JP2019200090A - Embedding tray and manufacture method for biological tissue sample - Google Patents

Abstract

An object of the present invention is to provide a method for preparing as many sections as possible from a fixed amount of biological tissue sample in order to obtain as much information as possible from a valuable biological tissue sample. According to the present invention, there is provided an embedding tray for preparing a biological tissue specimen, the embedding material accommodating portion being formed by a bottom surface and side walls standing upright around the embedding material accommodating portion, and an upper portion of the embedding material accommodating portion. There is provided an embedding tray including a cassette mounting portion, in which at least a bottom surface of the embedding material storage portion has a flat surface with unevenness within ± 3 μm. [Selection diagram] Fig. 6

Description

  The present invention relates to an embedding tray used for preparing an embedding specimen from a living tissue (specimen) such as a cut human body tissue. Furthermore, the present invention relates to an embedded specimen, specifically, an embedding block including a living tissue, and a method for manufacturing a section including the living tissue.

  Based on information on important molecules involved in cancer invasion, metastasis, and proliferation identified in recent research, emphasis has been placed on the development of cancer molecular target drugs in recent drug development. In developing such molecular targeting drugs, not only genomic analysis but also information from morphology such as the localization of tumor cells and immune cells is important. In particular, a technique for efficiently and quantitatively analyzing the expression level of a specific gene has been developed, and it is possible to analyze an intracellular event actually acting in a tissue at a molecular level.

  In order to obtain useful information for drug development, analysis of pathological tissue specimens is important. A histopathological specimen cut out from a human body is valuable, and a method for efficiently preparing a histopathological specimen is necessary to enable various analyses.

  In general, a pathological tissue specimen is prepared by immobilizing a biological tissue sample with formalin or alcohol, and then preparing an embedded block in which the biological tissue sample is embedded in a material such as resin. Sections containing are produced and used for actual analysis. Various reports have been made on a method for preparing an embedding block for preparing a section and an instrument used for the preparation (Patent Documents 1 to 12).

  In general, section preparation can be performed by the following procedure. First, the embedding tray is placed on a suitable table, the biological tissue sample is accommodated in the embedding tray, and the cassette is placed on the embedding tray. Then, an embedding material such as molten paraffin is poured from above the cassette. There are a plurality of holes in the bottom wall of the cassette, and the molten embedding material is filled through the holes into an embedding tray containing a biological tissue sample. The embedding material is used in such an amount that the cassette and the embedding block are combined after solidification. The embedding material is solidified by cooling of the embedding tray, and the cassette and the embedding block are integrated. The embedding block can be removed.

  The obtained embedded block is cut and molded so as to have an appropriate shape, and then the tip of the embedded block is cut into a prismatic shape having a predetermined cross-sectional shape using a microtome or the like to produce a slice. The obtained section is used for microscopic analysis and other analysis (Patent Document 10, paragraph numbers [0003] to [0008], FIG. 1 and the like).

JP 2001-116669 A JP 2003-106963 A JP 2006-105839 A JP 2006-300755 A JP 2008-145118 A JP 2008-180625 A JP 2008-216235 A JP 2009-042075 A JP 2009-250782 A JP 2011-053230 A JP 2014-182130 A JP-A-2006-512603

  A histopathological specimen obtained in the clinical field is used to obtain information for grasping a patient's medical condition. In particular, biological tissue specimens from patients whose medical conditions change cannot be obtained again in the same state, and since the amount is limited, it is possible to obtain as much information as possible from biological tissue specimens. There is a need for a technique for producing as many sections as possible from a certain amount of biological tissue sample.

  As a result of intensive studies in view of the above problems, the present inventors have found that a section can be efficiently produced from a small amount of a biological tissue sample by using an embedding tray having a specific shape. Was completed.

One aspect of the present invention provides the following inventions [1] to [14].
[1] An embedding tray for producing a biological tissue specimen, wherein an embedding material accommodating portion formed by a bottom surface and a side wall provided around the bottom, and a cassette mounting portion on an upper portion of the embedding material accommodating portion Prepared,
An embedding tray in which at least a part of the bottom surface of the embedding material accommodating portion is a flat surface having an unevenness of within ± 3 μm.

