Staining condition evaluation method and computer-readable program

Abstract

The staining condition evaluation method comprises: a step for imparting a first primary antibody to a test specimen under a first staining condition (step S11); a step (step S13) for capturing an image of the test specimen to acquire a first image; a step (step S15) for imparting a second primary antibody to the test specimen under a second staining condition; a step (step S17) for capturing an image of the test specimen and acquiring a second image; and a step (step S18) for determining whether or not the first staining condition and the second staining condition are appropriate on the basis of the detection results of the stained cells in the first image and the second image. In step S18, when the cells on the test specimen are specifically stained in the first image, the cells on the test specimen different from the first image are specifically stained in the second image, and the cells on the test specimen stained in the first image are not stained in the second image, it is determined that the first staining condition and the second staining condition are 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 JP2023-101954, 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] Method 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 a first antibody to a test specimen under a first 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 taking a picture of the test specimen to obtain a first image after step b); d) a step of inactivating or removing the first antibody from the test specimen after step c); e) a step of applying a second antibody to the test specimen under a second staining condition after step d); f) a step of applying the second antibody and the chromogenic agent to the test specimen after step e); g) a step of taking a picture of the test specimen to obtain a second image after step f); and h) a step of determining whether the first staining condition and the second staining condition are appropriate based on the detection results of stained cells in the first image and the second image after step g). The cells stained using the first antibody are different from the cells stained using the second antibody. In step h), if the cells on the test specimen in the first image are specifically stained, and the cells on the test specimen in the second image that are different from those in the first image are specifically stained, and if the cells on the test specimen stained in the first image are unstained in the second image, then the first staining condition and the second staining condition are deemed appropriate.

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

[0011] In the second embodiment of the present invention, based on the staining condition evaluation method of the first embodiment, the detection of stained cells in the first image and the detection of stained cells in the second image are performed using learned models created by machine learning in the h) step.

[0012] In embodiment 3 of the present invention, based on the staining condition evaluation method of embodiment 1 or 2, a slide containing the test specimen and other test specimens is prepared before step a). In step a), the first primary antibody is applied to the other test specimens under the third staining condition. In step b), the secondary antibody and the chromogenic agent are also applied to the other test specimens. In step c), the other test specimens are photographed to obtain a third image. In step d), the first primary antibody on the other test specimens is also inactivated or removed. In step e), the second primary antibody is applied to the other test specimens under the fourth staining condition. In step f), the secondary antibody and the chromogenic agent are also applied to the other test specimens. In step g), the other test specimens are photographed to obtain a fourth image. In step h), the appropriateness of the third and fourth staining conditions is determined based on the detection results of stained cells in the third and fourth images. In step h), cells on the other test specimens in the third image are specifically stained, and cells on the other test specimens in the fourth image that are different from those in the third image are specifically stained. If cells on the other test specimens that are stained in the third image are unstained in the fourth image, then the third staining condition and the fourth staining condition are deemed appropriate.

[0013] In embodiment 4 of the present invention, based on the staining condition evaluation method of embodiment 1 or 2 (or any one of embodiments 1 to 3), the first primary antibody and the second primary antibody are used in sequential multiplex immunostaining for a single specimen. In the sequential multiplex immunostaining, staining using the second primary antibody is performed after staining using the first primary antibody.

[0014] Embodiment 5 of the present invention is a computer-readable program that enables a computer to evaluate staining conditions for specimens based on immunostaining, wherein the computer performs the following steps: i) storing an image of the stained test specimen, i.e., a first image, and an image of the test specimen stained under conditions different from the first image, i.e., a second image; and j) determining whether the staining conditions are appropriate based on the detection results of stained cells in the first and second images. The first image is obtained by applying a first primary antibody to the test specimen under first staining conditions, and also by applying a secondary antibody and a chromogenic agent. The second image is obtained by applying a second primary antibody to the test specimen under second staining conditions, and also by applying the secondary antibody and the chromogenic agent, after acquiring the first image and inactivating or removing the first primary antibody from the test specimen. The cells stained with the first primary antibody are different from the cells stained with the second primary antibody. In step j), the cells on the test specimen in the first image are specifically stained, and the cells on the test specimen in the second image that are different from those in the first image are specifically stained. If the cells on the test specimen that are stained in the first image are unstained in the second image, it is determined that the first staining condition and the second staining condition are appropriate.

