JP7450547B2 - 定量的フローサイトメトリー - Google Patents
定量的フローサイトメトリー Download PDFInfo
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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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—Electro-optical investigation, e.g. flow cytometers
- G01N15/1425—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its control arrangement
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- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1429—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its signal processing
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- 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
- G01N15/06—Investigating concentration of particle suspensions
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- 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
- G01N15/1012—Calibrating particle analysers; References therefor
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- G—PHYSICS
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- G01N33/483—Physical analysis of biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G01N33/487—Physical analysis of biological material of liquid biological material
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Description
(a)サンプル中に存在する各母集団中の細胞を細胞標識と既知数の蛍光微小粒子で標識するために、1つ又はそれ以上の細胞標識を含んでいるような希釈剤を含むチューブに、サンプルを加える;
(b)蛍光トリガーをセットして、母集団中に存在するカウントしなければならない実質的に全ての微小粒子と細胞を算入する;
(c)各細胞標識と微小粒子を識別するために、1つ又はそれ以上の蛍光ゲートをセットする;
(d)蛍光トリガーに衝突したり又は該トリガーを乗越えたりしている工程(c)からの細胞の数をカウントする;及び
(e)工程(d)からの各蛍光ゲートに対する微小粒子1個当たりの細胞数を計算し、そのそれぞれに微小粒子の濃度を掛けて、単位体積当たりの細胞数の絶対カウントを得る。
(1)フローサイトメーターを用いて、複数の既知濃度の標準試料中の細胞濃度を測定する工程、
(2)工程(1)で測定した測定値と既知濃度とに基づいて検量線を作成する工程、
(3)フローサイトメーターを用いて、未知濃度の試料中の細胞濃度を測定する工程、及び
(4)工程(2)で作成した検量線を用いて、工程(3)で測定した測定値から細胞濃度を決定する工程。
項2.2種以上の既知濃度の標準試料を使用する、項1に記載の方法。
項3.各既知濃度に対して1個以上の標準試料を使用する、項1又は2に記載の方法。
項4.工程(2)において、既知濃度に対して誤差±30%以内に収まった測定値を検量線の作成に使用する、項1~3のいずれか一項に記載の方法。
項5.工程(1)で測定した測定値の既知濃度に対する正確度が、全て±30%以内である、項1~4のいずれか一項に記載の方法。
項6.前記試料が血液である、項1~5のいずれか一項に記載の方法。
項7.前記細胞がT細胞である、項1~6のいずれか一項に記載の方法。
(1)フローサイトメーターを用いて、複数の既知濃度の標準試料中の細胞濃度を測定する工程、
(2)工程(1)で測定した測定値と既知濃度とに基づいて検量線を作成する工程、
(3)フローサイトメーターを用いて、未知濃度の試料中の細胞濃度を測定する工程、及び
(4)工程(2)で作成した検量線を用いて、工程(3)で測定した測定値から細胞濃度を決定する工程。
工程(1)では、検量線を作成するために、フローサイトメーターを用いて、複数の既知濃度の標準試料中の細胞濃度の測定を行う。
工程(2)では、工程(1)で測定した測定値と既知濃度とに基づいて検量線の作成を行う。
工程(3)では、フローサイトメーターを用いて、未知濃度の試料中の細胞濃度の測定を行う。
工程(4)では、工程(2)で作成した検量線を用いて、工程(3)で測定した測定値から細胞濃度を決定する。
50μLのC.B-17 SCIDマウスの血液を検体とし、そこに10、100、1,000及び10,000細胞のヒトT細胞(末梢血T細胞、Cryo-T8: Human CD8+ Negatively Selected (5-8M cells)、Precision Bioservices社)をそれぞれ添加したサンプル、並びにヒトT細胞溶解液非添加のサンプルを調製した。これらのサンプルを、市販のチューブ(TruCOUNT Tubes、BD Biosciences社)内で、BD TritestTM CD3/CD8/CD45 reagent (BD Biosciences社)と混合させた。本試薬には、fluorescein isothiocyanate (FITC)-labeled anti-CD3 antibody (clone SK7), phycoerythrin (PE)-labeled anti-CD8 antibody (clone SK1)及びperidinin chlorophyll protein (PerCP)-labeled anti-CD45 antibody (clone 2D1)が含まれている。
検量線の作成の結果を表1に示す。検量線の直線性は良好であり、回収率は概ね100%であった。日内及び日間の正確度(accuracy, Relative error)及び精度(precision, Coefficient of variation)も良好であった。定量下限は30 cells/50μLであった。
Claims (7)
- 以下の工程を含む、試料中の細胞濃度を測定する方法:
(1)フローサイトメーターを用いて、複数の既知濃度の標準試料中の細胞濃度を測定する工程、
(2)工程(1)で測定した測定値と既知濃度とに基づいて検量線を作成し、細胞の回収率と正確度とを求める工程であって、ここでの回収率とは前処理及び測定の工程において標準試料中の細胞が回収される割合であって、既知濃度に対する測定値の比率又は検量線の傾きにより示される、工程、
(3)フローサイトメーターを用いて、未知濃度の試料中の細胞濃度を測定する工程、及び
(4)工程(2)で作成した検量線を用いて、工程(3)で測定した測定値から細胞濃度を決定する工程。 - 2種以上の既知濃度の標準試料を使用する、請求項1に記載の方法。
- 各既知濃度に対して1個以上の標準試料を使用する、請求項1又は2に記載の方法。
- 工程(2)において、既知濃度に対して誤差±30%以内に収まった測定値を検量線の作成に使用する、請求項1~3のいずれか一項に記載の方法。
- 工程(1)で測定した測定値の既知濃度に対する正確度が、全て±30%以内である、請求項1~4のいずれか一項に記載の方法。
- 前記試料が血液である、請求項1~5のいずれか一項に記載の方法。
- 前記細胞がT細胞である、請求項1~6のいずれか一項に記載の方法。
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JP2018205368 | 2018-10-31 | ||
JP2018205368 | 2018-10-31 | ||
PCT/JP2019/042470 WO2020090852A1 (ja) | 2018-10-31 | 2019-10-30 | 定量的フローサイトメトリー |
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JPWO2020090852A1 JPWO2020090852A1 (ja) | 2021-09-16 |
JP7450547B2 true JP7450547B2 (ja) | 2024-03-15 |
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US (1) | US20210356379A1 (ja) |
EP (1) | EP3875956A4 (ja) |
JP (1) | JP7450547B2 (ja) |
KR (1) | KR20210082469A (ja) |
CN (1) | CN112912724A (ja) |
WO (1) | WO2020090852A1 (ja) |
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CN116794313B (zh) * | 2023-08-18 | 2023-11-03 | 江西赛基生物技术有限公司 | 基于流式细胞仪同时检测三项肿瘤标志物的试剂盒及方法 |
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WO2007136025A1 (ja) | 2006-05-19 | 2007-11-29 | Bml, Inc. | 感染症の検出方法 |
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US9097712B2 (en) * | 2013-03-15 | 2015-08-04 | Allied Innovative Systems Llc | Flow-through cell counting assay |
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WO2007136025A1 (ja) | 2006-05-19 | 2007-11-29 | Bml, Inc. | 感染症の検出方法 |
JP2009186459A (ja) | 2008-01-10 | 2009-08-20 | Becton Dickinson & Co | 高速な粒子検出分析 |
WO2016013395A1 (ja) | 2014-07-22 | 2016-01-28 | 株式会社日立ハイテクノロジーズ | 細胞分散計測機構及びそれを用いた細胞継代培養システム |
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WO2020090852A1 (ja) | 2020-05-07 |
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