JP5566115B2 - 生物材料の固定 - Google Patents
生物材料の固定 Download PDFInfo
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- JP5566115B2 JP5566115B2 JP2009551197A JP2009551197A JP5566115B2 JP 5566115 B2 JP5566115 B2 JP 5566115B2 JP 2009551197 A JP2009551197 A JP 2009551197A JP 2009551197 A JP2009551197 A JP 2009551197A JP 5566115 B2 JP5566115 B2 JP 5566115B2
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- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
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- 235000002906 tartaric acid Nutrition 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 229950003937 tolonium Drugs 0.000 description 1
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 1
- PFURGBBHAOXLIO-PHDIDXHHSA-N trans-cyclohexane-1,2-diol Chemical compound O[C@@H]1CCCC[C@H]1O PFURGBBHAOXLIO-PHDIDXHHSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
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- 238000013519 translation Methods 0.000 description 1
- 229960002622 triacetin Drugs 0.000 description 1
- DQWPFSLDHJDLRL-UHFFFAOYSA-N triethyl phosphate Chemical compound CCOP(=O)(OCC)OCC DQWPFSLDHJDLRL-UHFFFAOYSA-N 0.000 description 1
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- FAKLAEQNIGOLGL-UHFFFAOYSA-N undecane-1,2,11-triol Chemical compound OCCCCCCCCCC(O)CO FAKLAEQNIGOLGL-UHFFFAOYSA-N 0.000 description 1
- LONLGEZTBVAKJF-UHFFFAOYSA-N undecane-1,2,3-triol Chemical compound CCCCCCCCC(O)C(O)CO LONLGEZTBVAKJF-UHFFFAOYSA-N 0.000 description 1
- BUMVVNKGNPPUME-UHFFFAOYSA-N undecane-1,2-diol Chemical compound CCCCCCCCCC(O)CO BUMVVNKGNPPUME-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
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Classifications
-
- 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
- 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
- G01N2001/305—Fixative compositions
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Description
i) 生物材料を提供する工程、および
ii) 該生物材料を、
(a1) 10〜90容量%のメタノール、および
(a2) 少なくとも1つのさらなる添加剤、および
(a3) 任意に酸、
を含む第一の非水性組成物と接触させる工程、
iii) 該生物材料を最高99容量%のエタノールを含む第二の組成物(B)中に移動させる工程
を含む方法によって提供される。
(α1) 10以上80容量%未満のメタノール、および
(α2) 少なくとも一つのさらなる添加剤、および
(α3) 酸
を含むものである。
iv) 生物材料の手作業での処理、生物材料のマイクロ波エネルギーによる処理または生物材料のあらゆる組織処理装置による処理から選択される、
さらなる処理工程
が続くことが好ましくあり得る。
(b1) 上記の本発明の方法の文脈において記載された、工程ii)の第一の組成物として使用可能な組成物、および
(b2) 組成物(B)
(b3) 任意に、包埋材料(C)、および/または、生物材料中のまたは生物材料の生体分子の分析または生物材料の形態分析のための試薬
を含むキットも、上記の目的達成に寄与する。
(b1) 組成物(A)
(b2) 任意に、組成物(B)
(b3) 任意に、包埋材料(C)、および/または、生物材料中のまたは生物材料の生体分子の分析または生物材料の形態分析のための試薬
を含むキットもまた、本発明の目的達成に寄与する。
(c1) 方法工程i)、ii)、iii)および任意にiv)を含む本発明の方法を実施する工程、および
(c2) 処理された生物材料を分子生物学的および/または組織学的に分析する工程
を含む、生体外での疾患の診断方法が、上記の目的達成に寄与する。
組成物Aに従った種々の試薬で安定化された組織試料からのRNAの単離
ラットの肝組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、5mlのポリプロピレン製の回収容器中、組成物A(表1)に従った2〜4mlの固定化溶液中に完全に浸した。組織試料を周囲温度で24時間保存した。
組成物Aに従った種々の試薬および主成分としてエタノールを含有する試薬で安定化した組織からのRNAの単離
ラットの肝組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、5mlのポリプロピレン製の回収容器中、組成物Aに従ったエタノール(表2、4〜6)またはメタノール(表2、1〜3)を含有する2〜4mlの固定化溶液中に完全に浸した。組織試料を周囲温度で24時間保存した。
主成分としてメタノールを含有する、含水または無水の種々の試薬で安定化した組織からのRNAの単離
ラットの肝組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、2〜4mlの、水溶液中のメタノール含有固定化溶液(表3、1)または組成物Aに従った無水試薬中のメタノール含有固定化溶液(表3、2)中に完全に浸した。組織試料を周囲温度でかなりの期間である4日間保存した。
種々の試薬で安定化し、本発明の組成物B を有する第二の試薬中へ移すか移さない組織からのRNAの単離
ラットの腸組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、組成物Aに従った種々の試薬5ml中に完全に浸し、周囲温度で保存した。試料は、7日間保存するか(表4、1〜4)、または、4時間後に組成物Bに従った試薬5ml中に移して、この試薬中で7日間保存した(表4、5〜8)。
アガロースゲル電気泳動により、7日間の保存後であっても、本発明に従って組成物AおよびBに従った試薬中で保存し、処理した組織試料から、損傷を受けないRNAを単離できたことが示された。移動を実施しなかった場合(図4)、ゲルによって低収量が確認された。
本発明の組成物AおよびBに従った試薬で安定化した組織からのRNAの単離
ラットの肝組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、10mlの組成物Aに従った試薬中に完全に浸し、周囲温度で保存した。1つの試料は3日間保存し(表5、10)、他のものは2時間後に10mlの組成物Bに従った種々の試薬中に移し、これらの試薬中で3日間保存した(表5、1〜8)。対照として、1つの試料をRNAlater中で保存した(表5、9)。
本発明に従って安定化した組織からのRNAの単離
ラットの肝組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、2mlの工程ii)の組成物に従った試薬中に完全に浸し、周囲温度で4時間保存し、組成物Bに従った種々の試薬中に移し、さらに2日間保存した(表6、3〜9)。加えて、1つの試料は移さずに、工程ii)に従った試薬組成物中に保存し(表6、10)、一つの試料は100%エタノール中に移し(表6、1)、一つの試料は70%エタノール中に移した(表6、2)。
本発明に従って安定化した組織からのRNAの単離、クロロホルムの置換
ラットの肝組織を、解剖直後に約5x4x4mmの小片に切り分けた。試料は、それぞれ、5mlの、クロロホルム含有試薬、クロロホルムを対応量の水またはメタノールで置換した試薬、または工程ii)の組成物による従った試薬のいずれかに完全に浸し、周囲温度で48時間保存した(表7、1〜4)。加えて、1つの試料は、3時間後に、組成物Bに従った試薬中に移し、さらに45時間保存した(表7、5)。
本発明に従って組成物AおよびBに従った試薬で安定化し、慣用法で処理した、パラフィン包埋組織からのRNAの単離
ラットの脾臓組織を、解剖直後に約4x4x4mmの小片に切り分けた。試料は、5mlの、組成物Aに従った試薬中に完全に浸し、周囲温度で24時間保存した。このインキュベーション期間の後、試料は、直接処理するか(表8、1および4)、または、組成物Bに従った試薬中に移し、周囲温度で(表8、2および5)または4℃で(表8、3および6)さらに4日間保存して、最終的に処理した。
本発明に従って安定化し、マイクロ波エネルギーで処理した、パラフィン包埋組織からのRNAの単離
ラットの脾臓組織を、解剖直後に約4x4x4mmの小片に切り分けた。試料は、5mlの、組成物Aに従った試薬中に完全に浸した。試料は、周囲温度で24時間保存するか(表9、2および3)、または30分後に組成物Bに従った試薬中に移し、周囲温度で24時間保存した(表9、4および5)。並行して、1つの試料を5mlのBoonfixに浸した(表9、1)。
処理には、試料の100%エタノール中での脱水およびマイクロ波エネルギーによる65℃の加熱を含む、4工程の標準的プロトコルを適用した。エタノールをイソプロパノールで置換し、真空加熱で風乾後、マイクロ波エネルギーによる熱と真空を同時に用いる最終工程において、試料を流動パラフィン(低融点パラプラスト(Paraplas)XTRA、ロス(Roth)社)に浸透させた(プロトコル工程は表10参照)。ミクロトーム法に必要な支持材を提供するため、試料を浸潤用に用いられる同一のパラフィン中に手作業で包埋した。
本発明に従って組成物AおよびBに従った試薬で安定化し、慣用法で処理した、組織試料の組織学的分析
ラットの小腸組織を、解剖直後に約6mmの長さの小片に切り分けた。試料は、5mlの、組成物Aに従った試薬中に完全に浸した。周囲温度で4時間後、試料を組成物Bに従った試薬中に移し、周囲温度で20時間保存した(表11、A)。並行して、1つの試料を5mlの10%中性緩衝ホルマリン中に浸した(NBF;表11、B)。24時間後、試料を標準的な処理カセット(ヒストセット(histosette))中に入れ、初めに100%エタノール中でそれぞれ180分間のインキュベーションを2回行う、標準的なプロトコルに従って、手作業で処理した。100%キシレン中で60分間のインキュベーションを2回行って、清浄した。65℃で約12時間、流動パラフィン(低融点パラプラスト(Paraplas)XTRA、ロス(Roth)社)への浸透を行った後、同一のパラフィン中に包埋した。
本発明の組成物Aに従って種々の試薬で安定化し、慣用法で処理した、組織試料の組織学的分析
ラットの脾臓および腎臓を、解剖直後に約3x5x5mmの小片に切り分けた。試料は、10mlの、組成物Aに従った試薬または10%中性緩衝ホルマリン中に完全に浸した(表13)。周囲温度で24時間後、試料を処理し、染色した。試料の処理は、ライカ(Leica)TP1020ティッシュ・プロセッサー(Tissue Processor)にて、解剖の約30時間後に実施した。パラフィン包埋組織試料を6μmの薄片に切った。10%ホルマリン中に保存した腎臓試料のみ、4μmの薄片に切った。実施例8および10に詳細に記載した方法と同一または類似の標準的プロトコルに従って、ライカ・オートステイナー(Autostainer)でヘマトキシリン-エオジン染色を実施した。
本発明に従って組成物Aに従った試薬で安定化し、慣用法で処理した、パラフィン包埋組織からのDNA単離
ラットの脾臓組織を、解剖直後に約2x5x5mmの小片に切り分けた。試料は、5mlの組成物Aに従った種々の試薬中に完全に浸し(表14)、周囲温度で24時間保存した。このインキュベーション期間の後、実施例7に記載した通り、試料を手作業で処理した。パラフィン包埋組織塊は、DNA抽出の前に、周囲温度で5週間保存した。
Claims (25)
- 核酸を含む生物材料を安定化する方法であって、該方法が、
i)前記生物材料(生物がヒトの場合は生物材料はヒトから採取されたものに限る。)を提供する工程、