[2] The embedding tray according to claim 1, wherein a bottom surface of at least a part of the embedding material accommodating portion is parallel to a surface specified by a point where the cassette placing portion contacts the cassette.
[3] The cassette placing portion has a bottom surface, and the variation in the distance between the surface formed by the cassette placing portion bottom surface and the bottom surface of at least a part of the embedding material accommodating portion is within ± 4 μm, [1] or [ 2].

[4] The embedding tray according to any one of [1] to [3], wherein the at least part of the bottom surface is 10% or more of the entire bottom surface.
[5] The embedding tray according to any one of [1] to [4], wherein the side wall has an inclination so that the upper opening of the embedding material container is wider than the bottom surface.

[6] The embedding tray according to any one of [1] to [4], which is formed of a material selected from stainless steel, titanium, gun metal, and a heat-shrinkable resin.
[7] storing the biological tissue sample in the embedding tray according to any one of [1] to [6] so that the biological tissue sample is in contact with the bottom surface of the embedding material storage unit;
Placing the cassette on the cassette placement section of the embedding tray and filling the embedding tray with the molten embedding material;
Solidifying the embedding material and removing the embedding block integrated with the cassette from the embedding tray to obtain the embedding block;
A method for producing a biological tissue specimen.

[8] The method according to [7], wherein the embedding material contains wax or paraffin having a melting point of 54 to 60 ° C.
[9] The biological tissue sample is attached to one surface of the embedding material cube, and the cube is placed on the bottom surface of the embedding tray so that the surface faces the bottom surface of the embedding tray and the biological sample is in contact with the bottom surface. The method according to [7] or [8], wherein the biological tissue sample is accommodated in the buried tray.

[10] The method according to any one of [7] to [9], further comprising a step of preparing a section from the embedded block.
[11] The method according to [10], wherein the section to be prepared has a thickness of 2 to 4 μm.

[12] The method according to any one of [7] to [11], wherein the biological tissue sample is a section having a thickness of 40 μm to 4 mm.
[13] The method according to any one of [7] to [12], wherein the biological tissue sample is an already embedded section.

[14] An embedded block containing a biological tissue sample,
A biological tissue sample is present on the upper surface of the embedding block,
An embedding block in which a section can be produced by cutting the upper surface of 10 μm or less.

According to another aspect of the present invention, the following inventions [2-1] to [2-7] are provided.
[2-1] preparing a cube of embedding material;
Place the biological tissue sample on one surface of the embedding material cube and place the cube on the embedding tray bottom surface with the surface facing the bottom surface of the embedding tray so that the biological sample is in contact with the bottom surface. Containing a biological tissue sample,
Placing the cassette on the cassette placement section of the embedding tray and filling the embedding tray with the molten embedding material;
Solidifying the embedding material and removing the embedding block integrated with the cassette from the embedding tray to obtain the embedding block;
A method for producing a biological tissue specimen.

[2-2] The method according to [2-1], wherein the embedding material contains wax or paraffin having a melting point of 54 to 60 ° C.
[2-3] The method according to any one of [2-1] or [2-2], further comprising a step of preparing a section from the embedded block.

[2-4] The method according to [2-3], wherein the section to be prepared has a thickness of 2 to 4 μm.
[2-5] The method according to any one of [2-1] to [2-4], wherein the biological tissue sample is a section having a thickness of 40 μm to 4 mm.

[2-6] The method according to any one of [2-1] to [2-5], wherein the biological tissue sample is an already embedded section.
[2-7] The cube of the embedding material is prepared by the method according to any one of [3-1] to [3-6], and any one of [2-1] to [2-6] The method described.

According to another aspect of the present invention, the following inventions [3-1] to [3-6] are provided.
[3-1] (1) Mold A having a U-shaped groove so as to pass through two parallel planes to a metal piece having two parallel planes and a plane perpendicular thereto, and the two parallel planes and the vertical plane Preparing a mold A and a mold B for making a metal embedding material, wherein the distance between two parallel surfaces of the mold A and the mold B is substantially the same,
(2) preparing a tray having a mold installation surface which is a plane;
(3) The mold A and the mold B are installed in combination on the mold installation surface of the tray so as to form a square shape, and a mold that can accommodate the molten embedding material is formed on the mold installation surface of the tray.
(4) filling the mold with molten embedding material and then solidifying the embedding material;
(5) removing unnecessary parts from the solidified embedding material;
(6) separating mold A and mold B to obtain an embedding material cube;
A method for manufacturing an embedding material cube.

