Dyeing condition evaluation method

Abstract

The present invention provides a staining condition evaluation method for evaluating staining conditions of a specimen by an immunostaining method, the method comprising: a step (step S11) for imparting a primary antibody to a test specimen under one staining condition; a step (step S12) for imparting a secondary antibody and a developer to the test specimen after 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 imparting a secondary antibody and a developer to the test specimen after the step S14; and a step (step S17) for determining, after the step S16, whether or not the one staining condition is appropriate by checking the presence or absence of stained cells in the test specimen. As a result, it is possible to easily determine whether or not the one dyeing condition in step S11 is appropriate.

Description

Technical Field

[0001] This invention relates to a technique for evaluating staining conditions for specimens based on immunostaining methods.

[0002] [Reference to relevant applications]

[0003] This application claims priority to Japanese Patent Application JP2023-101953, filed on June 21, 2023, and incorporates all disclosures of that application. Background Technology

[0004] In recent years, medical and biological research has focused on elucidating disease mechanisms, biological mechanisms, and drug action mechanisms, employing the analysis of single-cell units derived from biological specimens such as tissues and cells. For example, in immunostaining analysis, quantitative / qualitative analysis is performed by staining biological substances such as proteins and measuring the staining state.

[0005] In the aforementioned immunostaining analysis, the staining state of the specimen needs to be suitable for analysis. For example, in the blood smear specimen preparation apparatus disclosed in Japanese Patent Application Publication No. 2011-185895 (Reference 1), in order to reduce batch-to-batch variations and manufacturer differences in the staining solution and to homogenize the staining state of the specimen, when the staining solution is changed, the staining conditions (e.g., the dilution rate of the staining solution, staining time, etc.) are changed based on the difference between the average nuclear G value of leukocytes in the test-stained specimen and the target nuclear G value.

[0006] In addition, Japanese Patent Publication No. 2020-523614 (Document 2) proposes the following: In the process recording slide for immunostaining, a detection area is set up for detection based on immunostaining, and a reference area is set up for determining the color density of stained tissue, etc., qualitatively or quantitatively.

[0007] However, in immunostaining analysis, multiple immunostaining methods are known, which involve repeatedly staining, photographing, and removing color from the same specimen by changing the antibodies used in the immunostaining process. In multiple immunostaining methods, if the staining conditions, such as the concentration of an antibody or the reaction time based on that antibody, are inappropriate during the immunostaining process using that antibody, it can adversely affect subsequent immunostaining processes using other antibodies, potentially reducing the accuracy of the analysis. Summary of the Invention

[0008] The purpose of this invention is to easily determine whether the staining conditions are appropriate.

[0009] Embodiment 1 of the present invention is a staining condition evaluation method for evaluating staining conditions of a specimen based on immunostaining, comprising: a) a step of applying an antibody to a test specimen under a staining condition; b) a step of applying a second antibody and a chromogenic agent to the test specimen after step a); c) a step of inactivating or removing the first antibody from the test specimen after step b); d) a step of applying the second antibody and the chromogenic agent to the test specimen after step c); and e) a step of determining whether the staining condition is appropriate by confirming the presence or absence of stained cells in the test specimen after step d).

[0010] According to the present invention, it is easy to determine whether the staining conditions are appropriate.

[0011] The second method of the present invention, based on the staining condition evaluation method of the first method, further includes: between the steps b) and c), a step of determining whether a staining condition is appropriate by confirming the presence or absence of stained cells in the test specimen.

[0012] In the third method of the present invention, based on the staining condition evaluation method of method 1 or 2, the presence or absence of stained cells in step e) is confirmed using a learned model created by machine learning.

[0013] In the present invention, according to the staining condition evaluation method of method 1 or 2 (or any one of methods 1 to 3), in step e), in the first image obtained by taking the test specimen between steps b) and c), a staining position representing the position of the stained cells is obtained, and the presence or absence of stained cells at the staining position in the second image obtained after taking the test specimen in step d) is used as the presence or absence of stained cells in the test specimen.

[0014] In embodiment 5 of the present invention, based on the staining condition evaluation method of embodiment 1 or 2 (or any one of embodiments 1 to 4), a slide containing the test specimen and other test specimens is prepared before step a). In step a), the primary antibody is applied to the other test specimens under other staining conditions. In step b), the secondary antibody and the chromogenic agent are also applied to the other test specimens. In step c), the primary antibody on the other test specimens is inactivated or removed. In step d), the secondary antibody and the chromogenic agent are also applied to the other test specimens. In step e), the appropriateness of the other staining conditions is further determined by confirming the presence or absence of stained cells in the other test specimens.

