Staining condition evaluation method

Abstract

To easily determine suitability of a staining condition.SOLUTION: A staining condition evaluation method for evaluating a staining condition of a specimen by an immunostaining method includes: a step (step S11) for applying a primary antibody to a test specimen under one staining condition; a step (step S12) for applying a secondary antibody and a color developing agent to the test specimen after the step S11; a step (step S14) for inactivating or removing the primary antibody on the test specimen after the step S12; a step (step S16) for applying the secondary antibody and the color developing agent to the test specimen after the step S14; and a step (step S17) for determining suitability of the one staining condition by checking whether there is a stained cell in the test specimen after the step S16. Thus, the suitability of the one staining condition in the step S11 can be easily determined.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 primary antibody to a test specimen under certain staining conditions; b) applying a secondary antibody and a color developer to the test specimen after step a); c) inactivating or removing the primary antibody on the test specimen after step b); d) applying the secondary antibody and the color developer to the test specimen after step c); and e) determining the suitability of the certain staining conditions by checking the presence or absence of stained cells in the test specimen after step d).

[0009] A second aspect of the present invention is a staining condition evaluation method according to the first aspect, further comprising, between the b) step and the c) step, a step of determining the suitability of the one of the staining conditions by confirming the presence or absence of stained cells in the test specimen.

[0010] A third aspect of the present invention relates to the staining condition evaluation method according to the first or second aspect, wherein the confirmation of the presence or absence of stained cells in the step e) is carried out using a trained model created by machine learning.

[0011] Aspect 4 of the present invention is a staining condition evaluation method according to aspect 1 or 2 (which may be any one of aspects 1 to 3), wherein in step e), a staining position indicating the position of a stained cell is obtained in a first image obtained by imaging the test specimen between steps b) and c), and the presence or absence of a stained cell at the staining position in a second image obtained by imaging the test specimen after step d) is confirmed as the presence or absence of a stained cell in the test specimen.

[0012] Aspect 5 of the present invention is a staining condition evaluation method according to aspect 1 or 2 (which may be any one of aspects 1 to 4), in which, prior to step a), a slide on which the test specimen and another test specimen are arranged is prepared. In step a), the primary antibody is applied to the other test specimen under other staining conditions. In step b), the secondary antibody and the color developer are also applied to the other test specimen. In step c), the primary antibody on the other test specimen is also inactivated or removed. In step d), the secondary antibody and the color developer are also applied to the other test specimen. In step e), the suitability of the other staining conditions is also determined by checking the presence or absence of stained cells in the other test specimen.

[0013] Aspect 6 of the present invention is the staining condition evaluation method of Aspect 1 or 2 (which may be any one of Aspects 1 to 5), further comprising a step of applying another primary antibody to the test specimen under another staining condition between the step c) and the step d). The cells stained with the primary antibody are different from the cells stained with the other primary antibody. In the step e), if a cell is specifically stained in a first image obtained by imaging the test specimen between the step b) and the step c), a cell different from that in the first image is specifically stained in a second image obtained by imaging the test specimen after the step d), and the cell stained in the first image is unstained in the second image, the one staining condition is determined to be appropriate. Effect of the Invention

[0014] In the present invention, the suitability of the staining conditions can be easily determined. [Brief description of the drawings]

[0015] [Figure 1] FIG. 4 is a diagram showing a flow of staining condition evaluation according to the first embodiment. [Diagram 2] FIG. 13 is a diagram showing an example of a stained image. [Diagram 3] FIG. 13 is a diagram showing an example of a stained image. [Figure 4] FIG. 13 is a diagram showing an example of a stained image. [Diagram 5] FIG. 1 illustrates a configuration of a computer. [Figure 6] FIG. 11 is a plan view showing a slide used in the staining condition evaluation method according to the second embodiment. [Figure 7] FIG. 13 is a diagram showing a part of a flow of staining condition evaluation according to the third embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 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.

[0017] 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 in the Nth immunostaining (N is an integer of 1 or more) out of the multiple immunostainings performed in the sequential multiple immunostaining method are appropriate will be described.

[0018] In the staining condition evaluation method, first, a test specimen to be used for evaluating 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 that are being 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.

