JP4334601B2 - 組織固定−脱水−脱脂−含浸装置 - Google Patents
組織固定−脱水−脱脂−含浸装置 Download PDFInfo
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Description
(1.技術分野)
本発明は、迅速で、連続的な流れの、顕微鏡検査用の組織の、固定から含浸までの処理のための装置に関する。
従来の方法では、含浸の前に、固定用にリン酸バッファー10%ホルムアルデヒド、脱水用に1連の濃度を順に増加したエタノールおよび洗浄用にキシレンの、別々の溶液でインキュベーションすることによって、組織学用の組織を調製していた。通常8時間またはそれより長時間、この処理に時間を要するので、これら別々のステップ−固定、脱水、洗浄、および含浸−を完了するには通常、これらの仕事用にデザインされた自動機器で1夜を要している(例えば、米国特許第3,892,197号、第4,141,312号、および第5,049,510号を参照)。典型的な自動組織処理機器(TISSUE−TEK)は、8時間より多くを必要とし、そして以下のように、組織試料のバッチを処理するようにプログラムされている。
本発明の目的は、組織の処理および解析に必要な時間を減少させ、そして時間の減少、実験室設備のサイズ、使用される試薬の容積、および必要な人数によるコストを減少させる、組織処理のための組成物、およびそれらを利用するためのシステムを提供するにある。このことにより、手術を受けている患者に対する外科病理の現在の実務を、迅速な応答へと変換することが可能となり、そして手術室に近接した、病理学者による治療時点での診断を可能にするであろう。
組織の迅速な、連続的組織学的処理の処理方法および装置が開示される。固定、脱水、脂肪除去、および含浸のステップを、約2時間より少ない時間で完遂することができる;このことは、病理学者が、試料を受領後短時間に、多分患者が手術中か回復室にいる間に、評価することを可能にする。病理診断に必要な時間を減じることによって、患者の不安を減らすことができる。組織標本の厚さの低減、混合物を含む非水性溶液の使用、高められた温度および攪拌による溶液交換、マイクロ波放射による組織および溶液の均一な加熱、減圧下での含浸、またはそれらの組合せによって、迅速かつ連続処理が完遂される。
実施例1
新鮮または既に固定した組織の2mm厚あるいは薄片を組織カセットに保持し、以下の組成の非水性第1溶液中に置いた:
40%イソプロピルアルコール、
40%アセトン、
20%ポリエチレングリコール(平均分子量300)、および
1%ジメチルスルホキシド(DMSO)(即ち、上記混合物の10ml/l)。
新鮮あるいは固定組織切片標本、約1mm厚、の固定、脱水、脂肪除去、およびパラフィン含浸、はこれら組織切片標本を、以下の継続したステップに曝露することによって40分間で完了した。
本実施例において、第1溶液は以下からなっている:
60%イソプロピルアルコール、
10%アセトン、
30%ポリエチレングリコール(平均分子量300)、および
ジメチルスルホキシド(DMSO)を総容量の約1%の濃度に加える。この溶液1リッターは、組織カセットに保持された60試料を固定するのに充分である。試料を、第1溶液を含有する1連の3つのバスで、各々5分間市販のマイクロ波処理装置(H2500またはH2800、Energy Beam Sciences)で、55℃にて、インキュベーションした(総インキュベーション15分間);溶液の攪拌は溶液交換を加速するためバブリングによった。
試料を、70%イソプロピルアルコール、30%アセトン、およびDMSOを約1%濃度に加えた溶液中で、60℃でインキュベーションした。試料を、溶液を含有する2つのビーカー中で、各々5分間市販の組織マイクロ波処理装置(H2800、Energy Beam Sciences)で加熱し(総インキュベーション10分間)、バブリングによって攪拌した。
マイクロ波照射に続いて、60℃または70℃のグリセリンバス中に静置した、大型デシケーター中に置かれた25%ミネラルオイルおよび75%溶融パラフィンのワックス溶液中で、約200mmHgの減圧下、5分間、インキュベーションすることによって、含浸を開始した。パラフィンは、実施例1に記載したように、使用前脱気した。
含浸は、グリセリンバス中に静置した大型デシケーター内に置かれた溶融パラフィンの4つのバス中で、75℃にてインキュベーションすることによって、完了した。組織切片標本は、1つのパラフィンバスから、次のバスに、3分間隔で、総含浸時間12分間かけて、運搬された。各3分間間隔は、圧力の読みが約640mmHgである時間で測った。
約1ないし2mm厚までの、新鮮な、あるいは固定した組織切片標本の、固定、脱水、脂肪除去、およびパラフィン含浸は、これら組織切片標本を、以下のようにして、65分間で完了することができる。
本実施例において、第1溶液は以下からなっている:
40%イソプロピルアルコール、
40%アセトン、
20%ポリエチレングリコール(平均分子量300)、