[0015] In embodiment 6 of the present invention, based on the staining condition evaluation method of embodiment 1 or 2 (or any one of embodiments 1 to 5), a step is further provided by subjecting the test specimen to another primary antibody under other staining conditions between step c) and step d). The cells stained with the primary antibody are different from the cells stained with the other primary antibody. In step e), in the first image obtained by taking a picture of the test specimen between steps b) and c), the cells are specifically stained, and in the second image obtained by taking a picture of the test specimen after step d), the cells different from those in the first image are specifically stained. If the cells stained in the first image are unstained in the second image, then the staining condition is deemed appropriate.

[0016] The above-mentioned objects, as well as other objects, features, forms, and advantages, will become clear from the following detailed description of the invention with reference to the accompanying drawings. Attached Figure Description

[0017] Figure 1 This is a diagram illustrating the process of evaluating staining conditions in the first embodiment.

[0018] Figure 2 This is a diagram representing an example of a stained image.

[0019] Figure 3 This is a diagram representing an example of a stained image.

[0020] Figure 4 This is a diagram representing an example of a stained image.

[0021] Figure 5 It is a diagram representing the structure of a computer.

[0022] Figure 6 This is a top view of the glass slide used in the staining condition evaluation method of the second embodiment.

[0023] Figure 7 This is a diagram showing a part of the process for evaluating staining conditions in the third embodiment. Detailed Implementation

[0024] Figure 1 This is a diagram illustrating the evaluation process in the staining condition evaluation method according to the first embodiment of the present invention. This staining condition evaluation method is used when evaluating the staining conditions of specimens (e.g., biological specimens) based on immunostaining methods.

[0025] This method for evaluating staining conditions is used, for example, in the evaluation of staining conditions in sequential multiplex immunostaining. Sequential multiplex immunostaining refers to a method in which multiple immunostaining, observation (e.g., photography), and color removal are performed repeatedly on the same specimen while changing the antibodies or other materials used in the immunostaining. The following describes the procedure for evaluating the appropriateness of the staining conditions for the Nth (N is an integer greater than or equal to 1) immunostaining in a series of multiple immunostainings performed using sequential multiplex immunostaining.

[0026] In this method for evaluating staining conditions, firstly, a test specimen for evaluating staining conditions is prepared. This test specimen is, for example, obtained from the same tissue as the predetermined specimen stained and analyzed using the sequential multiple immunostaining method described above. Next, the test specimen is subjected to antigen activation treatment (e.g., heat treatment or enzyme treatment) and primary antibody administration (step S11) under the staining conditions used in the Nth immunostaining of the aforementioned sequential multiple immunostaining method. This primary antibody is the primary antibody used in the Nth immunostaining described above.

[0027] Primary antibodies are immunoglobulins that bind to specific antigens (e.g., proteins, peptides, small molecules, etc.) with high affinity and specificity. For example, antibodies derived from various animals such as mice, rabbits, sheep, or chickens can be used as primary antibodies.

[0028] The staining conditions described above in step S11 (hereinafter also referred to as "primary staining conditions") include, for example, at least one of the concentration of the primary antibody administered to the test specimen and the duration of administration of the primary antibody relative to the test specimen (i.e., reaction time). These primary staining conditions may, for example, include treatment conditions for antigen activation. In the case where the antigen activation treatment is a heat treatment, these 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.

[0029] When step S11 is completed, the test specimen is given a second antibody and chromogenic agent under the predetermined staining conditions (hereinafter also referred to as "secondary staining conditions") implemented in the Nth immunostaining described above (step S12). Thus, specific cells, etc., in the test specimen after the first antibody binding are stained. The secondary antibody and chromogenic agent are the same as those used in the Nth immunostaining described above.

[0030] The secondary antibody is an antibody that binds to the primary antibody used in step S11, corresponding to the animal class associated with the production of that primary antibody. For example, if the primary antibody used in step S11 is mouse-derived, the secondary antibody used in step S12 is an anti-mouse antibody. The chromogenic agent can be any of various chromogenic agents that develop color according to the label of the secondary antibody (e.g., AEC (3-amino-9-ethyl-carbazole), etc.).

[0031] Then, the stained test specimen is observed to confirm the presence or absence of stained cells (hereinafter also referred to as "stained cells"). Then, based on the confirmation of the presence or absence of stained cells, it is determined whether the staining conditions in step S11 are appropriate (step S13).

[0032] In step S13, for example, an image (hereinafter also referred to as "stained image") is obtained by photographing a part or the whole of the test specimen stained in steps S11 to S12. Figure 2 This is a diagram representing an example of a stained image 91. In Figure 2 In the illustrated staining image 91, parallel oblique lines are marked on the stained region 92 (i.e., the region of the stained cell aggregate, hereinafter also referred to as "stained region 92").

[0033] Then, the operator performing the immunostaining visually checks the staining image 91 to confirm the presence or absence of stained cells. If stained cells are confirmed to be present in the staining image 91, the operator determines that the staining conditions in step S11 are appropriate. On the other hand, if stained cells are not substantially confirmed to be present in the staining image 91 (i.e., if the stained area 92 is substantially absent), the operator determines that the staining conditions in step S11 are inappropriate (e.g., insufficient antibody concentration or insufficient reaction time). Furthermore, in step S13, even if the distribution of stained cells in the staining image 91 is not specific, the operator can still determine that the staining conditions in step S11 are inappropriate.

[0034] In step S13, if the staining conditions are deemed inappropriate, the test specimen is washed to remove the primary antibody, secondary antibody, and chromogenic agent. Alternatively, the primary and secondary antibodies on the test specimen can be inactivated (step S131). That is, in step S131, at least one of the primary antibody on the test specimen is inactivated or removed. The test specimen is washed, for example, using phosphate-buffered saline (PBS) or Tris-buffered saline (TBS). The test specimen can be washed multiple times. The primary and secondary antibodies are inactivated, for example, by heat treatment such as immersing the test specimen in hot water. The inactivation of primary and secondary antibodies can be performed using various other methods. The removal of the chromogenic agent (more precisely, the removal of the color of the chromogenic precipitate derived from the chromogenic agent) can also be performed by various methods. When the chromogenic agent is AEC, the chromogenic agent is removed, for example, by washing the test specimen by immersing it in alcohols such as ethanol of various concentrations.

[0035] Then, the primary staining conditions related to the application of the primary antibody are changed (step S132), and the process returns to step S11 to perform steps S11-S13. The change in the primary staining conditions in step S132 may include, for example, increasing the concentration of the primary antibody applied to the test specimen in step S11 and / or increasing the reaction time based on the primary antibody.

[0036] In step S13, if the staining conditions are deemed appropriate, the test specimen is also washed to remove the primary antibody, secondary antibody, and chromogenic agent. Alternatively, the primary and secondary antibodies on the test specimen may be inactivated (step S14). That is, in step S14, at least one of the primary antibody on the test specimen is inactivated or removed. The washing of the test specimen in step S14, and the removal and / or inactivation of the primary and secondary antibodies, are performed, for example, by the same method as in step S131 described above. Furthermore, the washing of the test specimen in step S14, and the removal and / or inactivation of the primary and secondary antibodies, are performed under the same predetermined washing and inactivation conditions as those performed between the Nth and N+1th immunostainings described above.

[0037] When step S14 is completed, antigen activation treatment of the test specimen is performed under the same staining conditions as the N+1th immunostaining in the above-described sequential multiplex immunostaining method (step S15). The antigen activation treatment in step S15 can be performed under the same conditions as the antigen activation treatment in step S11, or under different conditions. Furthermore, if both the antigen activation treatment in step S15 and the inactivation of the primary and secondary antibodies in step S14 are heat treatments, the inactivation of the primary and secondary antibodies (step S14) and the antigen activation treatment (step S15) can be performed in parallel with a single heat treatment.

[0038] When step S15 is completed, under the predetermined secondary staining conditions implemented in the Nth immunostaining, a secondary antibody and chromogenic agent are applied to the test specimen (step S16). The secondary antibody and chromogenic agent used in step S16 are the same as those used in the Nth immunostaining and are of the same type as those used in step S12. The secondary staining conditions in step S16 may be the same as or different from those in step S12.

[0039] When step S16 ends, the test specimen, which has been given a second antibody and chromogenic agent, is observed to confirm the presence or absence of stained cells in the test specimen. Then, based on the confirmation of the presence or absence of stained cells, it is determined whether the primary staining conditions in step S11 were appropriate (step S17).

[0040] In step S17, for example, the operator visually confirms the presence or absence of stained cells by examining the stained images obtained from photographing the test specimen. Figure 3 As illustrated, in the case where the presence of stained cells is not substantially confirmed in staining image 91, the primary antibody on the test specimen is substantially completely removed and / or inactivated by step S14, thus determining that the primary staining conditions in step S11 are appropriate.

[0041] On the other hand, such as Figure 4 As illustrated, if the presence of stained cells is confirmed in staining image 91 (i.e., if staining area 92 is present), the primary antibody remains in an active state on the test specimen even after the removal and / or inactivation of the primary antibody in step S14 (i.e., retains stainability). Therefore, it is determined that the primary staining conditions in step S11 are inappropriate (e.g., the concentration of the primary antibody is too high or the reaction time is too long).

