JP2010261762A - Specimen preparing device and specimen preparing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stably prepare a specimen under a condition desired by a user. <P>SOLUTION: A standard specimen preparing device 2 includes a standard specimen image data memory part 4 storing the image data of a standard specimen image obtained by imaging a standard specimen. An image extracting part 353 extracts the standard specimen image from the standard specimen image data memory part 4 on the basis of the respective values of a specimen specifying parameter obtained with respect to a specimen to be prepared and a control parameter. An image display processing part 354 subjects a standard dyed image, which is used for determining the propriety of the value of the control parameter, to display processing on a display part 32 on the basis of the extracted standard specimen image. A propriety selection requesting part 355 requests the propriety selection of the value of the control parameter. A control parameter determining part 372 determines the value of the control parameter according to the result of the propriety selection to the request due to the propriety selection requesting part 355. A specimen preparing part 5 prepares the specimen to be prepared using the determined value of the control parameter. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a specimen creation device and a specimen creation method for creating a specimen.

  For biological tissue specimens, especially pathological specimens, specimens obtained by organ excision or needle biopsy are sliced into several μm to prepare specimens, and are widely observed using a microscope to obtain various findings. ing. Above all, transmission observation using an optical microscope is one of the most popular observation methods because the equipment is relatively inexpensive and easy to handle and has been historically used for a long time. . Here, the specimen collected from the living body hardly absorbs and scatters light and is almost colorless and transparent. For this reason, it is common to perform dyeing with a dye when preparing a specimen (staining process).

  Various dyeing techniques have been proposed, and the total number reaches over 100 types. These staining methods can be roughly classified into three types: general staining, special staining, and immunobiohistochemical staining (hereinafter referred to as “immunostaining”). The procedure for staining the specimen is determined by the staining method.

  Here, general staining is a staining method carried out to obtain pathomorphological findings. For example, hematoxylin-eosin staining (hereinafter referred to as “H & E staining”) using two kinds of pigments of blue-purple hematoxylin and red eosin corresponds to this and is most widely used in histopathological examination. ing. In this H & E staining, first, hematoxylin staining is performed with a hematoxylin staining solution, and after washing with water for coloration, eosin staining is performed with an eosin staining solution.

  Special staining is developed as a staining method that supplements general staining, and is known as a staining method for detecting substances that are difficult to identify by general staining. For example, collagen fibers, elastic fibers, reticulofibers, glycogens, polysaccharides, tissue pathogens, nucleic acids, minerals, melanin, intracellular granules, fibrin, striated, amyloid, nerves, fat, etc. Dyeing methods are known. The procedure for special staining is basically the same as that for general staining, and the staining solution (reagent) differs depending on the staining method. For example, a Schiff reagent is used in PAS staining, which is known as a technique for specially staining linear epithelial cells.

  Immunostaining is a coloring operation for visualizing antigen-antibody reaction (immune reaction). Although immunostaining is not exactly different from staining, the presence of antigen (local) Present) can be observed. For this reason, it is treated in the same manner as general staining and special staining as one of staining methods in histopathological examination. For example, an enzyme antibody method is known in which the localization of an antibody bound to an antigen is visualized by color development by an enzyme reaction. In this enzyme antibody method, first, a specific antibody is reacted with an object (antigen) on a section. Then, an immunocomplex of a specific antibody and a labeling enzyme is formed by appropriately combining an antigen-antibody reaction and a chemical reaction, and finally the presence (localization) of the antigen is visualized by a coloring reaction of the labeling enzyme. In the coloring reaction, for example, DAB coloring is widely used.

  By the way, the staining state (degree of staining) of these samples by general staining, special staining, and immunostaining is the time required for staining (reaction) (hereinafter referred to as “reaction time”) and a staining solution (reagent or antibody). It depends on the production conditions of the diluted solution. For example, H & E staining differs depending on the reaction time with the hematoxylin staining solution, the conditions for producing the staining solution, the reaction time with the eosin staining solution, the conditions for producing the staining solution, and the like. In immunostaining, for example, in the case of an enzyme antibody method, the sensitivity varies depending on the reaction format such as direct method, indirect method, LSAB method, and high-sensitivity polymer reagent method.

  In addition to the above-described staining process, the specimen preparation process includes a paraffin block preparation process, a thin slice process, a deparaffinization process, an encapsulation process, and the like, and the conditions of these processes also affect the dyeing.

  Here, the specimen is usually created by a laboratory technician located in a medical facility. For this reason, individual differences such as the experience of clinical laboratory technicians have an effect, and it has been difficult to keep the staining state of the specimen uniform. In addition, the staining state may vary depending on the surrounding environment such as individual differences in the specimen itself and room temperature at the time of specimen preparation.

  In recent years, development of an apparatus for automatically performing a part or all of a series of specimen preparation processes has been promoted in order to suppress such variation in staining and stably prepare specimens (for example, Patent Document 1). , 2). For example, Patent Document 1 discloses an automatic apparatus that automates the deparaffinization, staining, and cover glass coating operations of biological specimens. Patent Document 2 discloses an automated molecular pathology apparatus capable of individually monitoring the temperature for each sample.

JP 2005-527811 A Japanese Patent No. 3847559

  By the way, since the prepared specimen is observed and diagnosed by the clinician, the required staining state of the specimen varies depending on the preference of the clinician. For this reason, if an automated device disclosed in Patent Documents 1 and 2 can be used, consistent control of the specimen preparation process can be realized, and a specimen with a uniform staining state can be stably produced. The state is not always the staining state desired by the clinician, and the diagnosis accuracy may be reduced. For example, it may lead to stress of a clinician at the time of observation / diagnosis of a stained specimen (hereinafter referred to as “stained specimen”), and a definite finding may be overlooked.

  The present invention has been made in view of the above, and an object of the present invention is to provide a specimen preparation device and a specimen preparation method capable of stably preparing a specimen under conditions desired by a user.

  In order to solve the above-described problems and achieve the object, a specimen preparation device according to an aspect of the present invention shows a specimen image obtained by imaging a specimen prepared in advance as a standard specimen, indicating the kind of the specimen. An image storage unit that stores the sample specifying parameter and each value of the control parameter used when creating the sample, a sample specifying parameter acquiring unit that acquires a value of the sample specifying parameter for the sample to be created, and A control parameter acquisition unit that acquires the value of the control parameter for a sample to be created, and a sample image stored in the image storage unit based on at least the value of the sample specification parameter acquired by the sample specification parameter acquisition unit An image extracting unit that extracts one or more sample images from the image, and the control based on the sample image extracted by the image extracting unit An image display processing unit that performs processing for displaying a reference image for determining whether or not a parameter value is acceptable on a display unit, a availability selection requesting unit that requests input of availability of the control parameter value, and the availability selection request A control parameter determining unit that determines a value of the control parameter according to an input result of whether or not to accept the request by the unit, and a sample that generates the sample to be generated using the control parameter value determined by the control parameter determining unit And a creation unit.

  According to the specimen creation device according to this aspect, a specimen image obtained by imaging a standard specimen is stored in the image storage unit in advance, and one sheet is stored from the image storage unit based on at least the specimen specifying parameter acquired for the specimen to be created. The above specimen image can be extracted. Then, based on the extracted sample image, a reference image for determining whether or not the value of the control parameter can be displayed can be displayed. Then, it is possible to determine the value of the control parameter according to the input result of the availability selection requested for the value of the control parameter, and to create a sample to be created according to the determined value of the control parameter. Therefore, it is possible to stably create a sample under conditions desired by the user.

  Further, in the specimen preparation method according to another aspect of the present invention, when a specimen image obtained by imaging a specimen prepared as a standard specimen in advance creates a specimen specifying parameter indicating the specimen type and the specimen. A sample creation method in a sample creation device stored in association with each value of a used control parameter, the sample specification parameter acquisition step for acquiring the value of the sample specification parameter for a sample to be created, and the creation target A control parameter acquisition step for acquiring the value of the control parameter for the sample, and at least one of the stored sample images based on the value of the sample specification parameter acquired in the sample specification parameter acquisition step An image extraction process for extracting a sample image, and the value of the control parameter can be determined based on the sample image extracted in the image extraction process. An image display processing step for performing a process of displaying a reference image for determining whether the control parameter value is acceptable, an availability selection requesting step for requesting input of availability selection of the value of the control parameter, and availability selection for the request in the availability selection requesting step. A control parameter determining step for determining the value of the control parameter according to an input result; and a sample generating step for generating the sample to be generated using the control parameter value determined in the control parameter determining step. It is characterized by.

  According to the present invention, a sample can be stably created under conditions desired by a user.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiments. Moreover, in description of drawing, the same code | symbol is attached | subjected and shown to the same part.

(Embodiment 1)
First, as a first embodiment, a case where a specimen (stained specimen) is created by performing H & E staining known as general staining on a specimen collected from a living body of a subject by organ excision or needle biopsy will be described.

  FIG. 1 is an explanatory diagram for explaining an outline of a procedure for preparing a sample and observing / diagnosis. First, a specimen is created from a specimen collected from a subject (hereinafter, the specimen to be created is referred to as a “target specimen”). Prior to the creation of the target specimen, the clinician uses FIG. As shown, data input necessary to create the target specimen is performed. When the clinician inputs data, a reference staining image (reference image) representing the staining density corresponding to the input data is displayed on the screen and presented to the clinician. This reference stained image is displayed as a reference for the user (here, a clinician) to judge whether or not the value of a control parameter (specifically, a correction target control parameter described later) that is used when creating a target specimen is acceptable. . In the first embodiment, a specimen having a standard staining density prepared in advance as a standard specimen is imaged to generate an observation image (specimen image) and stored as a standard specimen image. If there is a standard specimen image with a staining density according to the input data, the standard specimen image is displayed on the screen as a reference staining image. If there is no standard specimen image with a staining density according to the input data, the staining density of this standard specimen image is displayed. Is virtually changed to the staining density corresponding to the input data, and a virtual dye image is generated and displayed on the screen as a reference dye image.

  In the first embodiment, the clinician determines whether or not the value of the correction target control parameter is acceptable by adjusting the staining density represented by the reference staining image to a desired staining density while viewing the reference staining image. Specifically, at this time, a virtual image in which the staining density of the reference staining image is virtually changed according to the staining density adjusted by the clinician is generated and displayed on the screen as a staining simulation image (simulation image). The clinician adjusts the staining density to a desired staining density while viewing the staining simulation image.

  When the clinician determines the staining density, the value of the correction target control parameter is determined according to the staining density, and the specimen preparation process is automatically performed by the apparatus in which the specimen preparation process is automated according to the determined correction target control parameter value. Alternatively, a target specimen is created semi-automatically through the work of a clinical laboratory technician (FIG. 1 (b)). A clinical technologist observes a target specimen prepared using a microscope and confirms the staining state. At this time, the target specimen is imaged and an observation image is generated (FIG. 1C). The clinician observes the observation image of the target specimen obtained as described above and makes a diagnosis (FIG. 1 (d)). According to this, the clinician can observe and diagnose the specimen in a desired staining state.

  Here, a process of capturing an image of a specimen and generating an observation image (hereinafter referred to as “observation image generation process”), and a process of calculating the staining density of the specimen from the observation image (hereinafter referred to as “staining density calculation process”). )) And the principle of processing for generating a virtual image in which the calculated staining density is virtually changed (hereinafter referred to as “virtual image generation processing”) will be described. FIG. 2 is an explanatory diagram for explaining an outline procedure of the observation image generation process and the staining density calculation process. FIG. 3 is an explanatory diagram illustrating a schematic procedure of the virtual image generation process. Each process described here can be realized by using a known technique, and in the first embodiment, these processes are used as appropriate in order to realize the specimen preparation and observation / diagnosis procedure shown in FIG.

  As shown in FIG. 2, in the observation image generation process, first, a specimen is imaged (a1). Subsequently, based on the obtained image, the spectral transmittance t ^ (x, λ) at each point on the sample corresponding to each pixel position (x) is calculated (a3). Here, t ^ indicates that a symbol "^ (hat)" representing an estimated value is attached on t. As a method for calculating the spectral transmittance t ^ (x, λ), a technique disclosed in Japanese Patent Application Laid-Open No. 2009-14355 can be used. Note that the spectral transmittance t ^ (x, λ) may be configured such that the spectral transmittance at each point of the specimen is measured by a spectral imaging apparatus.

