Staining condition evaluation method and computer-readable program

Abstract

To easily determine suitability of a staining condition.SOLUTION: A staining condition evaluation method includes: a step (step S11) for applying a first primary antibody to a test specimen under a first staining condition; a step (step S13) for imaging the test specimen to acquire a first image; a step (step S15) for applying a second primary antibody to the test specimen under a second staining condition; a step (step S17) for imaging the test specimen to acquire a second image; and a step (step S18) for determining, on the basis of detection results for the stained cells in the first image and the second image, suitability of the first staining condition and the second staining condition. In the step S18, the first staining condition and the second staining condition are determined to be suitable when cells in the test specimen are specifically stained in the first image, cells different from the first image in the test specimen are specifically stained in the second image, and cells in the test specimen stained in the first image are unstained in the second image.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a technique for evaluating staining conditions for a specimen by immunostaining method. [Background technology]

[0002] In recent years, in medical and biological research, single-cell analysis of biological specimens such as tissue specimens and cell specimens has been performed for the purpose of elucidating disease mechanisms, biological mechanisms, drug action mechanisms, etc. For example, in immunostaining analysis, biological substances such as proteins are stained and quantitative and qualitative analysis is performed by measuring the staining state.

[0003] In the above-mentioned immunostaining analysis, the staining state of the specimen needs to be suitable for the analysis. For example, in the blood smear preparation device of Patent Document 1, in order to reduce the difference between the lots of the undiluted staining solution and the differences between the manufacturers and to equalize the staining state of the specimen, when the undiluted staining solution is replaced, the staining conditions (e.g., the dilution rate of the undiluted staining solution, the staining time, etc.) are changed based on the difference between the average nuclear G value of the white blood cells in the test-stained specimen and the target nuclear G value.

[0004] Furthermore, Patent Document 2 proposes providing a process record slide for immunostaining with a detection area, which is a space for detection by immunostaining, and a control area, which is a reference space for qualitatively or quantitatively determining the color density of stained tissue, etc. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2011-185895 A [Patent Document 2] Special Publication No. 2020-523614 Summary of the Invention [Problem to be solved by the invention]

[0006] In immunostaining analysis, a multiple immunostaining method is known in which immunostaining, imaging, and color removal are repeated multiple times for the same specimen while changing the antibody, etc. used for immunostaining. In the multiple immunostaining method, if the staining conditions, such as the concentration of an antibody or the reaction time with the antibody, are not appropriate in immunostaining using one antibody, this may adversely affect not only the immunostaining using that antibody, but also subsequent immunostaining using other antibodies, resulting in a decrease in analytical accuracy.

[0007] The present invention has been made in consideration of the above problems, and has an object to easily determine whether or not dyeing conditions are appropriate. [Means for solving the problem]

[0008] A first aspect of the present invention is a staining condition evaluation method for evaluating staining conditions of a specimen by an immunostaining method, comprising the steps of: a) applying a first primary antibody to a test specimen under first staining conditions; b) applying a secondary antibody and a color developer to the test specimen after step a); c) imaging the test specimen to obtain a first image after step b); d) inactivating or removing the first primary antibody on the test specimen after step c); e) applying a second primary antibody to the test specimen under second staining conditions after step d); f) applying the secondary antibody and the color developer to the test specimen after step e); g) imaging the test specimen to obtain a second image after step f); and h) determining the suitability of the first staining conditions and the second staining conditions based on detection results of stained cells in the first image and the second image after step g). The cells stained with the first primary antibody are different from the cells stained with the second primary antibody. In the step h), if the cells on the test specimen are specifically stained in the first image, and the cells on the test specimen that are different from those in the first image are specifically stained in the second image, and the cells on the test specimen that are stained in the first image are unstained in the second image, it is determined that the first staining conditions and the second staining conditions are appropriate.

[0009] A second aspect of the present invention is a staining condition evaluation method according to the first aspect, wherein in step h), the detection of stained cells in the first image and the detection of stained cells in the second image are each performed using a trained model created by machine learning.

[0010] Aspect 3 of the present invention is a staining condition evaluation method according to aspect 1 or 2, in which a slide on which the test specimen and another test specimen are arranged is prepared prior to step a). In step a), the first primary antibody is applied to the other test specimen under a third staining condition. In step b), the secondary antibody and the coloring agent are also applied to the other test specimen. In step c), the other test specimen is imaged to obtain a third image. In step d), the first primary antibody on the other test specimen is also inactivated or removed. In step e), the second primary antibody is applied to the other test specimen under a fourth staining condition. In step f), the secondary antibody and the coloring agent are also applied to the other test specimen. In step g), the other test specimen is imaged to obtain a fourth image. In step h), the suitability of the third staining condition and the fourth staining condition is also determined based on the detection results of stained cells in the third image and the fourth image. In step h), if cells on the other test specimen are specifically stained in the third image, and cells on the other test specimen that are different from those in the third image are specifically stained in the fourth image, and cells on the other test specimen that are stained in the third image are unstained in the fourth image, the third staining conditions and the fourth staining conditions are determined to be appropriate.

