JP2009002768A - Tissue microarray preparation method - Google Patents
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本発明は、組織マイクロアレイ（以下、単に「ＴＭＡ」という。）の作製方法に関し、ＴＭＡによる解析のための、薄片切片試料の新規作製方法に関する。より詳しくは、組織検体を含む最初のパラフィンブロックを有効に活用でき、対象の組織配列（組合せ）を任意に選択可能とする、薄片切片試料の新規作製方法に関する。 The present invention relates to a method for producing a tissue microarray (hereinafter simply referred to as “TMA”), and relates to a novel method for producing a sliced piece sample for analysis by TMA. More specifically, the present invention relates to a novel method for preparing a sliced piece sample that can effectively utilize the first paraffin block containing a tissue specimen and can arbitrarily select a target tissue arrangement (combination).
ヒトゲノムの解析が進み、塩基配列解析から、機能解析へと焦点が移ってきており、生体関連物質を解析するための多く手法が開発されている。生体関連物質を解析するための手法として、マイクロアレイの手法が良く用いられる。例えば、ｃＤＮＡマイクロアレイは、正常組織由来のｃＤＮＡを用いて、多変数の遺伝子発現を解析する方法である。多数の症例で確認するには、それぞれの症例ごとに検索する必要があり、また遺伝子抽出の為に保存された検体のみで実施が可能である。 With the progress of human genome analysis, the focus has shifted from base sequence analysis to functional analysis, and many methods for analyzing biologically relevant substances have been developed. A microarray technique is often used as a technique for analyzing biological materials. For example, a cDNA microarray is a method for analyzing multivariable gene expression using cDNA derived from normal tissue. In order to confirm in a large number of cases, it is necessary to search for each case, and it is possible to carry out only with a specimen stored for gene extraction.
免疫組織化学（Immunohistochemistry;ＩＨＣ）は、発現したタンパク質を認識する抗体を用いて、特定の遺伝子発現タンパク質を個々の症例において解析する方法であり、パラフィンブロックで解析が可能である。また in situ hybridization（ＩＳＨ）は、目的遺伝子の塩基配列に相補なプライマーを用いて、組織上で遺伝子発現を解析する手法である。ＩＨＣ同様、パラフィンブロックを用いて解析が可能である。例えば、トラスツズマブ（Trastuzumab）はヒト癌遺伝子HER2/neu（c-erbB-2）の遺伝子産物であるHER2タンパク質に特異的に結合することで、抗腫瘍効果を発揮するヒト化モノクロナール抗体を含む抗がん剤であるが、このようなヒト化モノクロナール抗体療法においても、ＩＨＣやＩＳＨの手法を用いて適応の判定が行われている。 Immunohistochemistry (IHC) is a method of analyzing specific gene-expressed proteins in individual cases using an antibody that recognizes the expressed protein, and can be analyzed with a paraffin block. In situ hybridization (ISH) is a technique for analyzing gene expression on a tissue using a primer complementary to the base sequence of the target gene. As with IHC, analysis is possible using paraffin blocks. For example, Trastuzumab, which contains humanized monoclonal antibodies that exert antitumor effects by specifically binding to the HER2 protein, the gene product of the human oncogene HER2 / neu (c-erbB-2) Although it is a cancer agent, even in such humanized monoclonal antibody therapy, the determination of indication is performed using IHC or ISH techniques.
免疫組織化学は組織構築を確認しつつ、目的タンパク質の発現を判断できる点が最大の利点であるが、各組織の発現が弱かったりむらが生じたりして、クリアカットに判別できないこともあり、正確でない解析結果（false negativeやfalse positive）を避ける為にはある程度の大きさが必要である。 Immunohistochemistry is the greatest advantage that the expression of the target protein can be judged while confirming the tissue structure, but the expression of each tissue is weak or uneven, and it may not be possible to distinguish it as a clear cut, In order to avoid inaccurate analysis results (false negative and false positive), a certain size is required.
ＴＭＡは、ひとつのプレパラート上に多症例の組織をのせたもので、これまでＩＨＣやＩＳＨを個々別々に行っていたものが、同一プレパラート上で同一条件のもと、同時に解析することができる手法である。今後、オーダーメイド医療の発展に伴うタンパク質発現の包括的解析において、重要な役割を果たす。 TMA is a method in which multiple cases of tissue are placed on a single preparation. What has been performed separately for IHC and ISH can be analyzed simultaneously under the same conditions on the same preparation. It is. In the future, it will play an important role in comprehensive analysis of protein expression accompanying the development of tailor-made medicine.
ＴＭＡの質は１）組織の大きさと数のバランス、２）よくデザインされた組合せ、３）各組織の情報量の多さ、などが要因となり決定される。ＴＭＡの質の向上のために、単に多くの種類の組織を集めればよいという問題ではない。また、直径０．６ｍｍという小さな切片では、質的判断を誤る可能性がある。 The quality of TMA is determined by factors such as 1) the balance between the size and number of organizations, 2) well-designed combinations, and 3) the amount of information in each organization. It is not just a matter of gathering many types of organizations to improve the quality of TMA. In addition, there is a possibility that a qualitative judgment is mistaken for a small section having a diameter of 0.6 mm.
従来のＴＭＡは、パラフィンブロックから被検組織を直径０．６〜２ｍｍ大にくりぬき、円筒状のくりぬいた組織切片（切片試料）を多数集めて、新たなパラフィンブロックに埋め込み、その後薄切し、切片が配置されたシート状の薄片切片試料をスライドグラス上にのせて解析用試料を作製していた（特許文献１）。しかし、最初のパラフィンブロックに穴をあけて円筒状にくりぬいて切片試料を得ることで、一度切片試料をくりぬくと最初のパラフィンブロックは利用不能となる。また、新たなパラフィンブロックに円筒状のくりぬいた切片試料を多数集めて埋め込み、薄片切片試料を作製することは、同一の切片試料の組合せで一度に多くの薄片切片試料を作製するには便利な手法であるが、一度切片試料の組合せが決定されると、同じ組合せの複製しか作製できず、一度シリーズを作製すると作り変えが難しい。しかも薄切を進めていくうちに腫瘍組織が無くなってしまう可能性もある。 In conventional TMA, a test tissue is cut from a paraffin block to a diameter of 0.6 to 2 mm, a large number of cylindrical hollow tissue sections (section samples) are collected, embedded in a new paraffin block, and then sliced. An analysis sample was prepared by placing a sheet-like thin slice sample on which a section was placed on a slide glass (Patent Document 1). However, by drilling a hole in the first paraffin block to obtain a section sample, the first paraffin block cannot be used once the section sample is hollowed out. In addition, collecting and embedding a large number of cylindrical section samples in a new paraffin block to prepare thin section samples is convenient for preparing many thin section samples at the same time by combining the same section samples. Although it is a technique, once a combination of section samples is determined, only a duplicate of the same combination can be produced, and once a series is produced, it is difficult to recreate it. Moreover, there is a possibility that the tumor tissue disappears as the slicing proceeds.
ところで、腫瘍細胞は、組織型により単一なものではなく、個々の症例により組織型が同じでも性質は各々異なり、また、薬剤の感受性も各々異なる。一塩基多型の解析が精力的になされているところである。また腫瘍の性質を知る上で、予後因子は重要なファクターであるにも関わらず、長期間の経過観察を必要とするため解析には大変な労力を必要とする。したがって、プレパラート上の被検試料の組合せも、必要に応じて自由に組合せができれば、より緻密な解析が可能となる。
本発明は、ＴＭＡの作製に関し、組織検体を含む最初のパラフィンブロックを有効に活用でき、対象の組織配列（組合せ）を任意に選択可能とする、薄片切片試料の新規作製方法を提供することを課題とする。 The present invention relates to the production of TMA, and provides a novel method for producing a sliced piece sample that can effectively utilize the first paraffin block including a tissue specimen and can arbitrarily select a target tissue arrangement (combination). Let it be an issue.
本発明者らは上記課題を解決するために鋭意検討を重ねた結果、最初のパラフィンブロックをまず薄切して薄切試料を得、該薄切試料から薄片切片試料を得ることで、上記課題を解決しうることを見出し、本発明を完成した。薄切試料を効果的に裁断するために、薄切試料を、弾力を有する合成樹脂製のプレート上にとり、該プレート上の薄切試料を水溶液に浮かべて広げ、該広げた薄切試料から薄片切片試料を得ることができることを確認し、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors first sliced the first paraffin block to obtain a sliced sample, and obtained a sliced piece sample from the sliced sample. The present invention was completed. In order to effectively cut a sliced sample, the sliced sample is taken on a plate made of a synthetic resin having elasticity, and the sliced sample on the plate is floated on an aqueous solution and spread. It was confirmed that a section sample could be obtained, and the present invention was completed.
すなわち、本発明は以下よりなる。
１．以下の工程を含むＴＭＡの作製方法：
１）摘出検体組織を固定する工程；
２）固定した検体組織を脱水し、パラフィン包埋する工程；
３）包埋試料を薄切して薄切試料を得る工程；
４）上記３）で得た薄切試料を、弾力を有する合成樹脂製のプレート上にとり、該プレート上の薄切試料を水溶液に浮かべて広げる工程；
５）上記４）を水溶液に浮かべた薄切試料を、弾力を有する合成樹脂製のプレート上にすくいとる工程；
６）上記５）のプレート上の薄切試料を裁断用具にて裁断して、薄片切片試料を得る工程。
７）上記６）の薄片切片試料をプレパラート上に配置する工程。
２．６）の工程の後、７）の工程の前に、さらに、薄片切片試料を水溶液に浮かべる工程を含む、前項１に記載のＴＭＡの作製方法。
３．７）の工程の前に、前記再度水溶液に浮かべた薄片切片試料を、弾力を有する合成樹脂製のプレート上にすくいとり、該すくいとった薄片切片試料を７）の工程のプレパラート上にのせる、前項２に記載のＴＭＡの作製方法。
４．前項１〜３の何れかに記載のＴＭＡの作製方法において使用する合成樹脂製プレートからなる薄片試料受け用具。
５．前項４に記載の薄片試料受け用具、および薄片試料裁断用具を含むＴＭＡの作製キット。
That is, this invention consists of the following.
1. TMA production method including the following steps:
1) a step of fixing the extracted specimen tissue;
2) Dehydrating the fixed specimen tissue and embedding it in paraffin;
3) Slicing the embedded sample to obtain a sliced sample;
4) A step of taking the sliced sample obtained in 3) above on a plate made of a synthetic resin having elasticity, and floating the sliced sample on the plate in an aqueous solution;
5) A step of scooping the sliced sample obtained by floating the above 4) in an aqueous solution onto a plate made of synthetic resin having elasticity;
6) A step of cutting the sliced sample on the plate of 5) above with a cutting tool to obtain a sliced piece sample.
7) The process of arrange | positioning the slice section sample of said 6) on a preparation.
2. The method for producing TMA according to item 1, further comprising a step of floating the sliced piece sample in an aqueous solution after the step of 2.6) and before the step of 7).
Before the step of 3.7), the thin slice sample floated again in the aqueous solution is scooped on a plate made of a synthetic resin having elasticity, and the scooped thin slice sample is placed on the preparation of the step of 7). 3. A method for producing TMA according to item 2 above.
4). A thin sample receiving tool comprising a synthetic resin plate used in the method for producing a TMA according to any one of items 1 to 3.
5). 5. A TMA preparation kit comprising the thin sample receiving tool and the thin sample cutting tool according to 4 above.
本発明のＴＭＡの作製方法によると、最初のパラフィンブロック試料から被検試料を円筒状にくりぬく必要がないため、くりぬきによるパラフィンブロックの欠損が生じない。また、従来の方法であれば、円筒状にくりぬいた切片試料を集めて、新たなパラフィンブロックを作製したものから薄片切片試料を作製し、プレパラートを作製するため、新たなパラフィンブロックから複数の薄片切片試料を作製する場合において、組織片の組合せを変更することができない。また、従来法であれば、円筒状にくりぬいた組織片を薄切して切片試料を得ているため、切片試料の大きさ（直径）は変更させることができないのに対し、本発明の方法では、適宜所望の大きさの、例えば２〜８ｍｍの大きさの切片試料を作製することが可能であり、組織構築を確認しやすい。さらに、従来法では２回のパラフィンブロックを作製する必要があるが、本発明の方法であれば、再度のパラフィンブロックを作製する必要がない。 According to the method for producing TMA of the present invention, it is not necessary to hollow out the test sample from the initial paraffin block sample in a cylindrical shape, so that the paraffin block is not lost due to the hollowing out. In addition, in the conventional method, a sliced sample is collected from a cylindrical shape, a sliced sample is prepared from a new paraffin block, and a slice is prepared from the new paraffin block. When producing a section sample, the combination of tissue pieces cannot be changed. Further, in the case of the conventional method, since the sliced sample is obtained by slicing a tissue piece hollowed into a cylindrical shape, the size (diameter) of the sliced sample cannot be changed, whereas the method of the present invention Then, it is possible to prepare a section sample having a desired size as appropriate, for example, 2 to 8 mm, and it is easy to confirm the tissue construction. Furthermore, in the conventional method, it is necessary to prepare the paraffin block twice, but in the method of the present invention, it is not necessary to prepare the paraffin block again.