Subsequently, a pixel signal value g (b) at each pixel position is calculated based on the spectral transmittance calculated for each pixel position, and an observation image is generated (a5). Here, the relationship of the following equation (1) based on the response model function of the camera is established between the pixel signal value g (b) in the band b of the image and the spectral transmittance t (λ) of the corresponding sample. . λ is the wavelength, s (λ, b) is the spectral sensitivity characteristic of the camera in band b, e (λ) is the spectral radiation characteristic of illumination, and n (b) is the observation noise in band b. b is a serial number for identifying the band, and b = 3 in the case of a normal RGB camera.

However, the number of bands can be selected as appropriate. Moreover, it is good also as multiband imaging of a sample using the filter from which spectral characteristics differ. For example, when a monochrome camera and a filter with a band number b are used and a sample is subjected to multiband imaging, a pixel signal value g (b) is obtained by the following equation (2). f (b, λ) represents the spectral transmittance of the b-th filter.

  In a5 of FIG. 2, the pixel signal value g (b) at each pixel position is calculated according to the above formula (1) or formula (2) to obtain an observation image of the sample. This observation image is displayed on the screen as necessary.

  As shown in FIG. 2, in the staining density calculation process, the spectral transmittance calculated for each pixel position is converted into absorbance (a7), and the dye amount at each point on the sample is calculated based on this absorbance. Thus (a9), the staining density at each point of the specimen is acquired.

In general, in a material that transmits light, a Lambert bale (Lambert) expressed by the following equation (3) between the intensity I ₀ (λ) of incident light and the intensity I (λ) of emitted light for each wavelength λ. -Beer) law is known to hold.

  k (λ) is a material-specific value determined depending on the wavelength, and d represents the thickness of the material. Since the dye is inherently dispersed in the specimen, the concept of thickness is not accurate, but how much dye is compared to the assumption that the specimen is stained with a single dye. It is an indicator of the relative amount of pigment (the amount of pigment) that indicates whether or not there is. Hereinafter, the thickness d of the substance in this Lambert-Beer law is treated as the amount (dye amount) of the dye constituting the specimen (for example, the dye of the staining solution staining the specimen). Here, the amount of dye corresponds to the staining density. Hereinafter, the dye amount and the staining density are treated as synonyms. That is, the calculation of the dye amount described here is synonymous with calculating the staining density. Note that a value obtained by multiplying the dye amount d by a value k (λ) specific to a substance is generally called the absorbance or absorption intensity of the dye.

When there are a plurality of types of dyes that stain the specimen, if a serial number of 1, 2,... N is assigned to each dye, the following equation (4) is obtained at each wavelength λ from the above-mentioned Lambert-Beer law. To establish.

In the first embodiment, an H & E stained specimen is an observation / diagnosis target. Therefore, if hematoxylin is abbreviated as dye H and eosin is abbreviated as dye E, the following expression (5) is established at each wavelength λ.

More specifically, since the absorption component (abbreviated as G) of the slide glass on which the specimen is placed is included as an absorption component other than these staining pigments, the spectral transmittance t ^ (x) is expressed by the following equation (6). can get. k _H (λ), k _E (λ), and k _G (λ) represent k (λ) corresponding to the dye H, the dye E, and the absorption component G other than these dyes, and each k (λ) The value of corresponds to the spectral characteristics of the corresponding dye (absorbing component).

Each k (λ) may vary depending on the organ or part from which the specimen (specimen) is collected and the tissue in the specimen. For this reason, according to these differences, a plurality of k (λ) is prepared for each dye, such as k (λ) ₁ , k (λ) ₂ ..., And the corresponding k (λ) is used. It is good. More specifically, k (λ) corresponding to each pigment is prepared for each organ, part, or tissue, and k (λ) corresponding to the organ, part, or tissue from which the target specimen is collected is appropriately selected. It may be used. In addition, depending on the staining solution used in the applied staining method, the spectral characteristics of the dye may change depending on the substance in the tissue. In such a case, as a spectral characteristic of the dye, a spectral characteristic k ′ (λ) that takes into account the amount of change may be applied to a predetermined spectral characteristic k (λ) of the corresponding dye.

  Moreover, although the absorption component G of the slide glass has been described as an absorption component other than the staining pigment, other than this, a tissue such as erythrocytes having an absorption component at the time of no staining may exist in the specimen. That is, erythrocytes have their own unique color even in the unstained state, and are observed as the color of erythrocytes themselves after H & E staining. Alternatively, the color of eosin changed in the staining process is observed superimposed on the color of red blood cells themselves. In consideration of the absorption component of red blood cells, the value obtained by multiplying the predetermined spectral characteristic k (λ) of the absorption component of red blood cells by the amount of the dye in Formula (6) described above is further added, and the spectral transmittance t ^ (X) may be calculated. In this way, the pixel signal value in the red blood cell region can be displayed with high accuracy.

Here, assuming that the spectral transmittance obtained for each pixel position (x) is t ^ (x, λ) and the spectral absorbance is a ^ (x, λ), the following equation (7) A relationship is established. Here, a ^ indicates that the symbol "^" is added on a.

Substituting equation (6) into equation (7) yields the following equation (8).

In Equation (8), if the unknown variables are d _H and d _E and Equation (8) is used simultaneously for at least two different wavelengths λ, these can be solved. In order to further improve the accuracy, the multiple regression analysis may be performed by simultaneous equation (8) for three or more different wavelengths λ. For example, when equation (8) is made simultaneous for _three wavelengths λ ₁ , λ ₂ , and λ ₃ , the matrix can be expressed as the following equation (9).

Here, the formula (9) is replaced with the following formula (10).

Assuming that the number of sample points in the wavelength direction is D, A ^ (x) is a matrix of D rows and 1 column corresponding to a ^ (x, λ), and K is D rows 3 corresponding to k (λ). A matrix of columns, d (x) is a matrix of 2 rows and 1 column corresponding to d _H and d _E at the pixel position (x), and ε is a matrix of D rows and 1 column corresponding to errors. A ^ indicates that the symbol "^" is attached on A.

For example, the dye amounts d _H and d _E are calculated according to the equation (10) using, for example, the least square method. The least square method is a method for determining d (x) so as to minimize the sum of squares of errors in a single regression equation, and can be calculated by the following equation (11).

  As shown in FIG. 3, in the virtual image generation process, first, an adjustment coefficient for the dye amount (staining density) is set (b1). Subsequently, the dye amount is adjusted by multiplying the dye amount calculated from the observation image in a9 of FIG. 2 by the adjustment coefficient set (b3). Then, the dye amount adjusted in this manner is substituted into the equation (8) to newly calculate the absorbance (b5), and the calculated absorbance is converted into the spectral transmittance according to the above equation (6) (b7). Thereby, since the spectral transmittance at each pixel position after adjusting the dye amount is obtained, the pixel signal value at each pixel position is calculated according to the above-described equation (1) or (2), and the virtual image is obtained. Is generated (b9). Thereby, a virtual image is obtained in which the staining density of the observation image is virtually changed according to the adjustment coefficient. This virtual image is displayed on the screen as necessary.

  Next, the configuration of the specimen preparation device according to the first embodiment will be described. FIG. 4 is a schematic diagram illustrating the overall configuration of the specimen creation system 1 including the specimen creation apparatus 2 according to the first embodiment. As shown in FIG. 4, the specimen creation system 1 according to the first embodiment includes a specimen creation device 2 and an observation device 6. Here, the sample preparation device 2 includes a control device 3, a standard sample image data storage unit 4 as an image storage unit, and a sample preparation unit 5, which are connected so as to be able to exchange data. The The observation apparatus 6 is for observing the specimen created by the specimen creation apparatus 2. For example, a microscope capable of observing a target specimen through transmission, a camera for capturing an observation image of the specimen by the microscope, and the like. Consists of.

  FIG. 5 is a block diagram showing a functional configuration of the specimen creation device 2. Here, the sample preparation unit 5 semi-automatically performs a device that fully automates a series of sample preparation processes, a device that automates the sample preparation processing for each part, and a sample preparation process for each part through the work of a clinical technician. The apparatus is configured by combining the devices to be processed, and the specimen is processed according to the value of a predetermined control parameter to create a specimen. For example, the specimen preparation unit 5 according to the first embodiment includes a paraffin block creation processing unit 51 that performs paraffin block creation processing, a thin slice processing unit 52 that performs thin slice processing, and a deparaffinization processing unit 53 that performs deparaffinization processing. , A staining processing unit 54 that performs a staining process, and an encapsulation processing unit 55 that performs an encapsulation process. Here, the specific apparatus configuration of the specimen preparation unit 5 differs for each medical facility, and depending on the apparatus to be employed, the processes shared by each apparatus are also different. Moreover, this apparatus configuration also differs depending on the staining method applied to the specimen. The protocol for the specimen preparation process and the control parameters used in this protocol also differ depending on the manufacturer of the apparatus to be employed, and may differ depending on the setting on the medical facility side. The configuration of the specimen creation unit 5 is not limited to the exemplified configuration, and control parameters used in the specimen creation process can be set as appropriate.

  The control device 3 gives an operation instruction for creating a sample to the sample creating unit 5 and is realized by a general-purpose computer such as a workstation or a personal computer. The control device 3 acquires each value of the sample specifying parameter and the control parameter input by the user (for example, a clinician) as data necessary for creating the target sample, and uses the acquired sample specifying parameter and the value of the control parameter. A reference staining image having a corresponding staining density is displayed on the screen. Then, the value of the correction target control parameter is determined according to the staining density adjusted by the user while viewing the reference staining image, and an operation instruction is given to the specimen creation unit 5.

  The control device 3 includes an operation unit 31, a display unit 32, a storage unit 33, a control unit 35, and an image processing unit 37.

  The operation unit 31 is realized by various input devices such as a keyboard, a mouse, a touch panel, and various switches, for example, and outputs an input signal corresponding to the operation input to the control unit 35. The display unit 32 is realized by a display device such as an LCD, an EL display, or a CRT display, and displays various screens based on a display signal input from the control unit 35.

  The storage unit 33 is realized by various IC memories such as ROM and RAM such as flash memory that can be updated and stored, a hard disk connected by a built-in or data communication terminal, an information storage medium such as a CD-ROM, and a reading device thereof. It is. The storage unit 33 stores a program for operating the control device 3 and realizing various functions of the control device 3, data used during execution of the program, and the like. The storage unit 33 also stores a specimen creation processing program 331. The specimen creation processing program 331 is a program for realizing a process for determining the value of the correction target control parameter according to the staining density adjusted and confirmed by the user and instructing the specimen creation section 5 to perform an operation.

  The control unit 35 is realized by hardware such as a CPU. The control unit 35 performs instructions, data transfer, and the like to each unit constituting the specimen preparation device 2 based on an input signal input from the operation unit 31 or a program or data stored in the storage unit 33. The overall operation of the specimen preparation device 2 is controlled. The control unit 35 includes a sample specifying parameter acquisition unit 351, a control parameter acquisition unit 352, an image extraction unit 353, an image display processing unit 354, an availability selection request unit 355, and a sample creation processing setting unit 356. .

  The sample specifying parameter acquisition unit 351 requests input of the value of the sample specifying parameter for the target sample, and accepts input of the value of the sample specifying parameter by the user via the operation unit 31. Here, the specimen specifying parameter is a value indicating the kind of specimen, and examples thereof include an organ and a part from which the specimen is collected, a subject's disease name, and the like. Note that the sample specifying parameter acquired by the sample specifying parameter acquiring unit 351 is not limited to this, and information on the sample can be used as appropriate. For example, the state of the tissue in the specimen may be used as the specimen specifying parameter. Specific examples include the number, density, and shape of cell nuclei. This assumes that the clinician makes a diagnosis by observing the number, density, shape, etc. of tissues such as cell nuclei in the target specimen, and these values may be acquired as specimen specifying parameters. .