ii)前記生物材料を、
(a1)10〜90容量%のメタノール、および
(a2)少なくとも1つのさらなる添加剤(クロロホルムおよびトリクロロエタンを含まない。)、および
(a3)任意に酸
を含む第一の非水性組成物と接触させる工程、および
iii)工程ii)の前記生物材料を最高99容量%のエタノールを含む第二の組成物(B)中に移動させる工程
を含む、方法。 - 前記生物材料の前記組成物との前記接触が、-80℃〜+80℃の範囲の温度で実施され、および任意に前記組成物が前記温度範囲でさらに保存される、請求項1に記載の方法。
- 前記生物材料が、前記工程ii)において前記組成物と接触させられた後、前記工程ii)の後または工程iii)の後、-80℃〜+80℃の範囲の温度で保存される請求項1または2に記載の方法。
- 移動工程iii)が、生物材料を工程ii)の組成物から取り出し、該材料を組成物(B)に浸すことによって、または、生物材料を含有する工程ii)の組成物を組成物(B)と混合することによって実施される、請求項1〜3のいずれかに記載の方法。
- 請求項1〜4のいずれかに記載の方法であって、該方法が、
iv)前記生物材料の手作業での処理、マイクロ波エネルギーによる前記生物材料の処理または任意の組織処理装置による前記生物材料の処理から選択される、
さらなる処理工程を含む、方法。 - 前記生物材料が細胞または組織を含む、請求項1〜5のいずれかに記載の方法。
- 前記生物材料が、生命体、単離細胞、オルガネラ、バクテリア、菌類または菌類の一部、ウイルス、ウイロイド、プリオン、組織、組織片、組織切片、体液、天然の、任意に単離されたタンパク質、合成または変性タンパク質、天然の、任意に単離した核酸、合成または変性核酸、脂質、炭水化物、代謝産物および代謝物等の他の生体分子、植物または植物の一部、糞便、スワブ、吸引物、食品試料、環境試料、法医学試料を含む、請求項6に記載の方法。
- 前記生物材料が、新鮮または冷凍された生物材料である請求項1〜7のいずれかに記載の方法。
- 工程iii)の後に、前記材料を包埋材料(C)中に、浸透および/または包埋させることをさらに含む、請求項1〜8のいずれかに記載の方法。
- 包埋材料(C)がパラフィン、鉱物油、非水溶性ワックス、セロイジン、ポリエチレングリコール、ポリビニルアルコール、寒天、ゼラチン、ニトロセルロース、メタクリレート樹脂、エポキシ樹脂、または他のプラスチック媒体より選択される請求項9に記載の方法。
- 前記の処理した生物材料を保存すること、
前記試料中に元来含有されていた少なくとも一つの生物学的成分の単離、
生物材料中のまたは生物材料の、少なくとも一つの生物学的成分の分析であって、
生物学的成分が核酸、タンパク質、ペプチドおよび/もしくはペプチド核酸を含む、分析
ならびに/または
生物材料の組織学的分析
をさらに含む、請求項1〜10のいずれかに記載の方法。 - 生物材料の前記組織学的分析が染色、顕微鏡検査、解剖、ハイブリダイゼーションおよび/もしくは免疫組織化学を含む請求項11に記載の方法。
- 成分(a2)が、1以上の洗浄剤、核酸および/またはタンパク質の分解を阻害する1以上の阻害剤、1以上の粘度調整剤、1以上の染料、1以上の緩衝化合物、1以上の保存料、1以上の錯形成剤、1以上の還元剤、細胞透過性を改善する1以上の物質、1以上のカオトロピック物質、1以上の固定剤、メタノールと異なる1以上のさらなる溶媒および少なくとも2つのこれらの添加物の混合物を含む群から選択される、請求項1〜12のいずれかに記載の方法。
- 成分(a2)が、1,3-ブタンジオール、1,4-ブタンジオール、1,3-プロパンジオール、1,2-プロパンジオール、3-メチル-1,3,5-ペンタントリオール、1,2,6-ヘキサントリオール、グリセリン、グリコール等のジオールおよび/またはトリオール等のC2〜C12ポリオール;PEGおよびDEGMEAより選択される、請求項13に記載の方法。
- 生物材料の保存のための非水性組成物(A)であって、
(α1)10〜80容量%未満のメタノール、
(α2)少なくとも一つのさらなる添加剤(クロロホルムおよびトリクロロエタンを含まない。)であって、1以上の洗浄剤、核酸および/またはタンパク質の分解を阻害する1以上の阻害剤、1以上の粘度調整剤、1以上の染料、1以上の緩衝化合物、1以上の保存料、1以上の錯形成剤、1以上の還元剤、細胞透過性を改善する1以上の物質、1以上のカオトロピック物質、1以上の固定剤、メタノールと異なる1以上のさらなる溶媒および少なくとも2つのこれらの添加物の混合物を含む群から選択されるか、または、1,3-ブタンジオール、1,4-ブタンジオール、1,3-プロパンジオール、1,2-プロパンジオール、3-メチル-1,3,5-ペンタントリオール、1,2,6-ヘキサントリオール、グリセリン、グリコール等のジオールおよび/またはトリオール等のC2〜C12ポリオール;PEGおよびDEGMEAより選択される添加剤、および
