JP4298555B2 - 血液分離法及び装置 - Google Patents
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Description
(1)以下の工程を含むことを特徴とする全血試料からの血清又は血漿の分離方法。
(a)全血試料を血球捕捉層に接触させて赤血球を凝集した後、凝集した赤血球を前記捕捉層にて担持させる工程、
(b)前記捕捉層を通過した血漿又は血清を、捕捉層の下に配置された血球分離層を通過させる工程、
及び(c)前記分離層を通過した血漿又は血清を、分離層の下に配置された受容層に移動させる工程。
(2)以下の工程を含むことを特徴とする全血試料からの血清又は血漿の分離方法。
(a)赤血球を凝集させた血液試料を血球捕捉層に接触させ、凝集した赤血球を前記捕捉層にて担持させる工程、
(b)前記捕捉層を通過した血漿又は血清を、捕捉層の下に配置された血球分離層を通過させる工程、
及び(c)前記分離層を通過した血漿又は血清を、分離層の下に配置された受容層に移動させる工程。
(3)血球捕捉層に、血球凝集剤を含有させることを特徴とする(1)〜(2)記載の方法。
(4)血球凝集剤がコンドロイチン硫酸類であることを特徴とする(3)記載の方法。
(5)血球分離層が、分離層の下に配置された展開層に向かって、連続的又は不連続的に小さくなる孔径分布の非対称性多孔質膜を有する多孔性シートである(1)〜(2)記載の方法。
(6)全血試料を血球捕捉層と接触させ、赤血球を前記捕捉層に担持させることによって、全血試料から血清又は血漿を分離するための装置であって、少なくとも、以下の構成要素を含むことを特徴とする装置。
(a)コンドロイチン硫酸を含有する血球捕捉層、
(b)非対称性多孔膜を有する多孔性シートからなる血球分離層、
及び(c)血清又は血漿受容層。
(7)さらに、開放口を有すフィルムを含むことを特徴とする(6)記載の装置。
コンドロイチン硫酸Cナトリウム( 和光純薬工業社製 )の0.5%溶液及びコンドロイチン硫酸Aナトリウム( 同社製 )の1%溶液を、各々0.1mol/LのHEPES緩衝液(pH7.4)にて作製し、各々150μLと真空採血管により採血した人の静脈血50μL(ヘマトクリット値45)をすばやく混和した後、速やかにキャピラリー管(全長75mm 内径1.1〜1.2mm)にて倒立静置し、血球が完全分離するまで時間と、30分後の血球移動距離と血漿部分の移動距離を測定した。
実施例1同様に、5%マンニトール、1%ポリブレン、0.1%ポリブレン、1%ポリビニルアルコール溶液を作製し、血球の分離時間と移動距離を測定した。
(1)血球捕捉層の作製
2%コンドロイチン硫酸Cナトリウム( 和光純薬工業社製 )溶液及び2%コンドロイチン硫酸Aナトリウム( 同社製 )溶液を、各々0.1mol/LのHEPES緩衝液にて作製し、室温で5分間ポリエチレンシートに含浸し、60℃にて1時間乾燥後、10mm×10mm角にカットして使用した。
(2)多孔性フィルター
10mm×10mm角のBTS−SP( 日本ポール社製 )を使用した。
(3)分離装置の作製
(1)で作製した血球捕捉層と(2)の多孔性フィルターを、受容層である10mm×30mmの濾紙( アドバンテック社製 No.1 )上に重ねて固定し、図1のように組み立てて分離装置を作製した。
(4)(3)の血球捕捉層に血液70μLを滴下し、2分経過後血漿を受容した濾紙のみを取り出し、500μLの0.1mol/LのHEPES緩衝液で2時間受容物を溶出後、以下の測定試薬及び測定機器を用いて、溶出液の波長550nmでの吸光度(O.D.)並びに総コレステロール(T−CHO)及び総蛋白(TP)を測定した。なお、波長550mmは赤色の吸収域であり、この吸光度が高い程、赤血球の混在又は赤血球内のヘモグロビンの溶出を示すため、該波長での吸光度測定は、本装置の分離能の指標ともなる。
測定試薬:エクディアL‘栄研’T−CHO
EAテスト TP‘栄研’
測定機器:日立製作所 7170S型 自動分析機
島津製作所 UV−PC2400 分光光度計
実施例2と同様に、5%マンニトール、1%ポリブレン、1%ポリビニルアルコール溶液を0.1mol/LのHEPES緩衝液にて作製し、室温でポリエチレンシートに含浸、60℃にて1時間乾燥後、10mm×10mm角にカットして使用した。さらに、実施例2と同様に、分離装置を組み立て、血球捕捉層に血液70μLを滴下し、2分経過後血漿を受容した濾紙のみを取り出し、500μLの0.1mol/LのHEPES緩衝液で2時間受容物を溶出、溶出液の吸光度並びに、T−CHO及びTPを測定した。
2:血球分離層(多孔性シート)
3:フィルム
4:血漿等の受容層
5:支持体
Claims (4)
- 以下の工程を含むことを特徴とする全血試料からの血漿又は血清の分離方法。
(a)全血試料を血球凝集剤としてコンドロイチン硫酸類を含有させた血球捕捉層に接触させて赤血球を凝集した後、凝集した赤血球を前記捕捉層にて担持させる工程、
(b)前記捕捉層を通過した血漿又は血清を、捕捉層の下に配置された血球分離層を通過させる工程、
及び(c)前記分離層を通過した血漿又は血清を、分離層の下に配置された受容層に移動させる工程。 - 以下の工程を含むことを特徴とする全血試料からの血漿又は血清の分離方法。
(a)血球凝集剤としてコンドロイチン硫酸類を用いて赤血球を凝集させた血液試料を血液捕捉層に接触させ、凝集した赤血球を前記捕捉層にて担持させる工程、
(b)前記捕捉層を通過した血漿又は血清を、捕捉層の下に配置された血球分離層を通過させる工程、
及び(c)前記分離層を通過した血漿又は血清を、分離層の下に配置された受容層に移動させる工程。 - 血球分離層が、分離層の下に配置された受容層に向かって、連続的又は不連続的に小さくなる孔径分布の非対称性多孔質膜を有する多孔性シートである請求項1〜2記載の方法。
- 全血試料を血球捕捉層と接触させ、赤血球を前記捕捉層に担持させることによって、全血試料から血漿又は血清を分離するための装置であって、少なくとも、以下の構成要素を含むことを特徴とする装置。
(a)コンドロイチン硫酸類を含有する血液捕捉層、
(b)非対称性多孔質膜を有する多孔性シートからなる血球分離層、
及び(c)血漿又は血清受容層。
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