JP2017194430A - Tumor site identification method, specimen holder - Google Patents

Abstract

A novel method for discriminating a tumor site from a non-tumor site is provided.
SOLUTION: The method of the present invention comprises a step (a) of irradiating porphyrins accumulated in a tumor site of a specimen with excitation light to detect fluorescence emitted from the porphyrins after excitation, and a cured specimen. And (b) performing a pathological diagnosis pre-processing. The step (a) includes a step (a1) of preparing a sample holder having a storage unit for storing a sample, a step (a2) of storing a sample in the storage unit, and emitting excitation light from the outside of the sample holder. The step (a3) of irradiating the specimen housed in the housing part and the step (a4) of receiving fluorescence emitted from porphyrins excited by irradiation with excitation light. The step (b) includes a step (b1) of immersing the specimen after completion of the step (a4) in a chemical solution, replacing the moisture contained in the specimen with a predetermined material, and curing the specimen; And a step (b2) of shredding.
[Selection] Figure 3

Description

  The present invention relates to a method for discriminating a tumor site and a specimen holder used in a tumor site discrimination device.

  In recent years, Japan is facing a rapidly aging society, and the number of cancer patients is increasing. In particular, since lymph node metastasis is one of the important prognostic factors, it is important to accurately diagnose the presence or absence of lymph node metastasis in determining the patient's treatment method. In particular, the lymph node to which cancer cells away from the primary lesion first arrive is called the sentinel lymph node. Since the number of cancer cells that reach the sentinel lymph node is initially limited, if the sentinel lymph node is examined and there is very little cancer cell metastasis, then it may be considered that it has almost not metastasized to the previous lymph node. Has been.

  CT (Computed Tomography) and FDG-PET (Fluorodeoxyglucose positron emission tomography) are known as preoperative methods for diagnosing lymph node metastasis, but the current state of diagnosis is still not sufficient. It is. Histopathological diagnosis is used to diagnose lymph node metastasis during and after surgery. Usually, small metastases are missed because the presence or absence of metastasis is diagnosed only from specimens obtained from a single facet. In some cases, the diagnostic accuracy is not sufficient. Furthermore, in the intraoperative rapid examination, it takes at least about 30 minutes to make a diagnosis, and a judgment by a pathologist is required. Therefore, there is a possibility that a difference in diagnostic results may occur due to differences in skills of individual pathologists. Furthermore, in view of the current situation where the pathologists themselves are lacking, a new method is required that can diagnose lymph node metastasis with high accuracy without seeking the pathologist's judgment.

  In recent years, photodynamic techniques using 5-aminolevulinic acid (5-ALA) have been applied to cancer detection in a wide range including the digestive organs. 5-ALA is a kind of amino acid that is also present in the living body, and is water-soluble and can be administered orally or locally. When 5-ALA is administered from outside the body, it is rapidly metabolized to heme in normal cells, but protoporphyrin IX (PpIX), which is a metabolite, selectively accumulates in cancer cells due to the difference in the activity of metabolic enzymes. Here, heme does not recognize fluorescence, but PpIX is a fluorescent substance, and thus it is possible to diagnose cancer by detecting this light (see Patent Document 1 and Non-Patent Document 1).

International Publication No. 2013/002350

“Diagnosis of metastatic lymph nodes of gastrointestinal cancer using 5-aminolevulinic acid (5-ALA)”, Kyoto Medical University Journal, Vol.122, No.4, (2013)

  The above contents are still at the laboratory level at the present time, and it is assumed that various problems will arise when actually applied to human diagnosis. The present applicants are currently developing an apparatus for discriminating between a tumor site and a non-tumor site by spectroscopically detecting fluorescence emitted from porphyrins such as PpIX present in a tumor site of a specimen.

  An object of the present invention is to provide a novel method for discriminating between a tumor site and a non-tumor site. Another object of the present invention is to provide a specimen holder that can be suitably used in this method.

The present invention is a method of discriminating a tumor site,
Irradiating the porphyrins accumulated in the tumor site of the specimen with excitation light, and detecting the fluorescence emitted by the porphyrins after excitation (a);
(B) performing a pretreatment for pathological diagnosis by chopping the cured specimen
The step (a)
A step (a1) of preparing a specimen holder having an accommodating portion for accommodating the specimen;
A step (a2) of storing the specimen in the storage portion;
Emitting the excitation light from the outside of the specimen holder, irradiating the excitation light through the predetermined region of the specimen holder and irradiating the specimen accommodated in the accommodating portion (a3);
Receiving the fluorescence emitted from the porphyrins excited by irradiation with the excitation light (a4),
The step (b)
(B1) a step of immersing the specimen after completion of the step (a4) in a chemical solution, replacing the moisture contained in the specimen with a predetermined material, and curing the specimen;
And (b2) a step of chopping the cured specimen.