[0015] 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

[0016] Figure 1 This is a diagram illustrating the process of evaluating staining conditions in the first embodiment.

[0017] Figure 2 This is a diagram representing an example of the first image.

[0018] Figure 3 This is a diagram representing an example of the second image.

[0019] Figure 4 It is a diagram representing the structure of a computer.

[0020] Figure 5 This is a diagram illustrating part of the evaluation process in the staining condition evaluation method.

[0021] Figure 6 This is a top view showing the glass slide used in the staining condition evaluation method of the second embodiment. Detailed Implementation

[0022] Figure 1This 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 based on immunostaining methods (e.g., specimens derived from organisms).

[0023] 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 in the Nth (N is an integer greater than or equal to 1) and N+1th immunostainings performed using sequential multiplex immunostaining.

[0024] In this method for evaluating staining conditions, firstly, a test specimen used for evaluating the 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, for this test specimen, under the staining conditions implemented in the Nth immunostaining of the aforementioned sequential multiple immunostaining method, antigen activation treatment (e.g., heat treatment or enzyme treatment) and primary antibody administration are performed (step S11). This primary antibody is the primary antibody used in the aforementioned Nth immunostaining, hereinafter also referred to as the "first primary antibody". Furthermore, the staining conditions in step S11 will also be referred to as the "first staining condition" or "first primary staining condition" below.

[0025] 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. The same applies to secondary antibodies, which will be discussed later.

[0026] The first staining conditions may include, for example, at least one of the concentration of the first antibody applied to the test specimen and the application time of the first antibody relative to the test specimen (i.e., the reaction time). The first staining conditions may, for example, include the treatment conditions for antigen activation. If the antigen activation treatment is a heat treatment, these treatment conditions may include, for example, at least one of the pH of the antigen activation solution in the antigen activation treatment, and the heating temperature and heating time of the test specimen. The same applies to the second staining conditions described later.

[0027] When step S11 is completed, the test specimen is treated with a secondary 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., bound by the primary antibody in the test specimen are stained. This secondary antibody and chromogenic agent are the same as those used in the Nth immunostaining described above.

[0028] A secondary antibody is an antibody that binds to a primary antibody and corresponds to the animal class associated with the production of that 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. Various chromogenic agents (e.g., AEC (3-amino-9-ethyl-carbazole)) can be used to develop color in accordance with the labeling of the secondary antibody.

[0029] Next, a portion or the entire stained test specimen from steps S11 to S12 is photographed to obtain a stained image (hereinafter also referred to as "the first image") (step S13). Figure 2 This is a diagram representing an example of image 91 in the first image. In Figure 2 In the illustrated first 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").

[0030] Furthermore, if stained cells cannot be identified in the first image 91, or if the distribution of stained cells in the first image is not specific, the first staining conditions described above are considered inappropriate. In this case, steps S11-S12 can be repeated. Specifically, the test specimen is washed to remove the primary antibody, secondary antibody, and chromogenic agent. Alternatively, after washing the test specimen, the primary and secondary antibodies on the test specimen can be inactivated. Then, the first staining conditions are changed, and steps S11-S13 are performed.

[0031] When step S13 ends, 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 S14). That is, in step S14, 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 primary and secondary antibodies can be inactivated by various other methods. The removal of the chromogenic agent (more precisely, the removal of the color from the chromogenic precipitate derived from the chromogenic agent) can also be performed by various methods. In the case of AEC as the chromogenic agent, the chromogenic agent is removed, for example, by washing the test specimen by immersing it in alcohols such as ethanol of various concentrations. Under the same conditions as the predetermined washing and inactivation performed between the Nth and N+1th immunostainings described above, the test specimen in step S14 is washed, and the first and second antibodies are removed and / or inactivated.

[0032] When step S14 is completed, under the staining conditions implemented in the N+1th immunostaining of the above-described sequential multiplex immunostaining method, antigen activation treatment and primary antibody conferment are performed on the test specimen (step S15). The primary antibody conferred in step S15 is the primary antibody used in the N+1th immunostaining described above, hereinafter also referred to as the "second primary antibody". Furthermore, the staining conditions in step S15 will also be referred to as the "second staining conditions" or "second primary staining conditions". The second primary antibody is an antibody derived from the same animal class as the first primary antibody described above, but of a different species. The second primary staining conditions may be the same as or different from the first primary staining conditions described above. Furthermore, if the antigen activation treatment 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 treatment (step S15) can be performed in parallel with a single heat treatment.

