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.

Contribution of Raman spectroscopy in nephrology: a candidate technique to detect hydroxyethyl starch of third generation in osmotic renal lesions.

BACKGROUND AND OBJECTIVES: HydroxyEthyl Starch (HES) has been one of the most commonly used colloid volume expanders in intensive care units for over 50 years. The first and second generation HES, with a high molecular weight (≥200 kD) and a high degree of substitution (≥0.5), has been associated with both renal dysfunction and osmotic nephrosis-like lesions in histological studies. Recently, third generation HES (130 kD/<0.5) has also been shown to impair renal function in critically ill adult patients although tubular accumulation of HES has never been proven in the human kidney. Our objective was to demonstrate the potential of Raman micro-imaging to bring out the presence of third generation-HES in the kidney of patients having received the volume expander. DESIGN: Four biopsies presenting osmotic nephrosis-like lesions originated from HES-administrated patients with impaired renal function were compared with HES-negative biopsies (n = 10) by Raman microspectroscopy. RESULTS: The first step was dedicated to the identification of a specific vibration of HES permitting the detection of the cellular and tissue accumulation of the product. This specific vibration at 480 cm(-1) is assigned to a collective mode of the macromolecule; it is located in a spectral region with a limited contribution from biological materials. Based on this finding, HES distribution within tissue sections was investigated using Raman micro-imaging. Determination of HES positive pixels permitted us to clearly distinguish positive cases from HES-free biopsies (proportions of positive pixels from the total number of pixels: 23.48% ± 28 vs. 0.87% ± 1.2; p = 0.004). CONCLUSIONS: This study shows that Raman spectroscopy is a candidate technique to detect HES in kidney tissue samples currently manipulated in nephrology departments. In addition, on the clinical aspect, our approach suggests that renal impairment related to third generation HES administration is associated with osmotic nephrosis-like lesions and HES accumulation in the kidney.

Histopathological characterization of primary cutaneous melanoma using infrared microimaging: a proof-of-concept study.

BACKGROUND: The diagnosis of malignant melanoma is based upon the histological evaluation of the lesion. As such, the morphological interpretation relies on the expertise of a dermatopathologist. Infrared microimaging is emerging as a new powerful tool to investigate tissue biochemistry. Infrared spectra probe the biochemical constitution of the sample and are real tissue-specific spectroscopic fingerprints. OBJECTIVES: To assess the potential of infrared microimaging to aid in the analysis of tissue sections from primary cutaneous melanomas. METHODS: Ten samples of melanoma sections from the main histological subtypes were investigated using infrared microimaging combined with multivariate statistical analyses. RESULTS: This methodology yielded highly contrasted colour-coded images that permitted to highlight tissue architecture without any staining. It was possible to discriminate tumour areas from normal epidermis automatically, and intratumoral heterogeneity as revealed by our approach was correlated with the aggressiveness of the tumour. CONCLUSIONS: This proof-of-concept study shows that infrared microimaging could help in the diagnosis of primary cutaneous melanoma.

Changes in adsorption and permeability of mitoxantrone on plasma membrane of BCRP/MXR resistant cells.

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191 pmol MTX/10(6) HCT-116 R incubated with or without BA). At 4 degrees C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42 pmol MTX/10(6) cells), restored with fluidizers (e.g., 342 pmol MTX/10(6) cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation.

Effects of a selective A1-adenosine receptor agonist on heart rate and heart rate variability during permanent atrial fibrillation.

BACKGROUND: Mean heart rate and irregularity of the rate, i.e., heart rate variability (HRV), are two aspects of heart rate during atrial fibrillation (AF). An important goal of AF therapy is to control mean heart rate during exercise; the determinants of HRV during AF remain poorly known although its prognostic value has been established. OBJECTIVES: To investigate the effects of a stable, long-acting, selective A1-adenosine receptor agonist, SDZ WAG994, on heart rate during exercise and on HRV. METHODS: In a multicenter, double-blind, randomized, placebo-controlled, parallel group study, patients with permanent AF performed a symptom-limited exercise test and underwent 24-hour ECG monitoring on day 1 during treatment with placebo, and on day 2 during treatment with either placebo or 2 mg SDZWAG994 orally. Changes in mean heart rate during exercise and changes in HRV indices between day 1 and day 2 were compared between the two groups. RESULTS: Thirty-two patients (64 +/- 8 years; 81% male; 25% in NYHA Class II; 38% with no structural heart disease) were included in the study. During active treatments, heart rate remained unchanged at rest and increased significantly during exercise. A significant daytime increase in short-term HRV indices (DpNN50 = 4.5% P = 0.01; DrMSSD = 6% P = 0.03; DSDNN Index = 6% P = 0.02) occurred during active treatment. CONCLUSIONS: Selective A1-adenosine receptor agonism with SDZ/WAG994 limits the increase in mean heart rate during exercise in patients with AF. In addition, this agonist selectively increases short-term HRV indices, suggesting that pNN50, rMSSD, and SDNN reflect vagal influences during AF.

Electrophysiological effects of vasoactive intestinal peptide in rabbit atrium: a modulation of acetylcholine activity.