  In the present specification, “porphyrins” refers to those having a substituent on the porphine ring. For example, PpIX and protoporphyrins such as photo-protoporphyrin (PPp) generated from PpIX exist.

  When detecting whether or not a specimen contains a tumor site using the tumor site discrimination device, first, a predetermined drug such as 5-ALA is administered to a patient as a subject. When 5-ALA is absorbed in normal cells, it is metabolized by intracellular mitochondria into protoporphyrin IX (hereinafter referred to as “PpIX” as appropriate), which is one of porphyrins, and biosynthesized into heme. However, malignant tumor cells have higher enzyme (PBG deaminase) activity during PpIX production than normal cells, and lower enzyme (ferrochelatase) activity that catalyzes heme biosynthesis from PpIX. For this reason, when 5-ALA is absorbed in malignant tumor cells, a large amount of PpIX is accumulated in these cells.

  PpIX is a fluorescent material, while heme is not a fluorescent material. For this reason, it is possible to discriminate between a tumor site and a non-tumor site by irradiating a specimen with predetermined excitation light and performing light reception analysis of fluorescence emitted from the specimen.

  FIG. 1A is a diagram showing an absorption spectrum of PpIX. Moreover, FIG. 1B is a figure which shows the fluorescence spectrum of PpIX. PpIX exhibits high absorbance with respect to excitation light having a predetermined wavelength. Specifically, as shown in FIG. 1A, it exhibits high absorbance for light with a wavelength of 370 nm or more and 450 nm or less, and particularly shows extremely high absorbance for light with a wavelength of 385 nm or more and 425 nm or less. When PpIX is irradiated with light in the above wavelength range, as shown in FIG. 1B, fluorescence having a wavelength component of 620 nm or more and 710 nm or less is emitted from PpIX as a peak in the vicinity of 635 nm.

  The tumor site discrimination device requires a light source unit that can emit excitation light having a wavelength included in the above-described wavelength band and a light receiving unit that receives fluorescence emitted from PpIX. In addition, a mechanism for setting the specimen in the apparatus is required.

  As the specimen, a biopsy material such as a lymph node section is assumed. When setting such a biopsy material in the apparatus, the applicants previously store the specimen in a predetermined holder so that the problem such as contamination of the apparatus does not occur. A method of setting the position was devised.

  According to the above step (a), excitation light is irradiated to the holder in a state where the specimen is placed in the storage section provided in the holder. As a result, porphyrins contained in the specimen accommodated in the holder are excited and fluorescence is emitted. By receiving this fluorescence and analyzing the intensity, it is possible to distinguish between a tumor site and a non-tumor site based on the fluorescence intensity.

  According to said method, after completion | finish of the discrimination | determination process (a) based on fluorescence intensity, it has the process (b) which hardens | cures and cleaves a test substance. For this reason, after the completion of the step (b), a pathologist can diagnose the minute specimen after shredding visually. That is, according to the method of the present invention, it is possible to efficiently perform the steps up to the diagnosis by the pathologist.

  The step (b1) may include a step of immersing the sample holder together with the chemical solution.

  According to the above method, after the step (a) is completed, when performing the step (b1) of replacing the moisture contained in the sample with a predetermined material, the sample needs to be moved from the sample holder to another holder. There is no. For this reason, processing efficiency can be improved.

  By the way, the fluorescence intensity is analyzed by the step (a) in many cases in a region close to the surface on the side irradiated with the excitation light in the specimen. In addition, the specimen before curing easily moves in the holder. For this reason, when performing the step (b1), when the sample is transferred from the sample holder to another holder, the possibility that the region of the sample in which the fluorescence intensity analysis is performed is likely to be mistaken. There is. If such a situation occurs, when a pathologist diagnoses the microsample after shredding, there is a possibility that a microsample in a region different from the region where the fluorescence intensity analysis was performed will be diagnosed. In this case, it cannot be denied that there is a possibility that the sample that is determined to have a tumor site in step (a) is erroneously determined by the pathologist as having no tumor site.

  According to the above method, since the step (b1) can be performed in a state where the sample is stored in the sample holder used in the step (a), the number of times of transferring the holder storing the sample is reduced. Can reduce the risk of misjudgment.