[0042] In step S17, if the staining conditions are deemed inappropriate, for example, a new test specimen is prepared, and steps S11-S17 are performed with the staining conditions changed. Then, steps S11-S17 are repeated while changing the test specimen until appropriate staining conditions are obtained. Alternatively, instead of preparing a new test specimen, steps S11-S17 can be performed, for example, by carefully washing and heat-treating (i.e., inactivation) the test specimen deemed inappropriate in step S17, thereby substantially removing and / or inactivating the primary antibody, secondary antibody, and chromogenic agent on the test specimen.

[0043] In step S17, when appropriate staining conditions are obtained for the Nth immunostaining, for example, using the test specimen used to obtain those conditions, the appropriateness of the staining conditions for the (N+1)th immunostaining in a series of immunostainings performed using sequential multiplex immunostaining is evaluated. Specifically, the test specimen after step S17 is subjected to washing and / or heat treatment to remove and / or inactivate the secondary antibodies and chromogenic agents, and then steps S11 to S17 are performed under the predetermined staining conditions for the (N+1)th and (N+2)th immunostainings. In the above-described staining condition evaluation method, the appropriateness of the staining conditions can be evaluated for all of the multiple immunostainings performed using sequential multiplex immunostaining, or for a portion of the multiple immunostainings.

[0044] The determination of whether the primary staining conditions are appropriate in step S17 does not necessarily need to be done visually by the operator; various changes can be made. For example, the appropriateness of the primary staining conditions can also be determined by a computer executing a staining condition evaluation program.

[0045] Figure 5 This diagram illustrates the structure of the computer 5 described above. The computer 5 has the structure of a general computer system, including a CPU 51, ROM 52, RAM 53, a hard 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 operations. The GPU 59 performs various image processing-related arithmetic operations. The ROM 52 stores the basic program. The RAM 53 stores various information. The hard disk 54 stores information. The display 55 is a display unit that displays images and other information.

[0046] The input unit 56 includes a keyboard 56a and a mouse 56b for receiving input from the operator. The reading device 57 reads information from a computer-readable recording medium 571, such as an optical disc, magnetic disk, optical disc drive, or memory card. The display 55, keyboard 56a, mouse 56b, and reading device 57 are connected to the bus 50 via an interface I / F. The communication unit 58 transmits and receives signals with external devices of the computer 5. The bus 50 is a signal circuit that connects the CPU 51, GPU 59, ROM 52, RAM 53, disk 54, display 55, input unit 56, reading device 57, and communication unit 58.

[0047] In computer 5, program 572 is read from recording medium 571 beforehand via reading device 57 and stored on disk 54. Program 572 may also be stored on disk 54 via network. CPU 51 and GPU 59 perform arithmetic processing using RAM 53 and disk 54 according to program 572 for evaluating chromatic conditions. CPU 51 and GPU 59 function as arithmetic units in computer 5. In addition to CPU 51 and GPU 59, other structures that function as arithmetic units may also be used.

[0048] In step S17, the presence or absence of the stained cells is confirmed by executing program 572 by computer 5, for example, using a learned model created through machine learning. Specifically, in step S17, a stained image is obtained by photographing the test specimen that was given secondary antibody and chromogenic agent in step S16. Then, the stained image is input into the learned model, and the learned model outputs the presence or absence of stained cells in the stained image.

[0049] In computer 5, if stained cells are detected in the stained image using a learned model, the presence of stained cells can be displayed on display 55 using text or similar means. Alternatively, the stained image can be displayed on display 55, emphasizing the detected stained cells. On the other hand, if no stained cells are detected in the stained image, the absence of stained cells can be displayed on display 55 using text or similar means.

[0050] The learned model described above is created by performing machine learning on an initial model for evaluating staining conditions using a set of images used for learning, namely a learning dataset. This learning dataset contains images of specimens with and without stained cells. The machine learning is, for example, deep learning using neural networks. Learning based on this deep learning is performed, for example, using U-Net. Alternatively, this machine learning can also be performed using methods other than deep learning. Furthermore, the confirmation of the presence or absence of stained cells in step S13 can also utilize the confirmation of the presence or absence of stained cells based on the learned model in step S17.

[0051] In step S17, by executing program 572 by computer 5, for example, two images of the test specimen can be compared to confirm the presence or absence of the stained cells. In this case, the first image is obtained by taking a picture of the test specimen stained in steps S11-S12 in step S13. Additionally, the second image is obtained by taking a picture of the test specimen that was given a secondary antibody and chromogenic agent in step S16 in step S17. Furthermore, the first image can be obtained either between steps S12 and S14, or before or after step S13.

[0052] In step S17, the staining location, representing the position of the stained cells in the first image, is obtained by executing program 572 on computer 5. Then, the presence or absence of stained cells at that staining location in the second image is confirmed as the presence or absence of stained cells in the test specimen. After aligning the first and second images, the coordinates of the staining location on the first image are used to confirm the presence or absence of stained cells in the second image. The alignment of the first and second images can be performed automatically, for example, using known image registration techniques, or manually by the operator. The presence or absence of stained cells in the second image is confirmed only at the staining location in the first image; areas outside the staining location in the first image are not confirmed. This shortens the time required to confirm the presence or absence of stained cells in the second image.

[0053] In computer 5, if stained cells are detected in the second image, the presence of stained cells can be displayed on display 55 using text or similar means. Alternatively, the second image can be displayed on display 55, emphasizing the detected stained cells. On the other hand, if no stained cells are detected in the stained image, the absence of stained cells can be displayed on display 55 using text or similar means. Furthermore, the detection of stained cells based on the comparison of the first and second images can also be performed visually by the operator.

[0054] As explained above, the method for evaluating staining conditions for specimens based on immunostaining includes: a step of applying an antibody to the test specimen under one staining condition (step S11); a step of applying a second antibody and chromogenic agent to the test specimen after step S11 (step S12); a step of inactivating or removing the first antibody from the test specimen after step S12 (step S14); a step of applying a second antibody and chromogenic agent to the test specimen after step S14 (step S16); and a step of confirming the presence or absence of stained cells in the test specimen after step S16, thereby determining whether the above staining condition is appropriate (step S17).

[0055] Therefore, it is easy to determine whether the staining condition (i.e., the primary staining condition) in step S11 is appropriate. Specifically, in step S17, if stained cells that should not be present are confirmed, the primary staining condition in step S11 is determined to be inappropriate, for example, the concentration of the primary antibody and / or the reaction time is too long. On the other hand, if stained cells are not confirmed in step S17, the primary staining condition in step S11 is determined to be appropriate.

[0056] The preferred method for evaluating staining conditions also includes the following step: between steps S12 and S14, the appropriateness of a single staining condition is determined by confirming the presence or absence of stained cells in the test specimen (step S13). In this way, by detecting inappropriate staining conditions that could already be determined at the end of steps S11-S12, staining condition evaluation can be performed efficiently, without needlessly performing steps S14-S17. Specifically, in step S13, if stained cells that should be present are not confirmed, the staining condition in step S11 is determined to be inappropriate, for example, due to insufficient antibody concentration and / or reaction time. On the other hand, if stained cells are not confirmed in step S17, the staining condition in step S11 is determined to be appropriate.

[0057] As described above, the confirmation of the presence or absence of stained cells in step S17 is preferably performed using a learned model created through machine learning. This allows for the automatic and highly accurate confirmation of the presence or absence of stained cells in the stained image.

[0058] As described above, in step S17, it is preferable to obtain the staining location, representing the position of the stained cells, in the first image obtained by photographing the test specimen between steps S12 and S14. Furthermore, it is preferable to confirm the presence or absence of stained cells at the aforementioned staining location in the second image obtained by photographing the test specimen after step S16 as the presence or absence of stained cells in the test specimen. This allows for high-precision confirmation of the presence or absence of stained cells in the second image. Additionally, by confirming the presence or absence of stained cells in the second image only at the aforementioned staining location, the time required for this confirmation can be shortened. Furthermore, when comparing the first and second images using a computer, the presence or absence of stained cells in the stained image can be automatically confirmed.

[0059] Next, the staining condition evaluation method of the second embodiment of the present invention will be described. Figure 6 This is a top view showing the slide 81 used in the staining condition evaluation method of the second embodiment. In this staining condition evaluation method, multiple single staining conditions are determined in parallel using one slide 81.

[0060] like Figure 6As shown, the aforementioned test specimen (hereinafter referred to as 82) and other test specimens 83 are placed on slide 81. Similar to test specimen 82, test specimen 83 is obtained from the same tissue as the predetermined specimen stained and analyzed using the aforementioned sequential multiple immunostaining method. Furthermore, more than three test specimens may be placed on slide 81.

[0061] The following is for reference Figure 1 The procedure for evaluating staining conditions using slide 81 is described. First, before step S11, by preparing... Figure 6 Prepare a slide 81 as illustrated. Next, in step S11, antigen activation treatment and primary antibody application for test specimen 83 are performed substantially in parallel with the antigen activation treatment and primary antibody application for test specimen 82. The primary antibody applied to test specimen 83 is the same as that applied to test specimen 82. In step S11, primary antibody is applied to test specimen 83 under different primary staining conditions than those applied to test specimen 82. These other primary staining conditions are, for example, conditions obtained by shifting predetermined parameters (e.g., primary antibody concentration, reaction time) by a predetermined amount from the primary staining conditions implemented in the Nth immunostaining of the above-described sequential multiplex immunostaining method.

