Fluorescence changes at flow cytometric analysis of samples from sentinel lymph nodes excised with indocyanine green and methylene blue guided mapping techniques

Introduction: Flow cytometry (FC) has been used recently to assess percentages of infiltration by mast cells in sentinel lymph nodes (SLN) of dogs with mast cell tumors (MCT). SLN mapping often includes the use of different dyes such as methylene blue (MB) and indocyanine green (ICG), which might influence fluo- rescence assessment by FC. This study aimed to assess whether the color given by mapping dyes might affect the baseline fluorescence of SLN aspirates for FC. Methods: Baseline fluorescence was calculated as Median Fluorescence Intensity (MFI) in 4 channels (FL1, FL2, FL3, FL4, respectively corresponding to 530/30 nm, 585/40 nm, >670 nm, 660/20 nm) with a cytometer equipped with two lasers (488 nm and 638 nm) with constant setting and compensation. SLN aspirates were suspended in RPMI medium. They were classified based on results of map- ping techniques in 4 dye classes (blue/fluorescent, fluorescent/non-blue, blue/ non-fluorescent, non-blue/non-fluorescent), and possible differences in MFI values among SLN dye classes were assessed. Results: Thirty-five SLNs from 17 dogs were assessed. Considering dye classes, 13 were blue/fluorescent, 16 were fluorescent/non-blue, 4 were blue/non-fluo- rescent, 2 were non-blue/non-fluorescent. In all fluorescence channels, except FL1, MFI varied among SLN dye classes. Blue/non-fluorescent SLNs showed the highest fluorescence, followed by blue/fluorescent, fluorescent/non-blue, and non-blue/non-fluorescent.Discussion: These results suggest that the use of dyes for SLN mapping may introduce a relevant bias when MFI is quantitatively assessed via FC.