[0019] A primary antibody is an immunoglobulin that binds to a specific antigen (e.g., a protein, peptide, small molecule, etc.) with high affinity and specificity to the antigen. Primary antibodies can be derived from various animal species, such as mouse, rabbit, goat, or chicken.

[0020] The staining conditions in step S11 (hereinafter also referred to as "primary staining conditions") include, for example, at least one of the concentration of the primary antibody applied to the test specimen and the time for which the primary antibody is applied to the test specimen (i.e., the reaction time). The 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.

[0021] 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.

[0022] The secondary antibody is an antibody that binds to the primary antibody used in step S11 and corresponds to the animal species related to the production of the primary antibody. For example, if the 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.

[0023] Thereafter, the stained test specimen is observed to confirm the presence or absence of stained cells, etc. (hereinafter, also referred to as "stained cells") in the test specimen. Then, based on the confirmation result of the presence or absence of stained cells, the suitability of the primary staining conditions in step S11 is judged (step S13).

[0024] In step S13, for example, a part or the whole of the test specimen stained in steps S11 and S12 is imaged to obtain an image (hereinafter also referred to as a "stained image"). FIG. 2 is a diagram showing an example of a stained image 91. In the stained 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.

[0025] Then, the operator performing the immunostaining visually checks the stained image 91 to confirm the presence or absence of stained cells. If the presence of stained cells is confirmed in the stained image 91, the operator determines that the primary staining conditions in step S11 are appropriate. On the other hand, if the presence of stained cells is not substantially confirmed in the stained image 91 (i.e., if the stained region 92 is substantially absent), the operator determines that the primary staining conditions in step S11 are inappropriate (for example, the concentration of the primary antibody is insufficient or the reaction time is insufficient). Note that in step S13, the primary staining conditions in step S11 may also be determined to be inappropriate if the distribution of stained cells in the stained image 91 does not have specificity, etc.

[0026] In step S13, if the primary staining conditions are determined to be inappropriate, the test specimen is washed, and the primary antibody, the secondary antibody, and the color developer on the test specimen are removed. In addition, the primary antibody and the secondary antibody on the test specimen may be inactivated (step S131). That is, in step S131, at least one of inactivation and removal is performed on the 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 primary antibody and the secondary antibody are inactivated, for example, by heat treatment in which the test specimen is immersed in boiling water. The 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. In the case where 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.

[0027] Thereafter, the primary staining conditions for applying the primary antibody are changed (step S132), and the process returns to step S11 to perform steps S11 to S13. The change in the primary staining conditions in step S132 includes, for example, increasing the concentration of the primary antibody applied to the test specimen in step S11 and / or increasing the reaction time with the primary antibody.

[0028] In step S13, even if the primary staining conditions are determined to be appropriate, the test specimen is washed and the primary antibody, secondary antibody, and coloring agent on the test specimen are removed. The primary antibody and secondary antibody on the test specimen may be inactivated (step S14). That is, in step S14, at least one of inactivation and removal is performed on the primary antibody on the test specimen. The washing of the test specimen and the removal and / or inactivation of the primary antibody and secondary antibody in step S14 are performed, for example, in the same manner as in step S131 described above. The washing of the test specimen and the removal and / or inactivation of the primary antibody and secondary antibody in step S14 are performed under the same processing conditions as the washing and inactivation to be performed between the Nth immunostaining and the N+1th immunostaining.

[0029] After step S14 is completed, an antigen activation process is performed on the test specimen under the primary staining conditions that are being considered for implementation in the N+1th immunostaining of the sequential multiple immunostaining method (step S15). The antigen activation process in step S15 may be performed under the same conditions as the antigen activation process in step S11, or may be performed under different conditions. When the antigen activation process in step S15 and the inactivation of the primary and secondary antibodies in step S14 are both heat treatments, the inactivation of the primary and secondary antibodies (step S14) and the antigen activation process (step S15) may be performed in parallel by a single heat treatment.

[0030] 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 Nth immunostaining (step S16). The secondary antibody and coloring agent used in step S16 are the secondary antibody and coloring agent used in the Nth 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.