  The control parameter acquisition unit 352 requests input of the value of the control parameter used when creating the target specimen, and accepts input of the value of the control parameter by the user via the operation unit 31. The control parameter acquisition unit 352 is a process performed by each part of the specimen preparation unit 5, that is, each process performed by the paraffin block creation processing unit 51, the slicing processing unit 52, the deparaffinization processing unit 53, the staining processing unit 54, and the encapsulation processing unit 55. A value of at least one control parameter to be used is acquired. Specific examples include, for example, control parameters such as the block size used by the paraffin block creation processing unit 51 in the paraffin block creation processing, the type of formalin-based fixative, and the time (fixing time) required for fixing the tissue with the formalin-based fixative, The control parameters such as the thickness of the specimen used by the cutting processing unit 52 in the thin cutting processing, the control parameters such as the staining method used for the staining processing by the staining processing unit 54 and the generation conditions of the staining liquid, and the time required for staining (reaction time). Can be mentioned. The generation condition of the staining liquid is, for example, the concentration of the staining liquid (staining reagent). In the first embodiment, the concentration of the hematoxylin staining liquid and the concentration of the eosin staining liquid correspond to this. The reaction time includes the reaction time with the hematoxylin staining solution and the reaction time with the eosin staining solution, and each reaction time corresponds to the time for immersing the specimen in the corresponding staining solution. Hereinafter, in the first embodiment, the control parameter acquisition unit 352 determines the values of two control parameters of the specimen thickness used by the slicing processing unit 52 and the reaction time used by the staining processing unit 54 among these control parameters. The acquisition target (hereinafter, these acquisition target control parameters are collectively referred to as “acquisition target control parameters”), and the value of each acquisition target control parameter is acquired in accordance with a user operation. For control parameters other than the acquisition target control parameter, the value is set in advance, for example, as a fixed value.

  The image extracting unit 353 extracts a corresponding standard sample image from the standard sample images stored in the standard sample image data storage unit 4 based on the sample specifying parameters acquired by the sample specifying parameter acquiring unit 351.

  The image display processing unit 354 is a standard image extracted by the image extraction unit 353 or a virtual image generated by performing virtual image generation processing on the standard sample image by the virtual image generation unit 371 of the image processing unit 37. A process of displaying the reference stained image on the display unit 32 is performed. The image display processing unit 354 performs virtual image generation processing on the reference staining image by the virtual image generation unit 371 and generates a staining simulation image that is a virtual image, and then generates the staining simulation image generated first. Is displayed on the display unit 32.

  The availability selection requesting unit 355 requests input of the selection of whether or not the value of the correction target control parameter is acceptable, and requests the adjustment of the staining density as a staining density adjustment requesting unit. In the first embodiment, the availability selection requesting unit 355 accepts the staining density adjustment operation by the user via the operation unit 31, and finally accepts the staining density confirmation operation, thereby accepting the value of the correction target control parameter. Accept selection input. The user performs a staining density determination operation when the staining density represented by the reference staining image or the staining simulation image is a desired staining density, and the value of the correction target control parameter is selected as “OK” by this determination operation. .

  The sample creation processing setting unit 356 notifies the sample creation unit 5 of the value of the control parameter acquired by the control parameter acquisition unit 352 or determined by the control parameter determination unit 372 of the image processing unit 37 and gives an operation instruction to each unit. Do. In response to this, the sample creation unit 5 performs a sample creation process using the control parameter acquired by the control parameter acquisition unit 352 and the value of the control parameter determined by the control parameter determination unit 372. As a result, a target specimen having a staining density desired by the user is obtained. In the first embodiment, the specimen thickness and the reaction time are acquired by the control parameter acquisition unit 352, and the reaction time is determined by the control parameter determination unit 372. Therefore, in the first embodiment, the specimen creation processing setting unit 356 notifies the slice processing unit 52 of the thickness of the specimen acquired by the control parameter acquisition unit 352, and is acquired and controlled by the control parameter acquisition unit 352. The reaction time determined by the parameter determination unit 372 is notified to the staining processing unit 54.

  The image processing unit 37 is realized by hardware such as a CPU. The image processing unit 37 includes a virtual image generation unit 371 and a control parameter determination unit 372.

  As a reference image generation unit, the virtual image generation unit 371 performs a virtual image generation process on the standard specimen image to generate a virtual image (reference stained image). In addition, the virtual image generation unit 371, as a simulation image generation unit, performs a virtual image generation process on the reference stained image to generate a virtual image (stained simulation image).

  The control parameter determination unit 372 performs display processing on the display unit 32 by the image display processing unit 354 when the acceptance / rejection selection request unit 355 accepts the confirmation operation of the staining concentration (that is, when the confirmation operation is accepted). The value of the correction target control parameter is determined in accordance with the staining density represented by the stained simulation image. When the staining density is not adjusted and the staining simulation image is not generated, the control parameter determination unit 372 stains the reference staining image whose display unit 32 displays the value of the correction target control parameter. The value is determined according to the concentration. The correction target control parameter includes at least one control parameter used in each process performed by each unit of the specimen creation unit 5. Here, if the correction target control parameter is a target to be acquired by the control parameter acquisition unit 352, correction is made by reflecting the determined staining density in the acquired control parameter value, and the value is determined. . At this time, if the staining density is not adjusted, the value of the correction target control parameter is determined as a value corresponding to the staining density of the reference staining image, that is, a value acquired by the control parameter acquisition unit 352. On the other hand, if the control parameter to be corrected is not an acquisition target by the control parameter acquisition unit 352 and the value is set as a fixed value in advance, the determined staining density is reflected in the value. To decide. If the staining density is not adjusted, it is determined as a fixed value that has been set.

  By the way, the staining density adjusted and determined by the user may be reflected in a plurality of control parameters. However, in this case, the calculation formula becomes complicated and the calculation time increases. In addition, since an error included in the relationship between the calculated value and the staining density generated when each value is calculated is amplified, there is a problem that the staining state of the obtained specimen is different from the staining state desired by the user. Here, examples of control parameters that greatly affect the staining state and can be easily set (calculated) include the reaction time used by the staining processing unit 54 and the concentration of the staining solution. In view of this, in the first embodiment, the control parameter determination unit 372 sets the reaction time as the correction target control parameter. Then, the determined staining density is corrected by reflecting it in the reaction time acquired by the control parameter acquisition unit 352. Note that the concentration of the staining liquid may be used as the correction target control parameter, and the determined staining concentration may be reflected. Further, the control parameter to be corrected is not limited to this, and can be appropriately selected according to the specimen diagnosis method. A plurality of control parameters may be combined as appropriate to be corrected.

  The standard specimen image data storage unit 4 stores image data of a standard specimen image obtained by imaging a standard specimen. In the first embodiment, standard specimens are prepared for all types of specimens determined by combinations of specimen specifying parameter values, and the prepared specimens are imaged to generate observation images, whereby the standard specimen image data storage unit 4 is prepared. The image data of at least one standard specimen image is stored for each specimen type. The standard specimen image data storage unit 4 is realized by, for example, a database device connected to the control device 3 via a network, and is installed in a separate place away from the control device 3 to store image data of the standard specimen image. ·to manage. In addition, it is not limited to the structure memorize | stored in a separate apparatus, It is good also as a structure memorize | stored in the memory | storage part 33 of the control apparatus 3. FIG.

  As described above, this standard specimen image is an observation image obtained by imaging a standard specimen prepared in advance. Here, a detailed method for generating the standard specimen image will be described. As described above, first, a sample corresponding to each combination of values that can be taken by the sample specifying parameter is prepared. Moreover, the value (standard value) of the control parameter used when creating the standard specimen is set in advance. This standard value may be set individually for each combination of sample specifying parameter values. Here, the control parameter in which the standard value is set includes at least a correction target control parameter (reaction time in the first embodiment). As a specific example, the standard value for the sample thickness used by the slicing processing unit 52 is set in advance, for example, 4 [μm], and the standard value for the reaction time used by the staining processing unit 54, for example, 1 minute. Keep it.

  Then, for each control parameter for which a standard value is set, the value is used, and for each of the other control parameters, each specimen prepared using a fixed value set in advance is processed to create a standard sample. Then, an observation image generation process is performed for each obtained standard specimen, and the generated observation image is obtained as a standard specimen image. Further, a staining density calculation process is performed on each standard specimen image to calculate the staining density (dye amount) of the standard specimen. Then, the standard specimen image generated as described above is associated with the specimen specifying parameters for the standard specimen, the control parameter values used when creating the standard specimen, the calculated staining density of the standard specimen, etc. And stored in the standard specimen image data storage unit 4.

  FIG. 6 is a flowchart illustrating a processing procedure performed by the control device 3 according to the first embodiment. Note that the processing described here is realized by the operation of each unit of the control device 3 in accordance with the specimen creation processing program 331 stored in the storage unit 33.

  First, the sample specifying parameter acquisition unit 351 acquires the value of the sample specifying parameter according to the user operation (step c1). For example, the sample specifying parameter acquisition unit 351 performs processing for displaying a sample specifying parameter input screen on the display unit 32 and notifying the input of the sample specifying parameter, and accepting input of the sample specifying parameter on the sample specifying parameter input screen. .

  7 and 8 are diagrams illustrating an example of the specimen specifying parameter input screen. In this specimen specifying parameter input screen, as shown in FIG. 7, spin boxes B11 to B13 for inputting values of specimen specifying parameters such as organs, parts, and disease names, and user operations for these spin boxes are confirmed. An OK button BTN11, a cancel button BTN12 for canceling the operation, and the like are arranged.

  Each of the spin boxes B11 to B13 presents a list of values that can be set as corresponding sample characteristic parameters as options, and accepts a selection operation. For example, in the spin box B11 for inputting an organ, as shown in FIG. 8, a drop-down list L11 presenting a list of organs from which specimens such as liver, kidney, prostate, and lung can be collected as options is displayed. Thus, the user selects a corresponding organ for the target specimen from the drop-down list L11. Note that the method for acquiring the sample characteristic parameter is not particularly limited, and for example, the configuration may be such that the value of each sample characteristic parameter is directly input. 7 and 8 exemplify the case where the values of each sample specifying parameter are individually input. However, combinations of the values of sample specifying parameters are presented as options, and the values of each sample specifying parameter are selected by selecting this option. It is good also as a structure acquired simultaneously.

  Subsequently, the control parameter acquisition unit 352 acquires the value of the acquisition target control parameter according to the user operation (step c3). For example, the control parameter acquisition unit 352 performs a process of displaying a control parameter input screen on the display unit 32 to notify an input request for the acquisition target control parameter, and receives an input of the acquisition target control parameter on the control parameter input screen.

  FIG. 9 is a diagram illustrating an example of a control parameter input screen. In the control parameter input screen shown in FIG. 9, spin boxes B21 and B22 for inputting the control parameter values of the specimen thickness and reaction time to be acquired in the first embodiment, and the spin boxes for these spin boxes are displayed. An OK button BTN21 for confirming the user operation, a cancel button BTN22 for canceling the operation, and the like are arranged. Each spin box B21, B22 presents a list of values that can be set as the value of the corresponding control parameter as an option, and accepts a selection operation. For example, as in the case of the sample specifying parameter shown in FIG. Show the drop-down list and present your choices. Note that the control parameter acquisition method is not limited to the exemplified method, and for example, a configuration may be adopted in which the value of the acquisition target control parameter is directly input. In the first embodiment, the acquisition target control parameters are fixed to two types (specimen thickness and reaction time), but may be configured to be selectable by the user. That is, a configuration may be adopted in which a control parameter selection operation to be acquired from the control parameters used by each unit of the specimen creation unit 5 is received and the value of the control parameter selected by the user as the acquisition target is acquired.

  Subsequently, the virtual image generation unit 371 extracts a standard sample image corresponding to each value of the sample specifying parameter acquired in step c1 from the standard sample images stored in the standard sample image data storage unit 4, The control parameter value and the staining density associated with the extracted standard specimen image are read from the standard specimen image data storage unit 4 (step c5). For example, one standard sample image corresponding to the combination of each value of the sample specifying parameter acquired in step c1 is read from the standard sample image data storage unit 4.

