Method for discriminating surface stained lymphocytes

Claim 1

A flow cytometry method for detecting a lymphocyte population of interest in a heterogeneous whole blood cell suspension containing various classes of leukocytes, and wherein the red blood cells are lysed prior to said measurement, which method comprises: (a) first reacting said whole blood cell suspension with a first reagent comprising up to about 7.5% formaldehyde and up to about 15% sugar for up to about 10 minutes to fix and stabilize the cells in said whole blood cell suspension; (b) then incubating said fixed cell suspension of step (a) with a second reagent comprising primary antibodies specific for T lymphocyte or B lymphocyte surface antigens of said lymphocyte population of interest to form a primary antibody-labeled-fixed-lymphocyte population in said cell suspension; (c) then reacting said primary antibody-labeled-fixed-lymphocyte population of step (b) with an enzyme-ligand-conjugate wherein said ligand is specific for the immunoglobin class of said primary antibodies to form a cell suspension wherein said enzyme-ligand-conjugate is bound to said primary antibody-labeled-fixed-lymphocyte population; (d) thereafter reacting said cell-suspension of step (c) with at least one composition which reacts with said enzyme to stain said lymphocyte population of interest; (e) subsequently passing said cell-suspension of step (d) substantially one cell at a time, through a light beam while measuring light scattered and light absorbed by each cell passing therethrough; and (f) discriminating between stained and unstained cells in part on the magnitude of the light scattering and adsorption responses, whereby the stained cells indicate the presence of the lymphocyte population of interest in said heterogeneous whole blood cell suspension.