Reagent and method for classifying leukocytes by flow cytometry

Claim 1

A reagent system for use in 5-part differentiation of leukocytes by flow cytometry that contains a fluorochrome dye for selectively staining eosinophils and a fluorochrome dye for selectively staining basophils.

Claim 8

A method for classifying leukocytes by flow cytometry that comprises the following steps (a) to (d): (a) lysing the erythrocytes in a fresh sample of anti­coagulated blood by adding it to a hypotonic first fluid composed of a fluorochrome dye for selectively staining eosinophils, a fluorochrome dye for selectively staining basophils, and a buffer for maintaining an acidic pH range; (b) staining the leukocytes in the so-treated blood sample by adding to it a second fluid that is composed of a buffer for neutralizing the acid in the buffer in the first fluid and maintaining the pH of the resulting dye solution at a staining pH, and an osmolarity compensating agent for adjusting the osmolarity of the dye solution to a value at which the leukocytes remain unchanged in shape; (c) permitting the stained sample to flow through a flow cytometer, differentiating leukocytes from all other corpuscles and ghosts by intensity of fluorescence, and measuring the signals of fluorescence and right-angle (rectangular) scattered light from leukocytes; and (d) identifying the type of each of the leukocytes based on said two signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.

Claim 9

A method for classifying leukocytes by flow cytometry that comprises the following steps (a) to (d): (a) lysing the erythrocytes in a fresh sample of anti­coagulated blood by adding it to a hypotonic first fluid composed of a fluorochrome dye for selectively staining eosinophils, a fluorochrome dye for selectively staining basophils, and a buffer for maintaining an acidic pH range; (b) staining the leukocytes in the so-treated blood sample by adding to it a second fluid that is composed of a buffer for neutralizing the acid in the buffer in the first fluid and maintaining the pH of the resulting dye solution at a staining pH, and an osmolarity compensating agent for adjusting the osmolarity of the dye solution to a value at which the leukocytes remain unchanged in shape; (c) permitting the stained sample to flow through a flow cytometer, differentiating leukocytes from all other corpuscles and ghosts by intensity of fluorescence, and measuring the signals of fluorescence and right-angle (rectangular) scattered light from leukocytes; and (d) identifying the type of each of the leukocytes based on said two signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.