  Then, the virtual image generation unit 371 performs virtual image generation processing on the standard specimen image, and changes the staining density of the standard specimen image to the staining density according to the value of the acquisition target control parameter acquired in step c3. Is generated as a reference stained image (step c7). For example, in step c3, it is assumed that the values of these acquisition target control parameters are acquired by setting the sample thickness to 4 [μm] and the reaction time to 2 minutes. Further, it is assumed that the value of each control parameter is read from the standard sample image data storage unit 4 with the sample thickness being 4 [μm] and the reaction time being 1 minute. In this example, with respect to the thickness of the sample, the value input by the user matches the value related to the standard sample image. On the other hand, for the reaction time, the user inputs a double value (2 minutes) with respect to the value related to the standard specimen image (1 minute). In this case, the adjustment coefficient is calculated based on a preset relationship between the reaction time and the staining concentration. As the relationship between the reaction time and the staining concentration, for example, a lookup table described later with reference to FIG. 13 can be used. For example, if the value of 1.2 times the staining concentration when the reaction time is 1 minute is set in this lookup table as the staining concentration when the reaction time is 2 minutes, the adjustment coefficient is “ 1.2 "is set, and processing for generating a virtual image from the standard specimen image (virtual image generation processing) is performed. Actually, the relationship between the thickness of the specimen and the staining density is also set separately. Then, an adjustment coefficient is set based on each value of the acquisition target control parameter acquired in step c3. If control parameter values other than sample thickness and reaction time are acquired as acquisition targets, an adjustment coefficient is set taking these values into consideration as appropriate, and a virtual image is generated from the standard sample image. As good as

  Then, the image display processing unit 354 performs processing for displaying the reference stained image generated in step c7 on the display unit 32 (step c9). If each value of the acquisition target control parameter acquired in step c3 matches the value associated with the standard specimen image extracted in step c5, the staining density of the standard specimen image is acquired in step c3. This corresponds to the staining density corresponding to the value of the acquisition target control parameter. For this reason, it is not necessary to perform the process of step c7, and the image display processing unit 354 performs a display process on the display unit 32 using the standard specimen image as a reference stained image. This processing prompts the user to check the staining density of the target specimen. When the user determines that adjustment is necessary, the user performs an operation for adjusting the staining density, and finally performs an operation for determining the staining density.

  Then, as shown in FIG. 6, the availability selection requesting unit 355 monitors the presence / absence of a staining density determination operation, and accepts an input of whether the correction target control parameter value is permitted or not by this determination operation. Further, the availability selection requesting unit 355 monitors the presence / absence of an operation for adjusting the staining density until the staining density is determined (step c11: No). In the first embodiment, an adjustment coefficient changing operation for adjusting the dye density is accepted as the dye density adjusting operation. When the staining density is adjusted (when the adjustment coefficient is changed) (step c13: Yes), the virtual image generation unit 371 performs virtual image generation processing according to the changed adjustment coefficient, and the staining density of the reference dyed image Is generated as a staining simulation image (step c15). Then, the image display processing unit 354 displays the staining simulation image generated in step c15 on the display unit 32 (step c17). Thereafter, the process returns to step c11.

  Here, an example of an operation performed by the user in the processing of Step c11 to Step c17 will be described. For example, the image display processing unit 354 performs processing for displaying a staining density confirmation screen including display of the reference staining image on the display unit 32 as processing of step c9. FIG. 10 is a diagram illustrating an example of a staining density confirmation screen. As shown in FIG. 10, the staining density confirmation screen includes an image display unit W31 that displays the reference staining image and the staining simulation image side by side, and the reference staining image I31 is displayed on the left side toward the image display unit W31. The dyeing simulation image I32 is displayed on the right side. Further, on this staining density confirmation screen, a staining density adjustment button BTN31 for inputting a staining density adjustment operation and a staining density confirmation button BTN32 for inputting a staining density confirmation operation are arranged.

  In this way, if the reference staining image and the staining simulation image are displayed side by side, the user can visually grasp the change in the staining density accompanying the change in the adjustment coefficient on the screen. Here, the dyeing simulation image is generated when the dyeing density adjustment button BTN31 is pressed and the adjustment coefficient is changed. While the dyeing density adjustment button BTN31 is not pressed, the image display unit W31 has a reference. Only the stained image I31 is displayed. It is not necessary to display them side by side. The reference dyed image is displayed until the dyeing simulation image is generated, and after the dyeing simulation image is generated, the earliest generated dyeing simulation image is displayed. It is good also as a screen structure to do. In addition, it may be configured to perform enlargement processing or reduction processing of the reference dyed image or the dyeing simulation image of the image display unit W31 according to user operation, or processing for moving the center position, and the operability by the user can be improved. Moreover, it is good also as a structure which calculates an image feature-value by image-processing a reference | standard dyeing image and a dyeing | staining simulation image, and can show more image information to a user. For example, the color component of the pixel designated by the user or the statistic of the color component may be calculated and displayed on the screen. Alternatively, a specific area in the image may be extracted by a known recognition process or classification process and displayed on the screen.

  When the staining density adjustment button BTN31 is pressed on this staining density confirmation screen, the availability selection requesting unit 355 displays, for example, an adjustment coefficient change screen on the display unit 32, and performs a process of notifying a request for adjusting the staining density by changing the adjustment coefficient. Do. Then, the availability selection requesting unit 355 accepts an adjustment coefficient change operation (staining density adjustment operation) on the adjustment coefficient change screen as the process of step c13. FIG. 11 is a diagram illustrating an example of the adjustment coefficient change screen. As shown in FIG. 11, two sliders S41 and S42 for individually changing the adjustment coefficient for the dye H and the adjustment coefficient for the dye E are arranged on the adjustment coefficient change screen. Further, on this adjustment coefficient change screen, an OK button BTN41 for confirming a user operation on the sliders S41 and S42, a cancel button BTN42 for canceling the operation, and the like are arranged. The user moves the upper slider S41 to change the desired adjustment coefficient for the dye H, and moves the lower slider S42 to change the desired adjustment coefficient for the dye E. For example, if it is desired to increase the staining density, the sliders S41 and S42 are moved to the right side toward the screen, and if it is desired to be decreased, the sliders S41 and S42 are moved to the left side. According to this, the user can change the adjustment coefficient easily and intuitively. As a result, the virtual image generation unit 371 generates a staining simulation image with a staining density corresponding to the changed adjustment coefficient as the processing of step c15, and the image display processing unit 354 converts the staining simulation image into the processing of step c17. Display processing is performed on the image display portion W31 of the staining density confirmation screen.

  As described above, the user appropriately adjusts the staining density while viewing the reference staining image and the staining simulation image, and if the reference staining image or the staining simulation of the image display unit W31 is a desired staining density, the staining density confirmation button BTN32 is used. Press.

  Note that, as described above as the observation image generation process and the virtual image generation process, in the first embodiment, the pixel signal value at each pixel position is calculated to generate the observation image and the virtual image. For this reason, the image information presented to the user on the staining density confirmation screen is a pixel signal value, but is not limited thereto. For example, the color component of any pixel, the statistic of the color component, the texture information of a specific area, or image information obtained by combining these appropriately may be displayed. According to this, it is possible to present more detailed information regarding dyeing of the target specimen to the user.

  In addition, the method for obtaining the staining density is not limited to this, and for example, the adjustment coefficient value or the staining density value may be directly input. It is also possible to adopt a configuration in which the staining density is adjusted for a predetermined region of interest in which a specific tissue in the reference staining image is reflected. FIG. 12 is a diagram showing an example of the staining density confirmation screen in this case. In the staining density confirmation screen shown in FIG. 12, an image display unit W51 is arranged at the center of the screen, and the reference staining image or the most recently generated staining simulation image is displayed. In addition, a staining density adjustment button BTN51 for inputting a staining density adjustment operation and a staining density confirmation button BTN52 for inputting a staining density confirmation operation are arranged on the staining density confirmation screen.

  For example, if it is desired to adjust the staining density for the cytoplasm, the user selects the regions E51 and E52 in which the cytoplasm is displayed from the reference staining image or the staining simulation image displayed on the image display unit W51 via the operation unit 31. To do. The selected areas E51 and E52 are individually enlarged and displayed on the selection area display sections W521 and W522 outside the image display section W51. Then, the user presses the staining density adjustment button BTN51 with the region of interest selected in this way, and changes the adjustment coefficient in the manner described with reference to FIG. 11, for example.

  According to this modification, for example, the staining density can be adjusted for a predetermined region of interest such as cytoplasm, so that a specific tissue can be dyed deeply or lightly. Therefore, it is possible to adjust the staining density characteristically for a specific tissue while confirming the staining state of the entire specimen.

  Then, as shown in FIG. 6, when the staining concentration is determined (step c11: Yes), the control parameter determination unit 372 sets the determined staining concentration to the correction target control parameter (reaction time in the first embodiment). The value is reflected and corrected, and the value is determined (step c19).

  For example, a lookup table (hereinafter abbreviated as “LUT”) that defines the relationship between the reaction time and the staining concentration (correlation equation) is generated in advance and stored in the storage unit 33, and this LUT is referred to. Then, the value of the correction target control parameter is corrected. Here, a method of generating the LUT will be described. In the first embodiment, a sample actually prepared by changing the reaction time is prepared to obtain the staining concentration, and the correspondence between the reaction time and the staining concentration is set in advance. Here, even when the specimen is prepared with the same reaction time, the staining density varies slightly. In order to reduce this error, multiple samples prepared with the same reaction time are prepared. Then, an observation image generation process is performed on each of the obtained samples, and a staining density calculation process is performed to calculate a measurement value of the staining density and obtain a measurement value for each reaction time.

  FIG. 13 is a diagram schematically showing an example of the correspondence relationship between the reaction time and the staining density based on the measured value of the staining density for each reaction time, where the horizontal axis represents the reaction time and the vertical axis represents the staining density. The staining concentration of specimens prepared with different reaction times is shown. As described above, in this example, since a plurality of specimens prepared with the same reaction time are prepared, a plurality of measured values are obtained for each reaction time, and each value is indicated by a broken line in FIG. The values are distributed over the range. The variance for each reaction time indicated by the broken line is an error included in the relationship between the corresponding reaction time and the staining concentration. Note that an error indicated by a broken line in FIG. 13 may be presented to the user. As an example of the presentation method, the error range of the staining density for each reaction time is converted into the error range of the adjustment coefficient, and the error range of the converted adjustment coefficient is displayed on the adjustment coefficient change screen shown in FIG. The method presented in

  Specifically, the error range of the staining density for each reaction time is converted in advance to the error range of the adjustment coefficient. For example, at the reaction time 110 shown in FIG. 13, the staining density varies (disperses) in the range of 112 to 113 with the staining density 111 being the average value on the solid line as a reference. If attention is paid to the reaction time 110, an adjustment coefficient for each of the staining densities 111, 112, and 113 is obtained based on the staining density of the reference staining image, so that the error range 112 to 113 of the staining density in the reaction time 110 and its reference are obtained. 111 can be converted into the error range of the adjustment coefficient and its reference. Then, when the adjustment coefficient change screen of FIG. 11 is displayed, based on the current adjustment coefficient value indicated by the sliders 41 and S42, the error range of the adjustment coefficient whose reference value is most similar to the current adjustment coefficient value. To get. Then, the error range of the obtained adjustment coefficient is identified and displayed on the sliders 41 and S42 by changing the display color, for example. As will be described later, when the operation is confirmed on the adjustment coefficient change screen of FIG. 11, the reaction time that is the correction target control parameter is determined according to the adjustment coefficient at the time of the confirmation operation, and the target specimen is created according to this reaction time. According to this modification, the user can grasp in advance the variation in the staining density of the target specimen obtained in this way.

  In the first embodiment, for example, as shown by a solid line in FIG. 13, the average value of the measured values is calculated for each reaction time, and the staining concentration corresponding to each reaction time is obtained as one point with respect to the reaction time. Then, a plot distribution indicated by a solid line in FIG. 13 is tabulated to create an LUT and stored in the storage unit 33. The LUT is not limited to the case where it is stored as a data table that defines the correspondence between the reaction time and the staining concentration. For example, it may be stored as a function expression of an approximate curve calculated based on measurement points.

The control parameter determination unit 372 refers to the LUT generated in advance in this way, and individually calculates the reaction time for each of the dye H and dye E staining solutions. For example, among the determined staining concentrations, the staining concentration (dye amount) of dye _H is d _H and the staining concentration (dye amount) d _{E of} dye _E is used. Then, based on the value of d _H, it is calculated according to the following equation The reaction time S _H by hematoxylin staining solution (12). Further, based on the value of d _E, the reaction time S _E by the eosin staining solution is calculated according to the following equation (13).

Here, when the determined staining density value is a numerical value that is not set in the LUT, linear conversion is performed using the set value of the LUT that has the closest value to obtain the reaction time. Then, the control parameter determination unit 372 calculates the reaction time acquired by the control parameter acquisition unit 352 according to the equations (12) and (13), the reaction time S _H with the hematoxylin staining solution, and the reaction time S _{E with the} eosin staining solution. By replacing with, the determined staining density is reflected in the reaction time and corrected. The calculation of the reaction time corresponding to the staining concentration is not limited to the method performed by referring to the LUT according to the above formulas (12) and (13). For example, the reaction time may be calculated using a conversion function with the staining density as a variable.

  Then, as illustrated in FIG. 6, the sample creation processing setting unit 356 notifies the sample creation unit 5 of the value of the acquisition target control parameter acquired in step c3 and the value of the correction target control parameter determined in step c19. Then, an operation instruction is given to each part of the specimen preparation unit 5 (step c21). That is, in the first embodiment, the specimen creation processing setting unit 356 notifies the slice processing unit 52 of the thickness of the specimen acquired in step c3, and the reaction time determined in step c19. Notify

  FIG. 14 is a flowchart showing the processing procedure of the sample creation process performed by the sample creation unit 5 in response to the operation instruction in step c21 shown in FIG. By this sample creation process, a target sample is created using the value of the control parameter acquired in step c3 of FIG. 6 and determined in step c19.

  That is, as shown in FIG. 14, in the specimen preparation process of the first embodiment, first, the paraffin block creation processing unit 51 performs the paraffin block creation process (step d1 to step d9), and the paraffin block is obtained from the sample collected from the subject. Create

  Specifically, first, the paraffin block creation processing unit 51 cuts out a block from the collected specimen (step d1). Here, the block size to be cut out varies depending on the organ or part from which the specimen is collected (extracted), the disease name, the state of the disease, or the like. This block size is a control parameter used by the paraffin block creation processing unit 51, and is a fixed value in the first embodiment. For example, a block size is set in advance in association with a combination of an organ, a part, a disease name, and a disease state. Then, the block size of the target sample is specified based on the sample specifying parameter acquired in step c3 of FIG. The block size may be acquired by the control parameter acquisition unit 352, or the control parameter determination unit 372 may determine the block size. In this case, the process of step d1 is performed according to the acquired or determined value of the block size.

  The actual block size is generally about 1 [cm] × 2 [cm] × several [mm], but for example, 1 [cm] × several [cm] as in the case of preparing a brain specimen. × It may be cut out in a relatively large size such as several [cm]. In needle biopsy or the like, the process of collecting the specimen block from the specimen is unnecessary, and the process proceeds to the next step d3.

  In the subsequent step d3, the paraffin block creation processing unit 51 fixes the tissue to the cut specimen block using a formalin-based fixing solution. The type and fixing time of the formalin-based fixing solution are control parameters used by the paraffin block creation processing unit 51 and are fixed values in the first embodiment. Here, since the penetration rate of formalin varies depending on the block size cut out in step d1, the type of tissue to be fixed, and the type of formalin-based fixing solution to be used, the fixing time is determined from these values. Usually, fixing takes 1-2 days. If it is soaked for a long time, it will be in a state called over-fixation, and it may be difficult to dye with a dyeing solution in the subsequent dyeing process. In addition, it is good also as a structure which the control parameter acquisition part 352 acquires about the kind and fixing time of formalin type fixing solution, and it is good also as a structure which the control parameter determination part 372 determines. In this case, the process of step d3 is performed according to these acquired or determined values.

  Subsequently, the paraffin block creation processing unit 51 degreases and dehydrates using alcohol (step d5). This is because the living tissue contains fat. This treatment takes 2 to 3 days, for example, while increasing the concentration of alcohol stepwise (for example, in three steps). The alcohol solution to be used, the alcohol concentration, and the degreasing / dehydrating time are control parameters used by the paraffin block creation processing unit 51 and are fixed values in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d5 is performed according to these acquired or determined values.

  Subsequently, the paraffin block creation processing unit 51 penetrates the melted paraffin into the tissue and hardens it (dehydration embedding: step d7). This process takes two to three days. Specifically, the tissue is sequentially immersed in each solution of anhydrous alcohol, methanol + chloroform, xylene, and paraffin. Anhydrous alcohol is used for finishing dehydration and xylene is used for dealcoholization. Each solution and the permeation time for each solution are control parameters used by the paraffin block creation processing unit 51 and are fixed values in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d7 is performed according to these acquired or determined values. For this dehydration embedding, automated devices are widely used and can be used as appropriate.

  And the paraffin block creation process part 51 completely covers the sample block which processed the process of step d1-step d7 by paraffin by being submerged in the melted paraffin, and further cooling and solidifying (paraffin embedding: step d9). The permeation time is a control parameter used by the paraffin block creation processing unit 51, and is a fixed value in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d9 is performed according to the acquired or determined value. By embedding the paraffin, the strength of the paraffin is given to the specimen block which is originally composed of soft biological tissue, and the necessary degree of hardness can be realized at the time of slicing. Moreover, since the paraffin-embedded specimen block can be hermetically sealed under ideal conditions, it can be stored semipermanently. However, since it is not actually a completely confidential state, a state change occurs depending on the airtight state. In addition, when it is necessary to create a specimen in a short time, such as when observation or diagnosis is performed during surgery, the shape is fixed by freezing the tissue, and the process proceeds to the subsequent slicing process. May be.

  Subsequently, the slice processing unit 52 performs slice processing (step d11). The slicing processing unit 52 is configured by, for example, an automated device (automatic slicing device) in which slicing processing is automated. Here, the predetermined thickness (specimen thickness) is a control parameter used by the slice processing unit 52, and in the first embodiment, the control parameter acquisition unit 352 acquires in accordance with a user operation, and the sample creation processing setting unit 356 is obtained by notifying the slice processing unit 52. Note that the control parameter adjustment unit 39 may adjust. The automatic slicing apparatus uses the notified specimen thickness as a control parameter, for example, a microtome, and slices the specimen block thinly to a predetermined thickness. The sliced slice is floated on water and then sprinkled on a slide. The slices scooped up in the preparation are dried at about 42 ° C to 43 ° C. This temperature is set so as not to make wrinkles or wrinkles in the section.

  Subsequently, the deparaffinization processing unit 53 flushes the paraffin (deparaffinization: step d13). Since the paraffin is unnecessary after the slicing process is completed, it is removed so as not to disturb the dyeing. First, paraffin is melted at a temperature of about 60 ° C. If the preparation is set up, most of the paraffin will flow down naturally. The processing here usually takes about one hour, but a dedicated automation device is known and can be used as appropriate. Subsequently, the remaining paraffin is dissolved using xylene as a solvent. Since xylene is contaminated by dissolving paraffin, for example, three xylene pools of different purity are prepared, and the xylene pool having low purity is sequentially immersed for about 5 minutes. The permeation time is a control parameter used by the deparaffinization processing unit 53 and is a fixed value in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d13 is performed according to the acquired or determined value.

  Subsequently, the deparaffinization processing unit 53 rinses off xylene using alcohol (pure ethanol) (dexylene: step d15). As in the case of deparaffinization, alcohol is contaminated by dissolving xylene. Therefore, for example, three tanks with different purity are prepared, and the alcohol pool with low purity is sequentially immersed for about 5 minutes. The permeation time is a control parameter used by the deparaffinization processing unit 53 and is a fixed value in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d15 is performed according to the acquired or determined value.

  Subsequently, the deparaffinization processing unit 53 flushes the alcohol (dealcoholization: step d17). Usually, tap water is used for water washing, and distilled water is sprinkled before being put into other dyeing solutions or solvents. In some cases, such as when there is a concern that a sudden change from alcohol to water may adversely affect the specimen, there may be cases where the alcohol is once washed through a concentration of about 70%.

  Subsequently, the dyeing processing unit 54 performs dyeing processing (step d19 to step d27) and performs H & E dyeing. First, the staining processing unit 54 performs hematoxylin staining of the specimen (step d19). In hematoxylin staining, a specimen is washed with distilled water and then immersed in a hematoxylin staining solution for a predetermined time (reaction time). Here, the concentration of the staining solution and the reaction time are control parameters used by the staining processing unit 54. In the first embodiment, the concentration of the staining solution is a fixed value. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d19 is performed according to the acquired or determined value. On the other hand, the reaction time is a value acquired by the control parameter acquisition unit 352 according to the user operation and corrected by the control parameter determination unit 372 according to the user operation, and the hematoxylin staining notified to the staining processing unit 54 by the specimen creation processing setting unit 356 Reaction time by liquid.

  Subsequently, the staining processing unit 54 performs water washing for coloration on the specimen stained with hematoxylin (step d21). Usually, it is immersed in 1% hydrochloric acid alcohol to shorten the time. By this treatment, excess hematoxylin staining solution adhering to the cytoplasm or the like is removed. When using tap water, care should be taken because the color of hematoxylin will be lost by the chlorine contained in the tap water if it is soaked in tap water for a long time. Since the dyeing is once stable, it can be left for a long time in distilled water. Here, the solution concentration and the washing time of hydrochloric acid alcohol are control parameters used by the dyeing processing unit 54, and are fixed values in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d21 is performed according to these acquired or determined values.

  After washing with distilled water, the staining processing unit 54 subsequently performs eosin staining (step d23). Here, the specimen is immersed in eosin staining solution. Here, the concentration of the staining solution and the reaction time are control parameters used by the staining processing unit 54. In the first embodiment, the concentration of the staining solution is a fixed value. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d23 is performed according to the acquired or determined value. On the other hand, the reaction time is a value acquired by the control parameter acquisition unit 352 according to the user operation and corrected by the control parameter determination unit 372 according to the user operation, similar to the reaction time used in the hematoxylin staining in step d19, and the sample creation processing setting This is the reaction time by the eosin staining solution notified to the staining processing unit 54 by the unit 356.

  Subsequently, the staining processor 54 performs a fractional dehydration process on the eosin-stained specimen (step d25). Usually, the specimen immersed in the staining liquid is in a state containing the staining liquid as a whole. However, after that, by washing with an appropriate solvent (cleaning liquid), the dyeing liquid other than the part to be originally dyed is washed away so that only the target part is dyed. This process is called classification. For fractionation of eosin, alcohol (pure ethanol) is usually used as a cleaning solution. Since the water adhering during dyeing is also removed by washing with alcohol, the separation step in this case also serves as a dehydration step. If the sample is left in alcohol, eosin dissolves, so the process of shaking 10 times in alcohol is performed about 4 times to complete the separation. Here, the type of cleaning liquid and the cleaning time are control parameters used by the dyeing processing unit 54 and are fixed values in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d25 is performed according to these acquired or determined values.

  If the eosin is left in the state where the separation is completed, the eosin will fall off due to the alcohol remaining or adhered to the specimen, so the staining processing unit 54 immediately performs the clearing (step d27). Transparent is a process of immersing the specimen in xylene, and is performed about 3 times with a penetration time of 5 minutes, for example. This process is the same process as dealcoholization, but has the effect of improving the color development of the specimen, and is particularly called clearing. The penetration time is a control parameter used by the staining processing unit 54 and is a fixed value in the first embodiment. However, the control parameter acquisition unit 352 may acquire the configuration, or the control parameter determination unit 372 may determine the configuration. In this case, the process of step d27 is performed according to the acquired or determined value.

  Subsequently, the encapsulation processing unit 55 performs a specimen encapsulation process to obtain a specimen (target specimen) (step d29). That is, a cover glass is placed on a slide and fixed with an adhesive. Encapsulation and embedding is automated by a dedicated device and can be used as appropriate. Since it is completely sealed by the adhesive, the obtained specimen can be stored semipermanently.

  As described above, according to the first embodiment, the value of the acquisition target control parameter used when creating the target specimen is acquired according to the user operation, and the reference staining with the staining density (dye amount) corresponding to the acquired value is obtained. The image can be displayed on the screen and presented to the user. Further, it is possible to generate a dyeing simulation image having a dyeing density adjusted by the user while viewing the reference dyeing image, display it on the screen, and present it to the user. Then, the value of the correction target control parameter can be corrected and determined according to the finally determined staining density, and the operation can be instructed by notifying the specimen creation unit 5. As a result, the specimen creation unit 5 uses the value of the acquisition target control parameter acquired according to the user operation or the value of the correction target control parameter corrected and determined so that the dyeing of the specimen becomes the staining density determined according to the user operation. Create a target specimen. According to this, the user can grasp in advance the dyeing of the specimen, and can determine whether or not the value of the control parameter used when creating the specimen before the specimen creating section 5 creates the specimen. Therefore, it is possible to provide a specimen preparation device that can stably prepare specimens under conditions desired by the user.