[0011] A fourth aspect of the present invention is the staining condition evaluation method according to the first or second aspect (which may be any one of the first to third aspects), in which the first primary antibody and the second primary antibody are used in sequential multiple immunostaining of one specimen. In the sequential multiple immunostaining, staining with the second primary antibody is performed after staining with the first primary antibody.

[0012] A fifth aspect of the present invention is a computer-readable program for causing a computer to evaluate staining conditions for a specimen by an immunostaining method, the program causing the computer to execute the steps of: i) storing a first image, which is an image of a stained test specimen, and a second image, which is an image of the test specimen stained under conditions different from those of the first image; and j) determining whether the staining conditions are appropriate based on detection results of stained cells in the first image and the second image. The first image is captured in a state in which a first primary antibody is applied to the test specimen under first staining conditions, and a secondary antibody and a coloring agent are further applied. The second image is captured in a state in which a second primary antibody is applied to the test specimen under second staining conditions, and the secondary antibody and the coloring agent are further applied, after the first image is obtained and the first primary antibody on the test specimen is inactivated or removed. Cells stained with the first primary antibody are different from cells stained with the second primary antibody. In step j), if cells on the test specimen are specifically stained in the first image, and cells on the test specimen that are different from those in the first image are specifically stained in the second image, and cells on the test specimen that are stained in the first image are unstained in the second image, the first staining conditions and the second staining conditions are determined to be appropriate. Effect of the Invention

[0013] In the present invention, the suitability of the staining conditions can be easily determined. [Brief description of the drawings]

[0014] [Figure 1] FIG. 4 is a diagram showing a flow of staining condition evaluation according to the first embodiment. [Diagram 2] FIG. 2 is a diagram showing an example of a first image. [Diagram 3] FIG. 13 is a diagram showing an example of a second image. [Figure 4] FIG. 1 illustrates a configuration of a computer. [Diagram 5]FIG. 1 is a diagram showing a part of an evaluation flow in a dyeing condition evaluation method. [Figure 6] FIG. 11 is a plan view showing a slide used in the staining condition evaluation method according to the second embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] 1 is a diagram showing a flow of evaluation in a staining condition evaluation method according to a first embodiment of the present invention, which is used when evaluating staining conditions of a specimen (e.g., a specimen derived from a living body) by an immunostaining method.

[0016] The staining condition evaluation method is used, for example, to evaluate staining conditions in a sequential multiple immunostaining method. The sequential multiple immunostaining method is a multiple immunostaining method in which immunostaining, observation (e.g., imaging), and color removal for the same specimen are repeated multiple times while changing the antibody or the like used for immunostaining. Below, a procedure for evaluating whether or not the staining conditions are appropriate in the Nth (N is an integer equal to or greater than 1) and N+1th immunostaining of the multiple immunostainings performed in the sequential multiple immunostaining method is described.

[0017] In the staining condition evaluation method, first, a test specimen to be used for evaluating the staining conditions is prepared. The test specimen is, for example, obtained from the same tissue as the specimen to be stained and analyzed by the sequential multiple immunostaining method described above. Next, the test specimen is subjected to an antigen activation treatment (for example, heat treatment or enzyme treatment) and a primary antibody is applied under staining conditions considered for implementation in the Nth immunostaining of the sequential multiple immunostaining method (step S11). The primary antibody is the primary antibody used in the Nth immunostaining, and is hereinafter also referred to as the "first primary antibody." In addition, hereinafter, the staining conditions in step S11 are also referred to as the "first staining conditions" or the "first primary staining conditions."

[0018] The first primary antibody is an immunoglobulin that binds to a specific antigen (e.g., a protein, a peptide, a small molecule, etc.) and binds to the antigen with high affinity and specificity. The first primary antibody can be derived from various animal species, such as mouse, rabbit, goat, or chicken. The same applies to the second primary antibody described below.

[0019] The first primary staining conditions include, for example, at least one of the concentration of the first primary antibody applied to the test specimen and the time for which the first primary antibody is applied to the test specimen (i.e., reaction time). The first primary staining conditions may include, for example, treatment conditions for an antigen activation treatment. When the antigen activation treatment is a heat treatment, the treatment conditions include, for example, at least one of the pH of the antigen activation solution in the antigen activation treatment, and the heating temperature and heating time of the test specimen. The same applies to the second primary staining conditions described below.

[0020] After step S11 is completed, a secondary antibody and a coloring agent are applied to the test specimen under the staining conditions (hereinafter also referred to as "secondary staining conditions") to be implemented in the Nth immunostaining (step S12). This causes specific cells, etc., to which the primary antibody is bound in the test specimen to be stained. The secondary antibody and coloring agent are the secondary antibody and coloring agent used in the Nth immunostaining.

[0021] The secondary antibody is an antibody that binds to the primary antibody and corresponds to the animal species related to the production of the primary antibody. For example, if the first primary antibody used in step S11 is derived from a mouse, the secondary antibody used in step S12 is an anti-mouse antibody. As the coloring agent, various ones that develop a color corresponding to the label of the secondary antibody (e.g., AEC (3-amino-9-ethyl-carbazole), etc.) can be used.

[0022] Thereafter, a part or the whole of the test specimen stained in steps S11 and S12 is imaged to obtain a stained image (hereinafter also referred to as a "first image") (step S13). FIG. 2 is a diagram showing an example of a first image 91. In the first image 91 shown in FIG. 2, a stained region 92 (i.e., a region where stained cells are gathered, hereinafter also referred to as a "stained region 92") is marked with parallel diagonal lines.

[0023] If the stained cells cannot be confirmed in the first image 91, and if the distribution of the stained cells in the first image does not have specificity, the above-mentioned first primary staining conditions are considered to be inappropriate. In this case, steps S11 to S12 may be repeated. Specifically, the test specimen is washed to remove the primary antibody, the secondary antibody, and the coloring agent on the test specimen. After washing the test specimen, the primary antibody and the secondary antibody on the test specimen may be inactivated. Thereafter, the first primary staining conditions are changed, and steps S11 to S13 are performed.

[0024] After step S13 is completed, the test specimen is washed, and the first primary antibody, the secondary antibody, and the color developer on the test specimen are removed. The first primary antibody and the secondary antibody on the test specimen may also be inactivated (step S14). That is, in step S14, at least one of inactivation and removal is performed on the first primary antibody on the test specimen. The test specimen is washed, for example, with phosphate buffered saline (PBS) or Tris buffered saline (TBS). The test specimen may be washed multiple times. The first primary antibody and the secondary antibody are inactivated, for example, by heat treatment in which the test specimen is immersed in boiling water. The first primary antibody and the secondary antibody may be inactivated by various other methods. The removal of the color developer (to be precise, removal of color due to color precipitate derived from the color developer) may also be performed by various methods. When the color developer is AEC, for example, the color developer is removed by immersing the test specimen in alcohol such as ethanol at multiple concentrations and washing it. The washing of the test specimen in step S14 and the removal and / or inactivation of the first primary antibody and the secondary antibody are performed under the same processing conditions as the washing and inactivation planned to be performed between the Nth immunostaining and the N+1th immunostaining.

[0025] When step S14 is completed, the test specimen is subjected to an antigen activation process and a primary antibody is applied under staining conditions that are being considered for implementation in the N+1th immunostaining of the sequential multiplex immunostaining method (step S15). The primary antibody applied in step S15 is the primary antibody used in the N+1th immunostaining, and is also referred to as the "second primary antibody" below. In addition, the staining conditions in step S15 are also referred to as the "second staining conditions" or the "second primary staining conditions" below. The second primary antibody is an antibody derived from the same animal species as the above-mentioned first primary antibody, but is an antibody of a different type from the first primary antibody. The second primary staining conditions may be the same as or different from the above-mentioned first primary staining conditions. Note that, when the antigen activation process in step S15 and the inactivation of the primary antibody and the secondary antibody in step S14 are both heat treatments, the inactivation of the primary antibody and the secondary antibody (step S14) and the antigen activation process (step S15) may be performed in parallel by a single heat treatment.

[0026] After step S15 is completed, the secondary antibody and coloring agent are applied to the test specimen under the secondary staining conditions to be performed in the above-mentioned N+1th immunostaining (step S16). The secondary antibody and coloring agent used in step S16 are the secondary antibody and coloring agent used in the above-mentioned N+1th immunostaining, and are the same type as the secondary antibody and coloring agent used in step S12. The secondary staining conditions in step S16 may be the same as or different from the secondary staining conditions in step S12.

[0027] On the test specimen, the cells to which the first primary antibody applied in step S11 binds (i.e., cells stained with the first primary antibody) are different from the cells to which the second primary antibody applied in step S15 binds (i.e., cells stained with the second primary antibody). Therefore, if the removal and / or inactivation of the first primary antibody in step S14 is performed appropriately, the region in which the cells stained by the application of the secondary antibody and color developer in step S16 exist is different from and does not overlap with the region in which the cells stained by the application of the secondary antibody and color developer in step S12 exist. On the other hand, if the removal and / or inactivation of the primary antibody in step S14 is not performed appropriately, a part of the region in which the cells stained by the application of the secondary antibody and color developer in step S16 exist overlaps with the region in which the cells stained by the application of the secondary antibody and color developer in step S12 exist.

