Simultaneous Five-Six Color Multiparameter Analysis

The development of simultaneous multiparameter analysis protocols has facilitated the use of flow cytometry in many diverse and complex research and clinical programs. This effort has been advanced by the availability of a wide range of fluorochrome-conjugated antibody reagents and the use of fluorescent particles and probes that can be employed for the analysis of numerous antigens that define cell type and function in many diverse applications. These include clinical diagnoses and monitoring of disease states (1 ,2 ), bone marrow progenitor cell isolation (3 ), ion flux measurements (4 ), nucleic acid quantitation (5 ,6 ), cell migration (7 ), metabolic activation (8 ) and routine phenotyping (9 ). In addition, technical advances in flow cytometric instrumentation have allowed investigators to perform more complex experiments requiring the acquisition of an increasing number of parameters. Correlation of the multiparametric data obtained from these studies significantly increases the accuracy in identifying and defining selected subpopulations in a hetergeneous cell suspension. On the other hand, multicolor immunofluorescence may also be advantageous when cell numbers are limited. Typically, peripheral blood or lymphoid tissues provide a high cell number with which multiparametric determinations can be obtained easily when stained for two-, three-, four-, or five-color analysis. However, extensive flow cytometric analysis of samples with relatively few cells (i.e., fine needle aspirates, lung lavages) would necessitate simultaneous multiparametric determinations consisting of five- and possibly six-color immunofluorescence.