A STRATEGY FOR MULTIPLE IMMUNOPHENOTYPING BY IMAGE CYTOMETRY - MODEL STUDIES USING LATEX MICROBEADS LABELED WITH 7 STREPTAVIDIN-BOUND FLUOROCHROMES

Multiple immunophenotyping is aimed at identifying several cell popula tions in a single labeling procedure by their ability to bind combinat ions of specific labeled antibodies, The present work demonstrates the simultaneous discrimination by using image cytometry of aminomethylco umarin acetate (AMCA), Lucifer yellow (LY), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Texas red tandem (Red613), peridinin -chlorophyll protein (PerCP), and allophycocyanin (APC), which were al l bound to latex beads as streptavidin-conjugated fluorochromes. This has been the result of a step-by-step optimization of the several fact ors affecting the sensitivity and specificity of multiple immunofluore scence analysis, First, 14 streptavidin-conjugated fluorochromes were evaluated by using spectrofluorometry. A primary selection was then ma de of ten spectrally separable dyes that could be evaluated by using i mage cytometry, These dyes were bound to latex particles, and specific filter combinations were assembled to minimize crosstalk between fluo rophores while preserving sufficient fluorescence intensity and counti ng statistics, Potential probe associations were then assessed by meas uring the emissions of all fluorochromes that were detected by each fi lter combination, The resulting crosstalk matrix served as the basic t ool both for final selection of the optimal filter combination and for dye set (composed, in this case, of the seven fluorochromes described above) and for mathematical correction of residual spectral overlap, Next, an image cytometry system was adapted to collect seven images of matched brightness with the selected combination of excitation/emissi on filters and dichroic mirrors, Finally, seven-parameter synthetic im ages were generated by digital image processing. (C) 1996 Wiley-Liss, Inc.