[0033] When step S15 is completed, under the predetermined secondary staining conditions performed in the N+1th immunostaining described above, 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 N+1th immunostaining described above, and are the same types as those used in step S12. The secondary staining conditions in step S16 may be the same as those in step S12, or they may be different.

[0034] In the test specimen, the cells that bind to the first antibody applied in step S11 (i.e., the cells stained with the first antibody) are different from the cells that bind to the second antibody applied in step S15 (i.e., the cells stained with the second antibody). Therefore, if the first antibody in step S14 is properly removed and / or inactivated, the area where cells are stained by the application of the second 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 the second antibody and chromogenic agent in step S12. On the other hand, if the first antibody in step S14 is not properly removed and / or inactivated, a portion of the area where cells are stained by the application of the second antibody and chromogenic agent in step S16 overlaps with the area where cells are stained by the application of the second antibody and chromogenic agent in step S12.

[0035] When step S16 ends, a part or the whole of the stained test specimen from steps S15 to S16 is photographed to obtain a stained image (hereinafter also referred to as "the second image"). (Step S17) Figure 3 This is a diagram representing an example of image 93 in the second image. In Figure 3 In the illustrated second image 93, parallel diagonal lines are marked on the stained region 94 (i.e., the region of the stained cell aggregate, hereinafter also referred to as "stained region 94").

[0036] Next, stained cells are detected in the first image 91 and the second image 93, respectively. Then, based on the detection results of stained cells, it is determined whether the first staining conditions in step S11 and the second staining conditions in step S15 are appropriate (step S18).

[0037] In step S18, for example, the distribution of stained cells in each of the first image 91 and the second image 93 is confirmed (i.e., detected) by the operator visually examining the first image 91 and the second image 93. Specifically, it is confirmed whether cells in the first image 91 are specifically stained, and also whether cells in the second image 93 are specifically stained. Furthermore, the first image 91 and the second image 93 are compared to confirm whether cells stained in the first image 91 are unstained in the second image 93.

[0038] Then, after confirming that the cells are specifically stained in the first image 91 and the second image 93 respectively (i.e., the stained cells are specifically distributed) and the stained cells in the first image 91 are unstained in the second image 93, it is determined that the first staining conditions related to the first antibody application in step S11 and the second staining conditions related to the second antibody application in step S15 are appropriate.

[0039] On the other hand, in other cases, the first staining conditions and / or the second staining conditions are deemed inappropriate. For example, if there are almost no stained cells in the first image 91, it is considered that the concentration of the first antibody applied in step S11 is insufficient and / or the reaction time is insufficient. Similarly, if there are almost no stained cells in the second image 93, it is considered that the concentration of the second antibody applied in step S15 is insufficient and / or the reaction time is insufficient. In the comparison of the first image 91 and the second image 93, if some or all of the stained cells in the first image 91 are also stained in the second image 93 (i.e., the stained area 92 of the first image 91 and the stained area 94 of the second image 93 overlap at least partially), it is considered that the concentration of the first antibody applied in step S11 and / or the second antibody applied in step S15 is excessive and / or the reaction time is excessive.

[0040] If the first staining condition is deemed inappropriate in step S18, for example, a new test specimen is prepared, and steps S11-S18 are performed after changing the first staining condition. Similarly, if the second staining condition is deemed inappropriate in step S18, for example, a new test specimen is prepared, and steps S11-S18 are performed after changing the second staining condition. Then, steps S11-S18 are repeated while changing the test specimen until appropriate first and second staining conditions are obtained. Alternatively, instead of preparing a new test specimen, steps S11-S18 can be performed by carefully washing and heat-treating (i.e., inactivation) the test specimen deemed inappropriate in step S18, almost completely removing and / or inactivating the primary antibody, secondary antibody, and chromogenic agent on the test specimen.

[0041] In step S18, when appropriate staining conditions are obtained for the Nth and N+1th immunostainings described above, for example, the appropriateness of the staining conditions for the N+1th and N+2th immunostainings in the multiple immunostainings performed by the sequential multiple immunostaining method is evaluated using the test specimen used to obtain these staining conditions. Specifically, the test specimen after step S18 is subjected to washing and / or heat treatment, for example, to remove and / or inactivate the primary antibody, secondary antibody, and chromogenic agent. Then, steps S11 to S18 are performed under the predetermined staining conditions implemented in the N+1th and N+2th immunostainings.

