primary antibody, insufficient concentration or activity of the secondary antibody, incompatible primary antibody, or excessive activation is suspected. If spot (HRP(+))6 develops strongly, residual staining reagent is suspected.

[0041] Figure 4 schematically shows the results of immunostaining of quality control slide 9 from Figure 3.

[0042] For example, as shown in Figure 4a, if spot (antigen(-))1 and spot (fixed(+++) / antigen(+))4 do not develop color, but spot (fixed(-) / antigen(+))2, spot (fixed(+) / antigen(+))3, spot (primary antibody(+))5, and spot (HRP(+))6 develop color, then normal progression of immunohistochemistry is presumed. In other words, there are no abnormalities, and the entire process is presumed to be normal.

[0043] As shown in Figure 4b, if none of the spots (antigen(-))1, (fixed(-) / antigen(+))2, (fixed(+) / antigen(+))3, (fixed(+++) / antigen(+))4, (primary antibody(+))5, and (HRP(+))6 develop color, a failure in reaction system preparation is suspected. In other words, a reagent dispensing failure is suspected as the abnormality.

[0044] As shown in Figure 4c, if spot (antigen(-))1, spot (fixed(-) / antigen(+))2, spot (fixed(+) / antigen(+))3, spot (fixed(+++) / antigen(+))4, spot (primary antibody(+))5, and spot (HRP(+))6 all develop strong coloration, residual staining reagent is suspected. In other words, insufficient washing is suspected as the abnormality.

[0045] As shown in Figure 4d, if spot (antigen(-))1 and spot (fixed(+++) / antigen(+))4 do not develop color, spot (fixed(+) / antigen(+))3 develops weak color, and spot (fixed(-) / antigen(+))2, spot (primary antibody(+))5, and spot (HRP(+))6 develop color, then incomplete retrieval is suspected. In other words, the abnormality is suspected to be insufficient retrieval.

[0046] As shown in Figure 4e, if spot (antigen(-))1 and spot (fixed(+++) / antigen(+))4 do not develop color, spot (fixed(-) / antigen(+))2 and spot (fixed(+) / antigen(+))3 develop weak color, and spot (primary antibody(+))5 and spot (HRP(+))6 develop color, it is presumed that the primary antibody concentration or activity is insufficient. In other words, the abnormality is presumed to be degradation of the primary antibody.

[0047] As shown in Figure 4f, if spot (antigen(-))1 and spot (fixed(+++) / antigen(+))4 do not develop color, spot (fixed(-) / antigen(+))2, spot (fixed(+) / antigen(+))3, and spot (primary antibody(+))5 develop weak color, and spot (HRP(+))6 develops color, then insufficient concentration or activity of the secondary antibody is suspected. In other words, the abnormality is suspected to be degradation of the secondary antibody.

[0048] As shown in Figure 4g, if spot (antigen(-))1 and spot (fixed(+++) / antigen(+))4 do not develop color, while spots (fixed(-) / antigen(+))2, spot (fixed(+) / antigen(+))3, spot (primary antibody(+))5, and spot (HRP(+))6 develop weak color, then insufficient concentration or activity of the chromogenic reagent is suspected. In other words, the abnormality is suspected to be deterioration of the chromogenic reagent.

[0049] As shown in Figure 4h, if spots (antigen(-))1, (fixed(-) / antigen(+))2, (fixed(+) / antigen(+))3, and (fixed(+++) / antigen(+))4 do not develop color, while spots (primary antibody(+))5 and spot (HRP(+))6 develop color, then a primary antibody mismatch is suspected. In other words, the abnormality is suspected to be a misselection of the primary antibody.

[0050] As shown in Figure 4i, if spot (antigen(-))1 does not develop color, but spots (fixed(-) / antigen(+))2, spot (fixed(+) / antigen(+))3, spot (fixed(+++) / antigen(+))4, spot (primary antibody(+))5, and spot (HRP(+))6 develop color, it is presumed that the fixed(+++) is being activated. In other words, the abnormality is presumed to be excessive activation.

[0051] Furthermore, the quality control slide 9 of the present invention enables more accurate quality control by providing multiple levels for the histological fixation strength of the spots and the concentration of antigens contained in the spots.

[0052] The types and number of levels are not particularly limited, but considering the area occupied by the spots on the slide, it is preferable to have between 3 and 6 types.