More specifically,
The specimen holder has a hole that communicates with the housing from the outside of the specimen holder,
The step (b1) may be a step of immersing the specimen in the chemical solution by guiding the chemical solution to the accommodating portion through the hole.

At this time,
The specimen holder includes a base portion provided with the accommodating portion in a partial area, and a lid portion for covering the upper portion of the accommodating portion,
The hole portion may be provided in at least one of the base portion or the lid portion.

Further, the present invention irradiates the specimen with excitation light emitted from the light source unit, and spectroscopically detects the fluorescence emitted by porphyrins present in the tumor site of the specimen, thereby detecting the tumor site and the non-tumor site. A specimen holder used in a tumor site discrimination device that performs discrimination,
A storage section for storing the specimen;
A window part for transmitting the excitation light emitted from the outside of the specimen holder and irradiating the specimen contained in the accommodation part with the excitation light;
And a hole portion communicated with the housing portion from the outside.

  Here, the hole portion may be configured such that a predetermined drug solution can be introduced into the storage portion from the outside of the specimen holder.

  The specimen holder has a housing portion, and the specimen is placed in the housing portion when used. The holder is provided with a window that transmits excitation light emitted from the light source in the apparatus. Excitation light is transmitted through the window and irradiated on the specimen. If the specimen contains porphyrins, the porphyrins are excited and emit fluorescence. By analyzing and analyzing the light received by the light receiving unit provided on the apparatus side, it is possible to discriminate between a tumor site and a non-tumor site according to the intensity of light having a wavelength derived from the fluorescence of porphyrins.

  And according to said specimen holder, the hole part connected to the accommodating part from the exterior is provided. For this reason, after the fluorescence analysis process is completed, the chemical solution can be guided into the storage unit by, for example, immersing the specimen holder in a predetermined chemical solution. Thereby, the moisture contained in the specimen can be replaced with another material without removing the specimen from the specimen holder. As a result, when proceeding to the next step of immobilizing and chopping the specimen, the number of holders that contain the specimen is reduced, and the risk of the above-described misdiagnosis is reduced.

The hole can be provided at any location of the specimen holder. As an example,
The specimen holder includes a base portion provided with the accommodating portion in a partial area, and a lid portion for covering the upper portion of the accommodating portion,
The hole portion may be provided in at least one of the base portion and the lid portion.

The specimen holder is
The apparatus may further include a sample fixing unit for suppressing the displacement of the sample stored in the storage unit.

  According to the diligent research of the present inventors, the holder containing the specimen is set at a predetermined position of the apparatus, irradiated with light, and while the fluorescence is received at the light receiving unit, the specimen contracts, It turned out that it may expand on the contrary.

  One of the reasons why such a phenomenon occurs is that a biopsy material such as a sentinel lymph node is assumed as a specimen. When a biopsy material such as the above sentinel lymph node is used as a specimen, it is generally washed with physiological saline or the like after being excised from a human body and before being accommodated in a holder. As described above, when a biopsy material containing a lot of water is used as a sample, the water is evaporated while the holder containing the sample is set at a predetermined position of the apparatus and irradiated with light, and the sample is It is thought that it contracted. In addition, the biopsy material contracts due to drying, but it may partially expand due to stress deformation caused by partial contraction that occurs at that time (movement in the expansion direction).

  Here, as described above, according to the method of discriminating a tumor site and a non-tumor site according to the intensity of light having a wavelength derived from the fluorescence of porphyrins, first, in the light receiving unit provided in the apparatus, A determination is made whether the light intensity is above a predetermined threshold. Then, in the apparatus, it is determined that a portion where the light intensity exceeds a predetermined threshold is a tumor site, and a location where the light intensity is equal to or less than the threshold is a non-tumor site.

  In the case of this method, for example, an area irradiated with light through a window is divided into predetermined small areas, and it is determined whether or not the light intensity exceeds a predetermined threshold value for each small area. Is called. Thereby, it is possible to determine in which region on the storage unit the position of the specimen is the tumor site.

  However, as described above, if the specimen expands / contracts, the specimen is misaligned during the determination of the amount of light in the light receiving unit. As a result, the tumor site is displaced depending on the sample displacement, and in some cases, the tumor site may be erroneously recognized.

  On the other hand, according to the above configuration, the holder is provided with the sample fixing unit for suppressing the displacement (positional change) of the sample stored in the storage unit. For this reason, after the sample is accommodated in the holder, the displacement of the sample is suppressed by the sample fixing unit. As a result, even if the sample expands / contracts, the amount of displacement of the sample position is suppressed as compared with the conventional case, and the possibility that the tumor site is erroneously recognized is reduced.