  In the first embodiment, a standard specimen prepared with a control parameter as a predetermined value for each combination of specimen specifying parameter values is prepared in advance, and a standard specimen image obtained by imaging the standard specimen is stored in the standard specimen image data storage unit 4. I decided to remember it. Then, a virtual image generation process is performed on the standard specimen image, and a reference stained image corresponding to the value of the acquisition target control parameter acquired according to the user operation is generated. On the other hand, assuming that every value that can be acquired as the value of the acquisition target control parameter is assumed, it is possible to prepare a standard sample created by varying the value of each acquisition target control parameter. The stained specimen image obtained by imaging the prepared standard specimen may be stored in the standard specimen image data storage unit 4 in association with each value of the specimen specifying parameter and the control parameter.

  In this modification, when each value of the specimen specifying parameter and the acquisition target control parameter is acquired according to the user operation, a standard specimen image is extracted based on the acquired values, and the extracted standard specimen image is used as a reference staining image. Display on the screen. In this case, it is necessary to prepare and store a standard sample image for each combination of the sample specifying parameter and the acquisition target control parameter value. However, as in the first embodiment, the virtual image generation process is performed each time. There is no need to go and generate a reference stained image.

  When a plurality of control parameter values are acquired as acquisition target control parameters, a plurality of standard specimen images that match any one of the values may be extracted. For example, as described in the first embodiment, if each control parameter of reaction time and specimen thickness is an acquisition target, the standard specimen image and specimen thickness associated with the value obtained as the reaction time are obtained. And the standard specimen image associated with the value acquired as.

  In addition, when a plurality of standard specimen images are extracted in this way, one reference stained image is finally selected from these. The selection method may be performed manually in accordance with a user operation, or one image may be automatically selected.

  In the case of performing manually, for example, a plurality of extracted standard specimen images are displayed on the screen as candidates for reference stained images. At this time, a plurality of extracted standard specimen images may be displayed as a list in a thumbnail format, or may be configured to be switched and displayed one by one in accordance with a user operation. Further, at this time, virtual image generation processing may be performed on each extracted standard specimen image, and the image may be displayed as an image changed to a staining density corresponding to the acquired value of each control parameter. For example, if the reaction time and specimen thickness are used as the acquisition target control parameters, the staining density of the standard specimen image extracted based on the obtained reaction time is changed in consideration of the obtained specimen thickness. A virtual image is generated. Similarly, for a standard sample image extracted based on the thickness of the sample, a virtual image is generated in which the staining density is changed in consideration of the acquired reaction time. Then, these virtual images may be displayed on the screen as candidates for the reference stained image and presented to the user. Finally, one reference stained image is selected according to the user operation.

  On the other hand, as a method of automatically selecting one reference stained image, for example, using one piece of reference stained image using feature information such as the type of tissue shown in the extracted standard specimen image and its state as a discrimination reference. A method of selecting is listed. In this case, for example, a plurality of specimens having different numbers of tissues and different densities are prepared in advance as standard specimens. Then, in association with the standard specimen image obtained by imaging the prepared standard specimen, the standard specimen image data storage section 4 indicates the type of tissue shown in the corresponding standard specimen image, or the state of the tissue such as the number and density thereof. Remember it.

  Then, from the plurality of standard specimen images extracted as candidates for the reference staining image, one standard specimen image associated with the characteristic information corresponding to the value of the acquired specimen specifying parameter is selected as the reference staining image. For example, if “cancer” is acquired as the disease name that is one of the specimen specifying parameters, whether or not the density of the tissue is equal to or higher than a predetermined threshold can be used as a determination criterion.

  Alternatively, the user (clinician) may set the observation conditions for the target specimen in advance. In this case, the type and number of tissues to be focused on, the degree of congestion, the presence or absence of specific tissues, and the like are set as observation conditions. For example, if the user wants to observe dense cell nuclei and places importance on dyeing of dense cell nuclei, “cell nuclei are dense” is set as an observation condition. In this case, according to the set observation conditions, the density of cell nuclei associated with the extracted standard specimen image as feature information is thresholded, and one reference stained image is automatically selected. Here, threshold processing using a predetermined discrimination criterion can be realized using a known technique. The threshold value may be configured to be set according to a user operation.

(Embodiment 2)
Next, a second embodiment will be described. FIG. 15 is a block diagram illustrating a functional configuration of the specimen creation device 2b according to the second embodiment. In FIG. 15, the same components as those of the specimen preparation device 2 described in the first embodiment are denoted by the same reference numerals.

  As illustrated in FIG. 15, the specimen creation device 2 b according to the second embodiment includes a control device 3 b, a standard specimen image data storage unit 4, and a specimen creation unit 5. The control device 3b includes an operation unit 31, a display unit 32, a storage unit 33b, a control unit 35b, and an image processing unit 37b.

  The storage unit 33b stores a specimen creation processing program 331b. This sample creation processing program 331b is a program for realizing a process of determining the value of the correction target control parameter in accordance with a user operation and instructing the sample creation unit 5 to perform an operation.

  In addition, the control unit 35b includes a sample specifying parameter acquisition unit 351, a control parameter acquisition unit 352, an image extraction unit 353, an image display processing unit 354, an availability selection request unit 355b, and a sample creation processing setting unit 356b. Including.

  In the second embodiment, as in the first embodiment, the control parameter acquisition unit 352 acquires two control parameters of the specimen thickness used by the slicing processing unit 52 and the reaction time used by the staining processing unit 54. It is assumed that the value of each acquisition target control parameter is acquired according to the user operation as a parameter.

  The availability selection requesting unit 355b requests input of whether or not the correction target control parameter value is selected, and requests the adjustment of the correction target control parameter value as a control parameter adjustment requesting unit. In the second embodiment, the availability selection requesting unit 355b receives the adjustment operation of the correction target control parameter value by the user via the operation unit 31, and finally receives the determination operation of the correction target control parameter value. The selection input of whether or not the value is possible is accepted. The user confirms the value of the correction target control parameter when the staining density represented by the reference staining image or the simulation simulation image is the desired staining density. To select. As a result, the value of the correction target control parameter is determined.

  The sample creation processing setting unit 356b notifies the sample creation unit 5 of the value of the correction target control parameter acquired by the control parameter acquisition unit 352 or determined by the acceptability selection request unit 355b accepting the confirmation operation. Give instructions. In the second embodiment, the specimen creation processing setting unit 356b notifies the slice processing unit 52 of the thickness of the specimen acquired by the control parameter acquisition unit 352. The specimen creation processing setting unit 356b notifies the staining processing unit 54 of the reaction time determined as the value adjusted by the user.

  The image processing unit 37b includes a virtual image generation unit 371b and a staining density calculation unit 372b.

  As in the first embodiment, the virtual image generation unit 371b performs a virtual image generation process on the standard specimen image to generate a virtual image (reference stained image) as a reference image generation unit. Further, the virtual image generation unit 371b, as a simulation image generation unit, performs a virtual image generation process on the reference stained image to generate a virtual image (stained simulation image). Specifically, the staining simulation image is generated by changing the staining density represented by the reference staining image to the staining density corresponding to the value of the correction target control parameter adjusted in response to the input request from the availability selection requesting unit 355b.

  The staining density calculation unit 372b calculates the staining density according to the adjusted correction target control parameter value when the availability selection requesting unit 355b receives an adjustment operation for the correction target control parameter value.

  FIG. 16 is a flowchart illustrating a processing procedure performed by the control device 3b according to the second embodiment. Note that the processing described here is realized by the operation of each unit of the control device 3b according to the specimen creation processing program 331b stored in the storage unit 33b. In FIG. 16, the same reference numerals are assigned to the same processing steps as those in the first embodiment.

  In the second embodiment, after the image display processing unit 354 performs the process of displaying the reference stained image on the display unit 32 in step c9, the availability selection requesting unit 355b determines whether or not the correction target control parameter value is confirmed. And accepting an input for selecting whether or not the value of the correction target control parameter is acceptable. Further, the availability selection requesting unit 355b determines whether or not there is an adjustment operation for the value of the correction target control parameter (reaction time in the second embodiment) until the value of the correction target control parameter is determined (step e11: No). Monitor. For example, as in the first embodiment, a GUI such as a slider is displayed on the screen and a reaction time adjustment operation is accepted. Note that the user may directly input the reaction time. When the value is adjusted (step e13: Yes), the staining density calculation unit 372b calculates the staining density according to the changed value (step e15).

For example, an LUT that defines the relationship between the reaction time and the staining concentration (correlation equation) is generated in advance and stored in the storage unit 33b, and the staining concentration is calculated with reference to this LUT. As the LUT, the LUT described with reference to FIG. 13 in the first embodiment can be used. Specifically, the staining concentration calculation unit 372b refers to the LUT and calculates the staining concentrations of the pigment H and the pigment E individually. First, the reaction time S _E by reaction time S _H and eosin staining solution by the respective hematoxylin staining solution modified reaction times. Then, according to the following equation (12) shown in the first embodiment, the values of reaction time S _H by hematoxylin staining solution to calculate the dyeing concentration d _H of the original dye H, according to the following equation (13), eosin Based on the value of the reaction time S _E with the staining solution, the staining concentration d _E of the dye _E is calculated.

  As in the first embodiment, when the reaction time after change is a numerical value that is not set in the LUT, linear conversion is performed using the set value of the LUT having the closest value to obtain a staining density. The calculation of the staining concentration corresponding to the reaction time is not limited to the method of referring to the LUT according to the above formulas (12) and (13), but using a conversion function with the reaction time as a variable, It is good also as a structure which calculates a density | concentration.

  Then, the virtual image generation unit 371b performs virtual image generation processing based on the calculated staining density, and virtually changes the staining density of the reference staining image to the staining density corresponding to the adjusted correction target control parameter value. The generated virtual image is generated as a staining simulation image (step e17). Then, the image display processing unit 354 displays the staining simulation image generated in step e17 on the display unit 32 (step e19). Thereafter, the process returns to step e11.

  Then, when the value of the correction target control parameter is confirmed (step e11: Yes), the value of the correction target control parameter (reaction time in the first embodiment) is determined as the confirmed value (step e21). Subsequently, the sample creation processing setting unit 356b notifies the sample creation unit 5 of the value of the acquisition target control parameter acquired in step c3 and the value of the correction target control parameter determined in step e21. An operation instruction is given to each unit (step e23). That is, in the second embodiment, the specimen creation processing setting unit 356b notifies the slice processing unit 52 of the thickness of the specimen acquired in step c3, and the reaction time determined in step e21 is the staining processing unit 54. Notify

  Depending on the user, it may be easier to imagine the dyeing of the specimen by adjusting the values of control parameters such as reaction time, rather than adjusting the staining density as in the first embodiment. According to the second embodiment, the value of the acquisition target control parameter used when creating the target specimen is acquired according to the user operation, and the reference staining image of the staining density (dye amount) corresponding to the acquired value is displayed on the screen. It can be presented to the user. Further, it is possible to generate a dyeing simulation image having a dyeing density corresponding to the value of the correction target control parameter adjusted by the user while viewing the reference dyeing image, display it on the screen, and present it to the user. Then, it is possible to determine the value of the correction target control parameter as a finally determined value and notify the sample creation unit 5 to perform an operation instruction. As a result, the sample creation unit 5 creates a target sample using the value of the acquisition target control parameter acquired according to the user operation or the value of the correction target control parameter adjusted by the user while viewing the staining simulation image. According to this, the user can grasp in advance the dyeing of the specimen, and can determine whether or not the value of the control parameter used when creating the specimen before the specimen creating section 5 creates the specimen. Therefore, it is possible to provide a specimen preparation device that can stably prepare specimens under conditions desired by the user.

  It should be noted that whether to adjust the staining density as described in the first embodiment or the value of the correction target control parameter as described in the second embodiment can be selected in advance according to a user operation. Good.

(Embodiment 3)
In the first and second embodiments, the case where a specimen subjected to H & E staining known as general staining is created has been described. On the other hand, various other dyeing methods are known. As described above, these staining methods are roughly classified into general staining, special staining, and immunostaining, but the present invention can be applied to any specimen staining method.