[0062] Next, in step S12, the application of secondary antibodies and chromogenic agents to test specimen 83 is performed approximately in parallel with the application of secondary antibodies and chromogenic agents to test specimen 82. Thus, even in test specimen 83, specific cells, etc., after primary antibody binding are stained. The secondary antibodies and chromogenic agents applied to test specimen 83 are the same as those applied to test specimen 82. In step S12, the application of secondary antibodies and chromogenic agents to test specimen 83 is performed, for example, under the same staining conditions as those for the secondary staining of test specimen 82.

[0063] In step S13, the presence or absence of stained cells in test specimen 83 is confirmed roughly in parallel with the confirmation of the presence or absence of stained cells in test specimen 82. Based on the confirmation result, it is determined whether the other primary staining conditions mentioned in step S11 (i.e., the primary staining conditions for test specimen 83) are appropriate. If it is determined in step S13 that the primary staining conditions for both test specimens 82 and 83 are inappropriate, the primary antibody, secondary antibody, and chromogenic agent on 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 to perform steps S11-S13.

[0064] In step S13, if it is determined that the primary staining conditions of at least one of test specimens 82 and 83 are appropriate, in step S14, the removal and / or inactivation of the primary antibody, secondary antibody and chromogenic agent on test specimen 83 are also performed substantially in parallel with the removal and / or inactivation of the primary antibody, secondary antibody and chromogenic agent on test specimen 82.

[0065] Next, in step S15, antigen activation treatment for test specimen 83 is performed substantially in parallel with the antigen activation treatment for test specimen 82. The antigen activation treatment for test specimen 83 is performed, for example, under the same conditions as the antigen activation treatment for test specimen 82.

[0066] Next, in step S16, the application of secondary antibodies and chromogenic agents to test specimen 83 is performed approximately in parallel with the application of secondary antibodies and chromogenic agents to test specimen 82. The secondary antibodies and chromogenic agents applied to test specimen 83 are the same as those applied to test specimen 82. In step S16, the application of secondary antibodies and chromogenic agents to test specimen 83 is performed, for example, under the same staining conditions as those for the secondary staining of test specimen 82.

[0067] Subsequently, in step S17, while determining the appropriateness of the primary staining conditions related to test specimen 82, the presence or absence of stained cells in test specimen 83 is confirmed. Based on the confirmation of the presence or absence of stained cells, the appropriateness of the primary staining conditions related to test specimen 83 is determined. Specifically, if no stained cells are substantially detected in test specimen 83, the aforementioned other primary staining conditions related to test specimen 83 are deemed appropriate. Conversely, if stained cells are detected in test specimen 83, the aforementioned other primary staining conditions related to test specimen 83 are deemed inappropriate (e.g., the concentration of the primary antibody is too high or the reaction time is too long). Furthermore, test specimens 82 and 83 can also be placed on different slides 81.

[0068] As described above, in the staining condition evaluation method of the second embodiment, a slide 81 containing test specimen 82 and other test specimens 83 is prepared before step S11. Furthermore, in step S11, a primary antibody is applied to the other test specimens 83 under other staining conditions (i.e., other primary staining conditions). In step S12, a secondary antibody and chromogenic agent are also applied to the other test specimens 83. In step S14, the primary antibody on the other test specimens 83 is also inactivated or removed. In step S16, a secondary antibody and chromogenic agent are also applied to the other test specimens 83. In step S17, the appropriateness of the aforementioned other staining conditions is determined by confirming the presence or absence of stained cells in the other test specimens 83. Thus, it is possible to determine the appropriateness of two primary staining conditions in parallel.

[0069] Alternatively, three or more test specimens can be prepared on slide 81. Then, in step S11, the three or more test specimens can be treated with an antibody under three or more different primary staining conditions. This allows for the simultaneous determination of whether three or more primary staining conditions are appropriate.

[0070] Next, the staining condition evaluation method of the third embodiment of the present invention will be described. Figure 7 This diagram illustrates a portion of the staining condition evaluation process according to the third embodiment. In this staining condition evaluation method, firstly, similar to the first embodiment, a process is performed... Figure 1 The steps S11 to S15 are shown. Next, between steps S15 and S16, the following steps are performed. Figure 7 After step S21 shown, the process is largely the same as in the first embodiment. Figure 1 Steps S16-S17 are shown.