  In particular, the sample fixing unit is configured to be able to suppress displacement of the sample stored in the storage unit in a direction parallel to the bottom surface of the storage unit, more specifically in the horizontal direction. be able to.

More specifically,
The specimen holder includes a base portion provided with the accommodating portion in a partial area, and a lid portion for covering the upper portion of the accommodating portion,
The specimen fixing part is constituted by a convex part provided on at least one of the base part or the lid part,
By opening and closing the lid, the first state where the convex part comes into contact with the specimen and the second state where the convex part is separated from the specimen may be transitioned.

  According to this configuration, since the convex portion comes into contact with the sample by closing the lid after the sample is stored in the storage unit, the movement of the sample in the horizontal direction is suppressed. The tip of the convex portion may be in contact with the specimen when the lid portion is closed.

  More specifically, the specimen fixing part may be configured by a plurality of the convex parts discretely provided on the lid part. Various arrangements are possible for the arrangement of the plurality of convex portions. As an example, a mode in which each convex part has a square shape and is arranged in a vertical and horizontal direction, or a mode in which the convex parts are distributed in a staggered arrangement are possible. In addition, as the shape of each convex portion, a hemispherical shape, a conical shape, a truncated cone shape, a polygonal pyramid shape, a polygonal frustum shape, a corrugated shape, and other shapes can be adopted.

  According to the method or the specimen holder of the present invention, the tumor site can be identified efficiently and accurately.

It is a figure which shows the absorption spectrum of PpIX. It is a figure which shows the fluorescence spectrum of PpIX. It is a figure which shows typically the external appearance of a tumor site | part discrimination | determination apparatus. It is a block diagram which shows typically the internal structure of a tumor site | part discrimination | determination apparatus. It is a typical perspective view in one example of a sample holder. It is a typical top view of a base part in one example of a sample holder. It is a typical top view of the lid part in one example of a specimen holder. It is a typical perspective view of the base | substrate part in one Example of the holder for specimens. It is a typical top view of a base part in one example of a sample holder. It is a typical top view of a base part in one example of a sample holder. It is a typical top view of the lid part in one example of a specimen holder. It is a typical top view of the lid part in one example of a specimen holder. It is a typical top view of the lid part in one example of a specimen holder. It is a typical top view of the lid part in one example of a specimen holder.

[Device Overview]
Prior to the description of the specimen holder, the configuration of a tumor site determination apparatus using this holder will be described.

  FIG. 2 is a drawing schematically showing the appearance of the apparatus. FIG. 3 is a block diagram schematically showing an internal configuration of the apparatus. 2 and 3 are drawings showing an example of a tumor site determination device, and the device using the holder of the present invention is not limited to the configuration shown in FIGS. 2 and 3.

  As shown in FIG. 2, the tumor site determination apparatus 10 (hereinafter sometimes referred to as “apparatus 10” as appropriate) includes a holder mounting opening 11 and a display unit 12. The holder mounting port 11 is a mechanism for mounting the sample holder 1. The display unit 12 corresponds to a monitor on which the result determined by the tumor site determination device 10 is displayed. Here, the configuration in which the display unit 12 is provided in the main body of the tumor site determination device 10 is shown, but the main body of the device 10 does not include the display unit 12 and the determination result is displayed on another monitor. You may employ | adopt the structure to make.

  As shown in FIG. 3, the apparatus 10 includes a light source unit 21, a filter 22, a dichroic mirror 23, an objective lens 24, a filter 25, a light receiving unit 26, and an arithmetic processing unit 27. In the example of FIG. 3, a configuration in which the device 10 includes the display unit 12 is assumed as in FIG. 2.

  The light source unit 21 includes, for example, a mercury lamp, a light emitting diode element, a laser diode element, or the like. The filter 22 has a function of selectively transmitting light of a specific wavelength from the light emitted from the light source unit 21, and can be configured of, for example, a dielectric multilayer film. Here, the filter 22 is described as having a function of selectively transmitting light with a wavelength of 390 nm, but has a function of selectively transmitting light of a specific wavelength band that can excite porphyrins. It only has to be.

  The dichroic mirror 23 has a function of reflecting light of a predetermined wavelength band and transmitting light of another predetermined wavelength band, and can be formed of, for example, a dielectric multilayer film. Here, it is assumed that the dichroic mirror 23 has a function of reflecting light having a wavelength of 390 nm and transmitting light having a wavelength of 620 nm or more. The dichroic mirror 23 only needs to have a function of reflecting light having a wavelength selected by the filter 22 and transmitting at least light near the peak wavelength of fluorescence emitted from the specimen 2.

  The excitation light 31 having a wavelength of 390 nm emitted from the light source unit 21 and transmitted through the filter 22 is reflected by the dichroic mirror 23 and guided to the objective lens 24. Then, the light that has passed through the objective lens 24 passes through a predetermined region of the holder 1 (hereinafter referred to as “window portion 52” as appropriate) and is irradiated onto the specimen 2 accommodated in the holder 1. When porphyrins such as PpIX are accumulated in the specimen 2, the porphyrins are excited by the excitation light 31 having a wavelength of 390 nm and emit fluorescence 32. The fluorescence 32 passes through the window portion 52 of the holder 1, travels in the direction opposite to the excitation light, and is guided to the objective lens 24. Then, the light passes through the dichroic mirror 23 and enters the filter 25.

  The filter 25 has a function of selectively transmitting light having a predetermined wavelength from incident light. Here, the filter 25 is described as having a function of selectively transmitting light having a wavelength of 635 nm. However, the filter 25 has a function of selectively transmitting light in the vicinity of the peak wavelength of fluorescence emitted from porphyrins. It only has to be.

  The fluorescence having a wavelength of 635 nm transmitted through the filter 25 is received by the light receiving unit 26. The light receiving unit 26 can be configured by an imaging device such as a CCD camera, for example. The light receiving unit 26 outputs the intensity of the received light to the arithmetic processing unit 27 together with the position information in the sample 2. The arithmetic processing unit 27 is configured by, for example, a microcomputer and determines whether or not the light intensity at each position exceeds a predetermined threshold value. Then, the arithmetic processing unit 27 determines that the part where the light intensity exceeds the predetermined threshold is a tumor part, and the part where the light intensity is equal to or less than the threshold is a non-tumor part. Then, the determination result is output to the display unit 12.

  Based on the coordinate information of the tumor site sent from the arithmetic processing unit 27, the display unit 12 displays, for example, a mark indicating a tumor site or image data that has been colored at a predetermined position on the image of the specimen 2. To do. Further, when there is no region that is determined to be a tumor site in the arithmetic processing unit 27, information to that effect may be displayed on the display unit 12.

  The inspector can easily recognize the presence or absence of the tumor site in the specimen 2 and the presence location of the tumor site by checking the display unit 12 visually. . Further, for example, by providing an operation button on the apparatus 10 and mounting the holder 1 containing the sample 2 on the apparatus 10 and pressing the operation button, excitation light is emitted from the light source unit 21. Thus, it is possible to automatically determine the tumor site of the specimen 2, and the variation in the determination result due to the skill of the inspector is eliminated.

  The dichroic mirror 23 is provided for the purpose of sharing part of the optical paths of the excitation light 31 and the fluorescence 32 in order to reduce the size of the device 10. However, the dichroic mirror 23 is not necessarily required in the device 10. It is not a configuration. Further, the filter 22 may be integrated with the light source unit 21. The filter 25 may be integrated with the light receiving unit 26. The configuration of the apparatus 10 illustrated in FIG. 3 is merely an example, and it is needless to say that various design changes are possible as long as the configuration achieves the same function.

[holder]
Next, the configuration of the holder 1 will be described.

  FIG. 4A is a perspective view of one embodiment of the holder 1. As shown in FIG. 4A, the holder 1 includes a base portion 42 and a lid portion 43. 4B is a plan view of the base portion 42 in the present embodiment, and FIG. 4C is an example of a plan view of the lid portion 43 in the present embodiment.

  The base portion 42 and the lid portion 43 are connected by a hinge, and the lid portion 43 is configured to be rotatable with respect to the base portion 42. The base portion 42 is provided with a storage portion 41 for storing the sample 2 at a predetermined location. The container 41 has a depth deeper than the periphery thereof, and even if the cover 43 and the base 42 are overlapped in a state where the sample 2 is stored in the container 41, the cover 43 (more specifically, The specimen 2 is not crushed by the portion of the lid portion 43 where the convex portion 44 is not formed.

  As shown in FIG. 4A, the accommodating portion 41 is provided with a window portion 52 for transmitting light. In FIG. 4A, the window portion 52 is provided in a predetermined region on the bottom surface of the storage portion 41, but may be provided on the entire bottom surface of the storage portion 41.