[0031] After step S16 is completed, the test specimen to which the secondary antibody and coloring agent have been applied is observed to confirm the presence or absence of stained cells in the test specimen. Then, based on the confirmation result of the presence or absence of stained cells, the suitability of the primary staining conditions in step S11 is judged (step S17).

[0032] In step S17, for example, the presence or absence of stained cells is confirmed by an operator visually inspecting the stained image obtained by imaging the test specimen. As illustrated in Fig. 3, if the presence of stained cells is not substantially confirmed in the stained image 91, this means that the primary antibody on the test specimen has been almost completely removed and / or inactivated by step S14, and therefore the primary staining conditions in step S11 are determined to be appropriate.

[0033] On the other hand, as illustrated in FIG. 4, if the presence of stained cells is confirmed in stained image 91 (i.e., if stained area 92 exists), this means that the primary antibody remained in an active state on the test specimen even after the removal and / or inactivation of the primary antibody in step S14 (i.e., stainability was carried over), and it is determined that the primary staining conditions in step S11 are inappropriate (e.g., the primary antibody concentration is too high or the reaction time is too long).

[0034] If the primary staining conditions are determined to be inappropriate in step S17, for example, a new test specimen is prepared, the primary staining conditions are changed, and steps S11 to S17 are performed. Then, steps S11 to S17 are repeated while replacing the test specimen until appropriate 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 S17 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 S17 may be performed.

[0035] In step S17, when the appropriate primary staining conditions for the Nth immunostaining are acquired, for example, the test specimen used for acquiring the primary staining conditions is used to evaluate the suitability of the primary staining conditions for the N+1th immunostaining among the multiple immunostainings performed by the sequential multiple immunostaining method. Specifically, for example, the test specimen after step S17 is washed and / or heat-treated to remove and / or inactivate the secondary antibody and coloring agent, and then steps S11 to S17 are performed under the staining conditions to be performed in the N+1th and N+2th immunostainings. In the staining condition evaluation method described above, the suitability of the primary staining conditions may be evaluated for all of the multiple immunostainings performed by the sequential multiple immunostaining method, or the suitability of the primary staining conditions may be evaluated for some of the multiple immunostainings.

[0036] The judgment of the suitability of the primary staining conditions in step S17 does not necessarily have to be performed by visual inspection by an operator, and may be modified in various ways. For example, the suitability of the primary staining conditions may be judged by executing a program for evaluating staining conditions by a computer.

[0037] 5 is a diagram showing the configuration of the computer 5 described above. The computer 5 has a configuration of a general computer system 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 a basic program. 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] In step S17, the program 572 may be executed by the computer 5, and the presence or absence of the stained cells described above may be confirmed using, for example, a trained model created by machine learning. Specifically, the test specimen to which the secondary antibody and the coloring agent have been applied in step S16 is imaged in step S17 to obtain a stained image. The stained image is then input to the trained model, and the presence or absence of stained cells in the stained image is output by the trained model.

[0041] In computer 5, when stained cells are detected in the stained image by the trained model, for example, the presence of stained cells is displayed by characters or the like on display 55. The stained image may also be displayed on display 55, and the detected stained cells may be highlighted. On the other hand, when no stained cells are detected in the stained image, for example, the absence of stained cells is displayed by characters or the like on display 55.

[0042] The trained model is created by performing machine learning on an initial model for staining condition evaluation using a training data set, which is a collection of images for training. The training data set includes images of specimens in which stained cells are present and images of specimens in which stained cells are not present. The machine learning is, for example, deep learning using a neural network. The learning by deep learning is performed using, for example, U-Net. Note that the machine learning may be performed by a method other than deep learning. In addition, the confirmation of the presence or absence of stained cells by the trained model in step S17 may also be used in the confirmation of the presence or absence of stained cells in step S13.

[0043] In step S17, the program 572 may be executed by the computer 5 to compare two images of the test specimen, for example, to confirm the presence or absence of the stained cells described above. In this case, the test specimen stained in steps S11 and S12 is imaged in step S13 to obtain a first image. Also, the test specimen to which a secondary antibody and a coloring agent have been applied in step S16 is imaged in step S17 to obtain a second image. Note that the first image may be obtained before or after step S13, as long as it is obtained between steps S12 and S14.