  Examples of special staining include Azan staining, Alcian blue / PAS heavy staining, Alcian blue staining, Elastica-Wangeson staining (EVG staining), Oil Red O staining, Orsein staining, Giemsa staining, Grimerius staining, Kluber Barrera (KB) staining, Grocot staining, Kossa (Ca) reaction, Gomori aldehyde fuchsin staining, colloidal iron staining, Congo red (DFS 4BS) staining, Safranin O staining, Sudan III staining, Sudan black B staining, Direct First Scarlet staining , Thielensen staining, Toluidine blue (Tb) staining, naphthol ASD chloroacetate method, Nissl staining, Victoria blue staining, Foilgen staining, Berlin blue (Fe) staining, Bodian staining, Masson trichrome staining ( T staining), Masson Fontana staining, Muticalmine staining, Methyl green pyronin staining, Hematoxylin phosphotungstic acid (PTAH) staining, Lendrum inclusion body staining, Gram staining, Weigert staining, Wangyson staining, Watanabe silver (Ag) staining, A bleaching method, eosinophil staining (Luna staining), Kossa method, LFB staining, TRAP staining, PAM staining, PAS reaction and the like can be mentioned and can be applied to each staining method. The same applies to dyeing methods not listed here.

  Also in the case of applying to these special stains, as in the first and second embodiments, a standard specimen stained by the corresponding staining method is prepared, an observation image is generated, and stored as a standard specimen image. Then, each value of the sample specifying parameter and the control parameter is acquired for the target sample, a standard sample image is extracted based on the acquired value of the sample specifying parameter and the control parameter, and a reference stained image corresponding to each value is displayed on the screen. indicate. Thereafter, in the same manner as in the first embodiment, the staining simulation image is displayed on the screen by virtually changing the staining density of the reference staining image according to the staining density adjusted by the user while viewing the reference staining image. Then, the value of the correction target control parameter is corrected and determined according to the finally determined staining density, and a target specimen is created using the determined value. Alternatively, in the same manner as in the second embodiment, the staining simulation image is displayed on the screen by virtually changing the staining density of the reference staining image according to the value of the correction target control parameter adjusted by the user while viewing the reference staining image. Then, the value of the correction target control parameter is determined as a finally determined value, and a target sample is created using the determined value.

  Examples of the control parameters used as the acquisition target control parameter and the correction target control parameter include the generation conditions and reaction time of the staining liquid used in the applied staining method. Here, the generation conditions of the staining liquid are the configuration and concentration of the staining liquid. In addition, the control parameter used in each step of the specimen preparation process in the staining method can be appropriately set as a correction target control parameter. Here, also in the special staining, the protocol for the specimen preparation process in the staining method and the control parameters used in this protocol differ depending on the manufacturer of the apparatus to be adopted as in the case of the H & E staining exemplified as the general staining. May vary depending on the setting. The present invention may adopt any protocol, and the control parameters described in the protocol may be appropriately used as acquisition target control parameters or correction target control parameters.

  For example, among special dyes, simple polysaccharides (glycogen, cellulose, dextran), neutral or acidic by oxidizing the polysaccharide with periodic acid and reacting the resulting aldehyde group with a Schiff reagent to cause coloration. Periodate that dyes mucus (glycoprotein, so-called epithelial mucus), glycolipid, thyroid colloid, basement membrane, reticulofiber, shigella amoeba, fungus, lipofuscin, etc. from magenta to red and dyes collagen fibers and fibrin in pink In the case of acid Schiff reaction staining (hereinafter referred to as PAS staining), for example, periodate water production conditions, Schiff reagent production conditions, reaction time with periodate water, reaction time with Schiff reagent, and the like are listed as control parameters. . Further, for example, the conditions for generating the Schiff reagent include a combination of the composition material and the mixing amount of each composition material.

  In PAS staining, staining with periodic acid is performed, followed by staining with a Schiff reagent, and then staining of nuclei using a hematoxylin staining solution. More specifically, after staining with the Schiff reagent, a step of washing away the staining solution other than the target site using sulfite water is performed. For this reason, the nucleus is oxidized at this time. Here, since the hematoxylin staining is an oxidative staining, due to the effect of the treatment using sulfite water, in the subsequent hematoxylin staining, the nucleus is compared with the hematoxylin staining in the H & E staining described in the first and second embodiments. It becomes easy to be dyed. For this reason, when the present invention is applied to PAS staining and the reaction time by the hematoxylin staining solution is used as the control parameter to be corrected, for example, different from the LUT used in the H & E staining described in the first embodiment, An LUT defining the reaction time of the hematoxylin staining solution taking into account the reaction time of the Schiff reagent may be prepared and used.

  In addition, in the case of EVG staining, which is a staining method that combines Weigert staining, which is a method for staining elastic fibers, and Wangieson staining, which is a method for staining collagen fibers, the production conditions and reactions of resorcin fuchsin solution, iron hematoxylin and Wangyson solution, respectively. Time is listed as a control parameter.

  In addition, in the case of MT staining, which is a heavy staining using acidic dyes with different molecular weights, the generation conditions and reaction time of iron hematoxylin, ponso xylidine, acid fuchsin, azofuroxin, orange G aqueous solution and aniline blue are controlled parameters. As mentioned.

  Further, depending on the dyeing method, the dyed dye is insolubilized, so that a mordant treatment may be performed using a mordant before washing, and the mordant generation conditions and reaction time are also control parameters.

  On the other hand, as an example of immunostaining, for example, an enzyme antibody method includes a direct method and an indirect method. In addition, for indirect methods, the presence or absence of sensitization can be selected. As sensitization methods, PAP method, ABC method, streptavidin method, Tyramide signal amplification system (TSA system), LSAB method, high sensitivity polymer reagent method, enzyme antibody method And multiple dyeing. These immunostainings can also be applied as in the first and second embodiments. Here, in immunostaining, these reaction formats and types of sensitization methods in the enzyme antibody method are control parameters. In addition, it can apply similarly about the dyeing | staining method which is not mentioned here.

  In addition, examples of the control parameters used as the acquisition target control parameter and the correction target control parameter include the primary antibody and the secondary antibody used in the corresponding staining method, the generation conditions of the color reagent, the reaction time, and the like. The generation conditions include the composition and concentration of the chemical used. Also in immunostaining, the protocol for preparing a specimen in the staining method and the control parameters used in this protocol vary depending on the manufacturer of the apparatus to be employed, and may vary depending on the setting on the medical facility side. The present invention may adopt any protocol, and the control parameters described in the protocol may be appropriately used as acquisition target control parameters or correction target control parameters.

  FIG. 17 is a flowchart illustrating a processing procedure of a specimen creation process when immunostaining is applied. First, the antigen is activated to reveal the recognition site or sequence (epitope) of the antibody (step f1). As a method for activation, a method using a proteolytic enzyme, a method using a surfactant, and the like are known. Subsequently, blocking is performed by placing a solution obtained by dissolving a protein in a buffer solution on a section and placing the solution for a predetermined time (step f3). This blocking is performed to prevent the antibody from recognizing molecules other than the antigen.

  Subsequently, the primary antibody is reacted (step f5). Thereafter, after washing (step f7), the secondary antibody is reacted (step f9). Then, after washing (step f11), it is determined whether or not sensitization is performed. When sensitization is performed (step f13: Yes), the sensitization reaction is started (step f15). Here, for example, a sensitization reaction is appropriately performed according to the type of the secondary antibody reacted in step f9. Then, when the sensitization is not performed (step f13: No), or after the sensitization reaction of step f15, the process proceeds to a coloring process, and a substrate for the enzyme is added to cause color development (step f17).

  In the first and second embodiments, the reaction time is described in detail as the control parameter to be corrected. On the other hand, when a control parameter other than the reaction time is to be corrected, a correspondence relationship between the value of the control parameter and the staining density is set in advance, as in the first and second embodiments. realizable.

  For example, when the specimen thickness is used as the correction target control parameter, an LUT defining a relation (correlation formula) between the specimen thickness and the staining density may be generated in advance and stored in the storage unit 33. . That is, for example, a sample actually prepared by changing the thickness of the sample is prepared to obtain the staining concentration, and the correspondence between the sample thickness and the staining concentration is preset in the same manner as in the first embodiment. Keep it. At this time, even when the specimen is prepared with the same specimen thickness, there is some variation in the staining density. In order to reduce this error, a plurality of specimens prepared with the same specimen thickness are prepared. Then, an observation image generation process is performed for each obtained specimen, and a staining density calculation process is performed to calculate a measurement value of the staining density to obtain a measurement value (estimation) for each specimen thickness.

  FIG. 18 is a diagram schematically showing an example of a correspondence relationship between the thickness of the specimen and the staining density based on the measured value of the staining density for each thickness of the specimen. The horizontal axis represents the thickness of the specimen and the vertical axis. The axis shows the staining density, and shows the staining density of the specimens prepared with different specimen thicknesses. Also in this case, a plurality of measurement values are obtained for each thickness of the specimen as in the first embodiment, and the values are dispersed in a range as indicated by a broken line in FIG. 18, for example. Therefore, for example, as in the first embodiment, as shown by the solid line in FIG. 18, the average value of the measured values is calculated for each thickness of the sample, and the staining concentration corresponding to the thickness of each sample is calculated. Obtained as one point for thickness. Then, a plot distribution indicated by a solid line in FIG. 18 is tabulated to create an LUT and stored in the storage unit 33 or the storage unit 33b.

Then, the control parameter determination unit 372 calculates the thickness of the sample with reference to the LUT generated in advance in this way. For example, among the determined staining densities, the staining density (dye amount) of the dye _E is defined as dE. Based on the value of d _E , the sample thickness T is calculated according to the following equation (14).

  Similarly, for example, if the concentration of the hematoxylin staining solution is used as the correction target control parameter, an LUT that defines a relationship (correlation formula) between the concentration of the hematoxylin staining solution and the staining concentration is generated in advance, and the storage unit 33 or What is necessary is just to memorize | store in the memory | storage part 33b. That is, for example, a sample prepared by actually changing the concentration of the hematoxylin staining solution is prepared to obtain the staining concentration, and the correspondence between the concentration of the hematoxylin staining solution and the staining concentration is obtained in the same manner as in the first embodiment. Set in advance.

  FIG. 19 is a diagram schematically showing an example of the correspondence between the concentration of the hematoxylin staining solution and the staining concentration based on the measured value of the staining concentration for each concentration of the hematoxylin staining solution, and the horizontal axis represents the hematoxylin staining solution. , The vertical axis represents the staining concentration, and the staining concentration of the sample prepared by varying the concentration of the hematoxylin staining solution is shown. Also in this case, a plurality of measured values are obtained for each concentration of the hematoxylin staining solution as in the first embodiment, and the values are dispersed within a range as indicated by a broken line in FIG. 19, for example. Therefore, as in the first embodiment, as shown by the solid line in FIG. 19, the average value of the measured values is calculated for each concentration of the hematoxylin staining solution, and the staining concentration corresponding to the concentration of each hematoxylin staining solution is calculated. Obtained as one point for the concentration of the staining solution. Then, a plot distribution indicated by a solid line in FIG. 19 is tabulated to create an LUT and stored in the storage unit 33 or the storage unit 33b.

Then, the control parameter determination unit 372 calculates the concentration of the hematoxylin staining solution with reference to the LUT generated in advance in this way. For example, among the determined staining concentrations, the staining concentration (dye amount) of the pigment _H is defined as dH. Based on the value of d _H , the concentration C _{H of the} hematoxylin staining solution is calculated according to the following equation (15).

  Although not shown, the block size used for the paraffin block creation processing by the paraffin block creation processing unit 51 (the block size used when a block is cut out from the collected specimen) and the fixation of formalin-based fixative used for fixing the tissue in the specimen. Similarly, when the time is set as the correction target control parameter, an LUT that defines the block size and the staining density or the relationship (correlation formula) between the fixed time and the staining density is generated in advance and stored in the storage unit 33 or the storage unit 33b Just remember.

  In the first and second embodiments described above, the case has been described in which the control parameter to be corrected is the reaction time and correction is performed by reflecting the staining density determined according to the user operation. On the other hand, the reaction time may be corrected in consideration of the thickness of the sample.

  In a general H & E staining step, the dye H stains the nucleus. On the other hand, the dye E stains almost the entire region of the tissue including the nucleus. Depending on the staining process, the dye H may stain the entire tissue. Here, when a specimen having a certain thickness is stained with the dye E, the staining density of the dye E when the specimen having a thickness twice that of the specimen is stained with the dye E is almost doubled. This is because the dye E stains the entire tissue (mainly cytoplasm). That is, when the thickness of the specimen is doubled, the volume of the cytoplasm mainly stained with the dye E in the tissue occupying the specimen is also doubled. On the other hand, when a specimen having a certain thickness is stained with the dye H, the staining concentration of the dye H when the specimen having a thickness twice that of the specimen is stained with the dye H hardly changes. This is because the dye H mainly stains nuclei, and normally, even if the thickness of the specimen is doubled, the possibility that another nucleus exists in the specimen is extremely low.