総容量の約0.5%の濃度に加えた氷酢酸、および
総容量の約1%の濃度に加えたジメチルスルホキシド(DMSO)。
この溶液1リッターは、組織カセットに保持された60試料を固定するのに充分である。試料を、第1溶液を含有する1500mビーカー中で、15分間市販のマイクロ波処理装置(H2500またはH2800、Energy Beam Sciences)で、65℃にて、インキュベーションする;溶液の攪拌は、溶液交換を加速するためバブリングによる。
試料を、55%イソプロピルアルコール、25%アセトン、10%ポリエチレングリコール(平均分子量300)、10%低粘度ミネラルオイル、総容量の約0.5%の濃度に加えた氷酢酸、および約1%の濃度に加えたDMSOの溶液中で、インキュベーションする。試料を、溶液を含有する1500mlビーカー中で、15分間、市販の組織マイクロ波処理装置(H2800、Energy Beam Sciences)で、65℃にて加熱し、バブリングによって攪拌する。
試料を、55%イソプロピルアルコール、25%アセトン、20%低粘度ミネラルオイル、総容量の約0.5%の濃度に加えた氷酢酸、および約1%の濃度に加えたDMSOの溶液中で、インキュベーションする。試料を、溶液を含有する1500mlビーカー中で、5分間、市販の組織マイクロ波処理装置(H2800、Energy Beam Sciences)で、65℃にて加熱し、バブリングによって攪拌する。
マイクロ波照射に続いて、60℃のグリセリンバス中に静置した、大型デシケーター中に置かれた、70%溶融パラフィンのワックス溶液の2つのバス中で、約640mmHgの減圧下、5分間、各バス中でインキュベーションすることによって、含浸を開始する。
含浸は、グリセリンバス中に静置した大型デシケーター内に置かれた溶融パラフィンの4つのバス中で、約75℃ないし80℃、および約640mmHgの減圧にて、おのおの5分間、インキュベーションすることによって完了する。組織切片標本は、1つのパラフィンバスから、次のバスに、5分間隔で、総含浸時間20分間かけて、運搬された。各5分間隔は、圧力の読みが約640mmHgである時間で測った。
パラフィン切片標本を3ミクロンの厚さにミクロトームで切削し、水浴に置き、そしてガラススライドの上に浮かせる。パラフィンを、58℃のオーブン、あるいは好ましくは37℃のオーブン中で約18時間または1夜、スライドを置くことによって融解した。次いで、キシレンバス中で10分間脱ワックスした。スライドを、各1分間エタノール分を減らしていった溶液(無水アルコールで2バス、95%で2バス、90%で1バス)で再水和した。そして流水中に浸すことによって、2分間リンスした。
2つの6ミクロン組織切片標本を1.5ml微量遠心チューブに入れ、800μlキシレンを加え、そして振り混ぜて混合し、400μl無水エタノールを加え、振り混ぜて混合し、チューブを高速微量遠心器で5分間遠心分離し、そして、上清を捨てた。ペレットに800μl無水エタノールを加え、振り混ぜて混合した。
パラフィンブロックの10切片標本(各々7μm)をディスポーザブル刃を使用して切削した;ブロックは本発明に従って調製され、従来の組織処理は実施例5に記載により調製された。それらを、50mlFalconチューブに置き、キシレン20mlで脱パラフィン化し、残余の組織を無水アルコールで、30分間、2回洗滌した。組織は、4Mグアニジンチオシアネート、25mMクエン酸ナトリウムpH7.0、0.5%N−ラウリルザルコシン、および0.1M−2−メルカプトエタノールを含有する溶液に0.5g/ml懸濁した。溶液は、かき混ぜて混合し、DNAは18ないし22ゲージの注射針を通して、剪断した。
Claims (7)
- 連続的処理による組織標本の迅速処理のための装置であって、該装置は
(a)(i)組織標本を固定し、脱水し、アルコールとケトンを含む非水性溶液で組織標本から脂肪を除去し、(ii)該組織標本と非水性溶液を第1の容器中で攪拌し、(iii)該第1の容器中の組織標本と非水性溶液をマイクロ波エネルギーに暴露し、固定し、脱水し、脂肪を除去した組織標本を作製するマイクロ波プロセッサー;
及び該第1の容器と液体的に連結している非水性溶液源、
(b)(i)該固定し、脱水し、脂肪を除去した組織標本を第2の容器中ワックス溶液で含浸し、(ii)該固定し脱水し、脂肪を除去した組織標本と該ワックス溶液を該第2の容器中で攪拌し、(iii)該固定し、脱水し、脂肪を除去した組織標本と該ワックス溶液を真空下で高温に暴露して、固定し、脱水し、脂肪を除去し、含浸した組織標本を作製する含浸ユニット;
及び該第2の容器と液体的に連結しているワックス溶液源、
を含み、
組織標本は連続的に第1の容器に、次いで第2の容器に移送される、上記装置。 - 前記第1の容器又は前記非水性溶液源は、ケトンに対してアルコールを1と3の間の容量比で含む、請求項1記載の装置。
- 前記非水性溶液がさらに100から500の平均分子量のポリマーを含んでなる、請求項1又は2記載の装置。
- 前記非水性溶液が、アセトン、イソプロピルアルコール、およびポリエチレングリコールを含んでなる、請求項1〜3のいずれか一項記載の装置。
