Safety and performance analysis of acriflavine and methylene blue for in vivo imaging of precancerous lesions using fibered confocal fluorescence microscopy (FCFM): an experimental study

Abstract

Background: Fibered confocal fluorescence microscopy (FCFM) allows in vivo investigation of pulmonary microstructures. However, the bronchial epithelium can only be imaged using exogenous fluorophores. The objective of this study is to compare methylene blue (MB) and acriflavine genotoxicity and to assess FCFM performance for in vivo imaging of precancerous lesions. Methods: Genotoxicity was assessed using the comet assay on both cultured human lymphocytes and NCI-H460 cells, which had been exposed to MB or acriflavine before being illuminated at 660 or 488nm, respectively. FCFM was performed on precancerous lesions in the hamster cheek pouch model, following topical application of the fluorophores. FCFM data were analyzed according to histology. Results: No genotoxicity was found using 0.01% (w/v) MB after illumination at 660nm for 2 and 15min (5mW). Acriflavine exposure (0.025%) led to DNA damages, increasing from 2 to 15min of light exposure at 448nm in both lymphocytes (83.4 to 88%, p=0.021) and NCI H460 cell populations (79.9 to 84.6%, p=0.045). In total, 11 invasive carcinoma, 24 reserve cell hyperplasia, and 17 dysplasia lesions were imaged using FCFM in vivo. With both fluorophores, the cellular density increased from hyperplasia to high-grade dysplasia (p<0.05). With MB, the cellular diameter significantly decreased (48.9 to 13.9μm) from hyperplasia to carcinoma (p<0.05). In this model, a cut-off diameter of 30μm enabled the diagnosis of high-grade lesions with a sensitivity of 94.7% and a specificity of 97%. Conclusion: Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.