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1. WO2004104164 - METHOD AND APPARATUS FOR PREPARING CELL SAMPLES FOR INTRACELLULAR ANTIGEN DETECTION USING FLOW CYTOMETRY

Publication Number WO/2004/104164
Publication Date 02.12.2004
International Application No. PCT/US2004/012761
International Filing Date 26.04.2004
IPC
G01N 1/34 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
1Sampling; Preparing specimens for investigation
28Preparing specimens for investigation
34Purifying; Cleaning
G01N 15/14 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
15Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
10Investigating individual particles
14Electro-optical investigation
G01N 33/50 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
G01N 33/536 2006.1
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
53Immunoassay; Biospecific binding assay; Materials therefor
536with immune complex formed in liquid phase
CPC
G01N 1/30
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
1Sampling; Preparing specimens for investigation
28Preparing specimens for investigation ; including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
30Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
G01N 1/405
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
1Sampling; Preparing specimens for investigation
28Preparing specimens for investigation ; including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
40Concentrating samples
405by adsorption or absorption
G01N 15/147
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
15Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
10Investigating individual particles
14Electro-optical investigation, e.g. flow cytometers
1468with spatial resolution of the texture or inner structure of the particle
147the analysis being performed on a sample stream
G01N 33/5005
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
48Biological material, e.g. blood, urine
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
5005involving human or animal cells
G01N 33/536
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
48Biological material, e.g. blood, urine
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
53Immunoassay; Biospecific binding assay; Materials therefor
536with immune complex formed in liquid phase
Y10S 435/971
YSECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
10TECHNICAL SUBJECTS COVERED BY FORMER USPC
STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
435Chemistry: molecular biology and microbiology
971Capture of complex after antigen-antibody reaction
Applicants
  • BECKMAN COULTER, INC. [US]/[US]
Inventors
  • RUBIO, Oilda
  • APARICIO, Carlos
  • MAPLES, John, A.
  • WILKINSON, Julie
  • SMITH, Cecilia
  • LUCAS, Frank, J.
Agents
  • ALTER, Mitchell, E.
Priority Data
10/437,69514.05.2003US
Publication Language English (en)
Filing Language English (EN)
Designated States
Title
(EN) METHOD AND APPARATUS FOR PREPARING CELL SAMPLES FOR INTRACELLULAR ANTIGEN DETECTION USING FLOW CYTOMETRY
(FR) METHODE ET APPAREIL PERMETTANT DE PREPARER DES ECHANTILLONS CELLULAIRES POUR DETECTER DES ANTIGENES INTRACELLULAIRES PAR CYTOMETRIE EN FLUX
Abstract
(EN) A method and apparatus for preparing biological cell samples for intracellular analysis. The invention is based upon the recognition that many of the steps of the conventional methods for such sample preparation can be eliminated, leading to a process that readily lends itself to automation and the advantages associated therewith. The method of the invention comprises the steps of (a) cell-fixation, (b) permeabilization and (c) staining (or labeling) of intracellular molecules of interest by probes that are readily detectable by flow cytometric techniques, all without any intervening cell-washing (and re-suspension) steps. Rather, the single cell-washing step is affected after these three steps have been carried out. Preferably, the washing step is carried out by passing the fixed, permeabilized and stained cell sample through a semi-permeable membrane that serves to filter out (by transmission) interferants to waste while retaining the cells of interest.
(FR) L'invention concerne une méthode et appareil permettant de préparer des échantillons cellulaires afin d'effectuer une analyse intracellulaire. Cette invention est basée sur la découverte que la plupart des méthodes classiques de préparation de ces échantillons peuvent être supprimées, ce qui conduit à un processus qui se prête à l'automation et aux avantages qui lui sont associés. Le procédé de l'invention consiste: a) en une étape de fixation cellulaire, b) en une étape de perméabilisation et c) en une étape de coloration (ou un marquage) de molécules intracellulaires d'intérêt à l'aide de sondes facilement détectables au moyen de techniques cytométriques de flux, tout cela sans faire intervenir d'étapes de lavage de cellules (et de resuspension). L'étape de lavage de cellules seule intervient plutôt après l'exécution des trois premières étapes. L'étape de lavage s'exécute, de préférence, par passage de l'échantillon cellulaire fixe, perméabilisé et coloré à travers une membrane semi-perméable qui sert à éliminer par filtrage (par transmission) des substances interférantes à évacuer tout en retenant les cellules d'intérêt.
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