[0044] In step S17, the program 572 is executed by the computer 5 to obtain a staining position indicating the position of the stained cell in the first image. Then, the presence or absence of the stained cell at the staining position in the second image is confirmed as the presence or absence of the stained cell in the test specimen. The presence or absence of the stained cell in the second image is confirmed using the coordinates of the staining position on the first image after the first image and the second image are aligned. The alignment of the first image and the second image may be performed automatically by a known image registration technique, for example, or may be performed manually by an operator. The presence or absence of the stained cell in the second image is confirmed, for example, only at the staining position in the first image, and is not confirmed in the area other than the staining position in the first image. This can shorten the time required to confirm the presence or absence of the stained cell in the second image.

[0045] In computer 5, when stained cells are detected in the second image, for example, the presence of stained cells is displayed on display 55 using text or the like. Also, the second image may be displayed on display 55, and the detected stained cells may be highlighted. On the other hand, when stained cells are not detected in the stained image, for example, the absence of stained cells is displayed on display 55 using text or the like. Note that the detection of stained cells by comparing the first image with the second image described above may be performed visually by an operator.

[0046] As described above, the above-mentioned staining condition evaluation method for evaluating the staining conditions of a specimen using the immunostaining method includes a step of applying a primary antibody to a test specimen under one staining condition (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 inactivating or removing the primary antibody on the test specimen after step S12 (step S14), a step of applying a secondary antibody and a color developer to the test specimen after step S14 (step S16), and a step of determining the suitability of the one staining condition by checking the presence or absence of stained cells in the test specimen after step S16 (step S17).

[0047] This makes it possible to easily determine whether the one staining condition (i.e., the primary staining condition) in step S11 is appropriate. Specifically, if stained cells that should not be present are confirmed in step S17, it is determined that the primary staining conditions in step S11 are inappropriate, for example, that the concentration of the primary antibody and / or the reaction time are excessive. On the other hand, if no stained cells are confirmed in step S17, it is determined that the primary staining conditions in step S11 are appropriate.

[0048] The staining condition evaluation method preferably further comprises a step (step S13) between steps S12 and S14 of determining whether the primary staining conditions are appropriate by confirming the presence or absence of stained cells in the test specimen. In this way, by detecting the inadequacy of the primary staining conditions, which can already be determined at the end of steps S11 to S12, the staining condition evaluation can be performed efficiently without performing steps S14 to S17 unnecessarily. Specifically, in step S13, if stained cells that should be present are not confirmed, it is determined that the primary staining conditions in step S11 are inappropriate, for example, that the concentration of the primary antibody and / or the reaction time are insufficient. On the other hand, if stained cells are not confirmed in step S17, it is determined that the primary staining conditions in step S11 are appropriate.

[0049] As described above, the confirmation of the presence or absence of stained cells in step S17 is preferably performed using a trained model created by machine learning. This makes it possible to automatically and accurately confirm the presence or absence of stained cells in a stained image.

[0050] As described above, in step S17, it is preferable to obtain a staining position indicating the position of the stained cell in the first image obtained by imaging the test specimen between steps S12 and S14. It is also preferable to confirm the presence or absence of the stained cell at the staining position in the second image obtained by imaging the test specimen after step S16 as the presence or absence of the stained cell in the test specimen. This makes it possible to accurately confirm the presence or absence of the stained cell in the second image. Also, by confirming the presence or absence of the stained cell in the second image only at the staining position, it is possible to shorten the time required for the confirmation. Furthermore, when the comparison between the first image and the second image is performed using a computer, the presence or absence of the stained cell in the stained image can be automatically confirmed.

[0051] 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, one slide 81 is used to judge the suitability of a plurality of primary staining conditions in parallel.

[0052] 6, the above-mentioned test specimen (hereinafter, designated by reference numeral 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.

