Objective To evaluate the potential of autofluorescence spectroscopy as a real-time intraoperative tool for cholesteatoma detection and differentiation from surrounding non-cholesteatoma middle-ear tissues.

Study Design Prospective ex vivo study.

Setting Besançon University Hospital, France (tertiary care center).

Methods Autofluorescence spectral scans were obtained from middle ear biopsies following 405-nm laser excitation. Histopathologic analysis confirmed tissue classification. Spectral data were analyzed using principal component analysis and classified using a quadratic discriminant analysis model.

Results In a cohort of 23 patients (36 tissue samples, 3,787 fluorescence spectra), the classification model achieved an accuracy of 94.5%, with a sensitivity of 94.7% and a specificity of 94.2%. All biopsies were correctly classified as cholesteatoma or non-cholesteatoma.

Conclusion Autofluorescence spectroscopy demonstrated high diagnostic accuracy for cholesteatoma detection. The simplicity and real-time applicability of the method suggest its potential for intraoperative integration, aiming to enhance surgical precision and reduce recurrence rates. Future in vivo validation will be necessary to assess its feasibility in clinical practice.

The authors have declared no competing interest.

This work was supported by Agence Nationale de la Recherche (ANR-17-CE19-0005).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This prospective ex vivo study, "CONCERTO", was deemed to fall outside the scope of the Jardé Law and does not meet the definition of research involving human participants as specified in Article L1122-1 of the French Public Health Code. As such, approval from an ethics committee was not required. The study was registered by the University Hospital of Besançon (CHU de Besançon). Data collection and processing were carried out by a team from the CHU de Besançon in compliance with the European General Data Protection Regulation (GDPR) and in accordance with the guidelines of the French Data Protection Authority (CNIL). All patient data were fully anonymized prior to analysis. The Director General of the CHU of Besançon certifies the guarantee of compliance with regulations regarding storage of data.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Competing interest disclosures: None reported.

Funding source: This work was supported by Agence Nationale de la Recherche (ANR-17-CE19-0005).

Role of the funder/sponsor: The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

All data produced in the present study are available upon reasonable request to the authors