JP2009080122A - 赤芽球の分類計数方法 - Google Patents
赤芽球の分類計数方法 Download PDFInfo
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- JP2009080122A JP2009080122A JP2008278267A JP2008278267A JP2009080122A JP 2009080122 A JP2009080122 A JP 2009080122A JP 2008278267 A JP2008278267 A JP 2008278267A JP 2008278267 A JP2008278267 A JP 2008278267A JP 2009080122 A JP2009080122 A JP 2009080122A
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- erythroblasts
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- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1477—Multiparameters
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
【解決手段】(i)抹消血液に、白血球に特異的に結合する蛍光標識抗体を添加して白血球を蛍光染色し、(ii)工程(i)で得られた試料中の赤血球を溶血し、(iii)工程(ii)で得られた試料のpHおよび浸透圧を調整し、(iv)赤芽球の核を、前記蛍光標識抗体における蛍光と区別可能な蛍光スペクトルを有し、かつ通常細胞膜を透過しない核酸蛍光色素で蛍光染色し、(v)次いで、得られた試料をフローサイトメータに供して個々の細胞の少なくとも2つの蛍光信号を測定し、(vi)これら蛍光強度差に基づいて、抹消血液に含まれる赤芽球を分類し、分類された赤芽球を計数することからなる赤芽球の分類計数方法。
【選択図】図1
Description
実施例1まず、以下の組成の試薬を調製した。
蛍光標識抗体・FITC標識抗CD45抗体第1液(pH3.0、浸透圧:16mOsm/kg・H2O)
・緩衝剤 クエン酸一水和物 2.10g/L リン酸二ナトリウム 0.56g/L ・核酸蛍光色素 プロピジウムアイオダイド 100mg/L ・精製水第2液(pH7.5、浸透圧:420mOsm/kg・H2O)
・緩衝剤 リン酸一ナトリウム二水和物 0.95g/L リン酸二ナトリウム 6.24g/L ・浸透圧調整剤 塩化ナトリウム 10.2g/L ・精製水
蛍光標識抗体・FITC標識抗CD45抗体第1液(pH3.0、浸透圧:16mOsm/kg・H2O)
・緩衝剤 クエン酸一水和物 2.10g/L リン酸二ナトリウム 0.56g/L ・核酸蛍光色素 プロピジウムアイオダイド 1mg/L ・精製水第2液(pH7.5、浸透圧:420mOsm/kg・H2O)
・緩衝剤 リン酸一ナトリウム二水和物 0.95g/L リン酸二ナトリウム 6.24g/L ・浸透圧調整剤 塩化ナトリウム 10.2g/L ・精製水
NRBC 赤芽球
leu 白血球
Gho ゴースト
Ly リンパ球
Mo 単球
Gran 顆粒球
Claims (16)
- (i)抹消血液に、白血球に特異的に結合する蛍光標識抗体を添加して白血球を蛍光染色し、
(ii)工程(i)で得られた試料と、2.0〜5.0のpHおよび100mOsm/kg・H 2 O以下の浸透圧を有する第1液を混合して赤血球を溶血し、
(iii)工程(ii)で得られた試料と、pH5.0〜11.0のpHおよび300〜1000mOsm/kg・H 2 Oの浸透圧を有する第2液を混合してpHおよび浸透圧を調整し、
(iv)赤芽球の核を、前記蛍光標識抗体における蛍光と区別可能な蛍光スペクトルを有し、かつ通常細胞膜を透過しない核酸蛍光色素で蛍光染色し、
(v)次いで、得られた試料をフローサイトメータに供して個々の細胞の少なくとも2つの蛍光信号を測定し、
(vi)これら蛍光強度差に基づいて、抹消血液に含まれる赤芽球を分類し、分類された赤芽球を計数することからなる赤芽球の分類計数方法。 - 工程(i)における白血球に特異的に結合する蛍光標識抗体が、白血球表面に発現している抗原を認識し、その抗原に結合する蛍光標識抗体である請求項1記載の赤芽球の分類計数方法。
- 工程(i)の蛍光標識抗体の蛍光標識化合物が、フィコエリスリン、フルオレセインイソチオシアネート、アロフィコシアニン、テキサスレッド、CY5、ペリジニンクロロフィルコンプレックス及びそれらのフィコエリスリン結合体からなる群から選択される少なくとも1種の化合物である請求項1又は2のいずれかに記載の赤芽球の分類計数方法。
- 第1液または第2液が、核酸蛍光色素を含有する、請求項1〜3のいずれかに記載の赤芽球の分類計数方法。
- 第1液の浸透圧が、10〜60mOsm/kg・H 2 Oである、請求項1〜4のいずれかに記載の赤芽球の分類計数方法。
- 第1液のpHが、7.5〜10.0である、請求項1〜5のいずれかに記載の赤芽球の分類計数方法。
- 第2液を混合した後の試料の浸透圧が、400〜600mOsm/kg・H 2 Oである、請求項1〜6のいずれかに記載の赤芽球の分類計数方法。
- 分類された赤芽球を、核酸蛍光色素の蛍光強度に基づいて、少なくとも2つの異なる成熟度の赤芽球に分類する工程をさらに備える請求項1〜7のいずれかに記載の赤芽球の分類計数方法。
- 工程(iv)の核酸蛍光色素が、プロピジウムアイオダイド、N−メチル−
4−(1−ピレン)ビニル−プロピジウムアイオダイド、エチジウムブロマイド、TOTO-1、TOTO-3、YOYO-1、YOYO-3、BOBO-1、BOBO-3、エチジウムホモダイマー−1(EthD-1)、エチジウムホモダイマー−2(EthD-2)、POPO-1、POPO-3、BO-PRO-1、YO-PRO-1、TO-PRO-1からなる群から選択される少なくとも1種類の色素である請求項1〜8のいずれかに記載の赤芽球の分類計数方法。 - 測定された個々の細胞の蛍光信号から、白血球に特異的に結合する蛍光標識抗体に基づく蛍光及び核酸蛍光色素に基づく蛍光をそれぞれ2軸として2次元分布を得ることからなる請求項1〜9のいずれかに記載の赤芽球の分類計数方法。
- 2次元分布から赤芽球の分布領域を設定し、その領域の細胞数を計数する請求項10に記載の赤芽球の分類計数方法。
- 2次元分布から白血球及び赤芽球の分布領域を設定し、それぞれの領域
の細胞数を計数し、赤芽球細胞数を白血球細胞数で除算することにより白血球に対する赤芽球の比率を求める請求項10又は11に記載の赤芽球の分類計数方法。 - 核酸蛍光色素を、0.003mg/L〜10mg/Lの濃度範囲で使用する請求項1〜12に記載の赤芽球の分類計数方法。
- (1)測定された個々の細胞の蛍光信号から、白血球に特異的に結合する蛍光標識抗体に基づく蛍光及び核酸蛍光色素に基づく蛍光をそれぞれ2軸として2次元分布を得、
(2)2次元分布において、核酸蛍光色素に基づく蛍光の強度差で、赤芽球を少なくとも2つに分類する領域を設定し、各領域の細胞数を計数することによって、成熟度の異なる赤芽球を分類することからなる請求項8に記載の赤芽球の分類計数方法。 - 2次元分布において、全赤芽球及び少なくとも2つの成熟度の異なる赤芽球の各領域の細胞数を計数し、2つの成熟度の異なる赤芽球の細胞数を全赤芽球数でそれぞれ除算することにより全赤芽球に対する成熟度の異なる赤芽球の各比率を求める請求項14に記載の赤芽球の分類計数方法。
- 工程(i)において使用される蛍光標識抗体が、1種類である請求項1〜15に記載の赤芽球の分類計数方法。
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Cited By (5)
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JP2012525589A (ja) * | 2009-04-27 | 2012-10-22 | アボット・ラボラトリーズ | 自動血液分析器において、レーザーからの前方散乱を用いることによる、赤血球細胞を白血球細胞から判別する方法 |
JP2019529865A (ja) * | 2016-06-01 | 2019-10-17 | クアンタム−エスアイ インコーポレイテッドQuantum−Si Incorporated | パルス決定器及び塩基決定器 |
WO2020252692A1 (zh) * | 2019-06-19 | 2020-12-24 | 深圳迈瑞生物医疗电子股份有限公司 | 一种细胞分析仪、基于阻抗法对白细胞进行分类的方法及计算机可读存储介质 |
US11538556B2 (en) | 2018-01-26 | 2022-12-27 | Quantum-Si Incorporated | Machine learning enabled pulse and base calling for sequencing devices |
JP2023041904A (ja) * | 2016-12-15 | 2023-03-24 | シスメックス株式会社 | 前処理装置及び前処理方法 |
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JP4566299B2 (ja) * | 1998-04-09 | 2010-10-20 | シスメックス株式会社 | 赤芽球の分類計数方法 |
JP2007232382A (ja) * | 2006-02-27 | 2007-09-13 | Fuji Electric Systems Co Ltd | 微生物検出方法及び微生物検出装置 |
PL2202516T3 (pl) * | 2007-09-27 | 2015-02-27 | Sysmex Corp | Zestaw odczynników do analizy próbek i sposób analizy próbek |
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JPH04268453A (ja) * | 1991-02-22 | 1992-09-24 | Toa Medical Electronics Co Ltd | フローサイトメトリーによる赤芽球の分類方法 |
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JPH04268453A (ja) * | 1991-02-22 | 1992-09-24 | Toa Medical Electronics Co Ltd | フローサイトメトリーによる赤芽球の分類方法 |
JPH08501396A (ja) * | 1993-11-12 | 1996-02-13 | ベクトン・ディッキンソン・アンド・カンパニー | 希少細胞の絶対数を数える方法 |
JPH11326323A (ja) * | 1998-04-09 | 1999-11-26 | Sysmex Corp | 赤芽球の分類計数方法 |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2012525589A (ja) * | 2009-04-27 | 2012-10-22 | アボット・ラボラトリーズ | 自動血液分析器において、レーザーからの前方散乱を用いることによる、赤血球細胞を白血球細胞から判別する方法 |
JP2019529865A (ja) * | 2016-06-01 | 2019-10-17 | クアンタム−エスアイ インコーポレイテッドQuantum−Si Incorporated | パルス決定器及び塩基決定器 |
JP2023041904A (ja) * | 2016-12-15 | 2023-03-24 | シスメックス株式会社 | 前処理装置及び前処理方法 |
JP7503160B2 (ja) | 2016-12-15 | 2024-06-19 | シスメックス株式会社 | 前処理装置及び前処理方法 |
US11538556B2 (en) | 2018-01-26 | 2022-12-27 | Quantum-Si Incorporated | Machine learning enabled pulse and base calling for sequencing devices |
WO2020252692A1 (zh) * | 2019-06-19 | 2020-12-24 | 深圳迈瑞生物医疗电子股份有限公司 | 一种细胞分析仪、基于阻抗法对白细胞进行分类的方法及计算机可读存储介质 |
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