Article
Confocal Laser Endomicroscopy (CLE): Quantification of different fluorescent agents for brain and spine tumor diagnosis
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Authors
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Patra Charalampaki
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Christian Eyth
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Mirwais Morgan
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Ioannis Pechlivanis
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Mehran Mahvash
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Alhadi Igressa
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Friedrich Weber
- Department of Neurosurgery, Hospital Merheim, University of Witten-Herdecke, Cologne, Germany
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Bernhard Schick
- Department of Otorhinolaryngology, Saarland University Medical Center, Homburg/Saar, Germany
| Published: | May 13, 2014 |
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Outline
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Objective: Until today the diagnosis of neoplastic brain lesions is mainly based on histopathological analysis. Standard HE as well as special stains are highly precise but time consuming in processing. Rapid cryo-sections provide fast results but are prone to failure by artefact formation. Being able to achieve real-time imaging on a histological as well as cellular level would give the surgeon a powerful tool for diagnosis and treatment with an unprecedented accuracy. The aim of this work was to investigate potential new fluorescent agents for the use with CLE to enable a more precise differentiation between tumour entities. Furthermore we investigated how an optimal application of the tested might be possible in a clinical setting.
Method: Tissue samples gained from various intra- and extracranial neoplastic lesions (n=130) were collected and either analyzed immediately or fixed with 4% formaldehyde for storage and later processing. To perform analysis a small designed confocal endomicroscope was used (excitation wavelength 488 nm, collection bandwith 500 to 650 nm). Tissue samples were examined using different fluorescent agents such as acridine orange, cresyl violet and acriflavine.
Results: Tissue processing and fixation (either in ethanol, formaldehyde or native) has no relevant effect on autofluorescense. Dilution series show an optimal dilution of 0.01 to approximately 0.5 mg/ml for acriflavine and 0.01 to approximately 0.3 mg/ml for acridine orange. Using high concentrations of fluorophores leads to overframing and subsequently to a reduced visual discrimination. Furthermore, we could proof that high level autofluoresence is not an artefact of fixation. Finally, a wide range of concentrations can be used without diminution of image quality.
Conclusions: The use of fluorescent agents in confocal laser endomicroscopy is an indispensable tool to display histomorphological features to discriminate different tumor entities. Currently acriflavine and acridine orange are the most promising dyes. For clinical application and to provide a high specifity in tumor differentiation more fluorescent agents will have to be investigated. Using different fluorescent agents performing CLE we were able to observe characteristic histopathological findings to detect and differentiate immediately after tumor removal different entities in the field of neurosurgery.
