Discrimination of spectrally overlapping fluorochromes using Sony Corporation’s (SP6800) Spectral Analyzer

A maximum of seven colors can be separated using traditional 2-laser flow cytometers and it is required to have the necessary band pass filters to separate these fluorochromes. In addition, these systems cannot differentiate between spectrally overlapping fluorophores such as PerCP and PerCP/Cy5.5; or FITC, PE and Alexa Fluor 514® (A514). SP6800, a novel spectral analyzer from Sony Corporation, uses an innovative optics system that collects all emitted light and eliminates the need for band pass filters. We performed whole blood staining using a panel of 7-10 markers, which included significantly overlapping fluorochromes PerCP and PerCP/Cy5.5 and A514, PE and FITC and analyzed it on the SP6800. We were able to clearly identify CD56+ (PerCP/Cy5.5), CD19+ (PerCP) and CD3+ (A514) cells. Further comparison of a 7-color panel consisting of CD3 FITC, CD56 PE, CD19 PerCP, CD8 PE/Cy7, CD16 APC, CD4 Alexa Fluor 700® and CD62L APC/Cy7 on the BD LSRFortessa™ system and SP6800 gave similar results. Our data show that the SP6800 can perform all the analysis similar to a traditional cytometer with the added feature of separating spectrally overlapping fluorophores.