[3-2] The tray has an edge, and the combined mold A and mold B are pressed against the edge to stabilize, and a mold that can be accommodated is formed on the mold installation surface of the tray. [3-1] The method described in 1.
[3-3] The method according to [3-1] or [3-2], wherein the mold A has a comb shape so that a plurality of cubes can be created in one operation.

[3-4] Described in [3-3], the mold A and the mold B have a shape that can be combined with the mold B so that all the groove portions of the mold A having a comb shape have a square shape. the method of.
[3-5] The method according to [3-3] or [3-4], wherein the obtained cubes have the same shape.

  [3-6] The obtained cube is a rectangular parallelepiped or a cube, one side of the surface in contact with the tray at the time of manufacture is 5 to 30 mm, the other side is in the range of 5 to 30 mm, and the height is in the range of 2 to 4 mm. [3-1] The method according to any one of [3-5].

  By using the embedding tray or method provided by the present invention, it is possible to produce a large number of sections from a small amount of biological tissue sample. According to the present invention, for example, a section sample of about 40 μm can be re-embedded and about 10 or more sections of about 2.5 μm can be prepared therefrom. Furthermore, the embedding tray and method of the present invention are suitable for preparing a large number of sections from a small amount of a biological tissue sample, for example, when a biological tissue sample is obtained using a needle.

FIG. 1 is a photograph showing an example of an embodiment of an embedding tray of the present invention. FIG. 2 is a photograph showing an example of an embodiment of the embedding tray of the present invention, and shows a state where a cassette is placed on the embedding tray. FIG. 3 is a schematic cross-sectional view in the long side direction showing an example of the embodiment of the embedding tray of the present invention. FIG. 4-1 is a schematic cross-sectional view in the short side direction showing an example of the embodiment of the embedding tray of the present invention. FIG. 4-2 is a schematic cross-sectional view in the short side direction showing an example of the embodiment of the embedding tray of the present invention. FIG. 5 is a schematic cross-sectional view in the long side direction showing an example of the embodiment of the embedding tray of the present invention, and is a schematic view showing a state in which the embedding material cube and the biological tissue sample are accommodated in the tray. FIG. 6 is a schematic cross-sectional view in the long side direction showing an example of an embodiment of the embedding tray of the present invention, and shows a state in which an embedding material cube and a biological tissue sample are accommodated in the tray and a cassette is placed thereon. It is a schematic diagram. FIG. 7 is a top view showing an example of an embodiment of a cube tray for producing an embedding material cube. A cube-forming mold can be installed and used in the wavy line. FIG. 8 is a side view showing an example of an embodiment of a cube tray for producing an embedding material cube. FIG. 9 is a top view showing an example of an embodiment of a mold A for producing an embedding material cube. FIG. 10 is a front view showing an example of an embodiment of a mold A for producing an embedding material cube. FIG. 11 is a top view showing an example of an embodiment of a mold B for producing an embedding material cube. FIG. 12 is a photograph showing an example of a cassette that can be used in the present invention. FIG. 13 is a photograph showing an example of a conventional embedding tray.

Hereinafter, an embodiment of the embedding tray of the present invention will be described as an example. FIG. 1 shows a photograph of an embedding tray according to the present invention. FIG. 2 shows a picture of the embedding tray on which the cassette is placed.
The embedding tray of the present invention is used for preparing a biological tissue specimen, particularly for preparing an embedding block containing a biological tissue sample in an embedding material. The embedding tray of FIG. 1 and FIG. 2 is provided with a cassette mounting part for mounting a cassette. As shown in FIGS. 3 and 4, the cassette mounting portion (12, 15) is located above the embedding material accommodating portion formed by the side wall 14 and the bottom surface 13.

