JP2010517046A - 細胞計数及び分析のための方法、システム及び組成 - Google Patents
細胞計数及び分析のための方法、システム及び組成 Download PDFInfo
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Abstract
Description
多種多様の光学システムを、本発明で用いることができる。一般に、そのようなシステムは、明確な波長範囲で試料を連続的に照らすための1つ又は複数の照明ビームと、照明された試料からの画像データを記録するための画像収集デバイスと、画像データの組が連続的に収集されるように照明ビーム及び画像収集デバイスの操作を制御するコントローラとを提供する。
別の態様では、本発明は、試料内の複数の異なる被分析物の1つ又は複数のラベル付けで使用するための分別励起可能なプローブの組成を提供する。一般に、本発明のプローブ組成は、各々が異なる被分析物と特異的に結合することができる、被分析物−特異的プローブの混合物を含み、それぞれのプローブは、(a)結合条件のもとで細胞被分析物などの被分析物に特異的な結合化合物によって、及び(b)結合化合物に光ラベルを付けられることで特徴づけられ、それぞれの異なるプローブの光ラベルは、異なる励起帯域を有し、すべてのプローブの光ラベルは、同じ波長範囲内の光信号を放射する。普通、後者の波長範囲は、励起帯域のいずれとも重複しない。好ましくは、光ラベルは、蛍光信号を生成することができる蛍光染料などの蛍光ラベルである。しかしながら、暗視野照明条件のもとで使用されるときには、プラズモン共鳴粒子などの他の光ラベルが、本発明とともに使用されてもよい。一態様では、本発明のプローブ組成は、複数の異なる被分析物の各々に対して特異的な少なくとも1つのプローブを含む。別の態様では、そのような複数は、2から8の範囲内であり、又は別の態様では、2から4の範囲内であり、又は別の態様では、2から3の範囲内であり、別の態様では、そのような複数は、少なくとも3であり、又は3から4の範囲内である。本発明のプローブ組成の重要な特徴は、その組成の異なるプローブでラベルを付けられた試料内の被分析物が、そのような光ラベルに特異的な照明ビームを使用するそれぞれのプローブの光ラベルの連続的な励起によって、連続的に検出することができることである。通常、そのような連続的な励起は、それぞれの照明ビームが別々の時間間隔で試料に導かれるときという点において、時間的に重複しない。言い換えれば、照明ビームは、一度に1つずつ試料に連続的に導かれる。好ましくは、操作時には、それぞれの励起からの光信号は検出器の光応答性表面に撮像され、画像データが生成され、分析のために保存される。プローブの光信号が狭い波長範囲に制限されるときは、光路内のレンズの色収差に起因する画像劣化が減される又は除去される。
本発明の態様では、使い捨てキュベットは、全血について測定を行うための本発明のシステムとともに使用するために提供される。一実施形態では、そのようなキュベットは、決定できる容積内の所定の血球の種類、例えば非赤血球を数えるために使用され、従って、そのような所定の細胞種類の細胞計数又は濃度のどちらかを読み出し情報として与えることができる。一般に、本発明の使い捨てキュベットは、(a)全血の試料を受け取ることができる試料チャンバーであって、その試料チャンバーは、本体内に配置され、細胞被分析物に付けられたプローブによって生成される光信号が試料の赤血球によって妨害されないように、少なくとも1つの光学的に透過する壁及び分析されるべき非赤血球の直径と実質的に同等な垂直な寸法を有する、試料チャンバーと、(b)各々が非赤血球の異なる細胞被分析物と特異的に結合することができる複数の被分析物−特異的プローブを含むプローブ組成を形成するために試料と組み合わせることで溶解する、試料チャンバー内の乾燥試薬とを含み、それぞれのプローブは、(i)結合条件のもとで細胞被分析物に特異的な結合化合物によって、及び(ii)結合化合物に光ラベルを付けられることで特徴づけられ、それぞれの異なるプローブの光ラベルは異なる励起帯域を有し、すべてのプローブの光ラベルは同じ波長範囲内の光信号を放射する。好ましくは、そのような使い捨てキュベットは、上述のような光学システムとともに使用され、照明ビーム及び撮像光学系に関して固定位置を有するようにキュベットを受け取るための台を含む。そのような固定位置は、キュベットの試料チャンバーから光信号を収集することができるように、撮像光学系を整列させることになる。血液パラメータなどの生体液の特性を観察する又は測定するための使い捨て試料容器の設計及び加工は、参照により組み込まれる次の参考文献、即ち特許文献3〜8、及び同様のもので開示される。
一般に、別に特に定義されずに、本明細書で使用される術語は、次の専門書、即ち非特許文献21、22、29、30及び同様のもので表わされるなどの、分析化学、生化学、分子生物学、細胞生物学、顕微鏡法、画像分析、及び同様のものを含む、本発明に関連する分野でのそれらの慣例的用法に対応する意味を有する。
Claims (8)
- 検体の多重特徴を撮像するためのシステムであって、前記システムは、
各々が明確な波長帯域を有する照明ビームを連続的に生成することができる1つ又は複数の光源と、
それぞれの異なる特徴が、異なる分別励起可能なラベルを付けられるような、多重特徴を含む検体にラベルを付けることができる複数の分別励起可能なラベルと、
前記異なる分別励起可能なラベルの各々が、同じ波長帯域内の光信号を連続的に放射させられるように、照明ビームを前記検体上に連続的に導くための、前記1つ又は複数の光源と操作上関連するコントローラと、
そのような放射された光信号を収集し、前記検体の前記ラベル付き特徴に対応する連続的な画像を、画像データの連続的な組を形成するための光応答性表面に形成することができる光学システムとを含むことを特徴とするシステム。 - 血液検体内で複数の分別励起可能なラベルを付けられた非赤血球を分析するための装置であって、前記装置は、
血液検体を含み、赤血球の光妨害層の形成を妨げる光収集軸に沿った寸法を有することができる試料チャンバーと、
各々が、明確な波長帯域を有する照明ビームで前記血液検体を照らすことができる多重光源と、
前記複数の分別励起可能なラベルの各々が、同じ波長帯域内の光信号を連続的に放射させられるように、それぞれの光源の前記照明ビームを前記検体上に連続的に導くための前記多重光源に結合されたコントローラと、
そのような放射された光信号を収集し、それらに対応する連続的な画像を、画像データの連続的な組を形成するための光応答性表面に形成することができる光学システムとを含み、
画像データの前記連続的な組を分析することによって、前記血液検体内の非赤血球を数えることを特徴とする装置。 - 試料内の複数の異なる細胞被分析物の1つ又は複数のラベル付けで使用するためのプローブ組成であって、
各々が、異なる被分析物と特異的に結合することができる、被分析物−特異的プローブの混合物を含み、それぞれのプローブは、(a)結合条件のもとで細胞被分析物に特異的な結合化合物によって、及び(b)前記結合化合物に光ラベルを付けられることによって特徴づけられ、それぞれの異なるプローブの前記光ラベルは、異なる励起帯域を有し、すべてのプローブの前記光ラベルは、[前記プローブ組成の前記光ラベルの前記励起帯域から離れた]同じ波長範囲内の光信号を放射することを特徴とするプローブ組成。 - 非赤血球の光学的分析のための使い捨て血液収集キュベットであって、前記キュベットは、
全血の試料を受け入れるための入口を有する混合チャンバーであって、前記混合チャンバーはさらに、前記全血試料と接触して溶解することができる乾燥試薬を含み、各々が非赤血球の異なる細胞被分析物と特異的に結合することができる、複数の被分析物−特異的プローブを含むプローブ組成を含み、それぞれのプローブは、(a)結合条件のもとで細胞被分析物に特異的な結合化合物によって、及び(b)前記結合化合物に光ラベルを付けられることによって特徴づけられ、それぞれの異なるプローブの前記光ラベルは、異なる励起帯域を有し、すべてのプローブの前記光ラベルは、同じ波長範囲内の光信号を放射する、混合チャンバーと、
前記混合チャンバー内の試料が、毛管作用によって試料チャンバーに移されるように、前記混合チャンバーに流体的に接続される前記試料チャンバーであって、前記試料チャンバーは、細胞被分析物に付けられたプローブによって生成される光信号が、前記試料の赤血球によって妨害されないように、光学的に透過する壁及び垂直な非赤血球の直径と実質的に同等な寸法を有する試料チャンバーとを含むことを特徴とするキュベット。 - 前記寸法は、関心のある細胞と前記光学的に透過する壁との間での除核赤血球の光妨害層の形成を実質的に妨げることを特徴とする請求項4に記載のキュベット。
- 非赤血球の光学的分析のための使い捨て血液収集キュベットであって、前記キュベットは、
全血の試料を受け入れることができる試料チャンバーであって、前記試料チャンバーは、本体内に配置され、それらの細胞被分析物に付けられたプローブによって生成される光信号が、前記試料の赤血球によって妨害されないように、少なくとも1つの光学的に透過する壁及び垂直な、非赤血球の直径と実質的に同等な寸法を有する、試料チャンバーと、
各々が非赤血球の異なる細胞被分析物と特異的に結合することができる、複数の被分析物−特異的プローブを含むプローブ組成を形成するために、前記試料との組合せで溶解する、前記試料チャンバー内の乾燥試薬であって、それぞれのプローブは、(a)結合条件のもとで細胞被分析物に特異的な結合化合物によって、及び(b)前記結合化合物に光ラベルを付けられることによって特徴づけられ、それぞれの異なるプローブの前記光ラベルは、異なる励起帯域を有し、すべてのプローブの前記光ラベルは、同じ波長範囲内の光信号を放射する、乾燥試薬とを含むことを特徴とするキュベット。 - 複数の蛍光ラベルを付けられた検体を撮像するための装置であって、前記装置は、
それぞれの発光ダイオードが明確な波長帯域を有する照明ビームを生成し、前記検体を照らすことができる1つ又は複数の発光ダイオードと、
前記複数の蛍光ラベルの各々が光信号を連続的に放射させられるように照明ビームを前記検体上に導くための、前記発光ダイオードに結合されたコントローラと、
前記放射された光信号を収集し、対応する画像を、画像データを生成するための光応答性表面に形成することができる光学システムとを含み、前記光学システムは、異なる波長を有する多重光信号を獲得することができるカラーカメラを含むことを特徴とする装置。 - 前記1つ又は複数の発光ダイオードは、複数の発光ダイオードであり、前記光学システムは、複数の組の画像データを生成し、それぞれのそのような組は、前記発光ダイオードのうちの異なる1つによる照明に応答して生成される光信号に対応することを特徴とする請求項7に記載の装置。
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US20100261197A1 (en) | 2010-10-14 |
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