WO2013088693A1 - Staining kit and staining method - Google Patents

Staining kit and staining method Download PDF

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WO2013088693A1
WO2013088693A1 PCT/JP2012/007882 JP2012007882W WO2013088693A1 WO 2013088693 A1 WO2013088693 A1 WO 2013088693A1 JP 2012007882 W JP2012007882 W JP 2012007882W WO 2013088693 A1 WO2013088693 A1 WO 2013088693A1
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staining
tissue
biological tissue
methylene blue
biological
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PCT/JP2012/007882
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French (fr)
Japanese (ja)
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正規 長谷川
金田　次弘
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財団法人ヒューマンサイエンス振興財団
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6827—Total protein determination, e.g. albumin in urine
    • G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green

Definitions

  • the present invention relates to a biological tissue staining method and a staining kit used for the staining method.
  • Pathological diagnosis includes histopathological diagnosis using a permanent specimen and rapid pathological diagnosis during operation.
  • Intraoperative rapid pathological diagnosis determines whether the tumor is benign or malignant, whether the tumor has metastasized, whether the tumor remains at the resected margin, and the like.
  • the lesion tissue collected during the operation is rapidly frozen together with the embedding agent, sliced with a special device called a cryostat, and attached to a glass slide.
  • Frozen tissue slices are almost colorless, and various components are used to dye specific components to observe fine structures. In general, HE staining is used.
  • the mounting medium is dropped and covered with a cover glass to make a rapid pathological specimen.
  • the fine structure in the tissue can be observed with a transmitted light type (bright field) biological microscope.
  • the collected tissue is fixed with a fixative such as formalin.
  • Fixation is mainly performed by chemically cross-linking tissue and intracellular components.
  • tissue components such as proteins / nucleic acids / sugars are retained, and deterioration of tissue staining is prevented.
  • surgical tissue material is cut out by a pathologist. That is, a necessary part is selected by visual observation for preparing a pathological tissue specimen from a surgical tissue material, and is cut into a block shape of, for example, a size of 2 cm ⁇ 3 cm to 4 cm and a thickness of 0.5 cm. Because of observation with the naked eye, the tumor body and spread must be judged from the subtle differences in the properties of the lesion. When it cannot be visually recognized by the naked eye observation, it is necessary to block all the sites that may have a lesion, and the number of blocks becomes very large. The fixed tissue piece is non-uniform in hardness and is too soft to cut into thin sections of several ⁇ m.
  • an embedding agent such as paraffin is then infiltrated into the tissue, thereby producing a paraffin-embedded tissue block.
  • This process is composed of, for example, dehydration / defatting ⁇ removal of dehydrating agent and substitution with a companion agent ⁇ paraffin infiltration.
  • tissue is sliced.
  • a paraffin-embedded tissue block is sliced with a microtome to a thickness of microns.
  • the sliced slices are floated on a warm water surface, attached to a glass slide and dried.
  • Thin slices are almost colorless, and various components are used to dye specific components to observe fine structures. In general, HE staining is used.
  • the mounting medium is dropped and covered with a cover glass.
  • a fine structure in the tissue can be observed with a transmitted light (bright field) biological microscope.
  • cryostat the preparation of frozen specimens for rapid pathological diagnosis during surgery requires a special device called a cryostat, and the preparation itself requires high skill and skill. Since adipose tissue does not penetrate the embedding agent, frozen sections cannot be prepared. In addition, since a hard tissue such as a bone tissue cannot be cut, a frozen section cannot be prepared.
  • the present invention has been made in view of such problems, and can easily and rapidly perform pathological diagnosis during surgery without preparing frozen sliced sections even in a facility without a cryostat.
  • Staining kits and stains that allow rapid pathological diagnosis during surgery even in tissues where frozen sections cannot be prepared, such as pathological diagnosis at the stage of the excision process for preparation of surgical material permanent pathological tissue specimens It aims to provide a method.
  • a staining kit is a staining kit for staining a surface and a split surface of a biological tissue, wherein methylene blue for staining the biological tissue, hematoxylin for staining the biological tissue, and the methylene blue are used.
  • the staining method according to the present invention is a staining method for staining the surface and the split surface of a biological tissue, the first staining step for staining the biological tissue with methylene blue, and the organism stained with the methylene blue.
  • Decolorization step of decolorizing excessive staining in the tissue with a reducing agent second staining step of further staining the biological tissue with hematoxylin, and fading of the pigment in the biological tissue stained with the methylene blue as an oxidizing agent
  • a fading prevention step for preventing the discoloration is a staining method for staining the surface and the split surface of a biological tissue, the first staining step for staining the biological tissue with methylene blue, and the organism stained with the methylene blue.
  • the surface and split surface of unfixed tissue material submitted during surgery can be directly stained, and the normal tissue construction, tumor tissue construction, cellular findings, and nuclear findings can be visually confirmed. Therefore, in a facility without a cryostat or a facility without a skilled laboratory technician for preparing a frozen specimen, an intraoperative rapid pathological diagnosis can be easily performed. Intraoperative rapid pathological diagnosis is possible even in tissues where frozen sections such as adipose tissue and bone tissue cannot be prepared.
  • the surface and the split surface of the fixed biological tissue are directly stained at the stage of the cutting process for preparing the surgical material permanent pathological tissue specimen, and the normal tissue construction, tumor tissue construction, cytological findings are obtained. , And nuclear findings can be visually recognized. For this reason, it can cut out appropriately and leads to more accurate pathological diagnosis including reduction of the number of blocks and staging.
  • tumor diagnosis, progress determination, and stump retrieval can be performed at the stage of the cutting process for preparing a surgical material permanent pathological tissue specimen. Further, since the range of three-dimensional staining such as the lumen and the luminal surface of the cyst is wider than that of a sliced slice specimen, more information can be obtained.
  • a tissue construction including unfixed biological tissue material and cell findings and nuclear findings on the surface and face of the fixed biological tissue material. Can be observed.
  • FIG. 1 is a biological micrograph of a papillary thyroid cancer tissue in Example 1.
  • FIG. 4 is a biological micrograph of gastric cancer tissue in Example 2.
  • FIG. 4 is a biological micrograph of a blue skin nevus tissue in Example 3.
  • FIG. 4 is a biological micrograph of salivary adenoid cystic cancer tissue in Example 4.
  • FIG. 6 is a biological micrograph of chicken gizzard tissue in Example 5.
  • FIG. It is a stereoscopic microscope photograph figure of the chicken bone tissue in Example 6.
  • It is a biological-microscope photograph figure of the pig tongue tissue in Example 7.
  • It is a stereoscopic microscope photograph figure of the colon cancer tissue in a reference example.
  • the staining kit according to the present embodiment is a staining kit that stains a split surface of a biological tissue.
  • the biological tissue is, for example, a pathological tissue
  • the cleavage plane of the biological tissue is a cutting formed by dividing by a cutting blade along a line passing through a predetermined portion (for example, the central portion) of the biological tissue (for example, a tumor). Surface. It is also possible to stain the surface together with the split surface of the biological tissue.
  • the biological tissue to be stained can be either a biological tissue isolated from a living body or a biological tissue during surgery, and can be either a fixed or unfixed biological tissue.
  • the staining kit according to the present embodiment includes methylene blue for staining biological tissue, hematoxylin for staining biological tissue, and a reducing agent for decolorizing excessive staining in biological tissue stained with methylene blue, And an oxidizing agent for preventing fading of a pigment in a biological tissue stained with methylene blue.
  • methylene blue is used for the first staining of biological tissue.
  • the methylene blue stains mainly the cytoplasm of epithelial cells of the biological tissue. Although cell nuclei may be stained, dyeing unevenness often occurs and it is difficult to obtain clear nuclear staining.
  • Methylene blue includes methylene blue derivatives as long as they can stain biological tissues.
  • the methylene blue solvent is an aqueous solvent or an alcohol such as ethanol.
  • the pH of methylene blue is, for example, 3.0 to 7.0.
  • the concentration of methylene blue is not particularly limited as long as biological tissue can be stained, but it is preferably, for example, 0.5 g / L to 10 g / L.
  • the surface of the gastric mucosa or the surface of the large intestine mucosa, which is not a cleavage plane is stained with methylene blue in the endoscopic region, and the concentration of methylene blue is 0.1 g / L to 0.5 g / L. . Therefore, compared with the methylene blue staining used in the endoscope region, the staining concentration of methylene blue staining of 0.5 g / L to 10 g / L in this embodiment is higher.
  • the reducing agent is used after staining biological tissue with methylene blue.
  • methylene blue mainly stains the cytoplasm of epithelial cells, but the staining properties differ slightly depending on the site of the biological tissue.
  • This reducing agent is then used to standardize the degree of staining. That is, by staining the cytoplasm of epithelial cells with methylene blue and then decolorizing excessive staining of the tissue stroma with a reducing agent, the background staining of the stroma can be reduced while maintaining the staining state of the epithelial cells. As a result, good contrast can be obtained.
  • the reducing agent is not particularly limited as long as interstitial background staining can be reduced, and can be selected from, for example, oxalic acid, formic acid, hydrazine, ascorbic acid, and salts thereof.
  • the concentration of the reducing agent can be appropriately selected from the viewpoint of reducing the staining of the stroma, and can be, for example, 1 ⁇ 10 ⁇ 4 N to 1 ⁇ 10 ⁇ 2 N.
  • Hematoxylin is used for the second staining of biological tissue. With this hematoxylin, the nuclei of all cells of the biological tissue are stained. Hematoxylin also includes derivatives of hematoxylin as long as it can stain biological tissues.
  • the solvent for hematoxylin is an aqueous solvent, and the pH is, for example, 2.5 to 4.0.
  • Hematoxylin is not particularly limited as long as it can stain a biological tissue.
  • Mayer hematoxylin, gil hematoxylin, etc. are used, and preferably, caratach hematoxylin is used.
  • caratach hematoxylin contains glycerin, which improves the staining of cell nuclei and shortens the staining time.
  • Hematoxylin other than caratach hematoxylin can be preferably used by containing 10 to 20% (v / v) glycerin, which can improve staining of the cell nucleus and shorten the staining time.
  • an oxidizing agent is used to prevent discoloration of the pigment in the biological tissue stained with methylene blue. Since methylene blue fades quickly when irradiated with light or by the action of a reducing agent contained in the tissue, the use of an oxidizing agent prevents the methylene blue from fading and maintains clear staining. Although the biological tissue can be sufficiently observed even with the purple color of hematoxylin, the fading of methylene blue is prevented by the oxidizing agent, and the blue color is formed by changing hematoxylin into hematein.
  • the oxidizing agent is not particularly limited as long as the fading of methylene blue is prevented.
  • nitric acid, hydrochloric acid, sulfuric acid, and salts thereof can be selected.
  • the concentration of the oxidizing agent can be appropriately selected from the viewpoint of preventing the fading of methylene blue, and can be, for example, 1 ⁇ 10 ⁇ 4 N to 1 ⁇ 10 ⁇ 2 N.
  • the staining method according to the present embodiment stains a split surface of a biological tissue. It is also possible to stain the surface together with the split surface of the biological tissue.
  • the staining method according to the present embodiment includes a first staining step for staining a biological tissue with methylene blue, a decolorization step for decolorizing excessive staining in the biological tissue stained with methylene blue with a reducing agent, Furthermore, it has the 2nd dyeing
  • a sufficient amount of methylene blue in the above-described staining kit is dropped onto a fixed or unfixed biological tissue to stain mainly the cytoplasm of epithelial cells.
  • the dyeing by dropping methylene blue requires, for example, 0.1 to 5 seconds.
  • the methylene blue may be dripped a plurality of times at predetermined intervals in order to sufficiently perform methylene blue staining. Subsequently, the biological tissue is washed with water.
  • the excessive staining of the tissue is decolorized using the reducing agent in the staining kit described above.
  • This provides good staining contrast.
  • Decolorization using this reducing agent requires, for example, 0.1 to 5 seconds. Subsequently, the biological tissue is washed with water.
  • a sufficient amount of hematoxylin in the above-described staining kit is dropped onto a biological tissue to stain the cell nuclei of all cells. For example, 1 to 30 seconds are required for dyeing by dropping hematoxylin. Subsequently, the biological tissue is washed with water.
  • methylene blue color fading is prevented by using the oxidizing agent in the dyeing kit described above. Further, fading of methylene blue is prevented, and at the same time, hematoxylin can be colored.
  • the time required for staining the split surface of the biological tissue by the staining method in the present embodiment is, for example, 10 seconds to 40 seconds, and rapid pathological diagnosis is possible.
  • the stained biological tissue is encapsulated with a transparent gel-like substance and subjected to macro observation, stereoscopic microscope observation, biological microscope observation, or the like. Further, a cover glass may be placed on the gel substance for observation.
  • a transparent gel-like substance it is also possible to use a mixture of a transparent gel substance in the oxidizing agent, and in such a case, it becomes possible to include the stained biological tissue at the same time as preventing the fading of methylene blue. Therefore, pathological diagnosis becomes quicker and easier. Observation can also be facilitated by immersing the stained biological tissue in water and fixing it with a pin or the like without being included in a transparent gel substance.
  • methylene blue is used in the first staining step and hematoxylin is used in the second staining step.
  • the staining method according to this embodiment is not limited to such an order of use.
  • the biological tissue is stained with hematoxylin as the first staining step, and then the biological tissue is stained with methylene blue as the second staining step.
  • the staining of cells in the biological tissue stained with methylene blue in the decolorization step is decolorized with a reducing agent, and finally the color fading of the biological tissue stained with methylene blue in the anti-fading step is oxidized It is also possible to prevent this.
  • This staining method includes a staining step of staining a biological tissue with methylene blue, a decolorization step of decolorizing excessive staining of the tissue in the biological tissue stained with methylene blue with a reducing agent, and a biological tissue stained with methylene blue. And a fading prevention step for preventing fading of the dye in the case of using an oxidizing agent.
  • a staining step of staining a biological tissue with methylene blue a decolorization step of decolorizing excessive staining of the tissue in the biological tissue stained with methylene blue with a reducing agent, and a biological tissue stained with methylene blue.
  • a fading prevention step for preventing fading of the dye in the case of using an oxidizing agent.
  • the concentration of methylene blue may be lower than that in the above-described staining using methylene blue and hematoxylin, for example, 0.5 g / L to 5 g / L.
  • Example 1 Surgical material thyroid tumor tissue was used as the biological tissue. Surgical material Thyroid tumor tissue was fixed with formalin and split with a cutting blade in parallel at 5 mm intervals by vertical cutting to form a split surface. Next, a surgical material thyroid tumor tissue section containing the tumor was sliced to a thickness of 1 mm. Next, a sufficient amount (about 1 ml) of methylene blue (diluted 30-fold of a methylene blue stock solution of a vendor code 275-07895 manufactured by Wako Pure Chemical Industries, Ltd.) was dripped over the entire surface and dyed for 2 seconds. Next, the tissue was moved up and down in the water tank.
  • methylene blue diluted 30-fold of a methylene blue stock solution of a vendor code 275-07895 manufactured by Wako Pure Chemical Industries, Ltd.
  • tissue slice was moved up and down while observing in a 0.001 N oxalic acid water bath until the split surface had an appropriate color.
  • tissue was moved up and down in the water tank.
  • a 2x caratach hematoxylin solution (Wako Pure Chemical Industries, Ltd., sales code 039-17705) was dropped in a sufficient amount (about 1 ml) to reach the entire face and stained for 10 seconds.
  • the composition of the 2x caratach hematoxylin solution was 2.0 g of hematoxylin, 50 g of potassium alum, 0.4 g of sodium iodate, 200 mL of glycerin, and 800 mL of distilled water.
  • tissue was moved up and down in the water tank. Next, a sufficient amount (about 1 ml) of 0.001 N nitric acid solution was dripped over the entire cleavage surface for each tissue section.
  • the tissue was placed on a slide glass with the stained split surface to be observed facing up, and a sufficient amount of OCT compound (Tissue-Tek (registered trademark): SAKURA (registered trademark)) was coated on the split surface. It was covered with a cover glass (0.17 mm) and observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 1, papillary thyroid cancer cell nuclei, nuclear sulcus, and intranuclear inclusions were visible.
  • Example 2 Staining was carried out in the same manner as in Example 1 except that a formalin-fixed surgical material gastric tumor tissue was used as the biological tissue, and observation was performed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 2, poorly differentiated adenocarcinoma cells and signet ring cells were visible.
  • Example 3 Except for using a formalin-fixed surgical material skin tumor tissue as a biological tissue, staining was performed in the same manner as in Example 1 and observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 3, epidermal cell nuclei, intradermal capillaries, intradermal inflammatory cells, and blue nevi cells were visible.
  • Example 4 Except for using a formalin-fixed surgical material salivary gland tumor tissue as a biological tissue, staining was performed in the same manner as in Example 1 and observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 4, the sieve structure of the salivary gland adenoid cystic cancer tissue and the cancer cell nucleus were visible.
  • Example 5 Staining was carried out in the same manner as in Example 1 except that chicken gizzard tissue was used as the biological tissue and an unfixed tissue was used, and observation was performed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 5, glandular epithelial structures, glandular epithelial cell nuclei, and submucosal fibroblasts were visible.
  • Example 6 Chicken bone tissue was used as the biological tissue. Staining was performed in the same manner as in Example 1 except that an unfixed tissue was used, the slice thickness was set to 10 mm, and the sample was observed on a sample stage of a stereomicroscope, and observed with a stereomicroscope. The results are shown in FIG. As shown in FIG. 6, bone cells and bone marrow cells were visible.
  • Example 7 Staining was carried out in the same manner as in Example 1 except that porcine tongue tissue was used as a biological tissue and an unfixed tissue was used, and the tissue was observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 7, stratified squamous cell nuclei and cell walls were visible.
  • Surgical material colon tumor tissue was used as the biological tissue.
  • the colon tumor tissue was fixed with formalin, and the tumor component was split with a cutting blade in parallel at 5 mm intervals by vertical cutting to form a split surface.
  • a sufficient amount (about 2 ml) of methylene blue (diluted 30-fold of a methylene blue stock solution of a manufacturer's code 275-07895 manufactured by Wako Pure Chemical Industries, Ltd.) was dropped over the entire face and dyed for 3 seconds.
  • the tissue was moved up and down in the water tank.
  • the tissue slice was moved up and down while observing in a 0.001 N oxalic acid water bath until the split surface had an appropriate color.
  • the tissue was moved up and down in the water tank.
  • a sufficient amount (about 2 ml) of 0.001 N nitric acid solution was dripped over the entire cleavage surface for each tissue section. Place the tissue on the sample stage of the stereomicroscope with the stained split surface to be observed facing up, and then cover the split surface with a sufficient amount of OCT compound (Tissue-Tek (registered trademark): SAKURA (registered trademark)) Observed with a stereomicroscope.
  • OCT compound Tissue-Tek (registered trademark): SAKURA (registered trademark)
  • FIG. 8 tumor cell nuclei, fibroblast nuclei, and capillaries were visible.

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Abstract

Provided is a staining kit which does not require the production of a thin section, enables the staining of a biological tissue that cannot be cut into a thin section, and can achieve accurate and rapid pathological diagnoses. The staining kit comprises methylene blue for staining a biological tissue, haematoxylin for staining a biological tissue, a reducing agent for decolorizing excess tissue staining in a biological tissue stained with methylene blue, and an oxidizing agent for preventing the discoloration of a dye in a biological tissue stained with methylene blue. A cut face of a biological tissue material can be stained using the staining kit. Haematoxylin is Carrazi's haematoxylin. The biological tissue material may be in an immobilized form or a non-immobilized form, and is observed on a stereo microscope or a biological microscope.

Description

染色キット及び染色方法Staining kit and staining method
　本発明は、生物組織の染色方法及びその染色方法に使用される染色キットに関する。 The present invention relates to a biological tissue staining method and a staining kit used for the staining method.
　生体組織の顕微鏡等による観察は、病理診断において公知の検査方法である（例えば特許文献１～３）。病理診断には、永久標本による病理組織診断と、術中迅速病理診断が含まれる。 Observation of biological tissue with a microscope or the like is a well-known inspection method in pathological diagnosis (for example, Patent Documents 1 to 3). Pathological diagnosis includes histopathological diagnosis using a permanent specimen and rapid pathological diagnosis during operation.
　術中迅速病理診断は、手術中に、腫瘍の良性悪性の判定や、腫瘍の転移の有無、切除断端における腫瘍残存の有無等を判定する。手術中に採取された病変組織を、包埋剤と共に急速に凍結させ、クライオスタットという特別な装置で薄切をし、スライドガラスに貼り付ける。 Intraoperative rapid pathological diagnosis determines whether the tumor is benign or malignant, whether the tumor has metastasized, whether the tumor remains at the resected margin, and the like. The lesion tissue collected during the operation is rapidly frozen together with the embedding agent, sliced with a special device called a cryostat, and attached to a glass slide.
　次に凍結組織薄切切片を染色する。凍結組織薄切切片はほとんど無色であり、細かい構造を観察するには各種の色素を用いて特定の成分を染める。一般にはHE染色が用いられる。 Next, the frozen tissue slices are stained. Frozen tissue slices are almost colorless, and various components are used to dye specific components to observe fine structures. In general, HE staining is used.
　次に染色された凍結組織薄切切片を脱水処理後、封入剤を滴下し、カバーガラスで覆い、迅速病理標本とする。これにより例えば透過光式（明視野）生物顕微鏡にて組織内の微細構造を観察することができる。 Next, after dehydrating the sliced frozen tissue slices, the mounting medium is dropped and covered with a cover glass to make a rapid pathological specimen. Thereby, for example, the fine structure in the tissue can be observed with a transmitted light type (bright field) biological microscope.
　しかし、応急標本である為、パラフィン包埋の工程を経て作製された、いわゆる永久病理組織標本と比較して、細胞所見、核所見の判定が難しく、高い技能が要求される。更に術中迅速病理診断標本の作製も高い技能が必要であり、しかも10分程度で作製作業を完了することが必要である。 However, since it is an emergency specimen, it is difficult to determine cellular findings and nuclear findings compared to so-called permanent pathological tissue specimens prepared through a paraffin embedding process, and high skill is required. In addition, preparation of rapid pathological diagnosis specimens during surgery requires high skill, and it is necessary to complete the preparation work in about 10 minutes.
　一方、外科材料における永久病理組織標本の作製では、まず、採取された組織がホルマリン等の固定液にて固定される。固定は主として組織内・細胞内成分を化学的に架橋することでなされる。これにより組織・臓器の自家融解及び腐敗が停止し、蛋白質・核酸・糖質等の組織成分が保持され、組織染色性の低下が防止される。 On the other hand, in the preparation of a permanent pathological tissue specimen in a surgical material, first, the collected tissue is fixed with a fixative such as formalin. Fixation is mainly performed by chemically cross-linking tissue and intracellular components. As a result, self-melting and decay of tissues / organs is stopped, tissue components such as proteins / nucleic acids / sugars are retained, and deterioration of tissue staining is prevented.
　次に病理医による外科組織材料の切り出しが行われる。すなわち、外科組織材料から病理組織標本作製のために肉眼観察で必要箇所を選定し、例えば2cm×3cm～4cmまでの大きさ、薄さ0.5cmのブロック状に切り取る。肉眼での観察の為、病変部の微妙な性状の違いから、腫瘍本体や広がりを判断せざるを得ない。肉眼観察では視認できない場合、病変の可能性のある部位を全てブロックにする必要があり、ブロック数が非常に多くなる。固定の終わった組織片は、そのままでは硬さが不均一であるうえに柔らかすぎて数μｍの薄い切片に切ることは不可能である。そのため、次にパラフィン等の包埋剤が組織に浸透させられ、これによりパラフィン包埋組織ブロックが作製される。この過程は、例えば、脱水・脱脂→脱水剤の除去及び仲間剤への置換→パラフィンの浸透から構成される。 Next, surgical tissue material is cut out by a pathologist. That is, a necessary part is selected by visual observation for preparing a pathological tissue specimen from a surgical tissue material, and is cut into a block shape of, for example, a size of 2 cm × 3 cm to 4 cm and a thickness of 0.5 cm. Because of observation with the naked eye, the tumor body and spread must be judged from the subtle differences in the properties of the lesion. When it cannot be visually recognized by the naked eye observation, it is necessary to block all the sites that may have a lesion, and the number of blocks becomes very large. The fixed tissue piece is non-uniform in hardness and is too soft to cut into thin sections of several μm. Therefore, an embedding agent such as paraffin is then infiltrated into the tissue, thereby producing a paraffin-embedded tissue block. This process is composed of, for example, dehydration / defatting → removal of dehydrating agent and substitution with a companion agent → paraffin infiltration.
　次に組織の薄切がなされる。パラフィン包埋組織ブロックをミクロトームにてミクロン単位の厚さで薄く切る。 Next, the tissue is sliced. A paraffin-embedded tissue block is sliced with a microtome to a thickness of microns.
　次に薄切された切片を温水面に浮かべ、スライドガラスに貼り付け、乾燥させる。 Next, the sliced slices are floated on a warm water surface, attached to a glass slide and dried.
　次に薄切切片を染色する。薄切切片はほとんど無色であり、細かい構造を観察するには各種の色素を用いて特定の成分を染める。一般にはHE染色が用いられる。 Next, the sliced section is stained. Thin slices are almost colorless, and various components are used to dye specific components to observe fine structures. In general, HE staining is used.
　次に染色された薄切切片を脱水処理後、封入剤を滴下し、カバーガラスで覆う。これにより例えば透過光式（明視野）生物顕微鏡にて組織内の微細な構造を観察することができる。 Next, after the dehydrated thin sliced section, the mounting medium is dropped and covered with a cover glass. Thereby, for example, a fine structure in the tissue can be observed with a transmitted light (bright field) biological microscope.
特開２００２－０８２０２６号公報JP 2002-082026 A 特開２００４－１３２８３８号公報JP 2004-132838 A 特開２００８－２８６６９４号公報JP 2008-286694 A
　しかし、術中迅速病理診断における凍結標本の作製には、クライオスタットという特別な装置を必要とし、更に、作製自体に高い技能、熟練を必要とする。脂肪組織は包埋剤が浸透しない為、凍結切片の作製が出来ない。また、骨組織等の硬い組織においても、切れない為、凍結切片の作製が出来ない。 However, the preparation of frozen specimens for rapid pathological diagnosis during surgery requires a special device called a cryostat, and the preparation itself requires high skill and skill. Since adipose tissue does not penetrate the embedding agent, frozen sections cannot be prepared. In addition, since a hard tissue such as a bone tissue cannot be cut, a frozen section cannot be prepared.
　また、従来の外科材料の永久病理組織標本作製のための切り出し工程では、肉眼での観察の為、病変部の微妙な性状の違いから、腫瘍本体や広がりを判断せざるを得ず、腫瘍を視認できない場合、病変の可能性のある部位を全てブロックにする必要があり、ブロック数が非常に多くなる。更に、従来の外科材料永久病理組織標本作製には、顕微鏡による病理診断が可能となるまでに5～7日程度を必要とする。 In addition, in the conventional extraction process for the preparation of permanent pathological tissue specimens of surgical materials, because of the observation with the naked eye, it is necessary to judge the tumor body and spread from the subtle differences in the characteristics of the lesion, and the tumor is removed. When it cannot be visually recognized, it is necessary to block all the sites that may have a lesion, and the number of blocks becomes very large. In addition, the preparation of a conventional surgical material permanent pathological tissue specimen requires about 5 to 7 days before the pathological diagnosis by a microscope becomes possible.
　本発明はかかる問題点に鑑みてなされたものであって、クライオスタットのない施設においても、凍結薄切切片の作製をすることなく、簡易に、術中迅速病理診断が可能となり、脂肪組織や骨組織等の凍結切片の作製の不可能な組織においても術中迅速病理診断が可能であり、また外科材料永久病理組織標本作製のための切り出し工程の段階にて病理診断を可能とする、染色キット及び染色方法を提供することを目的とする。 The present invention has been made in view of such problems, and can easily and rapidly perform pathological diagnosis during surgery without preparing frozen sliced sections even in a facility without a cryostat. Staining kits and stains that allow rapid pathological diagnosis during surgery even in tissues where frozen sections cannot be prepared, such as pathological diagnosis at the stage of the excision process for preparation of surgical material permanent pathological tissue specimens It aims to provide a method.
　本発明にかかる染色キットは、生物組織の表面及び割面を染色する染色キットであって、前記生物組織を染色するためのメチレンブルーと、前記生物組織を染色するためのヘマトキシリンと、前記メチレンブルーにて染色された生物組織における過剰な染色を脱色するための還元剤と、前記メチレンブルーにて染色された生物組織における色素の退色を防止するための酸化剤と、を備える。 A staining kit according to the present invention is a staining kit for staining a surface and a split surface of a biological tissue, wherein methylene blue for staining the biological tissue, hematoxylin for staining the biological tissue, and the methylene blue are used. A reducing agent for decolorizing excessive staining in the stained biological tissue; and an oxidizing agent for preventing fading of the pigment in the biological tissue stained with the methylene blue.
　また、本発明にかかる染色方法は、生物組織の表面及び割面を染色する染色方法であって、前記生物組織をメチレンブルーにて染色する第１の染色工程と、前記メチレンブルーにて染色された生物組織における過剰な染色を還元剤にて脱色する脱色工程と、前記生物組織を更にヘマトキシリンにて染色する第２の染色工程と、前記メチレンブルーにて染色された生物組織における色素の退色を酸化剤にて防止する退色防止工程と、を有する。 The staining method according to the present invention is a staining method for staining the surface and the split surface of a biological tissue, the first staining step for staining the biological tissue with methylene blue, and the organism stained with the methylene blue. Decolorization step of decolorizing excessive staining in the tissue with a reducing agent, second staining step of further staining the biological tissue with hematoxylin, and fading of the pigment in the biological tissue stained with the methylene blue as an oxidizing agent And a fading prevention step for preventing the discoloration.
　本発明によれば、手術中に提出された未固定組織材料の表面及び割面を直接染色し、その正常組織構築、腫瘍組織構築、細胞所見、及び核所見を視認することができる。そのためクライオスタットのない施設や、凍結標本作製のための熟練した検査技師のいない施設においても、簡易に、術中迅速病理診断が可能となる。脂肪組織や骨組織等の凍結切片の作製の不可能な組織においても術中迅速病理診断が可能となる。 According to the present invention, the surface and split surface of unfixed tissue material submitted during surgery can be directly stained, and the normal tissue construction, tumor tissue construction, cellular findings, and nuclear findings can be visually confirmed. Therefore, in a facility without a cryostat or a facility without a skilled laboratory technician for preparing a frozen specimen, an intraoperative rapid pathological diagnosis can be easily performed. Intraoperative rapid pathological diagnosis is possible even in tissues where frozen sections such as adipose tissue and bone tissue cannot be prepared.
　また本発明によれば、外科材料永久病理組織標本作製のための切り出し工程の段階にて、固定された生物組織の表面及び割面を直接染色し、その正常組織構築、腫瘍組織構築、細胞所見、及び核所見を視認することができる。このため適切な切り出しが出来、ブロック数の低減やステージングを含めたより正確な病理診断につながる。また、外科材料永久病理組織標本作製のための切り出し工程の段階にて、腫瘍の診断及び進達度の判定、並びに断端検索が可能となる。更に管腔や嚢胞の内腔面等、薄切切片標本と比べ三次元的に染色される範囲が広いため、より多くの情報が得られる。 Further, according to the present invention, the surface and the split surface of the fixed biological tissue are directly stained at the stage of the cutting process for preparing the surgical material permanent pathological tissue specimen, and the normal tissue construction, tumor tissue construction, cytological findings are obtained. , And nuclear findings can be visually recognized. For this reason, it can cut out appropriately and leads to more accurate pathological diagnosis including reduction of the number of blocks and staging. In addition, tumor diagnosis, progress determination, and stump retrieval can be performed at the stage of the cutting process for preparing a surgical material permanent pathological tissue specimen. Further, since the range of three-dimensional staining such as the lumen and the luminal surface of the cyst is wider than that of a sliced slice specimen, more information can be obtained.
　更には本発明によれば、例えば学校等のミクロトームやクライオスタットのない施設においても、未固定生物組織材料や、固定された生物組織材料の表面及び割面おける、細胞所見、核所見を含む組織構築の観察が可能となる。 Furthermore, according to the present invention, even in a facility without a microtome or a cryostat such as a school, a tissue construction including unfixed biological tissue material and cell findings and nuclear findings on the surface and face of the fixed biological tissue material. Can be observed.
実施例１における甲状腺乳頭癌組織の生物顕微鏡写真図である。1 is a biological micrograph of a papillary thyroid cancer tissue in Example 1. FIG. 実施例２における胃癌組織の生物顕微鏡写真図である。4 is a biological micrograph of gastric cancer tissue in Example 2. FIG. 実施例３における皮膚青色母斑組織の生物顕微鏡写真図である。4 is a biological micrograph of a blue skin nevus tissue in Example 3. FIG. 実施例４における唾液腺腺様嚢胞癌組織の生物顕微鏡写真図である。4 is a biological micrograph of salivary adenoid cystic cancer tissue in Example 4. FIG. 実施例５における鶏砂嚢組織の生物顕微鏡写真図である。6 is a biological micrograph of chicken gizzard tissue in Example 5. FIG. 実施例６における鶏骨組織の実体顕微鏡写真図である。It is a stereoscopic microscope photograph figure of the chicken bone tissue in Example 6. 実施例７における豚舌組織の生物顕微鏡写真図である。It is a biological-microscope photograph figure of the pig tongue tissue in Example 7. 参考例における大腸癌組織の実体顕微鏡写真図である。It is a stereoscopic microscope photograph figure of the colon cancer tissue in a reference example.
　以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
　（染色キット）
　本実施形態にかかる染色キットは、生物組織の割面を染色する染色キットである。生物組織学的診断において、生物組織の割面を染色することは新規であり、本実施形態にかかる染色キットは、この新規染色方法に使用される。ここで生物組織は例えば病理組織であり、生物組織の割面とは、生物組織（例えば腫瘍）の所定部（例えば中心部）を通るラインに沿って切断刃による割を入れて形成される切断面である。生物組織の割面と一緒にその表面を染色することも可能である。染色対象となる生物組織は、生体から単離された生物組織又は手術中の生物組織のいずれとすることも可能であり、固定又は未固定のいずれの生物組織でも可能である。 (Dyeing kit)
The staining kit according to the present embodiment is a staining kit that stains a split surface of a biological tissue. In the biohistological diagnosis, it is novel to stain the cleavage plane of the biological tissue, and the staining kit according to the present embodiment is used for this new staining method. Here, the biological tissue is, for example, a pathological tissue, and the cleavage plane of the biological tissue is a cutting formed by dividing by a cutting blade along a line passing through a predetermined portion (for example, the central portion) of the biological tissue (for example, a tumor). Surface. It is also possible to stain the surface together with the split surface of the biological tissue. The biological tissue to be stained can be either a biological tissue isolated from a living body or a biological tissue during surgery, and can be either a fixed or unfixed biological tissue.
　本実施形態にかかる染色キットは、生物組織を染色するためのメチレンブルーと、生物組織を染色するためのヘマトキシリンと、メチレンブルーにて染色された生物組織における過剰な染色を脱色するための還元剤と、メチレンブルーにて染色された生物組織における色素の退色を防止するための酸化剤と、を備える。 The staining kit according to the present embodiment includes methylene blue for staining biological tissue, hematoxylin for staining biological tissue, and a reducing agent for decolorizing excessive staining in biological tissue stained with methylene blue, And an oxidizing agent for preventing fading of a pigment in a biological tissue stained with methylene blue.
　まず、メチレンブルーは、生物組織の１回目の染色に使用される。このメチレンブルーにて生物組織の主として上皮細胞の細胞質が染色される。なお細胞核が染色されることもあるが、染まりムラが生じることが多く明瞭な核染色が得られることは難しい。メチレンブルーには、生物組織を染色することができる限りメチレンブルーの誘導体も含まれる。メチレンブルーの溶媒は、水性溶媒又はアルコール例えばエタノールである。メチレンブルーのｐＨは例えば３．０～７．０である。 First, methylene blue is used for the first staining of biological tissue. The methylene blue stains mainly the cytoplasm of epithelial cells of the biological tissue. Although cell nuclei may be stained, dyeing unevenness often occurs and it is difficult to obtain clear nuclear staining. Methylene blue includes methylene blue derivatives as long as they can stain biological tissues. The methylene blue solvent is an aqueous solvent or an alcohol such as ethanol. The pH of methylene blue is, for example, 3.0 to 7.0.
　メチレンブルーの濃度は、生物組織を染色することができる限り特に限定されるものではないが、例えば０．５ｇ／Ｌ～１０ｇ／Ｌであることが好ましい。割面ではない例えば生体組織の胃粘膜表面や大腸粘膜表面のメチレンブルーによる染色は内視鏡領域で行われており、ここでのメチレンブルーの濃度は０．１ｇ／Ｌ～０．５ｇ／Ｌである。そのため内視鏡領域で使用されるメチレンブルー染色に比較すると、本実施形態での０．５ｇ／Ｌ～１０ｇ／Ｌのメチレンブルー染色は染色濃度が濃いことになる。これは、後述するように生物組織をメチレンブルーにて染色した後に還元剤で過剰な染色を脱色するところ、濃度の濃いメチレンブルーにて細胞質等を染色してから還元剤で過剰な染色を脱色したほうが、良好なコントラストが得られるからである。 The concentration of methylene blue is not particularly limited as long as biological tissue can be stained, but it is preferably, for example, 0.5 g / L to 10 g / L. For example, the surface of the gastric mucosa or the surface of the large intestine mucosa, which is not a cleavage plane, is stained with methylene blue in the endoscopic region, and the concentration of methylene blue is 0.1 g / L to 0.5 g / L. . Therefore, compared with the methylene blue staining used in the endoscope region, the staining concentration of methylene blue staining of 0.5 g / L to 10 g / L in this embodiment is higher. As described later, after staining a biological tissue with methylene blue, the excess staining is decolored with a reducing agent. However, it is better to stain the cytoplasm with dense methylene blue and then decolorize the excessive staining with a reducing agent. This is because good contrast can be obtained.
　還元剤は、生物組織をメチレンブルーにて染色した後に使用される。メチレンブルーは上述したように主として上皮細胞の細胞質を染色するが、染色性は生物組織の部位等により微妙に異なる。そこで染色の程度を標準化するためにこの還元剤が使用される。即ち、上皮細胞の細胞質をメチレンブルーにて染色した後に還元剤で組織の間質の過剰な染色を脱色することにより、上皮細胞の染色状態を維持しつつ間質のバックグラウンド染色を減少させることができるので、良好なコントラストが得られる。 The reducing agent is used after staining biological tissue with methylene blue. As described above, methylene blue mainly stains the cytoplasm of epithelial cells, but the staining properties differ slightly depending on the site of the biological tissue. This reducing agent is then used to standardize the degree of staining. That is, by staining the cytoplasm of epithelial cells with methylene blue and then decolorizing excessive staining of the tissue stroma with a reducing agent, the background staining of the stroma can be reduced while maintaining the staining state of the epithelial cells. As a result, good contrast can be obtained.
　還元剤は、間質のバックグラウンド染色を減少させることができる限り特に限定されるものではないが、例えばシュウ酸、ギ酸、ヒドラジン、アスコルビン酸及びこれらの塩類から選択することができる。還元剤の濃度は間質の染色を減少する観点から適宜選択することができ、例えば１×１０－４Ｎ～１×１０－２Ｎとすることができる。 The reducing agent is not particularly limited as long as interstitial background staining can be reduced, and can be selected from, for example, oxalic acid, formic acid, hydrazine, ascorbic acid, and salts thereof. The concentration of the reducing agent can be appropriately selected from the viewpoint of reducing the staining of the stroma, and can be, for example, 1 × 10 −4 N to 1 × 10 −2 N.
　次に、ヘマトキシリンは、生物組織の２回目の染色に使用される。このヘマトキシリンにて生物組織の全ての細胞の細胞核が染色される。ヘマトキシリンには、生物組織を染色することができる限りヘマトキシリンの誘導体も含まれる。ヘマトキシリンの溶媒は水性溶媒であり、ｐＨは例えば２．５～４．０である。 Next, hematoxylin is used for the second staining of biological tissue. With this hematoxylin, the nuclei of all cells of the biological tissue are stained. Hematoxylin also includes derivatives of hematoxylin as long as it can stain biological tissues. The solvent for hematoxylin is an aqueous solvent, and the pH is, for example, 2.5 to 4.0.
　ヘマトキシリンは、生物組織を染色することができる限り特に限定されるものではなく、例えばマイヤーヘマトキシリン、ギルヘマトキシリン等が使用され、好ましくはカラッチヘマトキシリンが使用される。カラッチヘマトキシリンにはグリセリンが含有されており、細胞核の染色性が向上すると共に染色時間を短縮できるからである。カラッチヘマトキシリン以外のヘマトキシリンでも、１０～２０％（ｖ／ｖ）のグリセリンを含有させることにより細胞核の染色性向上及び染色時間の短縮が可能となり、好適に使用することができる。 Hematoxylin is not particularly limited as long as it can stain a biological tissue. For example, Mayer hematoxylin, gil hematoxylin, etc. are used, and preferably, caratach hematoxylin is used. This is because carachach hematoxylin contains glycerin, which improves the staining of cell nuclei and shortens the staining time. Hematoxylin other than caratach hematoxylin can be preferably used by containing 10 to 20% (v / v) glycerin, which can improve staining of the cell nucleus and shorten the staining time.
　最後に、メチレンブルーにて染色された生物組織における色素の退色を防止するために酸化剤が使用される。メチレンブルーは光が照射されることや、組織に含まれる還元剤の作用により速やかに退色するため、酸化剤を使用することによりメチレンブルーの退色を防止して明瞭な染色を維持する。なお、ヘマトキシリンの紫色発色のままでも生物組織の観察は十分に可能であるが、酸化剤によりメチレンブルーの退色が防止されると共にヘマトキシリンがヘマテインに変化することにより青色発色がなされる。 Finally, an oxidizing agent is used to prevent discoloration of the pigment in the biological tissue stained with methylene blue. Since methylene blue fades quickly when irradiated with light or by the action of a reducing agent contained in the tissue, the use of an oxidizing agent prevents the methylene blue from fading and maintains clear staining. Although the biological tissue can be sufficiently observed even with the purple color of hematoxylin, the fading of methylene blue is prevented by the oxidizing agent, and the blue color is formed by changing hematoxylin into hematein.
　酸化剤は、メチレンブルーの退色が防止される限り特に限定されるものではないが、例えば硝酸、塩酸、硫酸、及びこれらの塩類から選択することができる。酸化剤の濃度はメチレンブルーの退色防止の観点から適宜選択することができ、例えば１×１０－４Ｎ～１×１０－２Ｎとすることができる。 The oxidizing agent is not particularly limited as long as the fading of methylene blue is prevented. For example, nitric acid, hydrochloric acid, sulfuric acid, and salts thereof can be selected. The concentration of the oxidizing agent can be appropriately selected from the viewpoint of preventing the fading of methylene blue, and can be, for example, 1 × 10 −4 N to 1 × 10 −2 N.
　（染色方法）
　本実施形態にかかる染色方法は、生物組織の割面を染色する。生物組織の割面と一緒にその表面を染色することも可能である。本実施形態にかかる染色方法は、生物組織をメチレンブルーにて染色する第１の染色工程と、メチレンブルーにて染色された生物組織における過剰な染色を還元剤にて脱色する脱色工程と、生物組織を更にヘマトキシリンにて染色する第２の染色工程と、メチレンブルーにて染色された生物組織における色素の退色を酸化剤にて防止する退色防止工程と、を有する。 (Dyeing method)
The staining method according to the present embodiment stains a split surface of a biological tissue. It is also possible to stain the surface together with the split surface of the biological tissue. The staining method according to the present embodiment includes a first staining step for staining a biological tissue with methylene blue, a decolorization step for decolorizing excessive staining in the biological tissue stained with methylene blue with a reducing agent, Furthermore, it has the 2nd dyeing | staining process dye | stained with hematoxylin, and the fading prevention process which prevents the fading of the pigment | dye in the biological tissue dye | stained with methylene blue with an oxidizing agent.
　まず、第１の染色工程において、上述した染色キットにおけるメチレンブルーを固定又は未固定の生物組織に十分量滴下して主として上皮細胞の細胞質を染色する。このメチレンブルーを滴下しての染色では、例えば０．１秒～５秒を要する。なお、生物組織の病巣部が大きい場合等ではメチレンブルーによる染色を十分に行うため、メチレンブルーの滴下は所定間隔を開けて複数回行われてもよい。続いて生物組織を水洗する。 First, in the first staining step, a sufficient amount of methylene blue in the above-described staining kit is dropped onto a fixed or unfixed biological tissue to stain mainly the cytoplasm of epithelial cells. The dyeing by dropping methylene blue requires, for example, 0.1 to 5 seconds. In addition, when the lesion part of a biological tissue is large or the like, the methylene blue may be dripped a plurality of times at predetermined intervals in order to sufficiently perform methylene blue staining. Subsequently, the biological tissue is washed with water.
　次に、脱色工程において、上述した染色キットにおける還元剤を使用して組織の過剰な染色を脱色する。これにより良好な染色のコントラストが得られる。この還元剤を使用しての脱色では、例えば０．１秒～５秒を要する。続いて生物組織を水洗する。 Next, in the decolorization step, the excessive staining of the tissue is decolorized using the reducing agent in the staining kit described above. This provides good staining contrast. Decolorization using this reducing agent requires, for example, 0.1 to 5 seconds. Subsequently, the biological tissue is washed with water.
　次に、第２の染色工程において、上述した染色キットにおけるヘマトキシリンを生物組織に十分量滴下して全ての細胞の細胞核を染色する。このヘマトキシリンを滴下しての染色では、例えば１秒～３０秒を要する。続いて生物組織を水洗する。 Next, in the second staining step, a sufficient amount of hematoxylin in the above-described staining kit is dropped onto a biological tissue to stain the cell nuclei of all cells. For example, 1 to 30 seconds are required for dyeing by dropping hematoxylin. Subsequently, the biological tissue is washed with water.
　最後に、退色防止工程において、上述した染色キットにおける酸化剤を使用してメチレンブルーの退色が防止される。なおメチレンブルーの退色が防止されると同時にヘマトキシリンの発色も可能となる。 Finally, in the color fading prevention step, methylene blue color fading is prevented by using the oxidizing agent in the dyeing kit described above. Further, fading of methylene blue is prevented, and at the same time, hematoxylin can be colored.
　これにより生物組織の割面の染色が可能となり、細胞所見、核所見を含む正常組織構築、腫瘍組織構築の観察が可能となる。また本実施形態における染色方法にて生物組織の割面の染色に要する時間は例えば１０秒～４０秒であり迅速な病理診断が可能となる。 This makes it possible to stain the fracture surface of biological tissue, and to observe normal tissue construction and tumor tissue construction including cellular findings and nuclear findings. In addition, the time required for staining the split surface of the biological tissue by the staining method in the present embodiment is, for example, 10 seconds to 40 seconds, and rapid pathological diagnosis is possible.
　その後は、観察を容易にするために、染色した生物組織を透明なゲル状物質にて包摂し、マクロ観察、実体顕微鏡観察、又は生物顕微鏡観察等に供する。更にカバーガラスをゲル状物質の上から被せて観察してもよい。ここで上記の退色防止工程において、酸化剤に透明なゲル状物質を混合して用いることも可能であり、かかる場合にあってはメチレンブルーの退色防止と同時に染色した生物組織の包摂が可能となるので病理診断がより迅速且つ容易となる。また透明なゲル状物質にて包摂せずに、染色した生物組織を水中に浸漬させてピン等で固定することによっても観察を容易にすることができる。 Thereafter, in order to facilitate observation, the stained biological tissue is encapsulated with a transparent gel-like substance and subjected to macro observation, stereoscopic microscope observation, biological microscope observation, or the like. Further, a cover glass may be placed on the gel substance for observation. Here, in the above fading prevention step, it is also possible to use a mixture of a transparent gel substance in the oxidizing agent, and in such a case, it becomes possible to include the stained biological tissue at the same time as preventing the fading of methylene blue. Therefore, pathological diagnosis becomes quicker and easier. Observation can also be facilitated by immersing the stained biological tissue in water and fixing it with a pin or the like without being included in a transparent gel substance.
　なお、上述の実施形態では、第１の染色工程にてメチレンブルーを使用し、第２の染色工程にてヘマトキシリンを使用したが、本実施形態にかかる染色方法はこのような使用順序に限定されない。例えば、メチレンブルー・ヘマトキシリンによる染色の程度が悪くなる虞があるものの、第１の染色工程として生物組織をヘマトキシリンにて染色し、次に第２の染色工程として生物組織をメチレンブルーにて染色し、次に脱色工程にてメチレンブルーにて染色された生物組織における細胞の間質の染色を還元剤にて脱色し、最後に退色防止工程にてメチレンブルーにて染色された生物組織における色素の退色を酸化剤にて防止することも可能である。 In the above-described embodiment, methylene blue is used in the first staining step and hematoxylin is used in the second staining step. However, the staining method according to this embodiment is not limited to such an order of use. For example, although there is a possibility that the degree of staining with methylene blue and hematoxylin may deteriorate, the biological tissue is stained with hematoxylin as the first staining step, and then the biological tissue is stained with methylene blue as the second staining step. The staining of cells in the biological tissue stained with methylene blue in the decolorization step is decolorized with a reducing agent, and finally the color fading of the biological tissue stained with methylene blue in the anti-fading step is oxidized It is also possible to prevent this.
　（メチレンブルーのみによる染色）
　メチレンブルーのみによる生物組織の割面の染色を説明する。この染色方法は、生物組織をメチレンブルーにて染色する染色工程と、メチレンブルーにて染色された生物組織における組織の過剰な染色を還元剤にて脱色する脱色工程と、メチレンブルーにて染色された生物組織における色素の退色を酸化剤にて防止する退色防止工程と、を有する。このメチレンブルーのみによる生物組織の染色では、上皮細胞核の染色は染色ムラがあるものの、上皮細胞質は迅速且つ安定して染色され、マクロ観察及びマクロ像撮影の場合に好適である。メチレンブルーのみによる生物組織の染色では、上述したメチレンブルー及びヘマトキシリンを使用する染色と比較して、メチレンブルーの濃度は低くしてもよく、例えば０．５ｇ／Ｌ～５ｇ／Ｌである。 (Dyeing with methylene blue only)
The staining of the cut surface of biological tissue with only methylene blue will be described. This staining method includes a staining step of staining a biological tissue with methylene blue, a decolorization step of decolorizing excessive staining of the tissue in the biological tissue stained with methylene blue with a reducing agent, and a biological tissue stained with methylene blue. And a fading prevention step for preventing fading of the dye in the case of using an oxidizing agent. In this staining of biological tissue with only methylene blue, although epithelial cell nuclei are stained unevenly, the epithelial cytoplasm is rapidly and stably stained, which is suitable for macro observation and macro image photography. In the staining of a biological tissue with only methylene blue, the concentration of methylene blue may be lower than that in the above-described staining using methylene blue and hematoxylin, for example, 0.5 g / L to 5 g / L.
　（実施例１）
　生物組織として外科材料甲状腺腫瘍組織を使用した。外科材料甲状腺腫瘍組織をホルマリンにて固定し、垂直断にて5mm間隔で平行に切断刃による割を入れて割面を形成した。
次に、腫瘍を含む外科材料甲状腺腫瘍組織切片を1mm厚にスライスした。次にメチレンブルー（和光純薬工業株式会社製の販売元コード275-07895のメチレンブルー原液を30倍希釈したもの）を割面全体に行き渡るに十分量（1ml程度）滴下し、2秒間染色した。次に、水槽中で組織を上下させた。次に、0.001規定シュウ酸水槽中で、観察しながら割面が適当な色合いになるまで、組織切片を上下させた。次に、水槽中で組織を上下させた。次に、2xカラッチヘマトキシリン液（和光純薬工業株式会社　販売元コード039-17705）を割面全体に行き渡るに十分量（1ml程度）滴下し、10秒間染色した。なお、2xカラッチヘマトキシリン液の組成は、ヘマトキシリン２．０ｇ、カリウムミョウバン５０ｇ、ヨウ素酸ナトリウム０．４ｇ、グリセリン２００ｍＬ、蒸留水８００ｍＬからなるものであった。次に、水槽中で組織を上下させた。次に、この組織切片につき0.001規定硝酸水を割面全体に行き渡るに十分量（1ml程度）滴下した。観察する染色した割面を上にして組織をスライドガラスに載せ、更に同割面に十分量のOCTコンパウンド（Tissue-Tek（登録商標）：SAKURA（登録商標））を被覆させた。その上にカバーガラス（0.17mm）を被せ、透過光式生物顕微鏡で観察した。結果を図１に示す。図１に示されるように、甲状腺乳頭癌細胞核、核溝、核内封入体が視認できた。 Example 1
Surgical material thyroid tumor tissue was used as the biological tissue. Surgical material Thyroid tumor tissue was fixed with formalin and split with a cutting blade in parallel at 5 mm intervals by vertical cutting to form a split surface.
Next, a surgical material thyroid tumor tissue section containing the tumor was sliced to a thickness of 1 mm. Next, a sufficient amount (about 1 ml) of methylene blue (diluted 30-fold of a methylene blue stock solution of a vendor code 275-07895 manufactured by Wako Pure Chemical Industries, Ltd.) was dripped over the entire surface and dyed for 2 seconds. Next, the tissue was moved up and down in the water tank. Next, the tissue slice was moved up and down while observing in a 0.001 N oxalic acid water bath until the split surface had an appropriate color. Next, the tissue was moved up and down in the water tank. Next, a 2x caratach hematoxylin solution (Wako Pure Chemical Industries, Ltd., sales code 039-17705) was dropped in a sufficient amount (about 1 ml) to reach the entire face and stained for 10 seconds. The composition of the 2x caratach hematoxylin solution was 2.0 g of hematoxylin, 50 g of potassium alum, 0.4 g of sodium iodate, 200 mL of glycerin, and 800 mL of distilled water. Next, the tissue was moved up and down in the water tank. Next, a sufficient amount (about 1 ml) of 0.001 N nitric acid solution was dripped over the entire cleavage surface for each tissue section. The tissue was placed on a slide glass with the stained split surface to be observed facing up, and a sufficient amount of OCT compound (Tissue-Tek (registered trademark): SAKURA (registered trademark)) was coated on the split surface. It was covered with a cover glass (0.17 mm) and observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 1, papillary thyroid cancer cell nuclei, nuclear sulcus, and intranuclear inclusions were visible.
　（実施例２）
　生物組織としてホルマリン固定外科材料胃腫瘍組織を使用した以外は、実施例１と同様にして染色を行い透過光式生物顕微鏡にて観察した。結果を図２に示す。図２に示されるように、低分化腺癌細胞、印環細胞が視認できた。 (Example 2)
Staining was carried out in the same manner as in Example 1 except that a formalin-fixed surgical material gastric tumor tissue was used as the biological tissue, and observation was performed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 2, poorly differentiated adenocarcinoma cells and signet ring cells were visible.
　（実施例３）
　生物組織としてホルマリン固定外科材料皮膚腫瘍組織を使用した以外は、実施例１と同様にして染色を行い透過光式生物顕微鏡にて観察した。結果を図３に示す。図３に示されるように、表皮細胞核、真皮内毛細血管、真皮内炎症細胞、青色母斑細胞が視認できた。 (Example 3)
Except for using a formalin-fixed surgical material skin tumor tissue as a biological tissue, staining was performed in the same manner as in Example 1 and observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 3, epidermal cell nuclei, intradermal capillaries, intradermal inflammatory cells, and blue nevi cells were visible.
　（実施例４）
　生物組織としてホルマリン固定外科材料唾液腺腫瘍組織を使用した以外は、実施例１と同様にして染色を行い透過光式生物顕微鏡にて観察した。結果を図４に示す。図４に示されるように、唾液腺腺様嚢胞癌組織の篩状構造、癌細胞核が視認できた。 (Example 4)
Except for using a formalin-fixed surgical material salivary gland tumor tissue as a biological tissue, staining was performed in the same manner as in Example 1 and observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 4, the sieve structure of the salivary gland adenoid cystic cancer tissue and the cancer cell nucleus were visible.
　（実施例５）
　生物組織として鶏砂嚢組織を使用し、未固定の組織を使用した以外は、実施例１と同様にして染色を行い透過光式生物顕微鏡にて観察した。結果を図５に示す。図５に示されるように、腺上皮構造、腺上皮細胞核、粘膜下組織線維芽細胞が視認できた。 (Example 5)
Staining was carried out in the same manner as in Example 1 except that chicken gizzard tissue was used as the biological tissue and an unfixed tissue was used, and observation was performed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 5, glandular epithelial structures, glandular epithelial cell nuclei, and submucosal fibroblasts were visible.
　（実施例６）
　生物組織として鶏骨組織を使用した。未固定の組織を使用し、スライス厚を10mmにし、実体顕微鏡の試料台の上で観察した以外は、実施例１と同様にして染色を行い実体顕微鏡にて観察した。結果を図６に示す。図６に示されるように、骨細胞、骨髄細胞が視認できた。 (Example 6)
Chicken bone tissue was used as the biological tissue. Staining was performed in the same manner as in Example 1 except that an unfixed tissue was used, the slice thickness was set to 10 mm, and the sample was observed on a sample stage of a stereomicroscope, and observed with a stereomicroscope. The results are shown in FIG. As shown in FIG. 6, bone cells and bone marrow cells were visible.
　（実施例７）
　生物組織として豚舌組織を使用し、未固定の組織を使用した以外は、実施例１と同様にして染色を行い透過光式生物顕微鏡にて観察した。結果を図７に示す。図７に示されるように、重層扁平上皮細胞核、細胞壁が視認できた。 (Example 7)
Staining was carried out in the same manner as in Example 1 except that porcine tongue tissue was used as a biological tissue and an unfixed tissue was used, and the tissue was observed with a transmitted light biological microscope. The results are shown in FIG. As shown in FIG. 7, stratified squamous cell nuclei and cell walls were visible.
　（参考例）
　生物組織として外科材料大腸腫瘍組織を使用した。大腸腫瘍組織をホルマリンにて固定し、腫瘍成分を垂直断にて5mm間隔で平行に切断刃による割を入れて割面を形成した。次にメチレンブルー（和光純薬工業株式会社製の販売元コード275-07895のメチレンブルー原液を30倍希釈したもの）を割面全体に行き渡るに十分量（2ml程度）滴下し、3秒間染色した。次に、水槽中で組織を上下させた。次に、0.001規定シュウ酸水槽中で、観察しながら割面が適当な色合いになるまで、組織切片を上下させた。次に、水槽中で組織を上下させた。次に、この組織切片につき0.001規定硝酸水を割面全体に行き渡るに十分量（2ml程度）滴下した。観察する染色した割面を上にして組織を実体顕微鏡の試料台に載せ、更に同割面に十分量のOCTコンパウンド（Tissue-Tek（登録商標）：SAKURA（登録商標））を被覆させた後、実体顕微鏡で観察した。結果を図８に示す。図８に示されるように、腫瘍細胞核、線維芽細胞核、毛細血管が視認できた。 (Reference example)
Surgical material colon tumor tissue was used as the biological tissue. The colon tumor tissue was fixed with formalin, and the tumor component was split with a cutting blade in parallel at 5 mm intervals by vertical cutting to form a split surface. Next, a sufficient amount (about 2 ml) of methylene blue (diluted 30-fold of a methylene blue stock solution of a manufacturer's code 275-07895 manufactured by Wako Pure Chemical Industries, Ltd.) was dropped over the entire face and dyed for 3 seconds. Next, the tissue was moved up and down in the water tank. Next, the tissue slice was moved up and down while observing in a 0.001 N oxalic acid water bath until the split surface had an appropriate color. Next, the tissue was moved up and down in the water tank. Next, a sufficient amount (about 2 ml) of 0.001 N nitric acid solution was dripped over the entire cleavage surface for each tissue section. Place the tissue on the sample stage of the stereomicroscope with the stained split surface to be observed facing up, and then cover the split surface with a sufficient amount of OCT compound (Tissue-Tek (registered trademark): SAKURA (registered trademark)) Observed with a stereomicroscope. The results are shown in FIG. As shown in FIG. 8, tumor cell nuclei, fibroblast nuclei, and capillaries were visible.
　薄切切片の作製を要しない生物組織、及び薄切切片の作製不能な生物組織の染色、病理診断に有益である。 This is useful for staining and pathological diagnosis of biological tissues that do not require the preparation of sliced sections and those that cannot be prepared.

Claims (4)

  1. 　生物組織の表面及び割面を染色する染色キットであって、
    　前記生物組織を染色するためのメチレンブルーと、
    　前記生物組織を染色するためのヘマトキシリンと、
    　前記メチレンブルーにて染色された生物組織における組織の過剰な染色を脱色するための還元剤と、
    　前記メチレンブルーにて染色された生物組織における色素の退色を防止するための酸化剤と、を備える染色キット。     A staining kit for staining a surface and a split surface of a biological tissue,
    Methylene blue for staining the biological tissue;
    Hematoxylin for staining the biological tissue;
    A reducing agent for decolorizing the excessive staining of the tissue in the biological tissue stained with the methylene blue;
    A staining kit comprising: an oxidizing agent for preventing fading of a pigment in a biological tissue stained with the methylene blue.
  2. 　前記メチレンブルーの濃度が、０．５ｇ／Ｌ～１０ｇ／Ｌである請求項１記載の染色キット。 The staining kit according to claim 1, wherein the concentration of the methylene blue is 0.5 g / L to 10 g / L.
  3. 　前記ヘマトキシリンは、カラッチヘマトキシリンである請求項１又は２記載の染色キット。 The staining kit according to claim 1 or 2, wherein the hematoxylin is caratach hematoxylin.
  4. 　生物組織の表面及び割面を染色する染色方法であって、
    　前記生物組織をメチレンブルーにて染色する第１の染色工程と、
    　前記メチレンブルーにて染色された生物組織における組織の過剰な染色を還元剤にて脱色する脱色工程と、
    　前記生物組織を更にヘマトキシリンにて染色する第２の染色工程と、
    　前記メチレンブルーにて染色された生物組織における色素の退色を酸化剤にて防止する退色防止工程と、を有する染色方法。     A staining method for staining a surface and a split surface of a biological tissue,
    A first staining step of staining the biological tissue with methylene blue;
    A decolorization step of decolorizing excessive staining of the tissue in the biological tissue stained with methylene blue with a reducing agent;
    A second staining step of further staining the biological tissue with hematoxylin;
    A fading prevention step of preventing fading of a pigment in a biological tissue stained with the methylene blue with an oxidizing agent.
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CN112414824A (en) * 2020-12-07 2021-02-26 重庆医科大学 Staining method for non-decalcified bone slice
WO2023235284A1 (en) * 2022-05-31 2023-12-07 Ultivue, Inc. Methods and systems for removing a histological stain from a sample

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CN112414824A (en) * 2020-12-07 2021-02-26 重庆医科大学 Staining method for non-decalcified bone slice
CN112414824B (en) * 2020-12-07 2021-06-15 重庆医科大学 Staining method for non-decalcified bone slice
WO2023235284A1 (en) * 2022-05-31 2023-12-07 Ultivue, Inc. Methods and systems for removing a histological stain from a sample

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