(α3)酸
を含む、請求項1〜14のいずれかに記載の方法において使用される、組成物。 - 成分(α1)が、20容量%〜80容量%未満の量で存在する、請求項15に記載の組成物。
- 成分(α2)が1〜50容量%の量で存在する、請求項15または16に記載の組成物。
- 成分(α3)が、ギ酸、酢酸およびプロピオン酸またはそれらの混合物等の弱有機酸等の有機酸の少なくとも一つである、請求項15〜17のいずれかに記載の組成物。
- 成分(a2)が1〜50容量%の量で存在する、請求項1〜14のいずれかに記載の方法。
- 成分(a3)が、ギ酸、酢酸およびプロピオン酸またはそれらの混合物等の弱有機酸等の有機酸の少なくとも一つである、請求項1〜14のいずれかに記載の方法。
- (b1)(a1)10〜90容量%のメタノール、
(a2)少なくとも1つのさらなる添加剤(クロロホルムおよびトリクロロエタンを含まない。)、および
(a3)任意に酸
を含む非水性組成物、
(b2)最高99容量%のエタノールを含む組成物(B)、および
(b3)任意に、包埋材料(C)、および/または、生物材料中のまたは生物材料の生体分子の分析または生物材料の形態分析のための試薬
を含む、請求項1〜14、19および20のいずれかに記載の方法に用いるためのキット。 - 以下(b1)と(b2)および(b3)から選択される少なくとも1つとを含む、生物材料(生物がヒトの場合は生物材料はヒトから採取されたものに限る。)の処理のためのキット:
(b1)(α1)10〜80容量%未満のメタノール、
(α2)少なくとも一つのさらなる添加剤(クロロホルムおよびトリクロロエタンを含まない。)であって1以上の洗浄剤、核酸および/またはタンパク質の分解を阻害する1以上の阻害剤、1以上の粘度調整剤、1以上の染料、1以上の緩衝化合物、1以上の保存料、1以上の錯形成剤、1以上の還元剤、細胞透過性を改善する1以上の物質、1以上のカオトロピック物質、1以上の固定剤、メタノールと異なる1以上のさらなる溶媒および少なくとも2つのこれらの添加物の混合物を含む群から選択されるか、または、1,3-ブタンジオール、1,4-ブタンジオール、1,3-プロパンジオール、1,2-プロパンジオール、3-メチル-1,3,5-ペンタントリオール、1,2,6-ヘキサントリオール、グリセリン、グリコール等のジオールおよび/またはトリオール等のC2〜C12ポリオール;PEGおよびDEGMEAより選択される添加剤、および
(α3)酸
を含む非水性組成物(A)
(b2)最高99容量%のエタノールを含む組成物(B)、
(b3)包埋材料(C)、および/または、生物材料中のまたは生物材料の生体分子の分析または生物材料の形態分析のための試薬。 - 処理された生物材料の製造方法であって、請求項15〜18のいずれかに記載の組成物(A)または請求項21または22に記載のキットを使用する、方法。
- 生体外の生物材料の分析方法であって、請求項1〜14,19、および20のいずれかに記載の方法、請求項15〜18のいずれかに記載の組成物(A)または請求項21または22に記載のキットを使用する、方法。
- 請求項1〜14,19、および20のいずれかに記載の方法によって、または、請求項15〜18のいずれかに記載の組成物(A)と生物材料を接触させることによって得られる、処理された生物材料。
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CN101636649B (zh) | 2014-11-12 |
US20210389216A1 (en) | 2021-12-16 |
CA2679172C (en) | 2017-03-14 |
EP2126542B1 (en) | 2016-11-02 |
US20100173295A1 (en) | 2010-07-08 |
CN101636649A (zh) | 2010-01-27 |
US20210381937A1 (en) | 2021-12-09 |
AU2008220769A1 (en) | 2008-09-04 |
EP2126542A1 (en) | 2009-12-02 |
WO2008104564A1 (en) | 2008-09-04 |
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