For this reason, the staining density of the specimen (specifically, the staining density of the dye E) varies depending on the thickness of the specimen. Therefore, the reaction time may be corrected (determined) in consideration of the thickness of the specimen. For example, in the case where the thickness of the specimen is an acquisition target as in the first and second embodiments described above, the reaction time is expressed by the following equations (16) and (17) in consideration of the thickness of the acquired specimen. It is good also as calculating according to. In this case, the control parameter determination unit 372 determines the relationship between the reaction time and the staining concentration (correlation equation) described in the first embodiment (corresponding equation), the LUT (for example, the LUT in FIG. 13), the specimen thickness, and the staining concentration. Referring to the LUT (for example, the LUT in FIG. 18) in which the relationship (correlation equation) is defined, the reaction time for each dye H and dye E staining solution is calculated individually. For example, among the determined staining concentrations, the staining concentration (dye amount) of dye _H is d _H and the staining concentration (dye amount) d _{E of} dye _E is used. Then, based on the value of d _H, it is calculated according to the following equation The reaction time S _H by hematoxylin staining solution (16). Further, based on the value of d _{E and} the value of the acquired sample thickness T, the reaction time S _E by the eosin staining solution is calculated according to the following equation (17).

The following describes calculation of reaction time S _E by eosin staining solution shown in Equation (17). As shown in the equation (17), in calculating the reaction time S _E using the eosin staining solution, first, the sample thickness is obtained according to the LUT (for example, the LUT in FIG. 18) that defines the relationship between the sample thickness and the staining concentration. The dyeing density is obtained for the value T. Once the value of the staining density (LUT (T)) corresponding to the specimen thickness T is obtained, the reaction time is then determined according to the LUT (eg, LUT in FIG. 13) that defines the relationship between the reaction time and the staining density. Get S _E. Specifically, at this time, the final staining concentration of the dye E becomes d _E by the staining concentration (LUT (T)) corresponding to the sample thickness T and the staining concentration corresponding to the reaction time S _E. The difference value d _E / LUT (T) is obtained. Then, according to the LUT defining the relationship between the reaction time and the staining density, the reaction time corresponding to the obtained difference value is obtained as S _E. As a result, it is possible to obtain the value of the reaction time S _E of the eosin staining solution so that the staining density d _{E of} which the staining density of the dye E is determined in the specimen created with the thickness of the specimen as T.

Here, the case where the value of the reaction time S _E is calculated (corrected) in consideration of the thickness of the sample is illustrated. On the other hand, by applying the same method, it is possible to correct the value of the desired correction target control parameter by adding the value of the desired control parameter to be acquired other than the thickness of the sample. Further, by applying the same method, it is possible to correct the value of the correction target control parameter by taking into account the values of the plurality of acquisition target control parameters. For example, if the reaction time is to be corrected, LUTs that define the relationship between the value of each control parameter to be added and the staining density are prepared, and the final dye H and / or dye is sequentially referred to these LUTs. The reaction times S _H and S _E may be determined so that the staining density of E becomes the determined values d _H and d _E.

Here, the case where the reaction time S _E of the eosin staining solution is calculated using the LUT that defines the relationship between the reaction time and the staining concentration and the LUT that defines the relationship between the specimen thickness and the staining concentration is illustrated. On the other hand, it is good also as preparing LUT which defined the relationship between the reaction time and the dyeing density | concentration of the pigment | dye E separately for every thickness of a sample. In this case, the LUT corresponding to the acquired specimen thickness T is referred to, and the reaction time corresponding to the staining concentration of the dye E determined according to the reference destination LUT may be acquired.

Alternatively, the reaction time can be corrected in consideration of the type of formalin-based fixing solution used for fixing the tissue in the specimen used by the paraffin block creation processing unit 51. In this case, it can be realized by multiplying each value obtained by Equations (12), (13), Equations (16), and (17) by a coefficient corresponding to the type of formalin-based fixing solution to be used. For example, the coefficient when using a certain formalin-based fixative A is set to “1.0”. On the other hand, the coefficient when using a formalin-based fixative B different from A is set to “0.95”. Then, in the case of using formalin-based fixative A, obtain the value of S _H calculated according to equation (12) or equation (16) as the reaction time by hematoxylin staining solution. Similarly, the value of S _E calculated according to equations (13) and (17) is obtained as the reaction time with the eosin staining solution. On the other hand, in the case of using formalin-based fixative B obtains a value obtained by multiplying the coefficient "0.95" to the value of S _H calculated according to equation (12) or equation (16) as the reaction time by hematoxylin staining solution. The reaction time with the eosin staining solution is calculated in the same manner using the equations (13) and (17).

  In addition, the specimen to be created is not limited to the pathological specimen used for the pathological examination, but can be any specimen.

It is explanatory drawing explaining the outline | summary of the procedure which produces a sample and observes and diagnoses. It is explanatory drawing explaining the schematic procedure of an observation image generation process and a staining density calculation process. It is explanatory drawing explaining the schematic procedure of a virtual image generation process. 1 is a schematic diagram illustrating an overall configuration of a specimen creation system including a specimen creation apparatus according to Embodiment 1. FIG. FIG. 3 is a block diagram illustrating a functional configuration of the specimen preparation device according to the first embodiment. 4 is a flowchart illustrating a processing procedure performed by the control device according to the first embodiment. It is a figure which shows an example of a sample specific parameter input screen. It is a figure which shows the other example of a sample specific parameter input screen. It is a figure which shows an example of a control parameter input screen. It is a figure which shows an example of a staining density confirmation screen. It is a figure which shows an example of an adjustment coefficient change screen. It is a figure which shows an example of the staining density confirmation screen in a modification. It is a figure which shows an example of the correspondence of reaction time and dyeing density. It is a flowchart which shows the process sequence of a sample preparation process. FIG. 6 is a block diagram illustrating a functional configuration of a specimen preparation device according to a second embodiment. 10 is a flowchart illustrating a processing procedure performed by the control device according to the second embodiment. It is a flowchart which shows the process sequence of the sample preparation process at the time of applying immunostaining. It is a figure which shows an example of the correspondence of the thickness of a sample, and dyeing | staining density | concentration. It is a figure which shows an example of the correspondence of the density | concentration of a hematoxylin dyeing | staining liquid, and a dyeing | staining density | concentration.

DESCRIPTION OF SYMBOLS 1 Specimen preparation system 2, 2b Specimen preparation apparatus 3, 3b Control apparatus 31 Operation part 32 Display part 33, 33b Storage part 331, 331b Specimen preparation processing program 35, 35b Control part 351 Specimen specific parameter acquisition part 352 Control parameter acquisition part 353 Image extraction unit 354 Image display processing unit 355, 355b Availability selection request unit 356, 356b Sample creation processing setting unit 37, 37b Image processing unit 371, 371b Virtual image generation unit 372 Control parameter determination unit 373b Staining density calculation unit 4 Standard sample image Data storage unit 5 Specimen creation unit 51 Paraffin block creation processing unit 52 Slicing processing unit 53 Deparaffinization processing unit 54 Staining processing unit 55 Encapsulation processing unit 6 Observation device

Claims (10)

An image storage unit that stores a sample image obtained by imaging a sample prepared in advance as a standard sample in association with each value of a sample specifying parameter indicating the type of the sample and a control parameter used when creating the sample When,
A sample specifying parameter acquisition unit for acquiring a value of the sample specifying parameter for a sample to be created;
A control parameter acquisition unit that acquires the value of the control parameter for the sample to be created;
An image extracting unit that extracts one or more sample images from the sample images stored in the image storage unit, based on at least the value of the sample specifying parameter acquired by the sample specifying parameter acquiring unit;
Based on the sample image extracted by the image extraction unit, an image display processing unit that performs processing for displaying a reference image on the display unit for determining whether the value of the control parameter is acceptable;
An availability selection requesting unit for requesting input of availability selection of the value of the control parameter;
A control parameter determination unit that determines a value of the control parameter according to an input result of the selection of availability for the request by the availability selection request unit;
A sample creation unit that creates the sample to be created using the value of the control parameter determined by the control parameter determination unit;
A specimen preparation device comprising: The specimen is a stained specimen stained with a predetermined staining solution,
A simulation image generation unit that generates a simulation image in which the staining density represented by the reference image is virtually changed based on the reference image,
The specimen creation apparatus according to claim 1, wherein the image display processing unit displays the simulation image on the display unit when a simulation image is generated by the simulation image generation unit. It has a dye density adjustment requesting section that requests dye density adjustment,
The simulation image generating unit generates the simulation image by changing the staining density represented by the reference image to a staining density adjusted in response to a request from the staining density adjustment requesting unit,
The control parameter determination unit corrects and determines a value of the control parameter to a value corresponding to the adjusted staining density according to an input result of availability selection for the request by the availability selection requesting unit. Item 3. The specimen preparation device according to Item 2. A control parameter adjustment requesting unit that requests adjustment of the value of the control parameter;
The simulation image generation unit generates the simulation image by changing the staining density represented by the reference image to a staining density corresponding to the value of the control parameter adjusted in response to the request by the control parameter adjustment request unit,
3. The control parameter determination unit according to claim 2, wherein the control parameter determination unit determines a value of the control parameter as the adjusted control parameter value in accordance with an input result of the selection of availability for the request by the availability selection requesting unit. Sample preparation device.   The said control parameter determination part determines the value which instruct | indicates to the said sample preparation part the reaction time and / or the production | generation conditions of a staining liquid by the staining liquid as a value of the said control parameter. Sample preparation device. The image storage unit is a sample of a type indicated by the value of the corresponding sample specifying parameter in association with a value that can be taken by the sample specifying parameter, and a sample created using the value of the control parameter as a predetermined value is the standard sample The specimen image obtained as
The image extraction unit extracts a sample image corresponding to the value of the acquired sample specifying parameter from the sample images stored in the image storage unit,
Based on the extracted specimen image, the reference image is generated by changing the staining density when the control parameter represented by the specimen image is a predetermined value to a staining density according to the value of the acquired control parameter. The specimen preparation device according to claim 2, further comprising a reference image generation unit configured to perform the reference image generation unit. The image storage unit is a type of sample indicated by the value of the corresponding sample specifying parameter in association with a combination of values that can be taken by the sample specifying parameter and the control parameter, and uses the value of the corresponding control parameter. Memorize the sample image obtained as the standard sample,
The image extraction unit extracts a sample image corresponding to the acquired sample specifying parameter and the acquired control parameter value from the sample image stored in the image storage unit,
The sample creation apparatus according to claim 1, wherein the image display processing unit performs display processing of the extracted sample image as the reference image.   The control parameter determination unit uses, as the value of the control parameter, at least one of a specimen thickness, a specimen block size, and a fixing time using a formalin-based fixing solution used for fixing a tissue in the specimen. 2. The specimen preparation device according to claim 1, wherein a value instructed to the preparation unit is determined. The sample is a sample of a sample collected from a subject,
2. The specimen specifying parameter acquisition unit acquires at least one of an organ, a region, and a disease name of a subject from which a specimen is collected as the value of the specimen specifying parameter. The specimen preparation device described. A specimen image obtained by imaging a specimen prepared in advance as a standard specimen is stored in association with each value of the specimen specifying parameter indicating the specimen type and the control parameter used when creating the specimen. A specimen preparation method in a preparation device,
A sample specifying parameter acquisition step of acquiring a value of the sample specifying parameter for a sample to be created;
A control parameter acquisition step of acquiring a value of the control parameter for the sample to be created;
An image extracting step of extracting one or more sample images from the stored sample images based on at least the value of the sample specifying parameter acquired in the sample specifying parameter acquiring step;
Based on the sample image extracted in the image extraction step, an image display processing step for performing a process of displaying a reference image for determining whether the value of the control parameter is acceptable;
A permission selection requesting step for requesting input of permission control for the value of the control parameter;
A control parameter determination step for determining a value of the control parameter according to an input result of the selection of availability for the request in the availability selection request step;
A sample creation step of creating the sample to be created using the value of the control parameter determined in the control parameter determination step;
A specimen preparation method characterized by comprising: