US 2003/0215892 A1 · Multidimensional leukocyte differential analysis

Abstract

This invention relates to an improved procedure for the multiparameter analysis of leukocyte subpopulations from peripheral blood, bone marrow or any body fluid containing blood nucleated cells, including the quantification of each subpopulation in terms of cells per microliter. The method uses a cocktail of at least four monoclonal antibodies labelled each with a different fluorescent tag, the measurement of at least two light scatter and four fluorescence emissions related parameters and at least one population of fluorescent beads to differentiate and enumerate between and among the different leukocyte subpopulations in the peripheral blood, the bone marrow or other body fluid. It includes: the incubation of the sample with a mixture of monoclonal antibodies conjugated to a minimum of four different fluorochromes, the addition of one or more populations of reference beads, the flow cytometry measurement of the fluorescence emissions associated to each cell and bead population and, the mutidimesional analysis of each cell and bead population present in the sample aimed at its identification, enumeration and characterization.

Claims26

Dependent claims

A method for the multiparameter identification and/or enumeration and/or characterization of cells in a body fluid comprising the following steps:

(a) Taking a peripheral blood or another body fluid from an individual;
(b) Adding said sample to a recipient containing a pre-measured amount of fluorescent beads (or to which any type of counting materials are added);
(c) Adding to said sample a cocktail of fluorescently labeled monoclonal antibodies wherein the fluorescent labels on said antibodies have peak emission spectra different from each other;
(d) Analyzing said cells in an instrument capable of detecting and recording at least four channels of fluorescence and at least two channels of light scatter for each of the cells in said sample, wherein total leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, total dendritic cells, CD16 positive dendritic cells/monocytes, myeloid derived dendritic cells and lymphoplasmacytoid dendritic cells are measured and identified;
(e) A software gating algorithm using logical combinations of gates whereby said total leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, total dendritic cells, CD16 positive dendritic cells/monocytes, myeloid derived dendritic cells and lymphoplasmacytoid dendritic cells can be characterized in terms of scattergram position and spread and quantitatively enumerated in terms of cells per microliter.
(f) A software algorithm identifying any clustering cell populations that are different from any of the regular leukocyte subpopulations.

3 more elements

A method for the multiparameter identification, analysis and/or enumeration of cells in a body fluid comprising the following steps:

(a) Taking a sample of peripheral blood from an individual;
(b) Adding said sample to TruCOUNT tube;
(c) Adding to said sample a cocktail of fluorescently labeled monoclonal antibodies consisting of anti-HLA-Dr, anti-CD33, anti-CD45 and anti-CD14, wherein the fluorescent labels on said antibodies have peak emission spectra different from each other;
(d) Analyzing said cells in an instrument capable of detecting and recording at least four channels of fluorescence and at least two channels of light scatter for each of the cells in said sample, wherein total leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, total dendritic cells, CD16 positive dendritic cells, myeloid derived dendritic cells and lymphoid derived dendritic cells are measured and identified;
(e) A software gating algorithm using logical combinations of gates whereby said total leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, total dendritic cells, CD16 positive dendritic cells, myeloid derived dendritic cells and lymphoid derived dendritic cells can be characterized in terms of scattergram position and spread and quantitatively enumerated in terms of cells per microliter.
(f) A software algorithm identifying any clustering cell populations that are different from any of the regular leukocyte subpopulations.

3 more elements

A method for the multi-parameter analysis and/or enumeration of cells in a body fluid comprising the following steps:

(a) Taking a sample of bone marrow from an individual;
(b) Adding said sample to TruCOUNT tube;
(c) Adding to said mixture a lysing solution;
(d) Adding to said sample a cocktail of fluorescently labeled monoclonal antibodies consisting of anti-HLA-Dr, anti-CD33, anti-CD45 and anti-CD14, wherein the fluorescent labels on said antibodies have peak emission spectra different from each other;
(e) Analyzing said cells in an instrument capable of detecting and recording at least four channels of fluorescence and at least two channels of light scatter for each of the cells in said sample, wherein total leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, total dendritic cells, CD16 positive dendritic cells, myeloid derived dendritic cells and lymphoid derived dendritic cells are measured and identified;
(f) A software gating algorithm using logical combinations of gates whereby said total leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, total dendritic cells, CD16 positive dendritic cells, myeloid derived dendritic cells and lymphoid derived dendritic cells can be characterized in terms of scattergram position and spread and quantitatively enumerated in terms of cells per microliter.
(g) A software algorithm identifying any clustering cell populations that are different from any of the regular leukocyte subpopulations.

4 more elements

Dependencies are inferred from the claim text.

Multidimensional leukocyte differential analysis

Life of this patent

Multidimensional leukocyte differential analysis

Life of this patent

Abstract

Claims26