  In the holder 1 of this embodiment, a plurality of holes 47 are provided in the lid 43. The hole 47 communicates with the outside of the holder 1. Even when the specimen 2 is accommodated in the accommodating part 41 and the lid part 43 is overlapped with the base part 42, the specimen 2 can be communicated from the outside of the holder 1 through the hole 47. The function of the hole 47 will be described later.

  In the present embodiment, as shown in FIGS. 4A and 4C, the lid portion 43 is provided with a plurality of convex portions 44. With this configuration, after the sample 2 is accommodated in the accommodating portion 41, the lid portion 43 is closed and the base portion 42 and the lid portion 43 are overlapped so that the tip of the convex portion 44 is brought into contact with the sample 2. Thereby, the movement of the sample 2 in the horizontal direction is suppressed. In the present embodiment, the convex portion 44 corresponds to the “specimen fixing portion”. In this embodiment, the hole 47 is provided in a region of the lid 43 where the convex portion 44 is not formed, but various aspects can be adopted for mounting the hole 47, This point will be described later.

  As described with reference to FIG. 3, in the apparatus 10, the excitation light 31 passes through the holder 1 and is irradiated to the specimen 2. Here, in FIG. 4A, when the excitation light 31 is irradiated to the base portion 42 from the side opposite to the lid portion 43, the excitation light is applied to the specimen 2 accommodated in the accommodation portion 41 via the window portion 52. Led.

  By the way, the holder 1 in which the specimen 2 is housed in the housing portion 41 is mounted in the holder mounting port 11 of the apparatus 10, the excitation light 31 emitted from the light source unit 21 is irradiated on the sample 2, and fluorescence is received by the light receiving unit 26. The sample 2 may expand / contract while receiving light. If the convex portion 44 does not exist in the holder 1 as in the present embodiment, the sample 2 is expanded / contracted in the holder 1 and moved in the horizontal direction while the light receiving portion 26 receives the fluorescence. As a result, when the calculation processing unit 27 determines that the location where the fluorescence intensity exceeds the predetermined threshold is not actually a tumor site, or conversely, the location where the fluorescence intensity is determined to be less than the predetermined threshold. May actually be a tumor site.

  On the other hand, in the configuration of the present embodiment, since the convex portion 44 exists in the lid portion 43 of the holder 1, the convex portion is obtained by closing the lid portion 43 after accommodating the specimen 2 in the accommodating portion 41. The tip of 44 contacts the sample 2. As a result, even if the specimen 2 tries to expand / contract, the movement of the specimen 2 in the horizontal direction is hindered by the frictional force accompanying the contact between the convex portion 44 and the specimen 2, and while the light receiving section 26 receives the fluorescence. The amount by which the position of the specimen 2 varies is greatly suppressed. As a result, the tumor site in the specimen 2 can be correctly identified by the apparatus 10.

  The holder 1 can adopt various shapes. An example is described below.

  <1> FIG. 5A is a perspective view schematically showing the configuration of the base portion 42 of the holder 1 of another embodiment. Like the base portion 42 shown in FIG. 5A, a hole 47 may be provided on the side surface of the base portion 42. ,

  5B and 5C are plan views schematically showing the configuration of the base portion 42 of the holder 1 of another embodiment. As shown in FIG. 5B, the hole 47 may be provided on the entire bottom surface of the base portion 42, or the hole 47 may be provided in a region other than the window portion 52 on the bottom surface of the base portion 42. I do not care. As described above with reference to FIG. 4B, the base portion 42 may not be provided with the hole 47.

  <2> FIGS. 6A to 6D are plan views showing the configuration of the lid portion 43 of the holder 1 of another embodiment.

  As shown in FIG. 6A, the hole 47 may be formed in the region of the lid 43 where the convex portion 44 is formed. Further, as shown in FIGS. 6B and 6C, the lid portion 43 may be configured such that the convex portion 44 can be attached / detached. That is, the lid 43 may have the configuration shown in FIG. 6B when the convex portion 44 is attached, and may have the configuration shown in FIG. 6C when the convex portion 44 is removed. .

  Furthermore, as shown in FIG. 6D, the lid portion 43 may not be provided with the hole portion 47.

  <3> When the convex portion 44 is provided in the holder 1, various selections are possible for the shape of the convex portion 44 itself, the number of the convex portions 44, and the arrangement shape of the convex portions 44. As the shape of the convex portion 44 itself, a hemispherical shape, a corrugated shape or the like can be adopted in addition to the prismatic shape as shown in FIG. 4A. As a mode of arrangement of the convex portions 44, in addition to a configuration in which the convex portions 44 are arranged in the vertical and horizontal directions as shown in FIG. 4A, they may be arranged in a staggered shape or a radial shape. The holder 1 may not have the convex portion 44.