- 前記ワックス溶液がパラフィンを含んでなる、請求項1〜4のいずれか一項記載の装置。
- 前記ワックス溶液が、異なる融点のワックスを含んでなる、請求項1〜5のいずれか一項記載の装置。
- 前記ワックス溶液は鉱物油とパラフィンを含んでなる、請求項1〜5のいずれか一項記載の装置。
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US6207408B1 (en) * | 1997-08-20 | 2001-03-27 | University Of Miami | High quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation method |
ATE268470T1 (de) * | 1998-06-30 | 2004-06-15 | Lamina Inc | Zytologische und histologische fixier- zusammensetzung und verfahren zur verwendung |
AU4812600A (en) * | 1999-04-29 | 2000-11-17 | Arcturus Engineering, Inc. | Processing technology for lcm samples |
US7951612B2 (en) * | 1999-07-08 | 2011-05-31 | Lee H. Angros | In situ heat induced antigen recovery and staining apparatus and method |
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WO2001004634A1 (en) * | 1999-07-08 | 2001-01-18 | Lee Angros | Antigen recovery and/or staining apparatus and method |
US7897106B2 (en) * | 1999-07-08 | 2011-03-01 | Lee Angros | Situ heat induced antigen recovery and staining apparatus and method |
US6916608B2 (en) * | 1999-09-10 | 2005-07-12 | Becton, Dickinson And Company | Composition for providing long term stability to cells for diagnostic testing |
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DE69839963D1 (de) | 2008-10-16 |
CA2635001C (en) | 2012-12-04 |
EP1005633B1 (en) | 2008-09-03 |
JP2008281573A (ja) | 2008-11-20 |
EP1005633A1 (en) | 2000-06-07 |
US8221996B2 (en) | 2012-07-17 |
US20010000487A1 (en) | 2001-04-26 |
KR20010023070A (ko) | 2001-03-26 |
JP4164232B2 (ja) | 2008-10-15 |
TW571100B (en) | 2004-01-11 |
EP2199774A1 (en) | 2010-06-23 |
US7547538B2 (en) | 2009-06-16 |
ES2313752T3 (es) | 2009-03-01 |
US6207408B1 (en) | 2001-03-27 |
ATE407353T1 (de) | 2008-09-15 |
CA2301924C (en) | 2008-12-09 |
WO1999009390A1 (en) | 1999-02-25 |
AU746497B2 (en) | 2002-05-02 |
CA2635001A1 (en) | 1999-02-25 |
US20040004075A1 (en) | 2004-01-08 |
DK1005633T3 (da) | 2008-12-15 |
CA2301924A1 (en) | 1999-02-25 |
JP2001516869A (ja) | 2001-10-02 |
EP2199774B1 (en) | 2018-10-03 |
US6586713B2 (en) | 2003-07-01 |
AU8900198A (en) | 1999-03-08 |
EP1005633A4 (en) | 2003-01-22 |
US20080153127A1 (en) | 2008-06-26 |
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