JP5156738B2 - 全血試料の特異的溶血 - Google Patents
全血試料の特異的溶血 Download PDFInfo
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- JP5156738B2 JP5156738B2 JP2009513584A JP2009513584A JP5156738B2 JP 5156738 B2 JP5156738 B2 JP 5156738B2 JP 2009513584 A JP2009513584 A JP 2009513584A JP 2009513584 A JP2009513584 A JP 2009513584A JP 5156738 B2 JP5156738 B2 JP 5156738B2
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- sample
- whole blood
- analyte
- membrane
- hemolysis
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
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Description
試料中に成分がより多く存在するほど、そこに含まれる標的分析物の分析はより困難である。赤血球は、全血のような生物学的液体から検出される分析物に潜在的に干渉する劇的な量のタンパク質および低分子量成分を含む。このことが主要な原因の1つであるため、臨床慣例において、好ましくは血液血漿(しばしば単純に血漿、すなわち、抗凝固全血試料と呼ばれる;細胞および赤血球を除く)または血液血清(しばしば単純に血清、すなわち、凝固全血と呼ばれる;細胞、赤血球および凝固系、特にフィブリン/フィブリノーゲンの大部分のタンパク質を除く)のそれぞれが使用される。また、全血試料は、例えば血清または血漿と比較して取り扱うのがより困難である傾向がある。全血は安定でない傾向があり、赤血球の緩やかな破壊は目的の多くの分析物の信頼性のある測定を損なう。
第一の態様において、本発明は、赤血球を含むことが既知であるまたは疑われる液体試料および真核生物細胞を含むことが疑われるまたは既知である液体試料中の分析物を検出する方法であって、赤血球の細胞膜を溶解し、同時に試料成分の沈殿を生じない適切な条件下で、該液体試料を膜可溶化剤で処理する工程、第一工程で得られた処理試料をクロマトグラフィー分離に供する工程、ならびに分析物を検出する工程を含む方法に関する。
本発明による方法は、ヒトまたは動物の体ではなく、インビトロで行われる。
(式中、mは0または1、nは4または6である)
から選択される。
(式中、mは0または1、nは4または6である)
から選択され、アニオンが好ましくはクロライド、テトラフルオロボレート、オクチルスルフェート、ヨージドおよびチオシアネートから選択される塩である。
1. 正常相クロマトグラフィーは、非極性(分散性)移動相と共に極性固定相の使用を要する。
2. 逆相クロマトグラフィー、反対の可能性は、非極性固定相および極性移動相(1つ以上の極性溶媒、例えば水、メタノール、アセトニトリルおよびテトラヒドロフランなどからなる)の使用を要する。
3. イオン交換クロマトグラフィーはイオン性の相互作用を必要とする。この場合、移動相は、イオン性溶質の溶解を確実にするためにイオン化を支持する必要がある。固定相も、ある程度の保留を促進するために部分的にイオン性である必要がある。結果的に、固定相との相互作用は強力であり、これは通常、長い分析時間および広いピークに反映される。
4. サイズ排除クロマトグラフィーは分子の大きさのみに基づいた分離を必要とし、理想的には溶質と固定相の強力な相互作用が存在しないことを必要とする。
5. アフィニティークロマトグラフィーは特異的な相互作用、例えば抗原と対応抗体、またはレセプターと対応リガンドのような特異的な結合ペアの間の相互作用に基づく。例えば、結合ペアの第1のパートナーは適当な固定相に結合し、結合ペアの第2のパートナーを捕捉するために使用される。第2のパートナーは適切な手段により放出および単離することができる。
実施例1
種々の候補溶血試薬の評価
実施例1.1 溶血の視覚評価
溶液A:新しいEDTA-安定化全血を、1:10の比で0.15Mの塩化ナトリウム溶液で希釈した(50μL EDTA-血液+450μL塩化ナトリウム溶液)。
溶液A:新しいEDTA-安定化全血を、1:10の比で0.15Mの塩化ナトリウム溶液で希釈する(50μL EDTA血液+450μL塩化ナトリウム溶液)。