  <4> The holder 1 is configured to include, for example, one base portion 42 of FIGS. 4B and 5A to 5C and one lid portion 43 of FIGS. 4C and 6A to 6D. Can do.

[Distinction method]
Hereinafter, a method for discriminating a tumor site using the holder 1 will be described.

(Step S1)
A holder 1 is prepared, and a specimen 2 to be examined is accommodated in the holder 1. The specimen 2 is a biopsy material (for example, a biological tissue including a sentinel lymph node) that is a target for determining a tumor site. If this biopsy material includes a tumor site, a predetermined measure is taken in advance so that porphyrins are accumulated in the site.

  The holder 1 in which the sample 2 is accommodated is attached to an apparatus 10 as shown in FIG. Then, predetermined excitation light 31 is irradiated from the light source unit 21 in the apparatus 10. At this time, the excitation light 31 is irradiated to the specimen 2 in the holder 1 through the window 52.

  If porphyrins are accumulated in the specimen 2, the porphyrins are excited by excitation light, and fluorescence 32 is emitted from the porphyrins. When the light receiving unit 26 receives the fluorescence 32, the light receiving unit 26 outputs information on the intensity to the arithmetic processing unit 27. In the arithmetic processing unit 27, it is mechanically determined whether or not a tumor site is included in the specimen 2 by performing the processing as described above.

  This step S1 corresponds to the step (a).

(Step S2)
Next, preprocessing for enabling visual diagnosis of the specimen 2 by a pathologist is performed. More specifically, this process corresponds to a process of curing and chopping the specimen 2. As an example, the following steps S2A to S2E are performed.

(Step S2A)
Water contained in the specimen 2 is removed. For example, when the apparatus 10 has an injection means for dehydrating chemicals such as ethanol, ethanol is supplied to the holder 1 from the injection means of the apparatus 10 with the holder 1 mounted in the apparatus 10. To do. When the apparatus 10 does not have the injection means, the holder 1 is taken out from the apparatus 10 and the holder 1 is made to land in a chemical liquid pool such as ethanol. Thereby, ethanol is immersed in the specimen 2 accommodated in the accommodating part 41 through the hole 47 provided in the holder 1. As a result, the water contained in the specimen 2 is replaced with alcohol, and dehydration is performed.

(Step S2B)
Next, the alcohol that has been included in the sample 2 in step S2A is removed from the sample 2. For example, when the apparatus 10 has a means for injecting a chemical solution for dealcohol such as xylene or chloroform, the chemical solution is placed in the holder from the injection means of the apparatus 10 with the holder 1 mounted in the apparatus 10. 1 is supplied. When the apparatus 10 does not have the injection means, the holder 1 and the holder 1 are made to land in a chemical pool such as xylene outside the apparatus 10. Thereby, xylene is immersed in the specimen 2 accommodated in the accommodating portion 41 through the hole 47 provided in the holder 1. As a result, the alcohol contained in the specimen 2 is replaced with xylene, and dealcoholization is performed. This process is also called a transparent process.

  In addition, as a chemical solution for dealcoholization, acetone, benzol, Tsedel oil, etc. can be used in addition to xylene and chloroform. This chemical solution is preferably miscible with both alcohol and paraffin.

(Step S2C)
Next, the substance such as xylene that is included in the specimen 2 in step S2B is replaced with paraffin. For example, when the apparatus 10 has means for injecting molten paraffin, molten paraffin is supplied to the holder 1 from the injection means of the apparatus 10 with the holder 1 mounted in the apparatus 10. . When the apparatus 10 does not have the injection means, the holder 1 is made to land on the molten paraffin outside the apparatus 10. Thereby, paraffin is immersed in the specimen 2 accommodated in the accommodating portion 41 through the hole 47 provided in the holder 1, and xylene contained in the specimen 2 is replaced with paraffin.

(Step S2D)
The specimen 2 infiltrated with paraffin is taken out of the holder 1 and placed in another container (embedding dish) containing molten paraffin. By reducing the temperature, the paraffin around the specimen 2 is cured and a paraffin block is formed.

(Step S2E)
Thereafter, the paraffin block is shredded using an instrument such as a microtome. Thereby, the part including the area | region where the excitation light 31 was irradiated is taken out among the test substances 2. FIG.

  This step S2 corresponds to the step (b). The micro sample obtained through step S2E is diagnosed by a pathologist, and the presence or absence of a tumor site is determined.

