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01.12.2015 | Research article | Ausgabe 1/2015 Open Access

BMC Pulmonary Medicine 1/2015

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

BMC Pulmonary Medicine > Ausgabe 1/2015
Bérengère Obstoy, Mathieu Salaun, Liana Veresezan, Richard Sesboüé, Pierre Bohn, François-Xavier Boland, Luc Thiberville
Wichtige Hinweise

Competing interests

LT received a grant research from Mauna Kea Technologies (€1000); was invited as speaker at the International Conference on Cellvzio® Users in 2010 and 2011 (no fee) by Mauna Kea Technologies; and travel to the ACCP congress was funded by Mauna Kea Technologies in 2007. LT is listed as co-author on a patent related to pCLE, shared between Rouen University and Mauna Kea Technologies.
MS was invited to attend the International Conference on Cellvizio® Users by Mauna Kea Technologies in 2011; has been member of a Medical Advisory Board for Mauna Kea Tech. (€750). None of the authors has a financial relationship with a commercial entity that has an interest with the subject of this manuscript.

Authors’ contributions

The authors contributed to this work as follows: BO, LT, and MS carried out the conception and design of the study, the acquisition, analysis and interpretation of the microimaging data as well as drafted the manuscript. BO and RS carried out the genotoxicity assessment. BO and LV participated in the histological process and assessment of the cheek pouch lesions. BO and PB participated in the animal and cellular experiments, and in drafting the manuscript. F-XB participated in setting up the animal model. All authors read and approved the final manuscript.



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.


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 488 nm, 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.


No genotoxicity was found using 0.01% (w/v) MB after illumination at 660 nm for 2 and 15 min (5 mW). Acriflavine exposure (0.025%) led to DNA damages, increasing from 2 to 15 min of light exposure at 448 nm 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 13.9 μm) from hyperplasia to carcinoma (p < 0.05). In this model, a cut-off diameter of 30 μm enabled the diagnosis of high-grade lesions with a sensitivity of 94.7% and a specificity of 97%.


Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.
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