[0053] Figure 5 schematically shows quality control slide 9, in which four levels of spot antigen concentration and fixation intensity were applied. Figure 6 schematically shows the results of immunostaining of quality control slide 9 from Figure 5.

[0054] For example, as shown in Figure 5, quality control slide 9, which has 16 spots with four levels each for histological fixation strength (-), (+), (++), (+++) and antigen concentration (-), (+), (+++), can also perform more accurate quality control by showing a gradual color development pattern as shown in Figure 6.

[0055] Furthermore, in the quality control slide 9 of the present invention, multiple sets of spots may be placed for each type of antigen. Alternatively, multiple types of antigens may be included in a single spot. This increases versatility for immunohistochemistry and allows for the estimation of errors in primary antibody selection.

[0056] The quality control slide 9 of the present invention allows for quality assurance testing of the slide itself by using a fluorescently labeled antigen. The fluorescently labeled antigen can be obtained by reacting an antigen with a fluorescent labeling reagent. The fluorescently labeled antigen can be introduced into the spot using the same method as the antigen.

[0057] The fluorescence intensity of the spot can be quantified using a fluorescence microscope, fluorometer, or fluorescence imaging device, allowing visualization of the antigen introduced into the spot before use. At this time, a quality assurance range can be set for the immunohistochemical intensity. If there is an excess or deficiency in the immunohistochemical intensity estimated from the fluorescence intensity using a calibration curve, it can be determined that the slide is unsuitable for use as a quality control slide (9). The quality assurance range is set appropriately by creating a calibration curve based on the correlation between fluorescence intensity and immunohistochemical intensity.

[0058] The quality control slide 9 of the present invention can also be used to detect signs of abnormalities in an automated immunohistochemical staining device.

[0059] By observing the changes in the color patterns of at least three types of spots over time, it is possible to detect early signs of malfunction in the automated immunohistochemical system.

[0060] The details will be explained below using Figures 7 and 8. Figure 7 schematically shows the results of immunostaining of quality control slide 9 from Figure 1 over time. Figure 8 schematically shows the results of immunostaining of quality control slide 9 from Figure 5 over time.

[0061] For example, by checking the daily changes in the spot color pattern, if the normal color pattern for the entire process (Figure 7a) shows a tendency to approach one of the color patterns shown in Figures 7b, 7c, 7d, and 7e, it can be confirmed that an abnormality corresponding to that color pattern is occurring.

[0062] If the normal color development pattern in Figure 7a changes over time to a pattern like Figure 7b, it can predict an abnormality in reagent dispensing. If the color development pattern becomes like Figure 7c, it can predict an abnormality in the washing mechanism. If the color development pattern becomes like Figure 7d, it can predict an abnormality in the activation mechanism. If the color development pattern becomes like Figure 7e, it can predict an abnormality in the staining reagent. In this way, by checking the trend of changes in the color development pattern over time, it is possible to predict an abnormality in the automated immunohistochemical system.

[0063] Even when using the quality control slide 9 of the present invention as a warning of abnormalities, more accurate warnings of abnormalities can be made by setting multiple levels for the histological fixation strength of the spot and the concentration of the antigen contained in the spot.

[0064] For example, by checking the daily changes in the spot color pattern, if the normal color pattern for the entire process (Figure 8a) shows a tendency to approach one of the color patterns shown in Figures 8b, 8c, 8d, 8e, and 8f, it can be confirmed that an abnormality corresponding to that color pattern is occurring.

[0065] The method for reading the color pattern of the spots constituting the precision control slide 9 of the present invention is not particularly limited. The color intensity of the spot color pattern may be qualitatively read and estimated by visual inspection, but it is preferable to read it quantitatively.

[0066] To quantitatively determine the color intensity, methods include photographing the entire slide with a camera and analyzing the resulting image, microscopically observing each spot and analyzing the resulting image, measuring the chromaticity of each spot, or measuring the absorbance at a specific wavelength.

[0067] For example, in a method where one slide is photographed with a camera and the captured image is analyzed, the color pattern on the slide can be quantified by inputting the image into a device that outputs color information for each coordinate on the captured image. In this case, it is preferable to have pre-colored color sample spots installed on the precision control slide 9 in order to compare the color intensity.

[0068] Furthermore, to avoid being affected by the shooting environment, it is preferable to fix the position of the slide glass and take the photograph in a dark box with a constant light source.

[0069] Furthermore, for example, in methods involving microscopic observation of each spot and image analysis of the captured images, or in methods involving chromaticity measurement of each spot, the RGB values ​​of the spots can be placed in an RGB spatial coordinate system before and after immunostaining, and the distance between those two points can be used as the color intensity.

[0070] Furthermore, in a method that measures the absorbance at a specific wavelength, for example, the absorbance of the dye used as a chromogenic reagent in immunohistochemistry can be used as the color intensity. Alternatively, the absorbance of the dye can be measured at two wavelengths, and the difference or ratio of the two absorbances can be used as the color intensity. When measuring the absorbance at a specific wavelength, the light source used may be a monochromatic light source, multiple monochromatic light sources, or a light source obtained by spectrally separating white light.

[0071] When detecting signs of an anomaly with the precision control slide 9 of the present invention, it is preferable to quantitatively read the color pattern of the spot.

[0072] Anomaly detection can be performed by recording the daily change in quantitative values ​​or the slope of change, and comparing them with pre-prepared educational data.

[0073] The type of anomaly can be estimated by observing which color pattern in the educational data the anomaly is approaching. The educational data can be prepared in advance by intentionally introducing various types of anomalies and quantifying the color intensity.

[0074] Furthermore, if signs of an anomaly are detected, for example, an alert will be sent to the user if the anomaly is related to a reagent, or to the equipment manufacturer's technician if the signs of an anomaly are related to the equipment, enabling a swift response to the anomaly.

[0075] It is preferable that the following functions be incorporated into the automated immunohistochemical system, or installed as ancillary equipment: the imaging function described above, the function to quantify the color intensity by analyzing the captured image, the function to predict abnormalities by comparing the quantitative values ​​with educational data, and the function to issue alerts according to the type of predicted abnormality.

[0076] The precision control slide 9 of the present invention can be realized based on the design guidelines described below.

[0077] As explained with reference to Figure 1, the quality control slide 9 of the present invention consists of at least three types of spots: spot 1 which does not contain an antigen (antigen(-)), spot 2 which does not undergo histological fixation and contains an antigen (fixed(-) / antigen(+)), and spot 3 which undergoes histological fixation and contains an antigen (fixed(+) / antigen(+)).

[0078] The antigens contained in the spots constituting the quality control slide 9 of the present invention are not particularly limited.

[0079] The type of antigen is not particularly limited, but examples include antigen proteins that are the target of pathological examination, or peptides or amino acid oligomers that have a partially homologous sequence to antigen proteins.

[0080] Examples of antigen proteins include Ki-67, p53, CK, D2-40, CD20, CD3, ER, PGR, HER2, CD34, CD79, CD10, αSMA, S100, CD68, CK7, BCL2, CD56, p63, CD5, CK20, Synaptophysin, Vimentin, Chromogranin A, TTF-1, Desmin, CK-HMW, CD31, EMA, CD117, Cyclin D1, CD30, Myeloperoxidase, CD4, and CEA. Antigen proteins can be either native or recombinant.

[0081] The method for histologically fixing the spots constituting the quality control slide 9 of the present invention preferably uses formaldehyde or glutaraldehyde. These may be used in combination, or other aldehyde compounds may be used. Histological fixation with formaldehyde or glutaraldehyde allows the spots to be made to closely resemble those of an actual specimen.

[0082] The method for determining the level of histological fixation of the spots constituting the quality control slide 9 of the present invention is not particularly limited.

[0083] For example, (fixation (-)) means no fixation with formaldehyde solution is performed, (fixation (+)) means immersion in a 4% formaldehyde solution (pH 7.4) for 8 hours, (fixation (++)) means immersion in a 4% formaldehyde solution (pH 7.4) for 3 days, and (fixation (+++)) means immersion in a 4% formaldehyde solution (pH 7.4) for 1 month.

[0084] The method for determining the level of antigen concentration of the spots constituting the quality control slide 9 of the present invention is not particularly limited.

[0085] For example, (antigen(-)) can be used if no immobilization procedure with formaldehyde solution is performed, (antigen(+)) can be used if the immobilization reaction is performed using a 25 μg / mL antigen protein solution, (antigen(++)) can be used if the immobilization reaction is performed using a 50 μg / mL antigen protein solution, and (antigen(+++)) can be used if the immobilization reaction is performed using a 100 μg / mL antigen protein solution.

[0086] The main composition of the spots constituting the quality control slide 9 of the present invention is preferably an amine-containing polymer compound. The amine-containing polymer compound has the function of distributing amino groups in the vicinity of the antigen. By histologically immobilizing the antigen and the amino groups in its vicinity, the spots can be made to resemble those of an actual specimen. The amine-containing polymer compound comprises at least one of the following: protein, peptide, polysaccharide, vinyl-based synthetic polymer, or allyl-based synthetic polymer. The amine-containing polymer compound has multiple amino groups to achieve sufficient histological immobilization of the antigen, and it is possible to chemically or physically immobilize the antigen.

[0087] Examples of materials that satisfy these conditions include materials made of proteins such as serum albumin, ovalbumin, gelatin, collagen, and casein from various animals; materials made of peptides such as polylysine and polyornithine; materials made of polysaccharides such as chitosan and chitin; vinyl-based synthetic polymers such as polyvinylamine; allyl-based synthetic polymers such as polyallylamine or compounds having polyallylamine as a partial structure; and copolymers having parts of these structures.

[0088] Among these, serum albumin is particularly preferred from the standpoint of spot formation, suppression of non-specific adsorption of staining reagents, etc., and availability, but is not limited to this. When using bio-derived materials such as proteins, they may be natural or recombinant.

[0089] The area occupied by each spot constituting the precision control slide 9 of the present invention is 1 mm². 2 Above 1000mm2 More preferably, 1 mm 2 500mm or more 2 The following is preferable: Considering the need to place multiple spots on the slide and the need to balance ease of observation and productivity during pathological examination, 5 mm 2 100mm or more 2 The following is more preferable:

[0090] The thickness of the spots constituting the quality control slide 9 of the present invention is preferably 0.5 μm or more and 500 μm or less. Considering that the thickness of specimens used in pathological examinations is generally about 5 μm, that the liquid level of the reagent during immunostaining is generally about 100 μm, and productivity, a thickness of 1 μm or more and 100 μm or less is more preferable.

[0091] The shape of the spots constituting the precision control slide 9 of the present invention is not particularly limited. They can be set as appropriate depending on the manufacturing method and inspection purpose, such as circular, elliptical, polygonal, rectangular, or linear, but considering uniform immunostaining, circular or elliptical shapes are more preferable.

[0092] The method for manufacturing the precision control slide 9 of the present invention is not particularly limited.

[0093] For example, methods include solidifying the spot precursor solution into a cylindrical shape by physical or chemical means, slicing it thinly, and attaching it to a slide; applying the spot precursor solution to a fluorine plate-shaped mold, solidifying it by physical or chemical means, cutting it out as a spot, and attaching it to a slide; or spraying the spot precursor solution onto a slide, similar to inkjet printing, and then solidifying it by physical or chemical means.

[0094] The physical or chemical method for solidifying the spot precursor solution is not particularly limited. A solidification method can be used depending on the properties of the compound used.

[0095] For example, when using serum albumin as a precursor solution for a spot, a gel-like solid can be obtained by physically heating an aqueous solution of serum albumin to a temperature of around 55°C to 100°C or by adding ethanol.

[0096] Additionally, when using gelatin or collagen as a precursor solution for spot treatment, a gel-like solid can be obtained by chemically solidifying the gelatin or collagen using a chemical crosslinking agent.

[0097] The method for introducing antigens to spots constituting the quality control slide 9 of the present invention is not particularly limited.

[0098] For example, one method involves solidifying the spot precursor solution into a cylindrical shape and then introducing the antigen solution by physical or chemical means, or one involves mixing the spot precursor solution and antigen solution beforehand and then introducing them by physical or chemical means.

[0099] The physical or chemical method used to introduce the antigen to the spot is not particularly limited. An introduction method can be used that is appropriate to the characteristics of the precursor solution used for the spot.

[0100] For example, when using serum albumin as a precursor solution for a spot, the antigen solution and serum albumin solution can be mixed beforehand, and a gel-like solid substance in which the antigen has been solidified can be obtained by physical means, such as applying strong heat of about 55°C to 100°C or adding ethanol.

[0101] Alternatively, a gel-like solid in which the antigen has been solidified can be obtained by a chemical method by immersing a cylindrical gel-like solid formed from serum albumin solution in a solution of a chemical crosslinking agent and the antigen.

[0102] The chemical crosslinking agent can be selected depending on the compound used. For example, when using serum albumin, gelatin, or collagen, DMT-MM, glutaraldehyde, dimethyl sveluiminate, genipin, (benzotriazole-1-yloxy)tris-(dimethylamino)phosphonium, benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate, polyfunctional ethylene glycol polymers, carbodiimide compounds, etc., can be applied.

[0103] Embodiments of the present invention will be described in more detail below. However, the present invention is not limited to the following embodiments. [Examples]

[0104] In Example 1, the precision control slide 9 of the present invention was manufactured using the following method.

[0105] 10 wt% bovine serum albumin was prepared and injected into a cylindrical mold with a diameter of 5 mm. The mold was heated to 90°C to produce a gel-like solid of bovine serum albumin.

[0106] The gel was immersed in a 0.1 wt% glutaraldehyde solution and left to stand at room temperature for 3 hours.

[0107] The gel was immersed in a 100 μg / mL Recombinant Human Vimentin (labeled with Alexa Fluor 680) solution (pH 7.4) and left to stand overnight at room temperature.

[0108] The gel was immersed in a 4% formaldehyde solution (pH 7.4) and left to stand at room temperature for 8 hours. The gel was then immersed in ethanol / water = 70v / 30v% and left to stand overnight, then immersed in ethanol / water = 95v / 5v% and left to stand for 1 hour, and finally immersed in ethanol and left to stand for 3 hours.

[0109] The gel was immersed in xylene / ethanol = 70v / 30v% and allowed to stand for 1 hour, then immersed in xylene / ethanol = 95v / 5v% and allowed to stand for 1 hour, and finally immersed in xylene and allowed to stand for 3 hours.

[0110] The gel was immersed in a xylene solution of 50 wt% paraffin and left to stand at 60°C for 1 hour. After immersion in paraffin and immersion at 60°C for 3 hours, the gel was cooled to 4°C to prepare a paraffin-embedded gel. Spots (fixation (+) / antigen (+)) (corresponding to spot 3 in Figure 1) were created on the paraffin-embedded gel thin film by sectioning with a microtome.

[0111] Using a similar method, PBS solution (pH 7.4) was used instead of 4 wt% formaldehyde solution (pH 7.4) and Recombinant Human Vimentin (labeled with Alexa Fluor 680) solution (pH 7.4) to prepare (antigen(-)) (corresponding to spot 1 in Figure 1) and (fixed(-) / antigen(+)) (corresponding to spot 2 in Figure 1).

[0112] A precision control slide 9 was created by attaching three different types of spots onto a single slide. The area of ​​each spot was 20 mm². 2 The thickness was 4 μm. [Examples]

[0113] In Example 2, the precision control slide 9 of the present invention was manufactured using a different method than in Example 1.

[0114] A 10 wt% bovine serum albumin was prepared and injected into a fluorine-based plate-shaped mold to a height of 200 μm. The mold was heated to 90°C to produce a gel-like thin film of bovine serum albumin.

[0115] A 0.1% glutaraldehyde solution was dropped into the mold and left to stand at room temperature for 3 hours.

[0116] A 100 μg / mL solution of Recombinant Human Vimentin (labeled with Alexa Fluor 680) (pH 7.4) was added dropwise to the mold and left to stand overnight at room temperature.

[0117] The gel film was immersed in a 4% formaldehyde solution (pH 7.4) and left to stand at room temperature for 8 hours.

[0118] A 5mm x 5mm thin gel film was cut to create a (fixed (+) / antigen (+)) (corresponding to spot 3 in Figure 1).

[0119] Using a similar method, PBS solution (pH 7.4) was used instead of 4 wt% formaldehyde solution (pH 7.4) and Recombinant Human Vimentin (Alexa Fluor 680 labeled) solution (pH 7.4) to prepare (antigen(-)) (corresponding to spot 1 in Figure 1) and (fixed(-) / antigen(+)) (corresponding to spot 2 in Figure 1).

[0120] A precision control slide 9 was created by attaching three types of spots to a single slide coated with a peel-preventing agent. The area of ​​each spot was 25 mm². 2 The thickness was 20 μm. [Examples]

[0121] In Example 3, the quality control slide 9 prepared in Example 1 was properly immunostained using an automated immunostaining apparatus to evaluate whether it could predict the normal progression of immunostaining. The staining intensity (fixed (+) / antigen (+)) at this time was standardized to 100.

[0122] The staining intensity for each sample was 5 for (antigen(-)), 110 for (fixed(-) / antigen(+)), and 100 for (fixed(+) / antigen(+)).

[0123] The color development pattern followed the trend shown in Figure 2a, confirming that it is possible to estimate the normal progression of immunohistochemistry. [Examples]

[0124] In Example 4, to evaluate whether the quality control slide 9 prepared in Example 1 could predict washing abnormalities, it was immunostained using an automated immunostaining apparatus while intentionally subjecting it to washing abnormalities. The washing step was performed manually by immersing it in 100 mL of pure water for 1 second.

[0125] The staining intensity was 200 for (antigen(-)), 250 for (fixed(-) / antigen(+)), and 250 for (fixed(+) / antigen(+)). The color development pattern followed the trend shown in Figure 2c, confirming that it is possible to estimate abnormalities related to washing. [Examples]

[0126] In Example 5, to evaluate whether the quality control slide 9 prepared in Example 1 could predict activation abnormalities, immunostaining was performed using an automated immunostaining apparatus while intentionally introducing activation abnormalities. Only the activation step was performed manually by heating at 40°C for 60 minutes.

[0127] The staining intensity was 5 for (antigen(-)), 100 for (fixed(-) / antigen(+)), and 20 for (fixed(+) / antigen(+)). The color development pattern followed the trend shown in Figure 2d, confirming that it is possible to estimate abnormalities related to insufficient activation. [Examples]

[0128] In Example 6, to evaluate whether the quality control slide 9 prepared in Example 1 could predict abnormalities in the staining reagent, immunostaining was performed using an automated immunostaining apparatus while intentionally introducing abnormalities in the staining reagent. This was done using antibody reagents that had been stored at room temperature for 3 months.

[0129] The staining intensity was 5 for (antigen(-)), 50 for (fixed(-) / antigen(+)), and 50 for (fixed(+) / antigen(+)). The color development pattern followed the trend shown in Figure 2e, confirming that it is possible to estimate abnormalities related to the staining reagent. [Examples]

[0130] In Example 7, we verified whether the quality control slide 9 prepared in Example 1 could detect signs of abnormality in the automated immunohistochemistry system. Immunostaining was performed once a day using the automated immunohistochemistry system for 30 days, and the immunohistochemistry intensity was measured.

[0131] On day 0, the immunohistochemical staining intensity was 5 for (antigen(-)), 110 for (fixed(-) / antigen(+)), and 100 for (fixed(+) / antigen(+)).

[0132] On day 10, the number of (antigen(-)) was 5, the number of (fixed(-) / antigen(+)) was 105, and the number of (fixed(+) / antigen(+)) was 95.

[0133] On day 20, the number of (antigen(-)) was 5, the number of (fixed(-) / antigen(+)) was 100, and the number of (fixed(+) / antigen(+)) was 90.

[0134] On day 30, the number of (antigen(-)) was 5, the number of (fixed(-) / antigen(+)) was 95, and the number of (fixed(+) / antigen(+)) was 85.

[0135] In this case, by confirming the trend shown in Figure 7e, we were able to detect early signs of abnormal deterioration of the staining reagent.

[0136] It should be noted that the present invention is not limited to the embodiments described above, and various modifications are included. For example, the embodiments described above are described in detail to make the present invention easier to understand, and are not necessarily limited to those having all the configurations described. Furthermore, it is possible to replace parts of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add configurations from other embodiments to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations. [Explanation of Symbols]

[0137] 1…A spot that does not contain the antigen (antigen-negative) 2…Spots containing antigens that have not undergone histological fixation (fixation-) / antigen-+) 3…Spots that have been histologically fixed and contain antigens (fixed (+) / antigen (+)) 4…Spots containing antigens due to excessive histological fixation (fixation (+++) / antigen (+)) 5…A spot containing a primary antibody that binds to the secondary antibody of the antibody reagent used in the test (primary antibody (+)) 6…A spot containing an enzyme that reacts with the color-developing reagent used in the test (HRP(+)) 7…Display unit (indicator) 8...Specimen carrying section 9…Precision control slides

Claims

1. Spots that do not contain antigens (antigen-negative) and Histological fixation was not applied, and the spots containing the antigen (fixation (-) / antigen (+)) Histological fixation is applied, and the spots containing the antigen (fixed (+) / antigen (+)) A precision control slide characterized by having the following features.