本発明のＴＭＡの作製方法について、以下詳述する。本明細書において、パラフィンブロックからミクロトームなどによりシート状の試料を得ることを「薄切する」といい、薄切して得た試料を「薄切試料」といい、薄切試料から裁断用具などで、所望の大きさに切り出した試料を、「薄片切片試料」ということとする。「薄片切片試料」を、単に「組織試料」という場合もある。また、パラフィンブロックから円筒状に切り出した組織試料を、「切片試料」といい、切片試料を含む薄切試料を、「薄片切片試料」ともいう。 The method for producing the TMA of the present invention will be described in detail below. In this specification, obtaining a sheet-like sample from a paraffin block with a microtome is referred to as “slicing”, and a sample obtained by slicing is referred to as a “thin slicing sample”. The sample cut out to a desired size is referred to as a “thin slice sample”. The “slice section sample” may be simply referred to as “tissue sample”. In addition, a tissue sample cut out in a cylindrical shape from a paraffin block is referred to as a “section sample”, and a sliced sample including a section sample is also referred to as a “thin section sample”.
本発明のＴＭＡの作製は、以下の１）〜７）の工程を含む。
１）摘出検体組織を固定する工程；
２）固定した検体組織を脱水し、パラフィン包埋する工程；
３）包埋試料を薄切して薄切試料を得る工程；
４）上記３）で得た薄切試料を、弾力を有する合成樹脂製のプレート上にとり、該プレート上の薄切試料を水溶液に浮かべて広げる工程；
５）上記４）を水溶液に浮かべた薄切試料を、弾力を有する合成樹脂製のプレート上にすくいとる工程；
６）上記５）のプレート上の薄切試料を裁断用具にて裁断して、薄片切片試料を得る工程。
７）上記６）の薄片切片試料をプレパラート上に配置する工程。
The production of the TMA of the present invention includes the following steps 1) to 7).
1) a step of fixing the extracted specimen tissue;
2) Dehydrating the fixed specimen tissue and embedding it in paraffin;
3) Slicing the embedded sample to obtain a sliced sample;
4) A step of taking the sliced sample obtained in 3) above on a plate made of a synthetic resin having elasticity, and floating the sliced sample on the plate in an aqueous solution;
5) A step of scooping the sliced sample obtained by floating the above 4) in an aqueous solution onto a plate made of synthetic resin having elasticity;
6) A step of cutting the sliced sample on the plate of 5) above with a cutting tool to obtain a sliced piece sample.
7) The process of arrange | positioning the slice section sample of said 6) on a preparation.
ここにおいて、工程１）の摘出検体組織については、ＴＭＡの手法に適用しうるあらゆる検体組織を適用することができる。さらに、工程１）および工程２）は、通常のＴＭＡの作製において、自体公知の方法を適用することができる。 Here, any sample tissue applicable to the technique of TMA can be applied to the extracted sample tissue in step 1). Further, in steps 1) and 2), a method known per se can be applied in the production of ordinary TMA.
従来では、３）の工程で、包埋試料から円筒状の試料を切り出して、まず切片試料を得ていたのに対し、本発明では包埋試料をまず薄切して薄切試料を得ることが、本発明の最大の特徴点である。薄切の手段は自体公知の方法、例えばミクロトームなどの薄切用機器を用いることができる。 Conventionally, in the step 3), a cylindrical sample was cut out from an embedded sample to obtain a sliced sample first, whereas in the present invention, an embedded sample is first sliced to obtain a sliced sample. Is the greatest feature of the present invention. As a means for slicing, a method known per se, for example, a slicing device such as a microtome can be used.
本発明は、３）で得た薄切試料を、弾力を有する合成樹脂製のプレート上にとり、該プレート上の薄切試料を水溶液に浮かべて薄切試料を広げる工程４）を含む。ここにおいて、弾力を有する合成樹脂製のプレートとは、ガラス状のような硬質素材ではない合成樹脂を素材とするプレートをいい、例えばカッティングマットなどに使用されている材質のプレートを挙げることができる。具体的には、発泡性フッ素樹脂、アクリル樹脂、軟質塩化ビニルや塩化ビニルの他、ポリプロピレン（ＰＰ）などの軟質オレフィン系樹脂や硬質オレフィン系樹脂などの素材からなるプレートを挙げることができる。 The present invention includes a step 4) of taking the sliced sample obtained in 3) on a plate made of a synthetic resin having elasticity, and spreading the sliced sample by floating the sliced sample on the plate in an aqueous solution. Here, the plate made of a synthetic resin having elasticity means a plate made of a synthetic resin that is not a hard material such as glass, and examples thereof include a plate made of a material used for a cutting mat or the like. . Specific examples include plates made of materials such as foaming fluororesins, acrylic resins, soft vinyl chloride and vinyl chloride, soft olefin resins such as polypropylene (PP), and hard olefin resins.
