Diagnosis Of The Chronic Lymphocytic Leukemia (CLL) Using A Raman-Based Scanner Optimized For Blood Smear Analysis (M3s Project)

Introduction/ Background In hematology, actual diagnosis of B chronic lymphocyte-leukemia (CLL) is based on the microscopic analysis of cell morphology from patient blood smear. However, new photonic technologies appear promising to facilitate and improve the early diagnosis, prognostic and monitoring of personalized therapy. The development of automated diagnostic approaches could assist clinicians in improving the efficiency and quality of health services, but also reduce medical costs. Aims The M3S project aims at improving the diagnosis and prognosis of the CLL pathology by developing a multimodal microscopy platform, including Raman spectrometry, dedicated to the automatic analysis of lymphocytes. Methods Blood smears were prepared on glass slides commonly used in pathology laboratories for microscopy. Two types of sample per patient were prepared: a conventional blood smear and a deposit of “pure” lymphocyte subtypes (i.e. normal B, CLL B, T and NK), sorted out in flow cytometry by using the negative double labeling technique. The second sample is used for the construction of a database of spectral markers specific of these different cell types. The preparations were analyzed with the multimodal machine which combines i) a Raman micro-spectrometer, equipped with a 532nm diode laser excitation source; ii) a microscope equipped with 40x and 150x lenses and a high precision xyz motorized stage for scanning the blood smear, and localizing x-y coordinates of representative series (~100 for each patient) of lymphocyte cells before registering three Raman spectra; these cells of interest being previously localized by an original method based on the morphology analysis. After the Raman acquisitions, the conventional blood smears were submitted to immunolabelling using specific antibodies. For the establishment of the Raman classifiers, this post-acquisition treatment was used as reference to distinguish the different lymphocyte sub-populations. Raman data were then analyzed using chemometric processing and supervised statistical classifiers in order to construct a spectral library of markers highly specific of the lymphocyte type and status (normal or pathological). Results Currently, a total of 60 patients (CLL and healthy) were included in the study. Various classification methods such as LDA (Linear Discriminant Analysis), PLS-DA (Partial Least Square Discriminant Analysis), RF (Random Forest) and SVM (Support Vector Machine), were tested in the purpose to distinguish tumoral B lymphocytes from other cell types. These classification algorithms were combined with feature selection approaches. The best performances were around 70% of correct identification when a three-class model (B-CLL vs B-normal vs T and NK lymphocytes) was considered, and 80% in case of a two-class model (B-CLL vs B-normal lymphocytes). These encouraging results demonstrate the potential of Raman micro-spectroscopy coupled to supervised classification algorithms for leukemic cell classification. The approach can find interest more generally in the field of cyto-hematology. Further developments will concern the integration of additional modality such as Quantitative Phase Imaging on one hand to speed the exploration process of cells of interest to be probed, and on the other hand to extract additional characteristics likely to be informative for CLL diagnosis. In addition, the identification of prognostic markers will be investigated by confronting the photonic data to clinical patient information.

Guiding Brain Tumor Resection Using Surface-Enhanced Raman Scattering Nanoparticles and a Hand-Held Raman Scanner

The current difficulty in visualizing the true extent of malignant brain tumors during surgical resection represents one of the major reasons for the poor prognosis of brain tumor patients. Here, we evaluated the ability of a hand-held Raman scanner, guided by surface-enhanced Raman scattering (SERS) nanoparticles, to identify the microscopic tumor extent in a genetically engineered RCAS/tv-a glioblastoma mouse model. In a simulated intraoperative scenario, we tested both a static Raman imaging device and a mobile, hand-held Raman scanner. We show that SERS image-guided resection is more accurate than resection using white light visualization alone. Both methods complemented each other, and correlation with histology showed that SERS nanoparticles accurately outlined the extent of the tumors. Importantly, the hand-held Raman probe not only allowed near real-time scanning, but also detected additional microscopic foci of cancer in the resection bed that were not seen on static SERS images and would otherwise have been missed. This technology has a strong potential for clinical translation because it uses inert gold-silica SERS nanoparticles and a hand-held Raman scanner that can guide brain tumor resection in the operating room

Discrimination between two different grades of human glioma based on blood vessel infrared spectral imaging

Gliomas are brain tumours classified into four grades with increasing malignancy from I to IV. The development and the progression of malignant glioma largely depend on the tumour vascularization. Due to their tissue heterogeneity, glioma cases can be difficult to classify into a specific grade using the gold standard of histological observation, hence the need to base classification on a quantitative and reliable analytical method for accurately grading the disease. Previous works focused specifically on vascularization study by Fourier transform infrared (FTIR) spectroscopy, proving this method to be a way forward to detect biochemical changes in the tumour tissue not detectable by visual techniques. In this project, we employed FTIR imaging using a focal plane array (FPA) detector and globar source to analyse large areas of glioma tumour tissue sections via molecular fingerprinting in view of helping to define markers of the tumour grade. Unsupervised multivariate analysis (hierarchical cluster analysis and principal component analysis) of blood vessel spectral data, retrieved from the FPA images, revealed the fine structure of the borderline between two areas identified by a pathologist as grades III and IV. Spectroscopic indicators are found capable of discriminating different areas in the tumour tissue and are proposed as biomolecular markers for potential future use of grading gliomas. Graphical Abstract Infrared imaging of glioma blood vessels provides a means to revise the pathologists' line of demarcation separating grade III (GIII) from grade IV (GIV) parts

Surface-enhanced Raman scattering and fluorescence spectroscopy reveal molecular interactions of all-trans retinoic acid and RAR gamma ligand-binding domain

International audienceSurface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the beta-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers

Biocompatibility of sol-gel hydroxyapatite-titania composite and bilayer coatings

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Biocompatibility of sol-gel hydroxyapatite-titania composite and bilayer coatings

International audienceTitania-Hydroxyapatite (TiO2/HAP) reinforced coatings are proposed to enhance the bioactivity and corrosion resistance of 316L stainless steel (316L SS). Herein, spin- and dip-coating sol-gel processes were investigated to construct two kinds of coatings: TiO2/HAP composite and TiO2/HAP bilayer. Physicochemical characterization highlighted the bioactivity response of the TiO2/HAP composite once incubated in physiological conditions for 7 days whereas the TiO2/HAP bilayer showed instability and dissolution. Biological analysis revealed a failure in human stem cells adhesion on TiO2/HAP bilayer whereas on TiO2/HAP composite the presence of polygonal shaped cells, possessing good behaviour attested a good biocompatibility of the composite coating. Finally, TiO2/HAP composite with hardness up to 0.6 GPa and elastic modulus up to 18 GPa, showed an increased corrosion resistance of 316L SS. In conclusion, the user-friendly sol-gel processes led to bioactive TiO2/HAP composite buildup suitable for biomedical applications