Bruno Wacogne

Objective: To evaluate whether autofluorescence spectroscopy (AFS) can reliably distinguish cholesteatoma from surrounding middle‐ear tissues, and to develop a real‐time intraoperative diagnostic tool. Study Design: Prospective ex vivo study. Setting: Besançon University Hospital, France (tertiary care center). Methods: In this prospective ex vivo study, middle‐ear tissue biopsies were collected during cholesteatoma surgeries and analyzed using autofluorescence following 405‐nm laser excitation. Each sample was classified based on its spectral signature and confirmed by histopathology. A computer‐assisted model was applied to differentiate cholesteatoma from noncholesteatoma tissues, and its performance was assessed using standard cross‐validation. Results: Thirty‐six tissue samples from 23 patients were analyzed, generating nearly 3800 fluorescence spectra. The model correctly classified all samples, achieving 94.5% accuracy, 94.7% sensitivity, and 94.2% specificity. The system provided consistent performance across tissue types and returned results in real time, with a response time of 0.1 seconds per sample. Each output included a probability score indicating the likelihood of cholesteatoma presence. Conclusion: AFS demonstrated high accuracy in distinguishing cholesteatoma from other middle‐ear tissues. Its speed, simplicity, and probabilistic feedback suggest strong potential for real‐time intraoperative use. Once validated in vivo, this technology could assist surgeons by improving tissue discrimination and potentially reducing recurrence rates.

Advanced therapy medicinal products (ATMPs), especially effective against cancer, remain costly due to their reliance on genetically modified T cells. Contamination during production is a major concern, as traditional quality control methods involve samplings, which can themselves introduce contaminants. It is therefore necessary to develop methods for detecting contamination without sampling and, if possible, in real time. In this article, we present a white light spectroscopy method that makes this possible. It is based on shape analysis of the absorption spectrum, which evolves from an approximately Gaussian shape to a shape modified by the 1/λ component of bacterial absorption spectra when contamination develops. A warning value based on this shape descriptor is proposed. It is demonstrated that a few hours are sufficient to detect contamination and trigger an alarm to quickly stop the production. This time-saving should reduce the cost of these new drugs, making them accessible to as many people as possible. This method can be used regardless of the type of contaminants, provided that the shape of their absorption spectrum is sufficiently different from that of pure T cells so that the shape descriptor is efficient.

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 production of advanced therapy medicinal products (ATMP) is a long and highly technical process, resulting in a high cost per dose, which reduces the number of eligible patients. There is a critical need for a closed and sample-free monitoring system to perform the numerous quality controls required. Current monitoring methods are not optimal, mainly because they require the system to be opened up for sampling and result in material losses. White light spectroscopy has emerged as a technique for sample-free control compatible with closed systems. We have recently proposed its use to monitor cultures of CEM-C1 cell lines. In this paper, we apply this method to T-cells isolated from healthy donor blood samples. The main differences between cell lines and human primary T-cells lie in the slightly different shape of their absorption spectra and in the dynamics of cell expansion. T-cells do not multiply exponentially, resulting in a non-constant generation time. Cell expansion is described by a power-law model, which allows for the definition of instantaneous generation times. A correlation between the linear asymptotic behavior of these generation times and the initial cell concentration leads to the hypothesis that this could be an early predictive marker of the final culture concentration. To the best of our knowledge, this is the first time that such concepts have been proposed.

Optical density measurement has been used for decades to determine the microorganism concentration and more rarely for mammalian cells. Although this measurement can be carried out at any wavelength, studies report a limited number of measurement wavelengths, mainly around 600 nm, and no consensus seems to be emerging to propose an objective method for determining the optimum measurement wavelength for each microorganism. In this article, we propose a method for analyzing the absorbance spectra of ESKAPEE bacteria and determining the optimum measurement wavelength for each of them. The method is based on the analysis of the signal-to-noise ratio of the relationships between concentrations and optical densities when the measurement wavelength varies over the entire spectral range of the absorbance spectra measured for each bacterium. These optimum wavelengths range from 612 nm for Enterococcus faecium to 705 nm for Acinetobacter baumannii. The method can be directly applied to any bacteria, any culture method, and also to any biochemical substance with an absorbance spectrum without any particular feature such as an identified maximum.

