JP2005526513A - 白血球数の測定方法および装置 - Google Patents
白血球数の測定方法および装置 Download PDFInfo
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Abstract
Description
a.生物学的流体サンプルからの妨害物質から白血球を、白血球を選択的に保持する保持体を用いて分離する;
b.妨害物質、例えば赤血球、酵素、酵素インヒビター、タンパク質、およびリピドを、水中に緩衝液およびオプションの添加物を含むかまたは極性有機液の水溶液を含む洗浄溶液を用いて洗浄して保持体から除去し、白血球のみを残す。
c.白血球中にある酵素が水不溶性色素を生成する色素基質を切断する色素反応の結果を、眼または機器によって読む。
a.サンプルおよび洗浄溶液を用いるための開放部を有するカバー;
b.固定化された色素を任意に有する白血球保持体;
c.サンプルを通って流れる過剰の洗浄溶液をすべて運び集める吸着層;
d.緩衝液、任意の反応促進剤、および任意の色素基質を含む洗浄溶液。
& Schuell 900フィルター紙のような高価でないセルロース材がこのために適当である。この紙は瞬時に捕獲膜から洗浄液300マイクロリットルまで処理する空隙率と容量をもつ。
5ミリグラムの3−アセチル−インドキシル基質は無水エタノールに溶解し、基質を用いてWBC捕獲膜(Leukosorb B,Pall,Inc,NY)に含浸させた。膜を次に15分間40ECでオーブンで乾燥させた。洗浄溶液は生理的食塩水mLあたり5mgのNBT(ニトロブルーテトラゾリウムクロリド一水和物)を溶解して調製した。デバイスは図1Aに示したように集めた。マイクロリットル当り8000個の白血球をもつクエン酸化した全血サンプルを食塩水で連続して希釈しマイクロリットル当り8,6,4,2および0.8千のWBCのものを用意した。30マイクロリットルの各サンプルをデバイスの頂部にピペットで取った。サンプルを各々添加後、250マイクロリットルの洗浄溶液をデバイスにピペットで取り、赤血球を洗浄し酵素反応を開始した。10分後に発現した色強度はデバイスの頭開口によってミノルタCR321クロモメーターで測定した。マイクロリットル当りの800ないし8000の範囲の機器により得られた白血球数に対する色測定のプロットは図4に示される。この製剤はマイクロリットル当り少なくとも800個の細胞の検出限度を示した。
ミルクはエステラーゼ活性を示す物質を含み、白血球数のためのエステラーゼテストの感度はこれら妨害物質の分量に従って変化する。ミルク中のエステラーゼ活性は、ミルク中にあるタンパク質、酵素インヒビター等によって、ミルクに実際にあるエステラーゼの量が約10%ないし90%の範囲である。従って、ミルク中の白血球数を数える前に妨害物質を除去することが重要である。表1はミルク中のエステラーゼ活性が減少した例を示す。
実施例2に従ってテストデバイスを作成した。尿路感染の患者からの4個のサンプルをテストデバイスを用いて、同じ試薬を用いる尿試験紙法によってアッセイした。表2は同じ製剤を用いる尿試験紙バージョンよりも、テストデバイスが尿中の白血球に遥かに感受性が高かったことを示している。
表2
ミノルタ色度計による呈色の読み
尿(マイクロリットル当りの細胞)200 600 1500 2000
テストデバイスの読み 4.0 10.7 26.8 37.8
尿試験紙の読み 6.3 7.1 8.0 10.2
1 デバイスの頂部ピース
2 デバイスの頂部ピースの入口穴
3 白血球捕獲膜
4 水吸収剤相
Claims (15)
- 次の工程からなるサンプル中の白血球数を測定する方法。
a.サンプルからの白血球を保持体に捕獲し;
b.洗浄溶液を用いて保持体を洗浄して保持体に捕獲された白血球からの妨害物質を除去し;
c.白血球に存在する酵素によって分解されて水不溶性色素を生成する色素原基質とサンプルを接触させ、前記基質は膜に固定化されているかまたは洗浄溶液の一部からなり;そして
d.白血球中に存在する酵素に起因する色彩変化を読み取る。 - 酵素がエステラーゼである請求項1記載の方法。
- サンプルが血液、ミルク、尿、唾液、および汗からなる群から選択される請求項1記載の方法。
- 洗浄溶液が緩衝液、任意の反応促進剤、および任意の基質を含む請求項1記載の方法。
- 緩衝液が約8から約11のpH範囲である請求項4記載の方法。
- 緩衝液が約9から約10.5のpH範囲である請求項5記載の方法。
- 洗浄溶液が水および極性有機溶媒の両方を含む請求項4記載の方法。
- 洗浄溶液が非イオン性界面活性物質を含む請求項4記載の方法。
- 白血球がCD4成分を含む請求項1記載の方法。
- 次の工程からなるミルクのサンプル中の白血球数を測定することからなる乳腺炎を検出する方法。
a.サンプルからの白血球を保持体に捕獲し;
b.洗浄溶液を用いて保持体を洗浄して保持体に捕獲された白血球からの妨害物質を除去し;
c.白血球に存在する酵素によって分解されて水不溶性色素を生成する色素原基質とサンプルを接触させ、前記基質は膜に固定化されているかまたは洗浄溶液の一部からなり;そして
d.白血球中に存在する酵素から得られた色彩変化を読み取る。 - 酵素がエステラーゼである請求項10記載の方法。
- 次の構成からなる白血球数を測定するデバイス。
a.色素基質を任意に固定化した白血球捕獲保持体および
b.サンプルを通って流れる過剰の洗浄溶液を毛管作用で運び取り出す吸収層。 - 保持体が約3から約15ミクロンの範囲のポアサイズおよび負正荷電を有する請求項12記載のデバイス。
- 保持体が有孔膜である請求項13記載のデバイス。
- さらにサンプルと洗浄溶液を適用するための開口を有するプラスチックカバーを含む請求項12記載のデバイス。
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US10/147,857 US6709868B2 (en) | 2002-05-20 | 2002-05-20 | Method and apparatus for measuring white blood cell count |
PCT/US2003/015739 WO2003100402A1 (en) | 2002-05-20 | 2003-05-20 | Method and apparatus for measuring white blood cell count |
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US (2) | US6709868B2 (ja) |
EP (1) | EP1525456B1 (ja) |
JP (1) | JP2005526513A (ja) |
AT (1) | ATE462967T1 (ja) |
AU (1) | AU2003239507B2 (ja) |
CA (1) | CA2486685A1 (ja) |
DE (1) | DE60331918D1 (ja) |
WO (1) | WO2003100402A1 (ja) |
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JP2012088055A (ja) * | 2010-09-21 | 2012-05-10 | Tokyo Univ Of Agriculture & Technology | 微量血液からの白血球ポピュレーション解析法 |
WO2013027611A1 (ja) | 2011-08-24 | 2013-02-28 | 栄研化学株式会社 | 白血球測定デバイス及び試薬キット |
WO2019151243A1 (ja) * | 2018-01-31 | 2019-08-08 | 株式会社キコーコーポレーション | 辺円対応変倍構造を有する生乳中の体細胞検査キット |
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Cited By (11)
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JP2012088055A (ja) * | 2010-09-21 | 2012-05-10 | Tokyo Univ Of Agriculture & Technology | 微量血液からの白血球ポピュレーション解析法 |
WO2013027611A1 (ja) | 2011-08-24 | 2013-02-28 | 栄研化学株式会社 | 白血球測定デバイス及び試薬キット |
CN103718040A (zh) * | 2011-08-24 | 2014-04-09 | 荣研化学株式会社 | 白细胞测定设备及试剂盒 |
JPWO2013027611A1 (ja) * | 2011-08-24 | 2015-03-19 | 栄研化学株式会社 | 白血球測定デバイス及び試薬キット |
CN103718040B (zh) * | 2011-08-24 | 2016-04-27 | 荣研化学株式会社 | 白细胞测定设备及试剂盒 |
US9523676B2 (en) | 2011-08-24 | 2016-12-20 | Eiken Kagaku Kabushiki Kaisha | Leukocyte measurement device and reagent kit |
WO2019151243A1 (ja) * | 2018-01-31 | 2019-08-08 | 株式会社キコーコーポレーション | 辺円対応変倍構造を有する生乳中の体細胞検査キット |
JPWO2019151243A1 (ja) * | 2018-01-31 | 2021-01-28 | 株式会社キコーコーポレーション | 辺円対応変倍構造を有する生乳中の体細胞検査キット |
JP7437736B2 (ja) | 2018-01-31 | 2024-02-26 | 株式会社キコーコーポレーション | 辺円対応変倍構造を有する生乳中の体細胞検査キット |
WO2021020520A1 (ja) * | 2019-07-31 | 2021-02-04 | 株式会社キコーコーポレーション | 検査用積層フィルタ |
JP2021023162A (ja) * | 2019-07-31 | 2021-02-22 | 株式会社キコーコーポレーション | 検査用積層フィルタ |
Also Published As
Publication number | Publication date |
---|---|
US6709868B2 (en) | 2004-03-23 |
AU2003239507B2 (en) | 2009-01-22 |
AU2003239507A1 (en) | 2003-12-12 |
EP1525456B1 (en) | 2010-03-31 |
WO2003100402A1 (en) | 2003-12-04 |
ATE462967T1 (de) | 2010-04-15 |
US20060088941A1 (en) | 2006-04-27 |
US20030215951A1 (en) | 2003-11-20 |
DE60331918D1 (de) | 2010-05-12 |
EP1525456A4 (en) | 2006-07-26 |
EP1525456A1 (en) | 2005-04-27 |
CA2486685A1 (en) | 2003-12-04 |
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