Identification of nasal eosinophils using two-photon excited fluorescence

Background: Eosinophils trigger symptoms in allergic rhinitis. New diagnostic methods for identifying nasal eosinophils based on autofluorescence of tlavin adenine dinucleotide in eosinophil granules could offer rapid monitoring without fixation or staining. Two-photon excitation is a powerful method for detecting this intrinsic fluorescence. Objectives: To demonstrate the use of 2-photon excited fluorescence (TPEF) to detect eosinophils from nasal mucosa in a proof-of-concept study for a future miniature in vivo imaging instrument. Methods: Thirty subjects with rhinitis were recruited. Results of our standard environmental panel were recorded. Fluorescence images were collected from nasal cytology smears with a 2-photon microscope. Cells were evaluated for intensity and size, and compared with Hansel stains. Correlation of cell count was made by linear regression, diagnostic performance was evaluated at various intensity thresholds, and correlation of nasal eosinophil count to allergic status was done through the Wilcoxon rank-sum test. Results: The fluorescence intensity of eosinophils compared with epithelial cells was 13.8 +/- 4.3 versus 3.7 +/- 1.8 (P < .01), and the size was 27.0 +/- 10.2 versus 392.0 +/- 214.6 mu m(2) (P < .01), respectively. Using both fluorescence intensity and size, a total accuracy of 100% is achieved. Eosinophil count on TPEF correlates with Hansel stain, R-2 = 0.91. Nasal eosinophil count correlates with allergic status on both TPEF (P = .008) and Hansel stain images (P = .027). Conclusions: TPEF is a promising novel technique for identifying and quantifying nasal eosinophils on nasal cytology specimens without the need for fixation or staining. Future development of a rhinoscope-compatible 2-photon microscope could be used as a clinical adjunct for the diagnosis and management of rhinitis patients in vivo. Ann Allergy Asthma Immunol. 2011;106:394-400.