[0053] Hereinafter, the flow of staining condition evaluation using the slide 81 will be described with reference to FIG. 1. First, before step S11, one slide 81 as exemplified in FIG. 6 is prepared by being created. Next, in step S11, substantially in parallel with the antigen activation treatment and application of the primary antibody to the test specimen 82, the antigen activation treatment and application of the primary antibody to the test specimen 83 are also performed. The primary antibody applied to the test specimen 83 is the same as the primary antibody applied to the test specimen 82. In step S11, the primary antibody is applied to the test specimen 83 under other primary staining conditions different from the primary staining conditions when the primary antibody is applied to the test specimen 82. The other primary staining conditions are, for example, those in which a predetermined parameter (for example, the concentration and reaction time of the primary antibody) is shifted by a predetermined amount from the primary staining conditions considered for implementation in the N-th immunostaining of the sequential multiple immunostaining method.

[0054] 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 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.

[0055] In step S13, the presence or absence of stained cells in the test specimen 83 is confirmed substantially in parallel with the confirmation of the presence or absence of stained cells in the test specimen 82, and the suitability of the other primary staining conditions in step S11 (i.e., the primary staining conditions for the test specimen 83) is determined based on the confirmation result. If the primary staining conditions for both the test specimen 82 and the test specimen 83 are determined to be inappropriate in step S13, the primary antibody, secondary antibody, and color developer on the test specimens 82 and 83 are removed and / or inactivated (step S131). Then, the primary staining conditions determined to be inappropriate are changed (step S132), and the process returns to step S11 and steps S11 to S13 are performed.

[0056] If in step S13, the primary staining conditions for at least one of test specimen 82 and test specimen 83 are determined to be appropriate, in step S14, the removal and / or inactivation of the primary antibody, secondary antibody and color developer on test specimen 83 is also performed substantially in parallel with the removal and / or inactivation of the primary antibody, secondary antibody and color developer on test specimen 82.

[0057] Next, in step S15, an antigen activation process is performed on the test specimen 83 substantially in parallel with the antigen activation process on the test specimen 82. The antigen activation process on the test specimen 83 is performed, for example, under the same conditions as the antigen activation process on the test specimen 82.

[0058] Next, in step S16, substantially in parallel with the application of the secondary antibody and coloring agent to test specimen 82, a secondary antibody and coloring agent are also applied to test specimen 83. The secondary antibody and coloring agent applied to test specimen 83 are the same as the secondary antibody and coloring agent applied to test specimen 82. In step S16, the secondary antibody and coloring agent are applied to test specimen 83 under staining conditions that are the same as the secondary staining conditions for test specimen 82, for example.

[0059] Thereafter, in step S17, substantially in parallel with the determination of the suitability of the primary staining conditions for the test specimen 82, the presence or absence of stained cells in the test specimen 83 is confirmed, and the suitability of the primary staining conditions for the test specimen 83 is determined based on the confirmation result of the presence or absence of stained cells. Specifically, if the presence of stained cells is not substantially confirmed in the test specimen 83, the other primary staining conditions for the test specimen 83 are determined to be appropriate. Also, if the presence of stained cells is confirmed in the test specimen 83, the other primary staining conditions for the test specimen 83 are determined to be inappropriate (for example, the concentration of the primary antibody is too high or the reaction time is too long). Note that the above-mentioned test specimens 82 and 83 may be provided on different slides 81, respectively.

[0060] 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 addition, in step S11, a primary antibody is applied to the other test specimen 83 under other staining conditions (i.e., other primary staining conditions). In step S12, a secondary antibody and a coloring agent are also applied to the other test specimen 83. In step S14, the primary antibody on the other test specimen 83 is also inactivated or removed. In step S16, a secondary antibody and a coloring agent are also applied to the other test specimen 83. In step S17, the suitability of the other staining conditions is also determined by checking the presence or absence of stained cells in the other test specimen 83. This allows the suitability of two primary staining conditions to be determined in parallel.

[0061] Furthermore, three or more test specimens may be placed on the slide 81. Then, in step S11, primary antibodies may be applied to the three or more test specimens under three or more different primary staining conditions. This allows the suitability of three or more primary staining conditions to be determined in parallel.