Vasoactive Intestinal Peptide (VIP) is a 28-amino acid peptide partially co-secreted with acetylcholine (Ach) in the atrial tissue. We studied the electrophysiological effects of VIP and Ach in rabbit isolated right atrium by the microelectrode technique. After a 10-min superfusion with VIP, action potential duration at 90% of repolarization (APD90) was lengthened by 23% (P = 0.01) at the concentration of 10(-8) M (n = 10), by 22% (P = 0.004) at 10(-7) M (n = 10) and by 33% (P = 0.03) at 2 x 10(-7) M (n = 5). To explain this APD90 lengthening, we performed 10 other experiments with VIP 10(-7) M, including five preparations pretreated with verapamil (10(-6) M) for 20 min. In the five preparations not pretreated, APD90 was increased by 27% (P = 0.04) after 10 min but remained unchanged in those previously exposed to verapamil, suggesting that VIP is a calcium current activator. Ach (1.4 x 10(-5) M) was superfused in five other experiments and we observed a 31% decrease in APD90 (P= 0.04) at 10 min. After washout, we simultaneously perfused, on the same preparations, Ach (same concentration) and VIP (10(-7) M) for 10 min. The decrease in APD90 (19%) was no longer significant. VIP (2 x 10(-7) M) lengthened cellular effective refractory periods (ERP) by 26% (P = 0.04) after 10 min (n = 5), whereas Ach (1.4 x 10(-5) M) decreased ERP by 33% (P = 0.04) at 10 min (n = 5). In conclusion, VIP lengthens atrial APD90, which may be the result of calcium current activation. In addition, VIP could modulate Ach activity in limiting APD90 shortening in the presence of Ach and because of its opposite effect on atrial ERP. Therefore, VIP could be involved in the control of vagal atrial arrhythmias.

Initial and long-term evaluation of escape rhythm after radiofrequency ablation of the AV junction in 50 patients.

Between 1986 and 1994, 50 patients (mean age 63 +/- 13 years), 25 of whom had organic heart disease and presenting with atrial arrhythmias refractory to 5.6 +/- 1.6 antiarrhythmic drugs, underwent radiofrequency ablation (5 +/- 3 pulses by procedure; duration of pulses 50.5 +/- 32 s) of the proximal AV junction to create complete and permanent AV block. The escape rhythm was studied immediately after the procedure and during long-term follow-up. Immediately after the procedure, an escape rhythm was observed in 80% of the patients (junctional in 92%). Over a mean follow-up of 36 +/- 16 months in 47 patients (2 patients died before assessment of escape rhythm and 1 was lost to follow-up), an escape rhythm was present in 39 patients (83%) and absent in the remaining 8 (17%). The only significant difference between the two groups was the initial presence of an escape rhythm (P = 0.008). However, three patients with an initial escape rhythm had none during long-term follow-up. The initial presence of an escape rhythm as a predictive factor of its presence during follow-up had a sensitivity of 87%, specificity of 63%, positive predictive value of 92%, and negative predictive value of 50%. Thus, the absence of an escape rhythm during long-term follow-up causing pacemaker dependency was noted in 1 of 6 patients. This represents a limitation to this palliative treatment, which should be reserved for patients suffering from supraventricular tachycardias refractory to other treatments.

[Right precordial leads and sudden death. Relation with arrhythmogenic right ventricular dysplasia]. / Dérivations en précordiales droites et mort subite. Relation avec la dysplasie ventriculaire droite arythmogène.

Non-coronary ST-segment elevation during right sided chest pain has been described in subjects with episodes of ventricular fibrillation at rest. This syndrome has been attributed to functional phenomena or to structural myocardial changes. A personal case has features belonging to two categories: ST-segment elevation observed before, during and after episodes of arrhythmia was compared to 11 previously recorded ECG recordings. Right ventricular dysplasia was shown by electrocardiography, electrophysiology and echocardiography. In addition, ST-segment elevation is classified in 3 categories: triangular and dome-shaped are the most commonly observed forms during the arrhythmias: the third form with "saddle"-shaped appearances has not been previously described and would seem to be a minor equivalent observed during intercritical periods. This form is found in 30% of clinically documented cases of arrhythmogenic right ventricular dysplasia.

Secretory non-pancreatic phopholipase A2 in severe sepsis: relation to endotoxin, cytokines and thromboxane B2.

Circulatory secretory non-pancreatic phospholipase A2 (snp-PLA2) was measured prospectively at the onset (day 0) of severe sepsis in 52 patients as well as on day 1 and 2 in 25 patients, in order to answer two questions: 1) does the snp-PLA2 plasma concentration differ according to the type and severity of infection? 2) what is the relation between snp-PLA2 and other mediators involved in severe sepsis, such as endotoxin, cytokines (TNF alpha, IL-1 beta, IL-6) and thromboxane B2 (the stable metabolite of thromboxane A2)? On day 0, the snp-PLA2 circulatory level was 78 +/- 17 nmol/min/ml in patients with severe sepsis as compared to 3.5 +/- 2 nmol/min/ml in 40 healthy volunteers. There was no statistical difference according to the outcome, the presence of shock, or the type of infection on day 0. However, snp-PLA2 remained elevated or even increased in patients who ultimately died, while it decreased in survivors (p = 0.01 by ANOVA). The cytokine profiles during the 2-day follow-up were similar to that of snp-PLA2, but the differences were not statistically significant between survivors and non-survivors. No correlation was found between snp-PLA2 and other mediators for either initial or peak values.