2. A precision control slide according to claim 1, A quality control slide characterized by having at least one of multiple spots to which histological fixation is applied at multiple levels of intensity, and multiple spots containing antigens at multiple levels of concentration.

3. A precision control slide according to claim 2, Excessive histological fixation was applied, resulting in spots containing antigens (fixation (+++) / antigen (+)), A spot containing a primary antibody that binds to the secondary antibody of the antibody reagent used in the test (primary antibody (+)), A spot (HRP(+)) containing an enzyme that reacts with the color-developing reagent used in the test, A precision control slide characterized by having the following features.

4. A precision control slide according to claim 3, The aforementioned excessive histological fixation is characterized by the fixation of 50% or more of the amino groups of the antigen, as is the case with quality control slides.

5. A precision control slide according to claim 1, A precision control slide characterized by having a color sample spot for comparing the color intensity with each of the aforementioned spots.

6. A precision control slide according to claim 1, A quality control slide characterized in that each of the aforementioned spots is subjected to histological fixation with formaldehyde or glutaraldehyde.

7. A precision control slide according to claim 1, Each of the aforementioned spots is a quality control slide characterized by having an amine-containing polymer compound as its main composition.

8. A precision control slide according to claim 7, The slide for quality control is characterized in that the amine-containing polymer compound comprises at least one of a protein, a peptide, a polysaccharide, a vinyl-based synthetic polymer, or an allyl-based synthetic polymer.

9. A precision control slide according to claim 7, The amine-containing polymer compound includes protein, The aforementioned protein is characterized by being one of serum albumin, ovalbumin, gelatin, collagen, or casein, and is used in quality control slides.

10. A precision control slide according to claim 7, The amine-containing polymer compound comprises a peptide, The aforementioned peptide is characterized by being either polylysine or polyornithine, and is used for quality control slides.

11. A precision control slide according to claim 7, The amine-containing polymer compound comprises polysaccharides, The aforementioned polysaccharide is characterized by being either chitosan or chitin, and is used as a quality control slide.

12. A precision control slide according to claim 7, The amine-containing polymer compound includes a vinyl-based synthetic polymer. The aforementioned vinyl-based synthetic polymer is polyvinylamine, and is characterized by being a slide for quality control.

13. A precision control slide according to claim 7, The amine-containing polymer compound includes an allyl-based synthetic polymer. The allyl-based synthetic polymer is a compound having polyallylamine or polyallylamine as a substructure, and is characterized by this quality control slide.

14. A precision control slide according to claim 1, A quality control slide characterized in that the antigen is one of the following: Ki-67, p53, CK, D2-40, CD20, CD3, ER, PGR, HER2, CD34, CD79, CD10, αSMA, S100, CD68, CK7, BCL2, CD56, p63, CD5, CK20, Synaptophysin, Vimentin, Chromogranin A, TTF-1, Desmin, CK-HMW, CD31, EMA, CD117, Cyclin D1, CD30, Myeloperoxidase, CD4, or CEA.

15. A precision control slide according to claim 1, The area of ​​each of the aforementioned spots is 1 mm ２ Above 1000mm ２ A precision control slide characterized by the following:

16. A precision control slide according to claim 1, A precision control slide characterized in that the thickness of each of the aforementioned spots is 0.5 μm or more and 500 μm or less.

17. A precision control slide according to claim 1, A quality control slide characterized in that the antigen is labeled with a fluorescent molecule.

18. A method for inspecting an automated immunohistochemical apparatus using a quality control slide as described in claim 1, A method for inspecting an automated immunohistochemical staining apparatus, characterized by determining whether or not there is an abnormality in the automated immunohistochemical staining apparatus, and if an abnormality occurs, estimating the cause thereof, based on the color development pattern of each spot.

19. A method for inspecting an automated immunohistochemical apparatus according to claim 18, A method for inspecting an automated immunohistochemical staining apparatus, characterized by detecting signs of an abnormality in the automated immunohistochemical staining apparatus based on the daily changes in the color development pattern of each of the aforementioned spots.

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