[0062] Next, a staining condition evaluation method according to a third embodiment of the present invention will be described. Fig. 7 is a diagram showing a part of the flow of staining condition evaluation according to the third embodiment. In the staining condition evaluation method, first, steps S11 to S15 shown in Fig. 1 are performed as in the first embodiment. Then, step S21 shown in Fig. 7 is performed between steps S15 and S16, and thereafter, steps S16 to S17 shown in Fig. 1 are performed approximately as in the first embodiment.

[0063] In step S21, a primary antibody different from the primary antibody used in step S11 is applied to the test specimen. The primary antibody is, for example, a primary antibody used in the N+1th immunostaining of the sequential multiplex immunostaining method. The primary antibody is an antibody of a different type from the primary antibody described above (i.e., the primary antibody used in the Nth immunostaining). The primary antibody is, for example, an antibody derived from the same animal species as the primary antibody described above. The primary staining conditions for the primary antibody in step S21 (hereinafter also referred to as "other staining conditions") are primary staining conditions that are being considered for implementation in the N+1th immunostaining. The other staining conditions may be different from or the same as the primary staining conditions in step S11. Note that step S21 is performed between step S14 and step S16 when the antigen activation process in step S15 is not performed. In step S16, the secondary antibody and color developer to be used in the N+1th immunostaining are used under the secondary staining conditions to be performed in the N+1th immunostaining of the sequential multiple immunostaining method.

[0064] In the test specimen that is the subject of the staining condition evaluation method according to the third embodiment, cells stained with the primary antibody applied in step S11 are different from cells stained with another primary antibody applied in step S21. Therefore, if the removal and / or inactivation of the primary antibody in step S14 is performed appropriately, the region in which cells stained by the application of the secondary antibody and coloring agent in step S16 exist is different from and does not overlap with the region in which cells stained by the application of the secondary antibody and coloring agent 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 cells stained by the application of the secondary antibody and coloring agent in step S16 exist overlaps with the region in which cells stained by the application of the secondary antibody and coloring agent in step S12 exist.

[0065] In the staining condition evaluation method according to the third embodiment, the test specimen stained in steps S11 and S12 is imaged in step S13 to obtain a first image. Also, the test specimen stained in steps S21 and S16 is imaged in step S17 to obtain a second image. Note that the first image may be obtained before or after step S13, as long as it is obtained between steps S12 and S14.

[0066] In step S17, for example, the program 572 shown in FIG. 5 is executed by the computer 5 to detect the position of each stained cell in the first image, and also detect the position of each stained cell in the second image. Then, it is confirmed whether or not the cell is specifically stained in the first image, and it is also confirmed whether or not the cell is specifically stained in the second image. Furthermore, the first image and the second image are compared, and it is confirmed whether or not the cell stained in the first image is unstained in the second image.

[0067] Then, if it is confirmed that cells are specifically stained in each of the first and second images and that the stained cells in the first image are unstained in the second image, it is determined that the primary staining conditions for applying the primary antibody in step S11 are appropriate.

[0068] On the other hand, in other cases, the primary staining conditions are determined to be inappropriate. For example, if some or all of the stained cells in the first image are also stained in the second image, the concentration of the primary antibody applied in step S11 may be too high and / or the reaction time may be too long. Also, if the cells are barely stained in the first image, the concentration of the primary antibody applied in step S11 may be insufficient and / or the reaction time may be insufficient. If the cells are barely stained in the second image, the concentration of the other primary antibodies applied in step S21 may be insufficient and / or the reaction time may be insufficient.

[0069] When the first image and the second image are compared, the first image and the second image are aligned. The alignment of the first image and the second image may be performed automatically by a known image registration technique, or may be performed manually by an operator.

[0070] The confirmation of whether or not the cells are specifically stained in each of the first image and the second image is performed, for example, using 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 training. 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, for example, using U-Net. Note that the machine learning may be performed by a method other than deep learning.

[0071] In step S17, the detection of the specificity of the above-mentioned stained cell distribution in the first image and the second image, and the detection of the above-mentioned overlap of stained cells by comparing the first image with the second image may be performed visually by an operator.