[0071] In step S21, the test specimen is treated with a different primary antibody than the primary antibody used in step S11. This other primary antibody is, for example, the primary antibody used in the N+1th immunostaining of the sequential multiplex immunostaining method described above. This other primary antibody is an antibody of a different class than the primary antibody described above (i.e., the primary antibody used in the Nth immunostaining). This other primary antibody is, for example, an antibody derived from the same animal class as the primary antibody described above. The primary staining conditions (hereinafter also referred to as "other staining conditions") related to this other primary antibody in step S21 are the primary staining conditions implemented in the N+1th immunostaining described above. These other staining conditions may be different from or the same as the primary staining conditions in step S11. Furthermore, step S21 is performed between steps S14 and S16 without performing the antigen activation treatment in step S15. In step S16, the secondary antibody and chromogenic agent used in the N+1th immunostaining are used under the predetermined secondary staining conditions implemented in the N+1th immunostaining of the sequential multiplex immunostaining method described above.

[0072] In the test specimens that are the subjects of the staining condition evaluation method of the third embodiment, the cells stained with the primary antibody applied in step S11 are different from the cells stained with other primary antibodies applied in step S21. Therefore, if the primary antibody in step S14 is properly removed and / or inactivated, the area where cells are stained by the application of secondary antibody and chromogenic agent in step S16 is different from and does not overlap with the area where cells are stained by the application of secondary antibody and chromogenic agent in step S12. On the other hand, if the primary antibody in step S14 is not properly removed and / or inactivated, a portion of the area where cells are stained by the application of secondary antibody and chromogenic agent in step S16 overlaps with the area where cells are stained by the application of secondary antibody and chromogenic agent in step S12.

[0073] In the staining condition evaluation method of the third embodiment, a first image is obtained by photographing the test specimen stained in steps S11-S12 in step S13. Additionally, a second image is obtained by photographing the test specimen stained in steps S21 and S16 in step S17. Furthermore, the acquisition of the first image can be performed between steps S12 and S14, or it can be performed before or after step S13.

[0074] In step S17, for example, by execution by computer 5 Figure 5The procedure 572 shown detects the position of each stained cell in the first image and also detects the position of each stained cell in the second image. Then, it confirms whether the cells in the first image are specifically stained, and also confirms whether the cells in the second image are specifically stained. Furthermore, it compares the first and second images to confirm whether cells stained in the first image are unstained in the second image.

[0075] Then, after confirming that the cells are specifically stained in the first image and the second image and that the stained cells in the first image are unstained in the second image, it is determined that the staining conditions related to the first antibody administration in step S11 are appropriate.

[0076] On the other hand, in other cases, the staining conditions are deemed inappropriate. For example, if some or all of the stained cells in the first image are also stained in the second image, it is considered that the concentration of the primary antibody applied in step S11 is too high and / or the reaction time is too long. Additionally, if the cells in the first image are almost unstained, it is considered that the concentration of the primary antibody applied in step S11 is insufficient and / or the reaction time is insufficient. If the cells in the second image are almost unstained, it is considered that the concentration of the other primary antibodies applied in step S21 is insufficient and / or the reaction time is insufficient.

[0077] When comparing the first and second images as described above, the first and second images are aligned. This alignment can be performed automatically, for example, using known image registration techniques, or manually by the operator.

[0078] Confirmation of whether cells are specifically stained in the first and second images is performed, for example, using a learned model created through machine learning. This learned model is created by performing machine learning on an initial model for the specific detection of stained cell distribution using a set of images used for training—a training dataset. This training dataset contains images of specimens with specifically stained cell distributions and images of specimens with non-specifically stained cell distributions. This machine learning is, for example, deep learning using neural networks. Learning based on this deep learning is performed, for example, using U-Net. Furthermore, this machine learning can also be performed using methods other than deep learning.

[0079] Furthermore, in step S17, the specific detection of the above-mentioned stained cell distribution in the first image and the second image, as well as the repeated detection of the above-mentioned stained cells based on the comparison between the first image and the second image, can also be performed by the operator's visual inspection.

[0080] In the above description, steps S11-S12 and S21, S16 are described as corresponding to two consecutive immunostainings (e.g., the Nth and N+1th) in a series of immunostainings performed using sequential multiplex immunostaining, but are not limited thereto. In this sequential multiplex immunostaining method, as long as steps S21, S16 are performed after steps S11-S12, immunostaining using a primary antibody different from that used in steps S11, S21 can also be performed between steps S11-S12 and steps S21, S16. In this case, similarly in the above-described staining condition evaluation method, between steps S14 and S15, the application of a primary antibody different from that used in steps S11, S21, the application of a secondary antibody and a chromogenic agent corresponding to that primary antibody, and the removal and / or inactivation of that primary antibody, etc., are performed. Furthermore, this primary antibody may be, for example, an antibody derived from an animal species different from the primary antibody used in steps S11, S21.

