One step method for detection and enumeration of absolute counts of one more cell populations in a sample
AssigneeBecton, Dickinson and Company
InventorsAnthony J. Ward, Thomas J. Mercolino, Diether J. Recktenwald
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Abstract
This invention comprises a one step method for the detection and enumeration of absolute counts of one or more cell populations in a blood sample. The method employs a reagent comprising a mixture of one or more cell markers, a fluorescent microparticle and a fixative. The reagent may be combined with unlysed whole blood and analyzed by means of flow cytometry.
Claims17
A method for determining the absolute counts of CD4.sup.+ lymphocytes within a whole blood sample by means of flow cytometry comprising the steps of:
- (a) adding the sample to a tube containing a diluent comprising an anti-CD4 monoclonal antibody bound to phycoerythrin, paraformaldehyde and a known concentration of a 2 .mu.m autofluorescent coumarin microparticle;
- (b) setting a fluorescence trigger in the second fluorescence channel to include at least 99% of all microparticles and CD4.sup.+ lymphocytes;
- (c) setting fluorescence gates in the first and second fluorescence channels to distinguish between the phycoerythrin fluorescence associated with the labelled CD4.sup.+ lymphocytes and the autofluorescence of the microparticle;
- (d) counting the number of CD4.sup.+ lymphocytes and microparticles from step (c);
- and
- (e) calculating the number of CD4.sup.+ lymphocytes per microparticle from step (d) and multiplying by the concentration of microparticles resulting in the absolute count of CD4.sup.+ lymphocytes per unit volume.
A method of staining cells in a sample containing said cells with one or more immunofluorescence markers, said immunofluorescence markers comprising a monoclonal antibody bound to a fluorochrome, wherein the sample is added directly to a tube containing a diluent, said diluent comprising said one or more immunofluorescence markers and a fixative.
A solution for staining cells comprising a diluent, said diluent comprising one or more immunofluorescence markers and a fixative.
A solution for staining unlysed whole blood comprising a diluent, said diluent comprising a fluorescently labelled monoclonal antibody, a fixative and a fluorescent microparticle.
A solution for staining CD4.sup.+ lymphocytes in an unlysed whole blood sample wherein the solution comprises an anti-CD4 monoclonal antibody bound to phycoerythrin, paraformaldehyde and an autofluorescent 2 .mu.m coumarin bead.
Dependencies are inferred from the claim text.