Fluorescence pH sensing has proven to be efficient but with the drawback that molecules photobleach, requiring frequent calibrations. Double-emission peak molecules allow ratiometric measurements and theoretically avoid calibration. However, they are often expensive and fragile and usually have very low quantum yields. Single emission peaks such as fluorescein and derivatives are inexpensive and have very high quantum yields. Because they are single emission peaks, the pH is assumed to be derived from the ratio of emitted intensities at measured pH and at high pH values, i.e., they require frequent calibration. However, the shape of their single emitted peak evolves slightly with pH. In this paper, we first demonstrate a simple method to calculate the emission spectrum shape of each prototropic form of fluorescein (and derivatives) as well as the values of the pKa_s. A complete model of the evolution of the emission spectrum shape with pH is then constructed. Second, we evaluate the potential of these molecules for pH sensing by fitting the experimental spectra with the complete emission model. The method is applied to fluorescein, FITC and FAM. Depending on the molecule, pH can be measured from pH 1.9 to pH 7.3 with standard deviations between 0.06 and 0.08 pH units. Estimating pH and pKas from shape instead of intensity allows calibration-free measurements even with single-emission peak molecules.

Despite numerous innovations, measuring bacteria concentrations on a routine basis is still time consuming and ensuring accurate measurements requires careful handling. Furthermore, it often requires sampling small volumes of bacteria suspensions which might be poorly representative of the real bacteria concentration. In this paper, we propose a spectroscopy measurement method based on a description of the absorption/attenuation spectra of ESKAPEE bacteria. Concentrations were measured with accuracies less than 2%. In addition, mixing the mathematical description of the absorption/attenuation spectra of mammalian T-cells and bacteria allows for the simultaneous measurements of both species’ concentrations. This method allows real-time, sampling-free and seeder-free measurement and can be easily integrated into a closed-system environment.

Advanced Therapy Medicinal Products are promising drugs for patients in therapeutic impasses. Their complex fabrication process implies regular quality controls to monitor cell concentration. Among the different methods available, optical techniques offer several advantages. Our study aims to measure cell concentration in real time in a potential closed-loop environment using white light spectroscopy and to test the possibility of simultaneously measuring concentrations of several species. By analyzing the shapes of the absorption spectra, this system allowed the quantification of T-cells with an accuracy of about 3% during 30 h of cultivation monitoring and 26 h of doubling time, coherent with what is expected for normal cell culture. Moreover, our system permitted concentration measurements for two species in reconstructed co-cultures of T-cells and Candida albicans yeasts. This method can now be applied to any single or co-culture, it allows real-time monitoring, and can be easily integrated into a closed system.

Based on the modification of biological substances, either directly from the patient to be treated or from healthy donors, Advanced Therapy Medicinal Products (ATMPs) are promising therapeutic solutions (among others, CAR T-cells represent the current and best-known ATMPs). However, at a few hundred thousand USD per dose, these products suffer from their prohibitive price, limiting the number of treated patients. The reasons for this can be summarized as follows. The whole process takes place in sterile facilities and requires trained staff (as well as onerous equipment) who perform sampling for the numerous rounds of quality control performed throughout production. Production can last more than one week, and each round of sampling further increases the risk of contamination and the need to fight against them. Therefore, there exists a need for online control of the content of the bioreactor during production, especially focused on concentration measurements and quality assessments. Meanwhile, the real-time detection of possible contamination would allow production to be stopped as soon as a problem arises, hence saving days of useless cultures and reducing the cost of global production. In this conference, we present simple and real-time white-light spectroscopy to simultaneously monitor T-cell growth, estimate the production quality and detect contamination. The mathematical description of the absorption spectra shape allows us to achieve these goals. The contactless nature of white-light spectroscopy prevents sampling of the bioreactor’s content, and consequently, reduces the risk of concomitant contamination. The possible integration of such online methods in bioreactors is also proposed with the ulterior motive of democratizing the use of ATMPs for as many people as possible.