Implementation of a classification strategy of Raman data collected in different clinical conditions: application to the diagnosis of chronic lymphocytic leukemia.

The literature is rich in proof of concept studies demonstrating the potential of Raman spectroscopy for disease diagnosis. However, few studies are conducted in a clinical context to demonstrate its applicability in current clinical practice and workflow. Indeed, this translational research remains far from the patient's bedside for several reasons. First, samples are often cultured cell lines. Second, they are prepared on non-standard substrates for clinical routine. Third, a unique supervised classification model is usually constructed using inadequate cross-validation strategy. Finally, the implemented models maximize classification accuracy without taking into account the clinician's needs. In this paper, we address these issues through a diagnosis problem in real clinical conditions, i.e., the diagnosis of chronic lymphocytic leukemia from fresh unstained blood smears spread on glass slides. From Raman data acquired in different experimental conditions, a repeated double cross-validation strategy was combined with different cross-validation approaches, a consensus label strategy and adaptive thresholds able to adapt to the clinician's needs. Combined with validation at the patient level, classification results were improved compared to traditional strategies.

Conformation changes in human hair keratin observed using confocal Raman spectroscopy after active ingredient application.

OBJECTIVE: In hair care cosmetic products' evaluation, one commonly used method is to evaluate the hair appearance as a gold standard in order to determine the effect of an active ingredient on the final state of the hair via visual appreciation. Although other techniques have been proposed for a direct analysis of the hair fibres, they give only surface or structural information, without any accurate molecular information. A different approach based on confocal Raman spectroscopy has been proposed for tracking in situ the molecular change in the keratin directly in the human hair fibres. It presents a high molecular specificity to detect chemical interactions between molecules and can provide molecular information at various depths at the cortex and cuticle levels. METHODS: To evaluate the potential of confocal Raman spectroscopy in testing the efficiency of cosmetic ingredients on keratin structure, we undertook a pilot study on the effectiveness of a smoothing shampoo on natural human hair, by analysing α-helix and ß-sheet spectral markers in the Amide I band and spectral markers specific to the cystin sulfur content. RESULTS: We confirmed that an active proved to be effective on a gold standard decreases α-helix keratin conformation and promotes ß-sheet keratin conformation in the hair fibres. We also showed that treatment with the effective active decreases the intensity of covalent disulfide (S-S at 510 cm-1 ) cross-linking bands of cysteine. These data confirm that the effective active also acts on the tertiary structure of keratin. CONCLUSION: From these experiments, we concluded that the effective active has a smoothing effect on the human hair fibres by acting on α-helix and ß-sheet keratin conformation and on the tertiary structure of keratin. Based on these results, confocal Raman spectroscopy can be considered a powerful technique for investigating the influence of hair cosmetic ingredients on keratin structure in human hair fibres. Moreover, this analytical technique has the advantage of being non-destructive and label free; in addition, it does not require sample extraction or purification and it can be applied routinely in cosmetic laboratories.

OBJECTIF: Dans l'évaluation des produits cosmétiques pour le soin des cheveux, une méthode communément utilisée consiste à évaluer l'aspect des cheveux afin de déterminer l'effet d'un principe actif sur l'état final des cheveux via l'appréciation visuelle. Bien que d'autres techniques ont été proposées pour une analyse directe de la fibre capillaire, elles ne donnent que des informations de surface ou de structure, sans aucune information moléculaire précise. Une approche différente par la spectroscopie confocale Raman a été proposée pour le suivi in situ du changement moléculaire de la kératine directement dans les fibres de cheveux humains. Il présente une grande spécificité moléculaire, détecter les interactions chimiques entre les molécules et peut fournir des informations moléculaires à différents niveaux de profondeur du cortex et de la cuticule. MÉTHODES: Afin d'évaluer le potentiel de la spectroscopie Raman confocale pour tester l'efficacité des ingrédients cosmétiques sur la structure de la kératine, nous avons entrepris une étude pilote sur l'efficacité d'un shampooing lissant sur cheveux naturels, en analysant les marqueurs spectraux de l'hélice α et du feuillet ß dans la bande Amide I et les marqueurs spectraux spécifiques au contenu en sulfure-cystine. RÉSULTATS: Nous avons confirmé qu'un actif s'avérant efficace sur un gold standard diminue la conformation de la kératine en hélice α et favorise la conformation de la kératine en feuillet ß dans les fibres des cheveux. Nous avons également montré que le traitement avec l'actif efficace diminue l'intensité des bandes de cystéine réticulant sous forme de ponts disulfures covalents (S - S à 510 cm-1). Ces données confirment que l'actif efficace agit également sur la structure tertiaire de la kératine. CONCLUSION: À partir de ces expériences, nous avons conclu que l'actif efficace a un effet lissant sur les fibres du cheveu humain en agissant sur la conformation hélice α et feuillet ß de la kératine et sur la structure tertiaire de la kératine. Sur la base de ces résultats, la spectroscopie confocale Raman peut être considérée comme une technique puissante pour étudier l'influence des ingrédients cosmétiques sur la structure de la kératine dans les fibres de cheveux. De plus, cette technique analytique a l'avantage d'être non destructive et ne nécessite pas de marquage; de plus, elle ne nécessite pas d'extraction ou de purification des échantillons et peut être appliquée en routine dans les laboratoires de cosmétiques.

Human skin penetration of hyaluronic acid of different molecular weights as probed by Raman spectroscopy.

BACKGROUND: Topical delivery of molecules into the human skin is one of the main issues in dermatology and cosmetology. Several techniques were developed to study molecules penetration into the human skin. Although widely accepted, the conventional methods such as Franz diffusion cells are unable to provide the accurate localization of actives in the skin layers. A different approach based on Raman spectroscopy has been proposed to follow-up the permeation of actives. It presents a high molecular specificity to distinguish exogenous molecules from skin constituents. METHODS: Raman micro-imaging was applied to monitor the skin penetration of hyaluronic acids (HA) of different molecular weights. The first step, was the spectral characterization of these HA. After, we have determined spectral features of HA by which they can be detected in the skin. In the second part, transverse skin sections were realized and spectral images were recorded. RESULTS: Our results show a difference of skin permeation of the three HA. Indeed, HA with low molecular weight (20-300 kDa) passes through the stratum corneum in contrast of the impermeability of high molecular weight HA (1000-1400 kDa). CONCLUSION: Raman spectroscopy represents an analytical, non-destructive, and dynamic method to evaluate the permeation of actives in the skin layers.

Diagnosis of hepatocellular carcinoma in cirrhotic patients: a proof-of-concept study using serum micro-Raman spectroscopy.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. The development of novel diagnostic methods is needed to detect tumours at an early stage when patients are eligible for curative treatments. The purpose of this proof-of-concept study was to determine if micro-Raman spectroscopy applied to the sera of cirrhotic patients may be an alternative method for rapidly discriminating patients with and without HCC. Serum samples were collected from 2 groups of patients: cirrhotic patients with HCC (n = 37) and without HCC (n = 34). Two different approaches were used, dried serum drops and freeze-dried serum, and micro-Raman spectra were acquired in the point-mode with a 785 nm laser excitation in the spectral range of 600-1800 cm(-1). Spectra were quality-tested and pre-processed (smoothing, baseline subtraction, vector normalization). Using principal component analysis, the 2 classes, corresponding to cirrhotic patients with and without HCC, could not be differentiated. In contrast, the support vector machine method using the leave-one-out cross validation procedure was able to correctly classify the two groups of patients with an overall rate of accuracy of 84.5% to 90.2% for dried serum drops and 86% to 91.5% for freeze-dried serum. These results are promising and support the concept that serum micro-Raman spectroscopy may become a useful diagnostic tool to detect biomarkers in the field of cancer, as described here for distinguishing between cirrhotic patients with and without HCC.