  The embedding tray of the present invention is characterized in that the shape of the bottom surface of the embedding material container has a very flat shape with little error. The embedding block produced by such an embedding tray has a flat shape with very little error on the surface in contact with the bottom surface, and the amount of cutting the upper surface of the embedding block before making a section from the embedding block It is possible to create a large number of slices with a reduced number.

  In one aspect of the present invention, the bottom surface of the embedding material container has a shape partially including a flat surface having irregularities of, for example, ± 6 μm or less, specifically ± 4 μm or less, more specifically ± 2 μm or less. Have. In one aspect of the present invention, the bottom surface of the embedding material container has a shape partially including a flat surface having irregularities of, for example, ± 6 μm or less, ± 5 μm or less, ± 4 μm or less, ± 3 μm or less, or ± 2 μm or less. Have.

  The planar shape with few irregularities may be part or all of the bottom surface. For example, the portion in contact with the side wall may be chamfered, and such processing is advantageous in the step of removing the embedding block from the embedding tray. In one aspect of the present invention, the planar shape with less unevenness is 5% or more of the entire bottom surface, for example, 10% or more, specifically 20% or more, and more specifically 30% or more. In one embodiment, the area of the planar shape with less unevenness is an area sufficient for the biological tissue sample to be accommodated in the embedding material accommodating portion in contact with the plane. In another aspect, the area of the planar shape with less unevenness is an area sufficient for the embedding material cube to be accommodated in the embedding material accommodating portion in contact with the plane.

  In one aspect of the present invention, at least a part of the bottom surface of the embedding material accommodating portion is parallel to a surface specified by a point where the cassette mounting portion is in contact with the cassette, It has a shape including a part of a plane in which the variation of the distance from the surface specified by the point where the mounting portion contacts the cassette is, for example, ± 6 μm or less, specifically ± 4 μm or less, more specifically ± 2 μm or less. . In one embodiment of the present invention, the variation of the distance is, for example, ± 6 μm or less, ± 5 μm or less, ± 4 μm or less, ± 3 μm or less, or ± 2 μm or less. Here, the cassette used in the present invention has a plate shape, and one surface is in contact with the embedding block when integrated with the embedding block, and when the embedding block is cut with a microtome. Secures the cassette part. The cassette placing portion has a shape that allows the cassette to be placed stably. The point at which the cassette placement unit contacts the cassette is at least three contact points, and the surface specified by the point at which the cassette placement unit contacts the cassette is a surface assumed as a surface including the contact point. There may be three or more points of contact, and the contacting part may be linear or planar.

  In one aspect of the present invention, the bottom surface of at least a part of the embedding material container is parallel to the bottom surface of the embedding tray, and the variation in the distance from the bottom surface of the embedding tray is, for example, ± It has a shape partially including a plane that is 6 μm or less, specifically ± 4 μm or less, and more specifically ± 2 μm or less. In one embodiment of the present invention, the variation in the distance is, for example, ± 6 μm or less, ± 5 μm or less, ± 4 μm or less, ± 3 μm or less, or ± 2 μm or less. In one embodiment, the bottom surface of at least a part of the embedding material container is parallel to a surface specified by the bottom surface of the embedding tray when the embedding tray is placed on the table, The variation of the distance from the surface specified by the bottom surface of the embedding tray is, for example, ± 6 μm or less, specifically ± 4 μm or less, more specifically ± 2 μm or less.

  In one aspect of the present invention, the cassette mounting portion has a bottom surface 12 as shown in FIGS. 3 and 4, and the cassette mounting portion bottom surface is parallel to at least a part of the bottom surface of the embedding material accommodating portion, Variation in the distance between the surface formed by the bottom surface of the cassette mounting portion and the bottom surface of at least a part of the embedding material container is, for example, ± 6 μm or less, specifically ± 4 μm or less, more specifically ± 2 μm or less. It has a shape partially including a certain plane. In one embodiment of the present invention, the variation in the distance is, for example, ± 6 μm or less, ± 5 μm or less, ± 4 μm or less, ± 3 μm or less, or ± 2 μm or less. The surface formed by the cassette mounting portion bottom surface is a surface assumed as a surface including the cassette mounting portion bottom surface.

  The cassette mounting part may be extended to the entire periphery of the upper opening of the embedding material container, may be extended only to the long side of the upper opening, or the short side of the upper opening You may be extended only in the part. The embedding tray of the present invention may include a sandwiching plate for fitting the cassette onto the embedding material accommodating portion.

The material for producing the embedding tray is not particularly limited, but from the viewpoint of ensuring a high level of flatness, resins (for example, polyester, polyamide, polycarbonate, polyacetal, ABS resin, urea resin, phenol resin, melamine resin, silicon) Resin, epoxy resin, polyethersulfone (PES), polyetherimide (PEI), polyamideimide (PAI), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE),
Polyphenylene sulfide (PPS), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), diallyl phthalate (PDAP), cycloolefin polymer (COP), cycloolefin copolymer (COC), polytetrafluoroethylene, etc.) and metals (for example, , Stainless steel, titanium, nickel, iron, gunmetal, etc.). As the resin, a heat-shrinkable resin such as polytetrafluoroethylene can be preferably used. From the viewpoint of resistance to wear and scratches, in one embodiment, the tray and mold can be made of metal (eg, stainless steel) or the like. Although the manufacturing method of an embedding tray is not specifically limited, For example, the embedding tray which has a highly accurate shape can be manufactured by methods, such as cutting out from a metal block (for example, stainless steel).

The error (unevenness) of the planar shape of the embedding tray of the present invention can be confirmed using, for example, a micrometer, a small level, a laser level, or the like.
The planar shape with a small error on the bottom surface of the embedding material accommodating portion only needs to have a sufficient area for accommodating the biological tissue sample. The biological tissue sample is accommodated in contact with the bottom surface of the embedding material accommodating portion as much as possible. As a result, in the prepared block, the biological tissue sample exists in a form exposed on the upper surface of the block that was in contact with the bottom surface of the embedding material container, and the chamfering and / or cutting of the section from the upper surface of the block is efficient. Can be done automatically.

  The side wall of the embedding material container may have an inclination so that the upper opening of the embedding material container is wider than the bottom surface. Moreover, the upper part of the embedding material container may have an inclination different from that of the lower part. In one embodiment, an angle formed by the side wall forming the upper part of the embedding material container and the bottom surface of the embedding material container (90 °). The larger angle) is larger than the angle (90 ° or more angle) formed between the side wall forming the lower part of the embedding material container and the bottom surface of the embedding material container.

  The embedding tray of the present invention may have a handle on the side surface (FIG. 1). The handle portion can be attached with a screw or an adhesive after the embedding tray is manufactured. The material of the handle portion is not particularly limited, but may be stainless steel, aluminum or the like. The handle portion can be used when gripping the embedding tray during the embedding block manufacturing operation, and is useful for efficient operation. Further, the embedding tray may have legs on the bottom surface. The leg can be attached with an adhesive or the like after the embedding tray is manufactured, and may be provided in a removable form. By providing the legs, the embedding block manufacturing operation can be performed without the bottom surface of the embedding tray being in direct contact with the work table or the like.

  In one embodiment of the present invention, an embedding material cube can be used for accommodating the biological tissue sample in the embedding material accommodating portion. As shown in FIG. 5, the biological tissue sample 22 is attached to one surface of the embedding material cube 21, and the cube is accommodated so that the surface is in contact with the bottom surface 13 of the embedding material accommodating portion. Thereby, it can be ensured that the biological tissue sample comes into contact with the bottom surface of the embedding material container. Further, as shown in FIG. 6, it is desirable that the upper surface of the cube is substantially at the same position as the lower surface of the cassette when the cassette is placed. In such an embodiment, when filling the embedding material container with the molten embedding material, the lower surface of the cassette can prevent the cube from being lifted, and the position and orientation of the biological tissue sample can be prevented from changing. .

  The embedding material cube can be manufactured using, for example, the embedding material cube tray of FIGS. 7 and 8, and the mold A of FIGS. 9 and 10 and the mold B of FIG. The mold A and the mold B are combined and installed on the mold installation surface of the tray. At this time, as indicated by a broken line in FIG. 7, the rod-shaped mold B is arranged so that the concave portion of the mold A forms a cube mold. After that, the mold formed by the mold A and mold B and the tray installation surface is filled with the molten embedding material, and after the embedding material is solidified by cooling, a sharp metal plate or the like matching the unnecessary portion with the mold And remove it from the mold to get the embedded material cube. Since the surface of the embedding material cube influences the state of the surface of the obtained embedding block, it is preferable that the cube tray or the surface of the mold is a flat surface with less error. Materials for producing cube trays and molds are not particularly limited, but from the viewpoint of ensuring high flatness, resins (for example, polyester, polyamide, polycarbonate, polyacetal, ABS resin, urea resin, phenol resin, melamine resin) , Silicone resin, epoxy resin, polyethersulfone (PES), polyetherimide (PEI), polyamideimide (PAI), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS) , Polyethylene terephthalate (PET), polybutylene terephthalate (PBT), diallyl phthalate (PDAP), cycloolefin polymer (COP), cycloolefin copolymer (COC), polytetrafluoroethylene Etc.) or a metal (e.g., stainless steel, titanium, nickel, iron, gunmetal) can be used. As the resin, a heat-shrinkable resin such as polytetrafluoroethylene can be preferably used. In one aspect, from the viewpoint of resistance to abrasion and scratches, and when the embedding material is poured in a state in which the mold A and the mold B are combined, it is fixed by its own weight on the mold installation surface of the tray and is easy to handle. The trays and molds for making the embedding material cubes are made of metal (eg, stainless steel).

  In the tray for embedding material cubes in FIG. 7, the combination of the mold A and the mold B can be stabilized by combining the mold A and the mold B in the shape indicated by the broken line and pressing the mold A and the mold B against the edges. The height of the edge part from the embedding material cube type | mold installation surface is formed lower than the height when the type | mold A and the type | mold B are installed in the embedding material cube type | mold installation surface. After the embedding material is solidified, it is easy to take out the cube from the mold, and the cube having a well-formed shape can be taken out from the mold. In addition, regarding the embedding material cube mold in which the mold A and the mold B are combined, the cube can be easily removed from the comb-shaped mold A by first removing the rod-shaped mold B when taking out the cube after solidification. The embedding material cube obtained in this way has a precise shape corresponding to the shape of the mold.

  In one embodiment of the present invention, the embedding material cube has a cubic or cuboid shape, one side of the surface in contact with the tray during manufacture is in the range of 5 to 30 mm, the other side is in the range of 5 to 30 mm, and the height is 2 mm. It is in the range of ˜4 mm.

  When the embedding material cube is installed on the bottom surface of the embedding material accommodation portion, the embedding material cube has a shape equal to or lower than the distance between the bottom surface of the embedding material accommodation portion and the bottom surface of the cassette placed on the cassette placement portion. The mold for producing the embedding material has a shape for producing such a cube. In one aspect of the present invention, the biological tissue sample is attached to one surface of the embedding material cube, the cube is placed with the surface facing the embedding material accommodation portion, and the biological tissue sample is in contact with the bottom surface of the embedding material accommodation portion. To accommodate. As a preferred embodiment, the height of the biological tissue sample and the embedding material cube when the biological tissue sample is attached to one surface of the embedding material cube is the same as that of the cassette placed on the cassette placing portion from the bottom of the embedding material accommodating portion. It has the same height as the bottom surface or a lower shape.

  The embedding material used in the present invention is not particularly limited as long as it is usually used. For example, a hydrocarbon having a melting point of 45 to 60 ° C., specifically, soft paraffin 45 to 52 ° C. or hard paraffin 54 to 58 ° C. For example, wax or paraffin can be used and may further contain additives. As the embedding material, for example, Surgipath Paraplast (registered trademark, manufactured by Leica), Paraplast Plus (registered trademark, manufactured by Leica) and the like can be used. The embedding material cube can be produced from the embedding material described above.

Hereinafter, the present invention will be described in more detail using reference examples and examples, but the present invention is not limited to these examples.
Production of embedding tray The embedding tray was produced by cutting stainless steel with Makino slice GF6. The bottom surface of the produced embedding material container has a rectangular shape of 30 × 25 mm, the portion in contact with the cassette mounting portion has a rectangular shape of 32 × 27 mm, the depth is 4 mm, and the cassette mounting portion has a long side direction. And a bottom surface having a width of about 1.5 mm in the short side direction. The error of the bottom surface of the embedding material container and the cassette mounting part is measured by setting Digimicro MFC-101A (Nikon) on DIGIMICRO STAND MS-11C (Nikon), ± 1.90 μm or less (n = 5, tray bottom 9 Standard deviation of measurement for each location), error between cassette placement part and embedding tray bottom is ± 1.74 μm or less (n = 5, standard deviation of difference between 3 left and right cassette installation parts and bottom center) It was confirmed that. A handle portion was formed on the side surface of the embedding tray using an aluminum angle (10 × 10 mm), and a screw (2.5 mmφ × 4 mm) was used on the side surface of the embedding plate and joined to both sides. The use of aluminum increases heat dissipation and retainability, thereby improving the operability of the experimenter when moving the embedding dish heated to 60 ° C. from the hot plate to the cold plate.

Production of embedded sample section (40 μm) The following samples were obtained as purchases as formalin-fixed biological tissue samples for preparation of embedded sample sections (40 μm).

(1) ILS 38167PT4 (breast cancer, 10mm x 10mm),
(2) ILS 36831PT1 (colorectal cancer, about 10mm x 16mm),
(3) ILS 38764PT2 (liver cancer, approximately 13mm x 13mm),
(4) Indivumed RW878-Tp12 (breast cancer, 6mm x 8mm)
(5) Indivumed H1111-Tp314 (liver cancer, 7mm x 14mm),
(6) Indivumed A4452-Tp13 (Colon cancer, 4mm x 9mm)
About 40 μm paraffin-embedded sample section (FFPE sample section) housed in a banking seal was prepared from the above sample using BioBank KIT (Pasology Laboratories, Inc.). The specific method was performed according to the instruction manual of BioBank KIT.

Paraffin (TissuePrep (registered trademark, manufactured by Fisher Scientific, model number: T580), melting point: 58 ° C.) was used as an embedding material from the section seal . The paraffin cube was manufactured by producing a paraffin cube mold matched to the shape of the paraffin cube to be used and a tray for preparing the paraffin cube. One side of the banking seal was removed, and the banking seal was placed on the hot plate kept at 65 ° C. with the section on top, and heated for about 15 seconds until the paraffin surrounding the tissue was dissolved. One side of the paraffin cube (15 × 15 × 3.9 mm) was slightly heated with a hot plate (65 ° C.), and the paraffin cube was crimped onto the banking seal tissue on a metal plate. At that time, it was confirmed that the paraffin was slightly melted. The metal plate was moved to a cold plate (−5 ° C.) and cooled for about 5 minutes. The paraffin cube was removed from the metal plate with the banking seal attached, and the banking seal was peeled off from the paraffin cube to obtain a paraffin cube having a 40 μm section attached.

The tissue block production cassette SFJ-4187-01 made by Sakura Finetech Japan was used as the production cassette for embedding blocks by re-embedding . Paraffin (thickness cut to 80 μm) was placed in the embedding tray on the hot plate and melted. It was placed on the bottom of the embedding tray so that the surface with the section of the paraffin cube was pressed down slightly. A cassette was set on the cassette mounting section, and molten paraffin was injected. The embedding tray was moved to a cold plate and cooled for about 12-13 minutes until the paraffin was fully solidified. Then, the cassette was grasped and the embedding block was removed from the embedding tray.
Section preparation by microtome The following instruments were used.

Sliding microtome: MICROM HM430;
Extension board: SAKURA PARAFFIN STRETCHER PS-52;
Glass slide: Fisher 12-550-15 Superfrost Plus, Fisher Scientific FRONTIER (Cat no .: 12-550-15);
Cover glass: Matsunami Glass 24 × 55mm;
Marking die: Pharma Green 302-401-1;
Hot water bath: Yamato BM400.

  After scraping off the unnecessary paraffin part of the block, attach the paraffin block to the microtome, adjust the mounting angle of the paraffin block, and make the embedded block face up so that the blade of the microtome and the top surface of the paraffin block are parallel. went. The embedding block with the surface facing down on the cooling ice produced in the pad was allowed to stand for about 6 minutes. The block was sliced with a microtome (microtome scale of 1.5 μm, using replaceable blade A35). The section was pinched with tweezers and transferred to a ball filled with water. The section was transferred to a slide (FRONTIER), moved to a hot water bath (41 ° C.), and stretched. The section was again taken on a slide (Superfrost Plus) and dried with an extension plate (41 ° C.). A plurality of sections were prepared by the same operation.

  The number of sections (2.5 μm) obtained from each biological tissue sample (40 μm section) used is shown in the following table.

  Out of 6 biological tissue samples, sections could be prepared by re-embedding. The average value of the number of slices prepared was 11.5. The thickness required for the chamfering when slicing was 10 μm or less. It has been confirmed that the method using the embedding tray of the present invention is useful even when the amount of biological tissue such as a biopsy sample obtained using a needle is very limited.

DESCRIPTION OF SYMBOLS 11 ... Embedded tray 12 ... Cassette mounting part bottom face 13 ... Embedded material accommodating part bottom face 14 ... Embedded material accommodating part side wall 15 ... Cassette mounting part side wall 16 ... Embedding tray bottom face 17 ... Embeding tray side surface 21 ... Packaging Embedded material cube 22 ... Biological sample 23 ... Cassette upper surface 24 ... Cassette lower surface 31 ... Tray edge for embedded material cube 32 ... Embedded material cube type installation surface

Claims (14)

An embedding tray for preparing a biological tissue specimen, comprising an embedding material container formed by a bottom surface and side walls erected around the bottom, and a cassette mounting unit at the top of the embedding material container,
An embedding tray in which at least a part of the bottom surface of the embedding material accommodating portion is a flat surface having an unevenness of within ± 3 μm.   The embedding tray according to claim 1, wherein a bottom surface of at least a part of the embedding material accommodating portion is parallel to a surface specified by a point where the cassette placing portion contacts the cassette.   The cassette mounting portion includes a bottom surface, and the variation in the distance between the surface formed by the cassette mounting portion bottom surface and the bottom surface of at least a part of the embedding material housing portion is within ± 4 μm. Embedding tray.   The embedding tray according to any one of claims 1 to 3, wherein the at least part of the bottom surface is 10% or more of the entire bottom surface.   The embedding tray according to any one of claims 1 to 4, wherein the side wall has an inclination so that the upper opening of the embedding material accommodating portion is wider than the bottom surface.   The embedding tray according to any one of claims 1 to 4, wherein the embedding tray is made of a material selected from stainless steel, titanium, gun metal, and heat-shrinkable resin. Storing the biological tissue sample in the embedding tray according to any one of claims 1 to 6 so that the biological tissue sample is in contact with the bottom surface of the embedding material storage unit,
Placing the cassette on the cassette placement section of the embedding tray and filling the embedding tray with the molten embedding material;
Solidifying the embedding material, and removing the embedding block integrated with the cassette from the embedding tray to obtain the embedding block;
A method for producing a biological tissue specimen.   The method according to claim 7, wherein the embedding material contains wax or paraffin having a melting point of 54 to 60 ° C.   Place the biological tissue sample on one surface of the embedding material cube and place the cube on the embedding tray bottom surface with the surface facing the bottom surface of the embedding tray so that the biological sample is in contact with the bottom surface. The method according to claim 7 or 8, wherein a biological tissue sample is accommodated.   The method according to any one of claims 7 to 9, further comprising the step of preparing a section from the embedded block.   The method of Claim 10 that the thickness of the slice to produce is 2-4 micrometers.   The method according to any one of claims 7 to 11, wherein the biological tissue sample is a section having a thickness of 40 µm to 4 mm.   The method according to any one of claims 7 to 12, wherein the biological tissue sample is an already embedded section. An embedded block containing a biological tissue sample,
A biological tissue sample is present on the upper surface of the embedding block,
An embedding block in which a section can be produced by cutting the upper surface of 10 μm or less.