[0028] After step S16 is completed, a part or the whole of the test specimen stained in steps S15 to S16 is imaged to obtain a stained image (hereinafter also referred to as a "second image") (step S17). Fig. 3 is a diagram showing an example of a second image 93. In the second image 93 shown in Fig. 3, a stained region 94 (i.e., a region where stained cells are gathered, hereinafter also referred to as a "stained region 94") is marked with parallel diagonal lines.

[0029] Thereafter, stained cells are detected in each of the first image 91 and the second image 93. Then, based on the detection results of the stained cells, the suitability of each of the first primary staining conditions in step S11 and the second primary staining conditions in step S15 is determined (step S18).

[0030] In step S18, for example, an operator visually checks the first image 91 and the second image 93 to confirm (i.e., detect) the distribution of stained cells in each of the first image 91 and the second image 93. Specifically, it is confirmed whether or not cells are specifically stained in the first image 91, and it is also confirmed whether or not cells are specifically stained in the second image 93. Furthermore, the first image 91 and the second image 93 are compared, and it is confirmed whether or not cells stained in the first image 91 are unstained in the second image 93.

[0031] Then, if it is confirmed that cells are specifically stained in each of the first image 91 and the second image 93 (i.e., the stained cells are specifically distributed) and that the stained cells in the first image 91 are unstained in the second image 93, it is determined that the first primary staining conditions for the application of the first primary antibody in step S11 and the second primary staining conditions for the application of the second primary antibody in step S15 are appropriate.

[0032] On the other hand, in other cases, it is determined that the first primary staining conditions and / or the second primary staining conditions are not appropriate. For example, when there are almost no stained cells in the first image 91, it is considered that the concentration of the first primary antibody applied in step S11 is insufficient and / or the reaction time is insufficient. When there are almost no stained cells in the second image 93, it is considered that the concentration of the second primary antibody applied in step S15 is insufficient and / or the reaction time is insufficient. When comparing the first image 91 and the second image 93, when some or all of the stained cells in the first image 91 are also stained in the second image 93 (i.e., when the stained area 92 of the first image 91 and the stained area 94 of the second image 93 at least partially overlap), it is considered that the concentration of the first primary antibody applied in step S11 and / or the reaction time of the second primary antibody applied in step S15 is excessive.

[0033] If the first primary staining conditions are determined to be inappropriate in step S18, for example, a new test specimen is prepared, the first primary staining conditions are changed, and steps S11 to S18 are performed. If the second primary staining conditions are determined to be inappropriate in step S18, for example, a new test specimen is prepared, the second primary staining conditions are changed, and steps S11 to S18 are performed. Then, steps S11 to S18 are repeated while replacing the test specimen until appropriate first primary staining conditions and second primary staining conditions are obtained. Note that, instead of preparing a new test specimen, for example, the test specimen determined to be inappropriate in step S18 may be thoroughly washed and heat-treated (i.e., inactivation treatment) to almost completely remove and / or inactivate the primary antibody, secondary antibody, and color developer on the test specimen, and then steps S11 to S18 may be performed.

[0034] In step S18, when appropriate staining conditions for the above-mentioned Nth and N+1th immunostainings are acquired, for example, the test specimen used to acquire the staining conditions is used to evaluate the suitability of the staining conditions for the N+1th and N+2th immunostainings among the multiple immunostainings performed by the sequential multiple immunostaining method. Specifically, for example, washing and / or heat treatment is performed on the test specimen after step S18 to remove and / or inactivate the primary antibody, secondary antibody, and color developer, and then steps S11 to S18 are performed under the staining conditions to be performed in the N+1th and N+2th immunostainings.

[0035] In the above-mentioned staining condition evaluation method, the suitability of the staining conditions may be evaluated sequentially for all of the multiple immunostainings performed in the sequential multiple immunostaining method, or the suitability of the staining conditions may be evaluated sequentially for some of the multiple immunostainings. Alternatively, the suitability of the staining conditions may be evaluated using one test specimen for some of the multiple immunostainings, and the suitability of the staining conditions may be evaluated using another test specimen for the other part of the immunostainings.

[0036] In step S18, for example, a program 572 shown in FIG. 4 may be executed by the computer 5 to determine the suitability of the first primary staining conditions in step S11 and the second primary staining conditions in step S15.

[0037] The computer 5 has a typical computer system configuration including a CPU 51, a ROM 52, a RAM 53, a fixed disk 54, a display 55, an input unit 56, a reading device 57, a communication unit 58, a GPU 59, and a bus 50. The CPU 51 performs various arithmetic processing. The GPU 59 performs various arithmetic processing related to image processing. The ROM 52 stores basic programs. The RAM 53 stores various information. The fixed disk 54 stores information. The display 55 is a display unit that displays various information such as images.

