Five-color immunophenotyping plus DNA content analysis by laser scanning cytometry

Laser scanning cytometry is a new laboratory technology similar to flow cytometry but with advantages for certain clinical and research applications. To date, laser scanning cytometry has been successfully used to perform three-color immunophenotypic analysis of hematologic specimens, single-color immunophenotyping plus DNA content analysis of numerous specimen types, and automated analysis of fluorescence in situ hybridization specimens. Several other interesting applications are also in development. In general, advantages of laser scanning cytometry include reduced specimen size requirements, simplified methodologies, and the ability to microscopically examine individual cells-allowing for the direct correlation of cytologic morphology with objective fluorescence measurements. In this report, we describe a method which more fully takes advantage of the laser scanning cytometer's capabilities for immunophenotypic analysis of hematologic specimens. Specifically, we have devised a method to increase the number of fluorescent parameters from three to a total of six, five representing binding of immunofluorescent antibodies and one for stoichiometric measurements of DNA content. As with most laser scanning cytometric applications, this technique can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology.