One step test for absolute counts

Claim 2

The method of claim 1 wherein the sample is unlysed whole blood.

Claim 3

The method of claim 1 wherein one or more of the cell markers comprise an immunofluorescence marker said immunofluorescence marker further comprising a monoclonal antibody bound to a fluorochrome.

Claim 4

The method of claim 3 wherein the monoclonal antibody is selected from the group consisting of anti-CD4, anti-CD8, anti-CD3, anti-CD14, anti-CD15, and anti-CD45 monoclonal antibodies.

Claim 5

The method of claim 4 wherein the fluorochromes are selected from the group consisting of phycobiliproteins, fluorescein derivatives, rhodamine, pthalocyanine derivatives, peridinin chlorophyll complex and coumarin derivatives.

Claim 6

The method of claim 1 wherein the microparticle is selected from the group consisting of fixed chicken red blood cells, liposomes containing a fluorescent dye, coumarin beads, fluoroscein beads, rhodamine beads, fixed fluorescent cells, fluorescent cell nuclei, microorganisms, surface labelled beads tagged with a fluorescent dye.

Claim 7

The method of claim 1 wherein one or more of the cell markers comprise a fluorescent tagging agent.

Claim 8

The method of claim 7 wherein the fluorescent tagging agents is selected from the group consisting of nucleic acid dye, 7-amino-actinomycin D, thiazole orange, thioflavin T, LDS-751 and acridine orange.

Claim 9

A method according to claim 1 for determining the absolute counts of one population of cells within an unlysed whole blood sample by means of flow cytometry comprising the steps of: (a) adding the sample to a tube containing a diluent wherein the diluent comprises an isotonic buffer, one immunofluorescent marker so that the cells in the sample are labelled with the immunofluorescent marker, a fixative and a known concentration of a microparticle having autofluorescence in two fluorescence channels and further wherein the ratio of autofluorescence of the microparticle in a first channel to second channel is distinguishable from the ratio of fluorescence emitted by the immunofluorescent marker in said first channel and second channel; (b) setting a fluorescence trigger in the second fluorescence channel to include essentially all the microparticles and cells of the population to be counted; (c) counting all microparticles and immunofluorescently labelled cells that meet or exceed the fluorescence trigger in the second fluorescence channel by means of flow cytometry; (d) setting fluorescence gates in the first and second fluorescence channels to distinguish between the immunofluorescent marker and the microparticle; and (e) calculating the number of cells per microparticle from step (d) and multiplying by the concentration of microparticles resulting in the absolute count of cells per unit volume.

Claim 10

A method according to claim 1 for determining the absolute counts of CD4⁺ 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 2µm autofluorescent microparticle; (b) setting a fluorescence trigger in the second fluorescence channel to include at least 99% of all microparticles and CD4⁺ lymphocytes; (c) setting fluorescence gates in the first and second fluorescence channels to distinguish between the phycoerythrin fluorescence associated with the labelled CD4⁺ lymphocytes and the autofluorescence of the microparticle; (d) counting the number of CD4⁺ lymphocytes and microparticles from step (c); and (e) calculating the number of CD4⁺ lymphocytes per microparticle from step (d) and multiplying by the concentration of microparticles resulting in the absolute count of CD4⁺ lymphocytes per unit volume.

Claim 11

A solution for staining unlysed whole blood comprising a diluent, said diluent further comprising one or more fluorescently labelled monoclonal antibodies, a fixative and a fluorescent microparticle.

Claim 12

The solution of claim 11 wherein one antibody is an anti-CD4 monoclonal antibody.

Claim 13

The solution of claim 11 wherein the fluorescent label is phycoerythrin.

Claim 14

The solution of claim 11 wherein the fixative is paraformaldehyde.

Claim 15

A solution for staining CD4⁺ and CD8⁺ lymphocytes in an unlysed whole blood sample wherein the solution comprises an anti-CD4 monoclonal antibody bound to a fluorochrome, an anti-CD8 monoclonal antibody bound to a second fluorochrome,. an anti-CD3 monoclonal antibody bound to a third fluorochrome, paraformaldehyde and an autofluorescent bead.