[0081] As explained above, the staining condition evaluation method of the third embodiment also includes a step (step S21) of applying another primary antibody to the test specimen under other staining conditions between steps S14 and S16. Furthermore, the cells stained with a primary antibody are different from the cells stained with other primary antibodies. In step S17, if the cells in the first image obtained by taking pictures of the test specimen between steps S12 and S14 are specifically stained, and the cells in the second image obtained by taking pictures of the test specimen after step S16 are specifically stained, and if the cells stained in the first image are unstained in the second image, then it is determined that one of the staining conditions (i.e., the primary staining condition in step S11) is appropriate.

[0082] Therefore, in two consecutive immunostaining processes, the staining conditions of the first immunostaining can be set so as not to adversely affect the subsequent immunostaining. Thus, this staining condition evaluation method is particularly suitable for evaluating staining conditions in sequential multiplex immunostaining.

[0083] Various modifications can be made to the above-mentioned staining condition evaluation method.

[0084] For example, determining the appropriateness of the staining conditions in step S13 may not be necessary. Furthermore, acquiring the first image in step S13 can be omitted. Acquiring the second image in step S17 may also not be necessary.

[0085] The antigen activation process in steps S11 and S15 can also be omitted.

[0086] The above-described methods for evaluating staining conditions are not necessarily required for evaluating staining conditions in sequential multiplex immunostaining; they can also be used for evaluating staining conditions in other immunostaining methods. Furthermore, the specimens and test specimens described above do not necessarily have to be derived from biological specimens; they can also be other types of specimens.

[0087] The structures in the above-described embodiments and their variations can be appropriately combined as long as they do not contradict each other.

[0088] The invention has been described and illustrated in detail, but the description is illustrative and not limiting. Therefore, various modifications and methods can be implemented without departing from the scope of the invention.

[0089] Explanation of reference numerals in the attached figures

[0090] 81 glass slide

[0091] Test specimens 82 and 83

[0092] Steps S11~S17, S21, S131, S132.

Claims

1. A staining condition evaluation method for evaluating staining conditions of specimens based on immunostaining, characterized in that, have: a) The procedure of conferring antibody to a test specimen once under a staining condition; b) A step following step a) of applying a second antibody and chromogenic agent to the test specimen; c) A step after step b) to inactivate or remove the primary antibody from the test specimen; d) The step of applying the secondary antibody and the chromogenic agent to the test specimen after step c); and e) After step d), a step to determine whether a staining condition is appropriate by confirming the presence or absence of stained cells in the test specimen.

2. The method for evaluating staining conditions according to claim 1, characterized in that, The staining condition evaluation method further includes a step between step b) and step c) to determine whether a staining condition is appropriate by confirming the presence or absence of stained cells in the test specimen.

3. The method for evaluating staining conditions according to claim 1 or 2, characterized in that, The presence or absence of stained cells in step e) is confirmed using a learned model created through machine learning.

4. The method for evaluating staining conditions according to claim 1 or 2, characterized in that, In step e), in the first image obtained by taking the test specimen between steps b) and c), the staining position representing the location of the stained cells is obtained, and the presence or absence of stained cells at the staining position in the second image obtained by taking the test specimen after step d) is used as the presence or absence of stained cells in the test specimen.

5. The method for evaluating staining conditions according to claim 1 or 2, characterized in that, Prior to step a), a glass slide containing the test specimen and other test specimens is prepared. In step a), the other test specimens are conditioned with the primary antibody under other staining conditions. In step b), the other test specimens are also treated with the secondary antibody and the chromogenic agent. In step c), the primary antibodies on the other test specimens are also inactivated or removed. In step d), the secondary antibody and the chromogenic agent are also applied to the other test specimens. In step e), the appropriateness of the other staining conditions is also determined by confirming the presence or absence of stained cells in the other test specimens.

6. The method for evaluating staining conditions according to claim 1 or 2, characterized in that, The staining condition evaluation method further includes a step between step c) and step d) of administering additional primary antibodies to the test specimen under other staining conditions. Cells stained with the aforementioned single antibody are different from cells stained with the aforementioned other single antibodies. In step e), cells are specifically stained in the first image of the test specimen taken between steps b) and c), and cells different from those in the first image are specifically stained in the second image of the test specimen taken after step d), and if cells stained in the first image are unstained in the second image, then the staining condition is deemed appropriate.

Images