Use of lissamine green before conjunctival impression cytology and flow cytometry in patients with sjögren’s syndrome

Abstract

AbstractPurpose Lissamine Green (LG) is a vital dye that stains damaged epithelial cells in ocular surface disease. Conjunctival Impression Cytology (CIC) is used with flow cytometry, to examine surface epithelial cells. We were concerned that, if used prior to CIC, cell uptake of LG might affect the fluorescent signal during flow cytometry. We therefore studied whether the use of LG before CIC, could cause fluorescence interference.Methods CIC was performed with two autoclaved Supor semicircular filters applied to the lateral bulbar conjunctiva of 6 patients with Sjögren's Syndrome. LG was eluted from a standard strip with a drop of anaesthetic and delivered into the inferior fornix. Two further filters were applied to the medial bulbar conjunctiva. Cells were recovered by agitation for 1 minute, washed, stained and analysed by flow cytometry.Results When compared with samples taken before LG instillation, cells isolated from all 6 samples taken after LG instillation, and excited at 630nm, showed an increase in fluorescence at 665nm. Some increased fluorescence was also noted at 680nm but was within the limits of spectral compensation. There was no observable effect of LG when cells were excited at 488nm.Conclusion Cell uptake of LG is detected by flow cytometry, predominantly in the 665nm channel when excited at 630nm. This persists despite cell washing. Whilst this excludes the use of fluorochromes that emit within this spectrum, the use of fluorochromes excited or emitting at other wavelengths, is feasible after LG staining.