[0042] In the above-described methods for evaluating staining conditions, the appropriateness of the staining conditions can be evaluated sequentially for all multiple immunostainings performed using the sequential multiple immunostaining method, or the appropriateness of the staining conditions can be evaluated sequentially for a subset of the multiple immunostainings. Alternatively, for a subset of the multiple immunostainings, the appropriateness of the staining conditions can be evaluated using one test specimen, while for another subset of immunostainings, the appropriateness of the staining conditions can be evaluated using other test specimens.

[0043] In step S18, for example, it could also be performed by computer 5. Figure 4 The procedure 572 shown is used to determine whether the first staining conditions in step S11 and the second staining conditions in step S15 are appropriate.

[0044] Computer 5 has the general structure of a computer system, including CPU 51, ROM 52, RAM 53, disk 54, display 55, input unit 56, reading device 57, communication unit 58, GPU 59, and bus 50. CPU 51 performs various arithmetic operations. GPU 59 performs various arithmetic operations related to image processing. ROM 52 stores the basic program. RAM 53 stores various information. Disk 54 stores information. Display 55 is a display unit for displaying images and other information.

[0045] 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 between the computer 5 and external devices. 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.

[0046] 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.

[0047] Figure 5This is a diagram illustrating the specific flow when the computer 5 performs step S18. First, the first image 91 obtained in step S13 and the second image 93 obtained in step S17 are stored in a storage unit implemented by RAM 53 and fixed disk 54, etc. (step S181).

[0048] Next, the determination unit, which is implemented by CPU 51, GPU 59, ROM 52, RAM 53, fixed disk 54 and their surrounding structures, detects stained cells in the first image 91 and the second image 93 respectively, and determines whether the first staining conditions and the second staining conditions are appropriate based on the detection results of stained cells (step S182).

[0049] Specifically, the determination unit confirms whether cells in the first image 91 are specifically stained, and also confirms whether cells in the second image 93 are specifically stained. Furthermore, the determination unit compares the first image 91 and the second image 93 to confirm whether cells stained in the first image 91 are unstained in the second image 93.

[0050] The confirmation of whether cells in each of images 91 and 93 are specifically stained (i.e., the detection of stained cells in images 91 and 93) is performed, for example, using a learned model created through machine learning. This learned model is created using a learning dataset, which is a set of images used for learning, based on an initial model for the specific detection of stained cell distribution. This learning 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. For example, U-Net is used for learning based on this deep learning. Furthermore, this machine learning can also be performed using methods other than deep learning.

[0051] Furthermore, in step S182, the confirmation of whether cells in each of the first image 91 and the second image 93 are specifically stained does not necessarily require the use of a learned model, and can also be performed by other methods executed by the computer 5.

[0052] During the comparison of the first image 91 and the second image 93, the determination unit performs alignment of the first image 91 and the second image 93. The alignment of the first image 91 and the second image 93 can be performed automatically, for example, by known image registration techniques, or manually by the operator.

[0053] In the above description, the staining using the first antibody (steps S11-S12) and the staining using the second antibody (steps S15-S16) are described as corresponding to two consecutive (e.g., the Nth and N+1th) immunostainings in multiple immunostainings performed by sequential multiplex immunostaining, but this is not a limitation. In this sequential multiplex immunostaining method, if the staining using the second antibody is performed after the staining using the first antibody, immunostaining using another primary antibody can also be performed between the staining using the first antibody and the staining using the second antibody. In this case, as in the staining condition evaluation method described above, between steps S14 and S15, the application of another primary antibody, the application of a secondary antibody and a chromogenic agent corresponding to that other primary antibody, and the removal and / or inactivation of that other primary antibody are performed. Furthermore, this other primary antibody may be, for example, an antibody derived from an animal species different from the first and second primary antibodies.

