title: 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 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.
  
  
  RESULTS
  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%.
  
  
  CONCLUSIONS
  Methylene blue can be used safely to image precancerous lesions in vivo. This study does not support the use of acriflavine in humans.
authors.affiliation: Quant.I.F Litis EA 4108, IRIB, Rouen University, Rouen, F-76000, France. berengere.obstoy@chu-rouen.fr.
authors.forename: Bérengère
authors.initials: B
authors.lastname: Obstoy
authors.affiliation: Quant.I.F Litis EA 4108, IRIB, Rouen University, Rouen, F-76000, France. mathieu.salaun@univ-rouen.fr.|Clinique Pneumologique and CIC INSERM U1404, IRIB, Rouen University Hospital, Rouen, F-76031, France. mathieu.salaun@univ-rouen.fr.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Salaun
authors.affiliation: Department of Pathology, H. Becquerel Cancer Center, Rouen, France. liana.veresezan@chb.unicancer.fr.
authors.forename: Liana
authors.initials: L
authors.lastname: Veresezan
authors.affiliation: INSERM U-1079, IRIB, Rouen University, Rouen, F-76000, France. richard.sesboue@univ-rouen.fr.
authors.forename: Richard
authors.initials: R
authors.lastname: Sesboüé
authors.affiliation: Quant.I.F Litis EA 4108, IRIB, Rouen University, Rouen, F-76000, France. pierre.bohn@chb.unicancer.fr.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bohn
authors.affiliation: Laboratory of Experimental Surgery, EA 3830 GRHV, IRIB, Rouen University, Rouen, F-76000, France. fboland@ch-dieppe.fr.
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Boland
authors.affiliation: Quant.I.F Litis EA 4108, IRIB, Rouen University, Rouen, F-76000, France. luc.thiberville@univ-rouen.fr.|Clinique Pneumologique and CIC INSERM U1404, IRIB, Rouen University Hospital, Rouen, F-76031, France. luc.thiberville@univ-rouen.fr.
authors.forename: Luc
authors.initials: L
authors.lastname: Thiberville
mesh_terms: D000167:Acriflavine / Q000494:pharmacology; D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000473:pathology*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D020552:Comet Assay; D004847:Epithelial Cells / Q000187:drug effects; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D000069416:Intravital Microscopy; D008168:Lung / Q000166:cytology; D008214:Lymphocytes / Q000187:drug effects; D008647:Mesocricetus; D008751:Methylene Blue / Q000494:pharmacology; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009061:Mouth Mucosa / Q000473:pathology*; D009062:Mouth Neoplasms / Q000473:pathology*; D009152:Mutagenicity Tests; D061848:Optical Imaging / Q000379:methods*; D011230:Precancerous Conditions / Q000473:pathology*
chemical_list: D005456:Fluorescent Dyes; D000167:Acriflavine; D008751:Methylene Blue