[0038] The input unit 56 includes a keyboard 56a and a mouse 56b for receiving input from an operator. The reader 57 reads information from a computer-readable recording medium 571 such as an optical disk, a magnetic disk, a magneto-optical disk, or a memory card. The display 55, the keyboard 56a, the mouse 56b, and the reader 57 are connected to the bus 50 via an interface I / F. The communication unit 58 transmits and receives signals to and from devices external to the computer 5. The bus 50 is a signal circuit that connects the CPU 51, the GPU 59, the ROM 52, the RAM 53, the fixed disk 54, the display 55, the input unit 56, the reader 57, and the communication unit 58.

[0039] In the computer 5, the program 572 is read in advance from the recording medium 571 via the reading device 57 and stored in the fixed disk 54. The program 572 may be stored in the fixed disk 54 via a network. The CPU 51 and the GPU 59 execute arithmetic processing using the RAM 53 and the fixed disk 54 in accordance with the program 572 for evaluating staining conditions. The CPU 51 and the GPU 59 function as a calculation unit in the computer 5. Other components functioning as a calculation unit other than the CPU 51 and the GPU 59 may be employed.

[0040] 5 is a diagram showing a specific flow when step S18 is performed by computer 5. First, first image 91 acquired in step S13 and second image 93 acquired in step S17 are stored in a storage unit realized by RAM 53, fixed disk 54, etc. (step S181).

[0041] Next, a judgment unit realized by the CPU 51, GPU 59, ROM 52, RAM 53, fixed disk 54 and their peripheral components detects stained cells in each of the first image 91 and the second image 93, and judges the suitability of the first primary staining conditions and the second primary staining conditions based on the detection results of the stained cells (step S182).

[0042] Specifically, the determination unit confirms whether or not a cell is specifically stained in the first image 91, and also confirms whether or not a cell is specifically stained in the second image 93. Furthermore, the determination unit compares the first image 91 and the second image 93, and confirms whether or not a cell that is stained in the first image 91 is unstained in the second image 93.

[0043] Confirmation of whether or not cells are specifically stained in each of the first image 91 and the second image 93 (i.e., detection of stained cells in the first image 91 and the second image 93) is performed using, for example, a trained model created by machine learning. The trained model is created by performing machine learning on an initial model for detecting the specificity of the stained cell distribution using a training dataset, which is a collection of images for learning. The training dataset includes images of specimens in which stained cells are specifically distributed and images of specimens in which stained cells are not specifically distributed. The machine learning is, for example, deep learning using a neural network. The learning by the deep learning is performed using, for example, U-Net. Note that the machine learning may be performed by a method other than deep learning.

[0044] In addition, in step S182, confirmation of whether or not cells are specifically stained in each of the first image 91 and the second image 93 does not necessarily have to be performed using a trained model, but may be performed by other methods executed by the computer 5.

[0045] When comparing the first image 91 and the second image 93 described above, the determining unit aligns the first image 91 and the second image 93. The alignment of the first image 91 and the second image 93 may be performed automatically by a known image registration technique, for example, or may be performed manually by an operator.

[0046] In the above, the staining using the first primary antibody (steps S11 to S12) and the staining using the second primary antibody (steps S15 to S16) are described as corresponding to two consecutive immunostainings (e.g., Nth and N+1th) in multiple immunostainings performed in the sequential multiple immunostaining method, but are not limited thereto. In the sequential multiple immunostaining method, if the staining using the second primary antibody is performed after the staining using the first primary antibody, immunostaining using another primary antibody may be performed between the staining using the first primary antibody and the staining using the second primary antibody. In this case, similarly in the above-mentioned staining condition evaluation method, between steps S14 and S15, the application of another primary antibody, the application of a secondary antibody and a coloring agent corresponding to the other primary antibody, and the removal and / or inactivation of the other primary antibody, etc. are performed. Note that the other primary antibody is, for example, an antibody derived from an animal species different from the first primary antibody and the second primary antibody.

[0047] As described above, the staining condition evaluation method for evaluating the staining conditions of a specimen by the immunostaining method includes a step of applying a first primary antibody to a test specimen under first staining conditions (i.e., first primary staining conditions) (step S11), a step of applying a secondary antibody and a color developer to the test specimen after step S11 (step S12), a step of imaging the test specimen to obtain a first image 91 after step S12 (step S13), a step of inactivating or removing the first primary antibody on the test specimen after step S13 (step S14), and a step of performing a second image 91 detection process after step S14. The method includes a step (step S15) of applying a second primary antibody to the test specimen under second staining conditions (i.e., second primary staining conditions) after step S15, a step (step S16) of applying the secondary antibody and the color developer to the test specimen after step S15, a step (step S17) of imaging the test specimen to obtain a second image 93 after step S16, and a step (step S18) of determining the suitability of the first staining conditions and the second staining conditions based on the detection results of stained cells in the first image 91 and the second image 93 after step S17.