[0072] In the above, steps S11 to S12 and steps S21 and S16 have been described as corresponding to two consecutive immunostainings (for example, Nth and N+1th) in multiple immunostainings performed in the sequential multiple immunostaining method, but the present invention is not limited thereto. In the sequential multiple immunostaining method, if steps S21 and S16 are performed after steps S11 to S12, immunostaining using a primary antibody different from that in steps S11 and S21 may be performed between steps S11 to S12 and steps S21 and S16. In this case, similarly in the above-mentioned staining condition evaluation method, between steps S14 and S15, a primary antibody different from that in the above-mentioned steps S11 and S21 is applied, a secondary antibody and a coloring agent corresponding to the primary antibody are applied, and the primary antibody and the like are removed and / or inactivated. The primary antibody is, for example, an antibody derived from an animal species different from that of the primary antibody used in steps S11 and S21.

[0073] As described above, the staining condition evaluation method according to the third embodiment further includes a step (step S21) of applying another primary antibody under another staining condition to the test specimen between steps S14 and S16. Also, cells stained with the primary antibody are different from cells stained with the other primary antibody. In step S17, if cells are specifically stained in the first image acquired by imaging the test specimen between steps S12 and S14, and cells different from those in the first image are specifically stained in the second image acquired by imaging the test specimen after step S16, and cells stained in the first image are unstained in the second image, it is determined that the above-mentioned one staining condition (i.e., the primary staining condition in step S11) is appropriate.

[0074] This allows the primary staining conditions of the first immunostaining to be set in two consecutive immunostainings so as not to adversely affect the second immunostaining, making the staining condition evaluation method particularly suitable for evaluating staining conditions in sequential multiple immunostaining methods.

[0075] The above-described method for evaluating dyeing conditions can be modified in various ways.

[0076] For example, it is not necessary to determine whether the primary staining conditions are appropriate in step S13. Also, acquisition of the first image in step S13 may be omitted. It is not necessary to acquire the second image in step S17.

[0077] The antigen activation processes in steps S11 and S15 may be omitted.

[0078] 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.

[0079] 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]

[0080] 81 Slides 82,83 Test specimen S11~S17, S21, S131, S132 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 primary antibody under a set of staining conditions; b) applying a secondary antibody and a color developer to the test specimen after step a); c) after step b), inactivating or removing the primary antibody on the test specimen; d) applying the secondary antibody and the color developer to the test specimen after step c); e) after the step d), determining whether the one staining condition is appropriate by confirming the presence or absence of stained cells in the test specimen; A dyeing condition evaluation method comprising:

2. The dyeing condition evaluation method according to claim 1, The staining condition evaluation method further comprises, between the step b) and the step c), a step of determining whether the one staining condition is appropriate by confirming the presence or absence of stained cells in the test specimen.

3. The method for evaluating dyeing conditions according to claim 1 or 2, A staining condition evaluation method in which the confirmation of the presence or absence of stained cells in the step e) is performed using a trained model created by machine learning.

4. The method for evaluating dyeing conditions according to claim 1 or 2, In the step e), a staining position indicating the position of a stained cell is obtained in a first image obtained by imaging the test specimen between the steps b) and c), and the presence or absence of a stained cell at the staining position in a second image obtained by imaging the test specimen after the step d) is confirmed as the presence or absence of a stained cell in the test specimen.

5. 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 primary antibody is applied to the other test specimen under other staining conditions; In the step b), the second antibody and the color developer are also applied to the other test specimen, In the step c), the primary antibody on the other test specimens is also inactivated or removed; In the step d), the second antibody and the color developer are also applied to the other test specimens, In the step e), the suitability of the other staining conditions is also judged by confirming the presence or absence of stained cells in the other test specimen.

6. The method for evaluating dyeing conditions according to claim 1 or 2, Between the steps c) and d), a step of applying another primary antibody to the test specimen under another staining condition is further included; The cells stained with the primary antibody are different from the cells stained with the other primary antibody, In the step e), if a cell is specifically stained in a first image obtained by imaging the test specimen between the steps b) and c), and a cell different from that in the first image is specifically stained in a second image obtained by imaging the test specimen after the step d), and a cell stained in the first image is unstained in the second image, the one staining condition is determined to be appropriate.

Images