溶血物を混合して、1滴を顕微鏡スライドガラス上に塗り、室温で風乾し、メイ-グリュンヴァルト染色試薬(Merkカタログ番号1.01424メイ-グリュンヴァルトエオシンメチレンブルー溶液)で染色する。メイ-グリュンヴァルト染色後、核は種々に変化する暗度の紫に染まり、細胞質は青〜明るいピンク色の色調に見え、鮮明な赤〜紅藤色の顆粒がいくつかの細胞型の細胞質中に存在する場合があり、好塩基球は細胞質中で暗い藍色の顆粒を示し、好酸球は細胞質中に明るいオレンジ色の顆粒を示し、赤血球はピンク〜オレンジ色に染まる。
処理された全血試料をトリパンブルー溶液(Merckカタログ番号1.11732;Trypanblau C.I. 23850)と混合し(1:1)、顕微鏡検査のためにNeugebauerチャンバーに分配する。油浸光学顕微鏡(倍率x630)により顕微鏡検査を行なう。
HPLCによる種々の候補溶血試薬の評価
溶解効率を評価するために、実施例1に従って調製した溶血全血試料をHPLCシステムに注入し、該システムの背圧をモニターする。
全血の試料由来のラパマイシンの測定
全血試料にラパマイシンを加えて溶血試薬と混合する。50%メタノール/水中の400μg/mLラパマイシンの50μLの溶液を450μLの新しいEDTA血液に添加し、ホモジナイズして室温で1時間インキュベートする。
全血の試料からの5-メチルテトラヒドロ葉酸の測定
全血試料に5-メチルテトラヒドロ葉酸を加えて溶血試薬と混合し、1%アスコルビン酸を有する水中の10μg/mL 5-メチルテトラヒドロ葉酸の10μLの溶液(4M水酸化ナトリウム溶液でpH 7に調整)を90μLの新しいEDTA血液に添加する。10μLの添加血液を90μLの水中0.15M塩化ナトリウムで希釈し、1%アスコルビン酸中の100μLの2mg 1-メチル-1-オクチルピロリジニウムクロライド/KSCNの溶液(4M水酸化ナトリウム溶液でpH 7に調整)と混合する。この溶血物をHPLC分析に使用する。
Claims (10)
- 赤血球を含むことが既知であるかまたは疑われ、かつ真核生物細胞を含むことが疑われるかまたは既知である液体試料中の分析物を検出する方法であって、該方法は
a)赤血球の細胞膜を溶解し、同時に試料成分の沈殿を生じない適切な条件下で、液体試料を膜可溶化剤で処理する工程、
b)工程(a)で得られた処理試料を高性能液体クロマトグラフィー(HPLC)によるクロマトグラフィー分離に供する工程、ならびに
c)分析物を検出する工程
を含み、
10μLの処理全血試料の50のアリコートを、2mmの直径および0.5μmの孔径を有するフィルターに適用でき、50回のインジェクションの間の背圧の増加が、第一インジェクションの背圧と比較して20 bar未満である場合に該条件は適切なものであり、ここで、該処理全血試料は、該膜可溶化剤と1:10希釈の全血試料とを1:1の比で混合し、30分間インキュベートすることによって得られ、該膜可溶化剤は、KBr、KI、KSCNまたは以下のカチオンおよびアニオンの1つ以上からなる塩、
ここで、カチオンは
(式中、mは0または1であり、nは4または6である)
から選択され、アニオンはクロライド、テトラフルオロボレート、オクチルスルフェート、ヨージド、およびチオシアネートから選択される、
から選択される化学物質を含む、方法。 - 前記クロマトグラフィー分離が、カラムクロマトグラフィーに基づいており、フリットおよび床物質を含むカラムの使用またはモノリシックカラムの使用によって行われる、請求項1記載の方法。
- 前記フリットが0.2または0.5μmの孔径を有する、請求項2記載の方法。
- 前記床物質が粒子状であり、粒子が1〜10μmの直径を有する、請求項2または3記載の方法。
- 前記分析物が質量分析によって検出される、請求項1記載の方法。
- 前記液体試料が脳脊髄液および全血から選択される、請求項1記載の方法。
- 前記分析物が赤血球内に少なくとも一部存在する、請求項1〜6いずれかに記載の方法。
- 前記分析物が免疫抑制薬である、請求項1〜7いずれかに記載の方法。
- 高性能液体クロマトグラフィー(HPLC)のための全血試料の処理における、
KBr、KI、KSCNまたは以下のカチオンおよびアニオンの1つ以上からなる塩、
ここで、カチオンは
(式中、mは0または1であり、nは4または6である)
から選択され、アニオンはクロライド、テトラフルオロボレート、オクチルスルフェート、ヨージド、およびチオシアネートから選択される、
から選択される化学物質を含む膜可溶化剤の使用。 - 液体クロマトグラフィー系分析における請求項1〜9記載の方法のいずれかによる膜可溶化剤を用いた特異的溶血によって得られた処理血液試料の使用。
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PCT/EP2007/004923 WO2007140961A1 (en) | 2006-06-06 | 2007-06-04 | Differential hemolysis of a whole blood sample |
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