[0054] As explained above, the method for evaluating staining conditions for specimens based on immunostaining includes: a step of applying a first antibody to the test specimen under the first staining condition (i.e., the first staining condition) (step S11); a step of applying a second antibody and a chromogenic agent to the test specimen after step S11 (step S12); a step of taking a picture of the test specimen to obtain a first image 91 after step S12 (step S13); and a step of inactivating or removing the first antibody from the test specimen after step S13 (step S14). Step S15: After step S14, the test specimen is given a second antibody under the second staining condition (i.e., the second primary staining condition); after step S15, the test specimen is given the above-mentioned secondary antibody and the above-mentioned chromogenic agent (step S16); after step S16, the test specimen is photographed to obtain the second image 93 (step S17); after step S17, the test specimen is photographed to determine whether the first staining condition and the second staining condition are appropriate based on the detection results of the stained cells in the first image 91 and the second image 93 (step S18).

[0055] In the test specimen, the cells stained with the first antibody are different from those stained with the second antibody. In step S18, the cells in the test specimen in the first image 91 are specifically stained, and the cells in the test specimen in the second image 93 that are different from those in the first image 91 are specifically stained. If the cells in the test specimen stained in the first image 91 are unstained in the second image 93, it is determined that the first and second staining conditions are appropriate. Thus, as described above, it is easy to determine whether the first and second staining conditions in the two sequentially performed immunostainings are appropriate.

[0056] As described above, in step S18, preferably, the detection of stained cells in the first image 91 and the detection of stained cells in the second image 93 are performed using learned models created through machine learning. This allows for the automatic and high-precision confirmation of the presence or absence of stained cells in the first image 91 and the second image 93.

[0057] As described above, it is preferable that the first and second antibodies are used in sequential multiplex immunostaining of a single specimen, wherein staining with the second antibody is performed after staining with the first antibody. This allows sequential multiplex immunostaining to be performed with appropriately set first and second staining conditions.

[0058] The above-described procedure 572 is a computer-readable program that enables a computer to evaluate the staining conditions of a specimen based on immunostaining. Procedure 572 causes the computer to perform: a step (step S181) of storing a first image 91 and a second image 93, wherein the first image 91 is an image of the stained test specimen, and the second image 93 is an image of the test specimen stained under conditions different from the first image 91; and a step (step S182) of determining whether the first and second staining conditions (i.e., the first primary staining conditions and the second primary staining conditions) are appropriate based on the detection results of stained cells in the first and second images 91 and 93. The first image 91 is an image captured under the first staining conditions where a first primary antibody is applied to the test specimen, and a second primary antibody and a chromogenic agent are also applied. The second image 93 is an image captured under the second staining conditions where a second primary antibody is applied to the test specimen, and the second primary antibody and the chromogenic agent are also applied, after the acquisition of the first image 91 and the inactivation or removal of the first primary antibody on the test specimen.

[0059] In the test specimen, the cells stained with the first antibody are different from those stained with the second antibody. In step S182, if the cells in the test specimen in the first image 91 are specifically stained, and the cells in the test specimen in the second image 93 that are different from those in the first image 91 are specifically stained, and the cells in the test specimen stained in the first image 91 are unstained in the second image 93, then the first and second staining conditions are deemed appropriate. Thus, as described above, it is easy to determine whether the first and second staining conditions in two sequentially performed immunostainings are appropriate.

[0060] Next, the staining condition evaluation method of the second embodiment of the present invention will be described. Figure 6This is a top view of the slide 81 used in the staining condition evaluation method of the second embodiment. In this staining condition evaluation method, for each combination of staining conditions in steps S11 and S15, one slide 81 is used to determine in parallel whether each of the two combinations is appropriate.

[0061] like Figure 6 As shown, the aforementioned test specimen 82 and other test specimens 83 are placed on slide 81. Like test specimen 82, test specimen 83 is obtained from the same tissue as the predetermined specimen stained and analyzed by the aforementioned sequential multiple immunostaining method. Furthermore, more than three test specimens may be placed on slide 81.

[0062] 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 first antibody application for test specimen 83 are performed substantially in parallel with the antigen activation treatment and first antibody application for test specimen 82. The first antibody applied to test specimen 83 is the same as the first antibody applied to test specimen 82. In step S11, the first antibody is applied to test specimen 83 under third staining conditions. The third staining conditions may be the same as or different from the first staining conditions when the first antibody was applied to test specimen 82. For example, the third staining conditions may be conditions that deviate from the first staining conditions by a predetermined amount of predetermined parameters (e.g., the concentration of the first antibody or the reaction time).