上記において、薄切試料を水溶液に浮かべて薄切試料を広げる工程を設けることは、本発明の方法を達成するために、必須である。従来、薄切試料を先に得て試料を加工することは、薄切試料の取扱い上非常に困難であることが問題であった。しかし、上記の弾力を有する合成樹脂製のプレートを用いて、薄切試料をすくい、水溶液表面で広げることで、薄切試料の取扱いが可能になり、続けて行う裁断処理も可能になった。水溶液は、試料を損傷させない溶液であればよく、特に限定されないが、水、生理食塩水、生物試料の取扱い分野で使用される一般的な緩衝液などを使用することができる。 In the above, it is indispensable to provide the step of spreading the sliced sample by floating the sliced sample in an aqueous solution in order to achieve the method of the present invention. Conventionally, it has been a problem that it is very difficult to handle a sliced sample by first obtaining a sliced sample and processing the sample. However, by using a plate made of synthetic resin having the elasticity described above, the sliced sample is scooped and spread on the surface of the aqueous solution, so that the sliced sample can be handled, and the subsequent cutting process is also possible. The aqueous solution is not particularly limited as long as it does not damage the sample, and water, physiological saline, general buffers used in the field of handling biological samples, and the like can be used.
本発明の５）の工程で、水溶液に浮かべた薄切試料を、弾力を有する合成樹脂製のプレート上にすくいとり、６）の工程で、上記プレート上の薄切試料を裁断用具にて裁断し、薄片切片試料を得る。弾力を有する合成樹脂製のプレート上で薄切試料を裁断することで、薄切試料に損傷を与えず裁断することができる。ここで裁断用具は、薄切試料を裁断しうる用具であればよく、特に限定されないが、例えば皮膚検体採取用の円筒形のカッターや、通常の切り出しナイフなどを挙げることができる。 The sliced sample floated in the aqueous solution in the step 5) of the present invention is scooped up on a plate made of synthetic resin having elasticity, and the sliced sample on the plate is cut with a cutting tool in the step 6). Then, a sliced piece sample is obtained. By cutting a sliced sample on a plate made of a synthetic resin having elasticity, the sliced sample can be cut without being damaged. Here, the cutting tool is not particularly limited as long as it is a tool capable of cutting a sliced sample, and examples thereof include a cylindrical cutter for collecting a skin specimen and a normal cutting knife.
本発明の７）の工程で、上記６）の工程で得た薄片切片試料をプレパラート上に配置し、所望の組合せのＴＭＡを作製する。薄片切片試料をプレパラートに配置するために、薄片切片試料を水溶液に浮かべる工程を含んでいてもよい。また、前記再度水溶液に浮かべた薄片切片試料を、弾力を有する合成樹脂製のプレート上にすくいとり、該すくい取った薄片切片試料をプレパラート上にのせることで、プレパラート上の所望の位置に、所望の組織試料を配置させることができる。プレパラート上に配置させた薄片切片試料は、通常の方法により、伸展させることができる。 In the step 7) of the present invention, the sliced piece sample obtained in the step 6) is placed on a preparation to produce a desired combination of TMAs. In order to place the sliced piece sample in the preparation, a step of floating the sliced piece sample in the aqueous solution may be included. In addition, the thin slice sample floated in the aqueous solution again is scooped on a plate made of a synthetic resin having elasticity, and the scooped thin slice sample is placed on the slide, so that the desired position on the slide is obtained. A desired tissue sample can be placed. The sliced piece sample placed on the preparation can be extended by a usual method.
本発明のＴＭＡの作製方法で、解析を所望する組織（被検組織）について、上述の１）〜６）の工程を各々繰り返して各薄片切片試料を作製し、上述の方法により、プレパラート上の所望の位置に、所望の薄片切片試料を配置させ、並べることにより所望のＴＭＡを得ることができる。 With the TMA production method of the present invention, for the tissue (test tissue) desired to be analyzed, each of the above-mentioned steps 1) to 6) is repeated to produce each sliced piece sample. A desired TMA can be obtained by arranging and arranging a desired sliced piece sample at a desired position.
また、従来と同様に、所望のＴＭＡを得るために、予め薄切試料を染色することで、ＴＭＡに使用する試料として適切な部位の組織試料を選択することができる。薄切試料の染色の方法は、本技術分野で用いられる通常の染色方法を適用することができる。 In addition, as in the prior art, in order to obtain a desired TMA, a tissue sample at an appropriate site can be selected as a sample to be used for TMA by staining a sliced sample in advance. A normal staining method used in this technical field can be applied as a method for staining a sliced sample.
本発明は、ＴＭＡの作製方法において使用する弾力を有する合成樹脂製のプレートからなる薄切試料受け用具も発明の範囲に含まれる。薄切試料受け用具は、薄片切片試料を受ける用具としても利用することができる。さらには、該薄片試料受け用具、および薄片試料裁断用具を含むＴＭＡの作製キットにも及ぶ。 The present invention also includes a thin-cut sample receiving tool made of a synthetic resin plate having elasticity that is used in the method for producing TMA. The sliced sample receiving tool can also be used as a tool for receiving a sliced piece sample. Further, the present invention extends to a TMA preparation kit including the thin sample receiving tool and the thin sample cutting tool.
以下に本発明の実施例を示し、本発明をさらに具体的に説明するが、本発明はこれらに限定されるものではなく、本発明の技術的思想を逸脱しない範囲内で種々の応用が可能である。 Examples of the present invention will be described below in more detail, but the present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention. It is.
（実施例１）
本実施例では、大腸癌のうち、高分化腺癌：５例、中分化腺癌：５例、低分化腺癌：５例、粘液癌：４、印環細胞癌：２例および正常大腸粘膜：２例について、本発明の方法によりＴＭＡを作製し、解析を行った。一枚のスライドグラス上に４ｍｍと３ｍｍの大腸癌２１症例の組織片(4mm-19片と3mm-2片)および正常大腸粘膜組織片２症例(4mm)が収載されている。
(Example 1)
In this example, among colon cancer, well differentiated adenocarcinoma: 5 cases, moderately differentiated adenocarcinoma: 5 cases, poorly differentiated adenocarcinoma: 5 cases, mucinous cancer: 4, signet ring cell carcinoma: 2 cases, and normal colon mucosa : For two cases, TMA was prepared and analyzed by the method of the present invention. Tissue pieces (4 mm-19 pieces and 3 mm-2 pieces) of 21 cases of colorectal cancer of 4 mm and 3 mm and 2 cases of normal colon mucosa tissue pieces (4 mm) are placed on one slide glass.
ＲＦＰ染色の結果、大腸癌症例２１例中１２例は陽性であり、ネガティブコントロールの正常大腸粘膜組織切片２片は陰性という良好な結果を得た（図１参照）。 As a result of RFP staining, 12 out of 21 colorectal cancer cases were positive, and 2 normal colon mucosa tissue sections of negative control were negative (see FIG. 1).
ＲＦＰ染色は、Beckman Coulter社製UltraTech HRP Streptavidin-Biotin Detection Systemを用い、添付文書の方法に従って染色した。ＲＦＰはRET finger proteinの略で、転写因子をつかさどる核タンパク質の一種であり、本実施例では該ＲＦＰのアミノ酸配列の一部を認識する抗体（rabbit polyclonal anti-RFP antibody）を用いて染色した。該染色した組織について、DAB発色（発現が見られる場合、濃茶色に発色がみられる）の有無を解析した。 For RFP staining, UltraTech HRP Streptavidin-Biotin Detection System manufactured by Beckman Coulter was used and stained according to the method of the package insert. RFP is an abbreviation for RET finger protein, and is a kind of nuclear protein that controls transcription factors. In this example, staining was performed using an antibody that recognizes part of the amino acid sequence of the RFP (rabbit polyclonal anti-RFP antibody). The stained tissue was analyzed for the presence or absence of DAB color development (when expression was observed, color development was observed in dark brown).
さらに、大腸癌の上記ＴＭＡ解析の２０倍拡大像を図２に示し、２００倍拡大像を図３に示した。濃く染まっているのが陽性の大腸癌の細胞核である。 Further, a 20-fold magnified image of the above-described TMA analysis of colon cancer is shown in FIG. 2, and a 200-fold magnified image is shown in FIG. The cell nuclei of positive colorectal cancer are darkly stained.
（実施例２）
本実施例では、胃癌のうち、高分化腺癌：４例、中分化腺癌：４例、低分化腺癌：４例、印環細胞癌：３例および正常大腸粘膜：２例について、本発明の方法によりＴＭＡを作製し、解析を行った。一枚のスライド上に胃癌症例１５例および正常胃粘膜組織片２例が収載されている。
(Example 2)
In this example, among gastric cancers, well differentiated adenocarcinoma: 4 cases, moderately differentiated adenocarcinoma: 4 cases, poorly differentiated adenocarcinoma: 4 cases, signet ring cell carcinoma: 3 cases, and normal colon mucosa: 2 cases, TMA was produced by the method of the invention and analyzed. On one slide, 15 cases of gastric cancer and 2 pieces of normal gastric mucosa tissue are listed.
ＲＦＰ染色の結果、胃癌症例１５例中９例は陽性であり、ネガティブコントロールの正常胃粘膜組織切片２片は陰性という良好な結果を得た（図４参照）。 As a result of RFP staining, 9 out of 15 cases of gastric cancer were positive, and 2 pieces of negative control normal gastric mucosa tissue sections were negative (see FIG. 4).
以上詳述したように、本発明のＴＭＡの作製方法によると、最初のパラフィンブロック試料から被検組織を円筒状にくりぬく必要がないため、くりぬきによるパラフィンブロックの欠損が生じない。また、従来の方法であれば、円筒状にくりぬいた切片試料を集めて、新たなパラフィンブロックを作製し、さらに薄片切片試料を作製してプレパラート上の組織試料を作製するため、新たなパラフィンブロックから複数の組織試料を作製する場合に、組織片の組合せを変更することができない。また、従来法であれば、円筒状にくりぬいた切片試料を薄切して薄片切片試料を得ているため、薄片切片試料の大きさ（直径）は変更させることができないのに対し、本発明の方法では適宜所望の大きさの、例えば２〜８ｍｍの大きさの薄片切片試料を作製することができ、組織構築を確認しやすい。 As described above in detail, according to the TMA production method of the present invention, it is not necessary to hollow out the test tissue from the initial paraffin block sample in a cylindrical shape, so that the defect of the paraffin block due to the hollowing does not occur. In addition, if the conventional method is used, a section sample that is hollowed out in a cylindrical shape is collected to produce a new paraffin block, and a slice section sample is further prepared to prepare a tissue sample on the slide. When producing a plurality of tissue samples from the above, the combination of tissue pieces cannot be changed. Further, according to the present invention, since the sliced sample is obtained by slicing a sliced sample cut into a cylindrical shape, the size (diameter) of the sliced sample cannot be changed. In this method, it is possible to prepare a sliced piece sample having a desired size, for example, a size of 2 to 8 mm, and it is easy to confirm the tissue construction.
本発明のＴＭＡの作製方法によると、ドナーブロック（最初のパラフィンブロック試料）を傷めない為、グレード別や臓器別、予後別など任意に組織配列の選択が可能である。また組織サイズとして適切な大きさが確保されているため組織構築の確認ができ、病理診断時のマスタースライドや教材としても利用することができる。 According to the TMA production method of the present invention, since the donor block (first paraffin block sample) is not damaged, it is possible to arbitrarily select the tissue arrangement by grade, organ, prognosis and the like. In addition, since an appropriate size is ensured as the tissue size, the organization construction can be confirmed, and it can be used as a master slide or teaching material for pathological diagnosis.
Claims (5)
１）摘出検体組織を固定する工程；
２）固定した検体組織を脱水し、パラフィン包埋する工程；
３）包埋試料を薄切して薄切試料を得る工程；
４）上記３）で得た薄切試料を、弾力を有する合成樹脂製のプレート上にとり、該プレート上の薄切試料を水溶液に浮かべて広げる工程；
５）上記４）を水溶液に浮かべた薄切試料を、弾力を有する合成樹脂製のプレート上にすくいとる工程；
６）上記５）のプレート上の薄切試料を裁断用具にて裁断して、薄片切片試料を得る工程。
７）上記６）の薄片切片試料をプレパラート上に配置する工程。 A method for producing a tissue microarray comprising the following steps:
1) a step of fixing the extracted specimen tissue;
2) Dehydrating the fixed specimen tissue and embedding it in paraffin;
3) Slicing the embedded sample to obtain a sliced sample;
4) A step of taking the sliced sample obtained in 3) above on a plate made of a synthetic resin having elasticity, and spreading the sliced sample on the plate in an aqueous solution;
5) A step of scooping the sliced sample obtained by floating the above 4) in an aqueous solution onto a plate made of synthetic resin having elasticity;
6) A step of cutting the sliced sample on the plate of 5) above with a cutting tool to obtain a sliced piece sample.
7) The process of arrange | positioning the slice section sample of said 6) on a preparation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011013050A (en) * | 2009-06-30 | 2011-01-20 | Pathology Institute | Method for preparing biosample specimen |
| CN102928279A (en) * | 2012-11-13 | 2013-02-13 | 无锡江南计算技术研究所 | Metallographical sample-filling secondary glue filling method |
| CN103439163A (en) * | 2013-09-04 | 2013-12-11 | 中南大学湘雅三医院 | Preparation method and preparation apparatus for typography type tissue chip |
| CN103940648A (en) * | 2014-04-04 | 2014-07-23 | 山西农业大学 | Preparation method for gill tissue paraffin section |
| CN108593380A (en) * | 2018-04-25 | 2018-09-28 | 复旦大学附属中山医院 | A kind of organization chip volume production production method |
| JP2021060279A (en) * | 2019-10-07 | 2021-04-15 | 学校法人藤田学園 | Specimen holder and trace element detection method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6380216A (en) * | 1986-09-24 | 1988-04-11 | Bio Meito:Kk | Plastic slide glass |
| JP2002122524A (en) * | 2000-10-13 | 2002-04-26 | Sumitomo Metal Mining Co Ltd | Method of making sample for transmission electron microscope |
| JP2004069666A (en) * | 2002-08-08 | 2004-03-04 | Tsuerun:Kk | Technique for cutting out and aligning part of paraffin section |
| JP2004258017A (en) * | 2003-02-07 | 2004-09-16 | Yasuhiko Kitayama | Array block preparation method, tissue scooping-out device used for the same, and tissue block |
| JP2005530504A (en) * | 2002-06-25 | 2005-10-13 | セケレシュ，チェルジー | Title of invention: Manual set for construction of tissue microarray |
| US20060269985A1 (en) * | 2005-05-31 | 2006-11-30 | Yasuhiko Kitayama | Method for constructing array blocks, and tissue punching instrument and tissue blocks used therefor |
| WO2007010924A1 (en) * | 2005-07-21 | 2007-01-25 | National University Corporation University Of Toyama | Gelatinized product of cell, and preparation system for high density cell or tissue array |
-
2007
- 2007-06-21 JP JP2007163417A patent/JP2009002768A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6380216A (en) * | 1986-09-24 | 1988-04-11 | Bio Meito:Kk | Plastic slide glass |
| JP2002122524A (en) * | 2000-10-13 | 2002-04-26 | Sumitomo Metal Mining Co Ltd | Method of making sample for transmission electron microscope |
| JP2005530504A (en) * | 2002-06-25 | 2005-10-13 | セケレシュ，チェルジー | Title of invention: Manual set for construction of tissue microarray |
| JP2004069666A (en) * | 2002-08-08 | 2004-03-04 | Tsuerun:Kk | Technique for cutting out and aligning part of paraffin section |
| JP2004258017A (en) * | 2003-02-07 | 2004-09-16 | Yasuhiko Kitayama | Array block preparation method, tissue scooping-out device used for the same, and tissue block |
| US20060269985A1 (en) * | 2005-05-31 | 2006-11-30 | Yasuhiko Kitayama | Method for constructing array blocks, and tissue punching instrument and tissue blocks used therefor |
| WO2007010924A1 (en) * | 2005-07-21 | 2007-01-25 | National University Corporation University Of Toyama | Gelatinized product of cell, and preparation system for high density cell or tissue array |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011013050A (en) * | 2009-06-30 | 2011-01-20 | Pathology Institute | Method for preparing biosample specimen |
| CN102928279A (en) * | 2012-11-13 | 2013-02-13 | 无锡江南计算技术研究所 | Metallographical sample-filling secondary glue filling method |
| CN102928279B (en) * | 2012-11-13 | 2015-07-08 | 无锡江南计算技术研究所 | Metallographical sample-filling secondary glue filling method |
| CN103439163A (en) * | 2013-09-04 | 2013-12-11 | 中南大学湘雅三医院 | Preparation method and preparation apparatus for typography type tissue chip |
| CN103940648A (en) * | 2014-04-04 | 2014-07-23 | 山西农业大学 | Preparation method for gill tissue paraffin section |
| CN103940648B (en) * | 2014-04-04 | 2016-02-24 | 山西农业大学 | The preparation method of fish gill tissue paraffin section |
| CN108593380A (en) * | 2018-04-25 | 2018-09-28 | 复旦大学附属中山医院 | A kind of organization chip volume production production method |
| JP2021060279A (en) * | 2019-10-07 | 2021-04-15 | 学校法人藤田学園 | Specimen holder and trace element detection method |
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