Methods for the identification and characterization of reticulocytes in whole blood

A method for identifying light-absorbing subclasses of cells of interest from other subclasses of cells in a whole blood sample by flow cytometry which comprises the steps of:

• (a) mixing an aliquot of said blood sample with an aqueous reagent composition to form a suspension, wherein the reagent composition comprises a dye compound which stains the ribonucleic acid of cells in the subclass of interest, a buffer for maintaining a pH of about 6 to about 9, and a sphering agent which is a zwitterionic surfactant and which does not precipitate said dye;
• (b) passing said suspension of step (a) substantially a cell at a time through an area of focused optical illumination;
• (c) detecting and measuring the light scattered and light absorbed by each cell;
• and
• (d) differentiating said cells of said subclass of interest at least in part on the basis of the measured magnitudes of said scattered said absorbed light.

A method for characterizing reticulocytes and erythrocytes in a whole blood sample by flow cytometry which comprises the steps of:

• (a) mixing an aliquot of said blood sample with a reagent composition comprising an organic cationic dye compound, a sphering agent which is a zwitterionic surfactant and does not precipitate said dye, and a buffer solution to form a suspension, wherein said dye compound stains the ribonucleic acid of the reticulocytes;
• (b) passing said suspension of step (a) substantially a cell at a time through an area of focused optical illumination;
• (c) detecting and measuring the light scattered and light absorbed by each cell;
• (d) identifying stained cells and unstained cells on the basis of the measured magnitudes of said light scattered and light absorbed;
• and
• (e) determining the number, the volume, hemoglobin concentration, and RNA concentration of the reticulocytes or erythrocytes on the basis of the measured magnitudes of said scattered and absorbed light.