[0063] 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. As a result, specific cells, etc., bound by the first antibody are also stained in test specimen 83. The secondary antibodies and chromogenic agents applied to test specimen 83 are the same as those applied to test specimen 82. In step S12, test specimen 83 is applied with secondary antibodies and chromogenic agents, for example, under the same staining conditions as those applied to test specimen 82.

[0064] In step S13, the test specimen 83 is photographed in roughly parallel with the acquisition of the first image 91 to obtain the third image. In step S14, the removal and / or inactivation of the primary antibody, secondary antibody, and chromogenic agent on the test specimen 83 is also performed in roughly parallel with the removal and / or inactivation of the primary antibody, secondary antibody, and chromogenic agent on the test specimen 82.

[0065] Next, in step S15, antigen activation treatment and second antibody application for test specimen 83 are performed substantially in parallel with the antigen activation treatment and second antibody application for test specimen 82. The second antibody applied to test specimen 83 is the same as the second antibody applied to test specimen 82. In step S15, the second antibody is applied to test specimen 83 under the fourth staining conditions. The fourth staining conditions may be the same as or different from the second staining conditions when the second antibody was applied to test specimen 82. For example, the fourth staining conditions may be conditions that deviate from the second staining conditions by a predetermined amount of predetermined parameters (e.g., the concentration of the second antibody or the reaction time).

[0066] Furthermore, if the fourth staining condition is the same as the second staining condition, the third staining condition is different from the first staining condition. Also, if the third staining condition is the same as the first staining condition, the fourth staining condition is different from the second staining condition. That is, at least the third staining condition is different from the first staining condition, or the fourth staining condition is different from the second staining condition.

[0067] 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 secondary antibodies and chromogenic agents are applied to test specimen 83, for example, under the same staining conditions as those applied to test specimen 82. In step S17, the fourth image is obtained by photographing test specimen 83 approximately in parallel with the acquisition of the second image 93.

[0068] Subsequently, in step S18, the appropriateness of the first and second staining conditions related to test specimen 82 is determined in roughly parallel with the appropriateness of the third and fourth staining conditions related to test specimen 83 based on the detection results of stained cells in the third and fourth images. Specifically, it is confirmed whether cells are specifically stained in the third image, and also confirmed whether cells are specifically stained in the fourth image. Furthermore, the third and fourth images are compared to confirm whether cells stained in the third image are unstained in the fourth image.

[0069] Then, if it is confirmed that the cells are specifically stained in both the third and fourth images (i.e., the stained cells are specifically distributed), and the stained cells in the third image are unstained in the fourth image, it is determined that the third staining conditions related to the application of the first antibody to test specimen 83 and the fourth staining conditions related to the application of the second antibody to test specimen 83 are appropriate. On the other hand, in other cases, similarly as described above, it is determined that the third staining conditions and / or the fourth staining conditions are inappropriate. Furthermore, test specimens 82 and 83 can also be placed on different slides 81.

[0070] As explained 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. In step S11, the other test specimens 83 are treated with a first primary antibody under the third staining condition. In step S12, the other test specimens 83 are also treated with a secondary antibody and a chromogenic agent. In step S13, the other test specimens 83 are photographed to obtain a third image. In step S14, the first primary antibody on the other test specimens 83 is also inactivated or removed. In step S15, the other test specimens 83 are treated with a second primary antibody under the fourth staining condition. In step S16, the other test specimens 83 are also treated with a secondary antibody and a chromogenic agent. In step S17, the other test specimens 83 are photographed to obtain a fourth image.

[0071] In step S18, the appropriateness of the third and fourth staining conditions is further determined based on the detection results of stained cells in the third and fourth images. In step S18, if cells on other test specimens 83 in the third image are specifically stained, and cells on other test specimens 83 in the fourth image that are different from those in the third image are specifically stained, and if cells on other test specimens 83 stained in the third image are unstained in the fourth image, then the third and fourth staining conditions are deemed appropriate. Therefore, it is possible to determine the appropriateness of two combinations of staining conditions in steps S11 and S15 in parallel.

[0072] Alternatively, more than three test specimens can be prepared on slide 81. Furthermore, the first and second primary antibodies can be applied to the three or more test specimens in different combinations of the staining conditions in steps S11 and S15. This allows for the simultaneous determination of the appropriateness of each of the three or more combinations of staining conditions in steps S11 and S15.

