Immunophenotyping of peripheral blood leukocytes by laser scanning cytometry

Many clinical situations demand repeated analyses of blood parameters but p ermit only minimal amounts of peripheral blood to be taken, e.g., in neonat es with low birth weight, during extensive operations of young children, or in patients with restricted bone marrow function. In these cases laser sca nning cytometry is the ideal tool to determine the distribution of differen t leukocyte-subsets. The purpose of this protocol is to describe stepwise a new method of immunophenotyping by laser scanning cytometry. In this assay nuclear DNA is stained by 7-aminoactinomycin-D (7-AAD) and surface antigen s are detected by direct three-colour immunofluorescence. For data acquisit ion, measurements are triggered on the 7-AAD-fluorescence. Data are obtaine d for forward scatter, green, orange, and long red fluorescence by excitati on with the argon-laser, and for far red fluorescence by excitation with th e helium-neon-laser. Using this protocol the amount of peripheral blood nee ded is minimised to 10 mul. Specimens can be stained a second time in a dif ferent way and analysed repeatedly and archived. (C) 2000 Elsevier Science B.V. All rights reserved.