  According to this method, after the tumor site is discriminated by fluorescence analysis, the medicinal solution injection process required until the specimen 2 is shredded while the specimen 2 is housed in the holder 1 can be performed. it can. Thereby, the process of transferring the sample 2 is reduced, and erroneous processing and erroneous determination can be prevented.

  Note that step S2C may be omitted and step S2D executed after step S2B.

[Another embodiment]
In the following, another embodiment will be described.

  <1> In the determination method described above, step S2C may be omitted, and step S2D may be executed after step S2B.

  <2> In the above-described embodiment, the holder 1 has been described as having a configuration including the base portion 42 and the lid portion 43. However, the holder 1 may be configured not to include the lid portion 43. In this case, the outer edge of the base portion 42 is surrounded by a wall so that the specimen 2 placed on the base portion 42 does not drop or liquid such as physiological saline contained in the specimen 2 does not flow out. May be adopted.

  <3> The holder 1 may not include the hole 47. In this case, the sample 2 may be transferred to a holder different from the holder 1 when the process proceeds from step S1 to step S2.

1: Specimen holder
2: Specimen
10: Tumor site discrimination device
11: Holder mounting opening
12: Display section
21: Light source
22: Filter
23: Dichroic mirror
24: Objective lens
25: Filter
26: light receiving section
27: Arithmetic processing part
31: Excitation light
32: fluorescence
41: accommodation section
42: Base portion
43: Lid
44: convex portion
45: Frame-shaped member
47: Hole
52: Window

Claims (10)

A method for identifying a tumor site,
Irradiating the porphyrins accumulated in the tumor site of the specimen with excitation light, and detecting the fluorescence emitted by the porphyrins after excitation (a);
(B) performing a pretreatment for pathological diagnosis by chopping the cured specimen
The step (a)
A step (a1) of preparing a specimen holder having an accommodating portion for accommodating the specimen;
A step (a2) of storing the specimen in the storage portion;
Emitting the excitation light from the outside of the specimen holder, irradiating the excitation light through the predetermined region of the specimen holder and irradiating the specimen accommodated in the accommodating portion (a3);
Receiving the fluorescence emitted from the porphyrins excited by irradiation with the excitation light (a4),
The step (b)
(B1) a step of immersing the specimen after completion of the step (a4) in a chemical solution, replacing the moisture contained in the specimen with a predetermined material, and curing the specimen;
And a step (b2) of chopping the cured specimen.   The method for discriminating a tumor site according to claim 1, wherein the step (b1) includes a step of immersing the sample holder together with the chemical solution. The specimen holder has a hole that communicates with the housing from the outside of the specimen holder,
3. The method for discriminating a tumor site according to claim 2, wherein the step (b1) is a step of immersing the specimen in the drug solution by guiding the drug solution to the container through the hole. The specimen holder includes a base portion provided with the accommodating portion in a partial area, and a lid portion for covering the upper portion of the accommodating portion,
The method according to claim 3, wherein the hole is provided in at least one of the base portion and the lid portion. Tumor site discrimination that discriminates between the tumor site and the non-tumor site by irradiating the sample with excitation light emitted from the light source unit and spectroscopically detecting fluorescence emitted by porphyrins present in the tumor site of the sample A specimen holder used in the apparatus,
A storage section for storing the specimen;
A window part for transmitting the excitation light emitted from the outside of the specimen holder and irradiating the specimen contained in the accommodation part with the excitation light;
A specimen holder having a hole portion communicated with the housing portion from the outside.   6. The specimen holder according to claim 5, wherein the hole portion is configured such that a predetermined drug solution can be introduced into the accommodating portion from the outside of the specimen holder. The specimen holder includes a base portion provided with the accommodating portion in a partial area, and a lid portion for covering the upper portion of the accommodating portion,
The specimen holder according to claim 5 or 6, wherein the hole is provided in at least one of the base portion or the lid portion.   The sample holder according to claim 5, further comprising a sample fixing unit for suppressing displacement of the sample stored in the storage unit. The specimen holder includes a base portion provided with the accommodating portion in a partial area, and a lid portion for covering the upper portion of the accommodating portion,
The specimen fixing part is constituted by a convex part provided on at least one of the base part or the lid part,
9. The opening and closing of the lid portion is configured to be able to transition between a first state in which the convex portion comes into contact with the specimen and a second state in which the convex portion is separated from the specimen. The specimen holder described in 1. The sample holder according to claim 9, wherein the sample fixing unit includes a plurality of the convex portions discretely provided on the lid portion.

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