[0073] Various changes can be made to the above-mentioned methods and procedures for evaluating staining conditions.

[0074] The antigen activation process in steps S11 and S15 can also be omitted.

[0075] The above-described methods for evaluating staining conditions are not necessarily required for evaluating staining conditions in sequential multiplex immunostaining methods; they can also be used for evaluating staining conditions in other immunostaining methods. Furthermore, the specimens and test specimens described above do not necessarily need to be derived from biological specimens; they can also be other types of specimens.

[0076] The structures in the above-described embodiments and their variations can be appropriately combined as long as they do not contradict each other.

[0077] Although the invention has been described and illustrated in detail, the description is illustrative and not limiting. Therefore, various modifications and methods can be implemented without departing from the scope of the invention.

[0078] Explanation of reference numerals in the attached figures

[0079] 5. Computers

[0080] 81 glass slide

[0081] 82 test specimens

[0082] 83 test specimens

[0083] 91 Image 1

[0084] 93 Image 2

[0085] 572 program

[0086] Steps S11~S18 and S181~S182.

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 the first antibody on the test specimen under the first staining conditions; b) A step following step a) of applying a second antibody and chromogenic agent to the test specimen; c) The step of taking a picture of the test specimen to obtain the first image after step b); d) A step after step c) to inactivate or remove the first antibody from the test specimen; e) A step of administering a second antibody to the test specimen under the second staining conditions following step d); f) A step following step e) of applying the secondary antibody and the chromogenic agent to the test specimen; g) The step of taking a picture of the test specimen to obtain a second image after step f); as well as h) A step following step g) to determine whether the first staining conditions and the second staining conditions are appropriate based on the detection results of stained cells in the first image and the second image. The cells stained with the first antibody are different from those stained with the second antibody. In step h), the cells on the test specimen in the first image are specifically stained, and the cells on the test specimen in the second image that are different from those in the first image are specifically stained. If the cells on the test specimen that are stained in the first image are unstained in the second image, it is determined that the first staining condition and the second staining condition are appropriate.

2. The method for evaluating staining conditions according to claim 1, characterized in that, In step h), the detection of stained cells in the first image and the detection of stained cells in the second image are performed using learned models created through machine learning.

3. 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 treated with the first antibody under the third staining condition. In step b), the other test specimens are also treated with the secondary antibody and the chromogenic agent. In step c), the other test specimens are photographed to obtain the third image. In step d), the first antibody on the other test specimens is also inactivated or removed. In step e), the other test specimens are treated with the second antibody under the fourth staining condition. In step f), the secondary antibody and the chromogenic agent are also applied to the other test specimens. In step g), the other test specimens are photographed to obtain the fourth image. In step h), the appropriateness of the third and fourth staining conditions is further determined based on the detection results of the stained cells in the third and fourth images. In step h), cells on the other test specimens in the third image are specifically stained, and cells on the other test specimens in the fourth image that are different from those in the third image are specifically stained. If cells on the other test specimens that are stained in the third image are unstained in the fourth image, then the third staining condition and the fourth staining condition are deemed appropriate.

4. The method for evaluating staining conditions according to claim 1 or 2, characterized in that, The first and second antibodies are used in sequential multiplex immunostaining of a single specimen. In the sequential multiple immunostaining, staining with the second antibody is performed after staining with the first antibody.

5. A computer-readable program that enables a computer to evaluate the staining conditions of specimens based on immunostaining methods, characterized in that, The program causes the computer to execute: i) The process of storing an image of the stained test specimen, i.e., the first image, and an image of the test specimen stained under conditions different from the first image, i.e., the second image; as well as j) The step of determining whether the staining conditions are appropriate based on the detection results of the stained cells in the first image and the second image. The first image was taken under the first staining conditions, after the test specimen had been treated with the first antibody, and also after the second antibody and chromogenic agent had been applied. The second image was taken after the first image was acquired and the first antibody on the test specimen was inactivated or removed, and the test specimen was given a second antibody under the second staining conditions, with both the second antibody and the chromogenic agent present. The cells stained with the first antibody are different from those stained with the second antibody. In step j), the cells on the test specimen in the first image are specifically stained, and the cells on the test specimen in the second image that are different from those in the first image are specifically stained. If the cells on the test specimen that are stained in the first image are unstained in the second image, it is determined that the first staining condition and the second staining condition are appropriate.

Images