[0048] In the test specimen, cells stained with the first primary antibody are different from cells stained with the second primary antibody. In step S18, if cells on the test specimen are specifically stained in the first image 91, and cells on the test specimen that are different from the cells in the first image 91 are specifically stained in the second image 93, and the cells on the test specimen that are stained in the first image 91 are unstained in the second image 93, it is determined that the first staining conditions and the second staining conditions are appropriate. This makes it possible to easily determine whether the first staining conditions and the second staining conditions in the two immunostainings that are performed sequentially, as described above, are appropriate.

[0049] As described above, in step S18, the detection of stained cells in the first image 91 and the detection of stained cells in the second image 93 are preferably performed using trained models created by machine learning. This makes it possible to automatically and accurately confirm the presence or absence of stained cells in the first image 91 and the second image 93.

[0050] As described above, the first primary antibody and the second primary antibody are used in sequential multiple immunostaining of one specimen, and in the sequential multiple immunostaining, it is preferable that staining with the second primary antibody is performed after staining with the first primary antibody. This allows sequential multiple immunostaining to be performed under appropriately set first and second staining conditions.

[0051] The above-mentioned program 572 is a computer-readable program that causes a computer to evaluate staining conditions of a specimen by an immunostaining method. The program 572 causes a computer to execute a step (step S181) of storing a first image 91, which is an image of a stained test specimen, and a second image 93, which is an image of a test specimen stained under conditions different from those of the first image 91, and a step (step S182) of determining whether the first staining conditions and the second staining conditions (i.e., the first primary staining conditions and the second primary staining conditions) are appropriate based on the detection results of stained cells in the first image 91 and the second image 93. The first image 91 is captured in a state in which a first primary antibody is applied to the test specimen under the first staining conditions, and further a secondary antibody and a coloring agent are applied. The second image 93 is taken after the first image 91 has been obtained and the first primary antibody on the test specimen has been inactivated or removed, after which a second primary antibody has been applied to the test specimen under second staining conditions, and further after the secondary antibody and color developer have been applied.

[0052] In the test specimen, cells stained with the first primary antibody are different from cells stained with the second primary antibody. In step S182, if cells on the test specimen are specifically stained in the first image 91, and cells on the test specimen that are different from those in the first image 91 are specifically stained in the second image 93, and the cells on the test specimen that are stained in the first image 91 are unstained in the second image 93, it is determined that the first staining conditions and the second staining conditions are appropriate. This makes it possible to easily determine whether the first staining conditions and the second staining conditions in the two immunostainings that are performed sequentially, as described above, are appropriate.

[0053] Next, a staining condition evaluation method according to a second embodiment of the present invention will be described. Fig. 6 is a plan view showing a slide 81 used in the staining condition evaluation method according to the second embodiment. In the staining condition evaluation method, the suitability of each of two combinations of primary staining conditions in steps S11 and S15 is judged in parallel using one slide 81.

[0054] 6, the above-mentioned test specimen 82 and another test specimen 83 are arranged on a slide 81. Like the test specimen 82, the test specimen 83 is obtained from the same tissue as the specimen to be stained and analyzed by the above-mentioned sequential multiple immunostaining method. Note that three or more test specimens may be arranged on the slide 81.

[0055] Hereinafter, a flow of staining condition evaluation using a slide 81 will be described with reference to FIG. 1. First, before step S11, one slide 81 is prepared by creating one slide 81 as shown in FIG. 6. Next, in step S11, substantially in parallel with the antigen activation process and the application of the first primary antibody to the test specimen 82, the antigen activation process and the application of the first primary antibody to the test specimen 83 are also performed. The first primary antibody applied to the test specimen 83 is the same as the first primary antibody applied to the test specimen 82. In step S11, the first primary antibody is applied to the test specimen 83 under a third primary staining condition. The third primary staining condition may be the same as or different from the first primary staining condition when the first primary antibody is applied to the test specimen 82. The third primary staining condition may be, for example, a condition in which a predetermined parameter (for example, the concentration or reaction time of the first primary antibody) is shifted by a predetermined amount from the first primary staining condition.

[0056] Next, in step S12, substantially in parallel with the application of the secondary antibody and coloring agent to the test specimen 82, the secondary antibody and coloring agent are also applied to the test specimen 83. As a result, specific cells, etc., to which the first primary antibody is bound are stained in the test specimen 83 as well. The secondary antibody and coloring agent applied to the test specimen 83 are the same as the secondary antibody and coloring agent applied to the test specimen 82. In step S12, the secondary antibody and coloring agent are applied to the test specimen 83 under staining conditions that are the same as the secondary staining conditions for the test specimen 82, for example.

[0057] In step S13, the test specimen 83 is imaged to obtain a third image, substantially in parallel with the acquisition of the first image 91. In step S14, substantially in parallel with the removal and / or inactivation of the primary antibody, the secondary antibody, and the color developer on the test specimen 82, the removal and / or inactivation of the primary antibody, the secondary antibody, and the color developer on the test specimen 83 is also performed.

[0058] Next, in step S15, substantially in parallel with the antigen activation process and application of the second primary antibody to the test specimen 82, the antigen activation process and application of the second primary antibody to the test specimen 83 are also performed. The second primary antibody applied to the test specimen 83 is the same as the second primary antibody applied to the test specimen 82. In step S15, the second primary antibody is applied to the test specimen 83 under fourth primary staining conditions. The fourth primary staining conditions may be the same as or different from the second primary staining conditions when the second primary antibody is applied to the test specimen 82. The fourth primary staining conditions may be, for example, the second primary staining conditions in which a predetermined parameter (for example, the concentration or reaction time of the second primary antibody) is shifted by a predetermined amount from the second primary staining conditions.

[0059] In addition, when the fourth primary dyeing condition is the same as the second primary dyeing condition, the third primary dyeing condition is different from the first primary dyeing condition. In addition, when the third primary dyeing condition is the same as the first primary dyeing condition, the fourth primary dyeing condition is different from the second primary dyeing condition. That is, at least the third primary dyeing condition is different from the first primary dyeing condition, or the fourth primary dyeing condition is different from the second primary dyeing condition.

[0060] Next, in step S16, substantially in parallel with the application of the secondary antibody and coloring agent to the test specimen 82, a secondary antibody and coloring agent are also applied to the test specimen 83. The secondary antibody and coloring agent applied to the test specimen 83 are the same as the secondary antibody and coloring agent applied to the test specimen 82. In step S16, the secondary antibody and coloring agent are applied to the test specimen 83 under, for example, the same staining conditions as the secondary staining conditions for the test specimen 82. In step S17, substantially in parallel with the acquisition of the second image 93, the test specimen 83 is imaged to acquire a fourth image.

[0061] Thereafter, in step S18, substantially in parallel with the judgment of the suitability of the first and second primary staining conditions for the test specimen 82, the suitability of the third and fourth primary staining conditions for the test specimen 83 is judged based on the detection results of the stained cells in the third and fourth images. Specifically, it is confirmed whether or not the cells are specifically stained in the third image, and whether or not the cells are specifically stained in the fourth image. Furthermore, the third and fourth images are compared, and it is confirmed whether or not the cells stained in the third image are unstained in the fourth image.

[0062] Then, when it is confirmed that the cells are stained specifically in each of the third and fourth images (i.e., the stained cells are distributed specifically), and the stained cells in the third image are unstained in the fourth image, the third primary staining conditions for applying the first primary antibody to the test specimen 83 and the fourth primary staining conditions for applying the second primary antibody to the test specimen 83 are determined to be appropriate. On the other hand, in other cases, the third primary staining conditions and / or the fourth primary staining conditions are determined to be inappropriate, as described above. Note that the above-mentioned test specimens 82 and 83 may each be provided on different slides 81.

[0063] As described above, in the staining condition evaluation method according to the second embodiment, the slide 81 on which the test specimen 82 and the other test specimen 83 are arranged is prepared before step S11. In step S11, a first primary antibody is applied to the other test specimen 83 under a third staining condition. In step S12, a secondary antibody and a coloring agent are also applied to the other test specimen 83. In step S13, the other test specimen 83 is imaged to obtain a third image. In step S14, the first primary antibody on the other test specimen 83 is also inactivated or removed. In step S15, a second primary antibody is applied to the other test specimen 83 under a fourth staining condition. In step S16, a secondary antibody and a coloring agent are also applied to the other test specimen 83. In step S17, the other test specimen 83 is imaged to obtain a fourth image.

[0064] In step S18, the suitability of the third staining condition and the fourth staining condition are also determined based on the detection results of the stained cells in the third image and the fourth image. In step S18, if the cells on the other test specimen 83 are specifically stained in the third image, and the cells on the other test specimen 83 that are different from those in the third image are specifically stained in the fourth image, and the cells on the other test specimen 83 that are stained in the third image are unstained in the fourth image, it is determined that the third staining condition and the fourth staining condition are appropriate. This allows the suitability of each of the two combinations of primary staining conditions in steps S11 and S15 to be determined in parallel.

[0065] Furthermore, three or more test specimens may be placed on the slide 81. The first and second primary antibodies may be applied to the three or more test specimens in different combinations of primary staining conditions in steps S11 and S15. This allows the suitability of each of the three or more combinations of primary staining conditions in steps S11 and S15 to be determined in parallel.

[0066] The above-mentioned staining condition evaluation method and program can be modified in various ways.

[0067] The antigen activation processes in steps S11 and S15 may be omitted.

[0068] The above-mentioned staining condition evaluation method does not necessarily have to be used for evaluating staining conditions in a sequential multiple immunostaining method, but may be used for evaluating staining conditions in other immunostaining methods. In addition, the above-mentioned specimen and test specimen do not necessarily have to be a specimen derived from a living body, but may be another specimen.

[0069] The configurations in the above-described embodiment and each of the modified examples may be combined as appropriate as long as they are not mutually inconsistent. [Explanation of symbols]

[0070] 5. Computers 81 Slides 82 Test specimen 83 Test specimen 91 1st image 93 2nd image 572 Programs S11~S18, S181~S182 Steps

Claims

1. A staining condition evaluation method for evaluating staining conditions of a specimen by an immunostaining method, comprising the steps of: a) subjecting a test specimen to a first primary antibody under first staining conditions; b) applying a secondary antibody and a color developer to the test specimen after step a); c) after step b), capturing an image of the test specimen to obtain a first image; d) after step c), inactivating or removing the first primary antibody on the test specimen; e) after step d), administering a second primary antibody to the test specimen under second staining conditions; f) applying the secondary antibody and the color developer to the test specimen after step e); g) after step f), capturing an image of the test specimen to obtain a second image; h) after the step g), determining whether the first staining condition and the second staining condition are appropriate based on detection results of stained cells in the first image and the second image; Equipped with The cells stained with the first primary antibody are different from the cells stained with the second primary antibody, In the step h), if cells on the test specimen are specifically stained in the first image, and cells on the test specimen that are different from those in the first image are specifically stained in the second image, and cells on the test specimen that are stained in the first image are unstained in the second image, the first staining conditions and the second staining conditions are determined to be appropriate.

2. The dyeing condition evaluation method according to claim 1, A staining condition evaluation method, in which in the step h), the detection of stained cells in the first image and the detection of stained cells in the second image are each performed using a trained model created by machine learning.

3. The method for evaluating dyeing conditions according to claim 1 or 2, Prior to the step a), a slide on which the test specimen and another test specimen are arranged is prepared; In the step a), the first primary antibody is applied to the other test specimen under a third staining condition; In the step b), the second antibody and the color developer are also applied to the other test specimen, In the step c), the other test specimen is imaged to obtain a third image; In the step d), the first primary antibody on the other test specimen is also inactivated or removed; In the step e), the second primary antibody is applied to the other test specimen under a fourth staining condition; In the step f), the second antibody and the color developer are also applied to the other test specimens; In the step g), the other test specimen is imaged to obtain a fourth image; In the step h), the suitability of the third staining condition and the fourth staining condition is also determined based on the detection results of the stained cells in the third image and the fourth image, In the step h), if cells on the other test specimen are specifically stained in the third image, and cells on the other test specimen that are different from those in the third image are specifically stained in the fourth image, and cells on the other test specimen that are stained in the third image are unstained in the fourth image, the third staining conditions and the fourth staining conditions are determined to be appropriate.

4. The method for evaluating dyeing conditions according to claim 1 or 2, The first primary antibody and the second primary antibody are used in sequential multiplex immunostaining of a specimen; The staining condition evaluation method, wherein in the sequential multiple immunostaining, staining with the second primary antibody is performed after staining with the first primary antibody.

5. A computer-readable program for causing a computer to evaluate staining conditions for a specimen by an immunostaining method, comprising: i) storing a first image, which is an image of a stained test specimen, and a second image, which is an image of the test specimen stained under conditions different from those of the first image; j) determining whether staining conditions are appropriate based on detection results of stained cells in the first image and the second image; Run the following on your computer: the first image is captured in a state in which a first primary antibody is applied to the test specimen under first staining conditions, and a secondary antibody and a color developer are further applied to the test specimen; the second image is captured in a state in which the first image is obtained and the first primary antibody on the test specimen is inactivated or removed, a second primary antibody is applied to the test specimen under second staining conditions, and the secondary antibody and the color developer are further applied to the test specimen; The cells stained with the first primary antibody are different from the cells stained with the second primary antibody, A computer-readable program in which, in step j), if cells on the test specimen are specifically stained in the first image, and cells on the test specimen that are different from those in the first image are specifically stained in the second image, and cells on the test specimen that are stained in the first image are unstained in the second image, the first staining conditions and the second staining conditions are determined to be appropriate.

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