Longitudinal Study of Cirrhosis Development in STAM and carbon tetrachloride Mouse Models Using Fourier Transform Infrared Spectral Imaging.

Animal models of cirrhosis are of great interest to investigate the pathological process leading to the final stage of cirrhosis. The aim of this study was to analyze the different steps involved in the progressive development of cirrhosis using Fourier transform infrared spectral histology in 2 mouse models of cirrhosis, the STAM model of metabolic cirrhosis, and the carbon tetrachloride-induced cirrhosis model. Formalin-fixed, paraffin-embedded liver samples were obtained from 3 mice at 5 time points in each model to analyze the course of hepatic lesions up to the formation of cirrhosis. For each time point, adjacent 3-µm-thick liver sections were obtained for histologic stains and spectral histology. Fourier transform infrared acquisitions of liver sections were performed at projected pixel sizes of 25 µm × 25 µm and 6.25 µm × 6.25 µm. Spectral images were then preprocessed with an extended multiplicative signal correction and analyzed with common k-means clustering, including all stages in each model. In both models, the 2- and 4-class common k-means clustering in the 1000 to 1350 cm-1 range showed that spectral classes characterized by higher absorbance peaks of glycogen were predominant at baseline, then decreased markedly in early stages of hepatic damage, and almost disappeared in cirrhotic tissues. Concomitantly, spectral classes characterized by higher absorbance peaks of nucleic acids became progressively predominant during the course of hepatic lesions. These results were confirmed using k-means clustering on the peaks of interest identified for glycogen and nucleic acid content. Our study showed that the glycogen depletion previously described at the stage of cirrhosis is an early event in the pathological process, independently of the cause of cirrhosis. In addition, there was a progressive increase in the nucleic acid content, which may be linked to increased proliferation and polyploidy in response to cellular lesions.

Investigation of Glypican-4 and -6 by Infrared Spectral Imaging during the Hair Growth Cycle.

The expression of glypicans in different hair follicle (HF) compartments is still poorly understood. Heparan sulfate proteoglycans (HSPGs) distribution in HF is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. Our previous study proposed a novel approach to assess hair histology and glypican-1 (GPC1) distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). We show in the present manuscript for the first time complementary data on the distribution of glypican-4 (GPC4) and glypican-6 (GPC6) in HF at different phases of the hair growth cycle using IR imaging. Findings were supported by Western blot assays focusing on the GPC4 and GPC6 expression in HFs. Like all proteoglycan features, the glypicans are characterized by a core protein to which sulfated and/or unsulfated glycosaminoglycan (GAG) chains are covalently linked. Our study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), GAG, and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs, as supported by Western blot. Thus, in one analysis, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a chemical and label-free manner. From a dermatological point of view, IRSI may constitute a promising technique to study alopecia.

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior.

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

Interference of hemolysis, hyperlipidemia, and icterus on plasma infrared spectral profile.

Recently, pre-analytical, analytical, and post-analytical issues have been addressed to implement biofluid FTIR spectroscopy as a novel diagnostic tool in the clinical setting. Although hemolysis, icterus, and hyperlipidemia are known to interfere with colorimetric and turbidimetric biochemical methods, there are no data on their impact on serum/plasma FTIR spectra. This study aimed at investigating the impact of hemoglobin, bilirubin, and triglycerides concentrations on plasma spectral analysis. Plasma samples with high concentrations of hemoglobin, conjugated bilirubin, or triglycerides were studied. To mimic the various concentrations observed in clinical setting, samples were diluted using normal plasma and analyzed using high-throughput FTIR spectroscopy. Hemolytic, icteric, and hyperlipidemic plasma spectra were compared with control plasma spectra. Unsupervised analysis of all spectra was performed using principal component analysis. The comparison between control and hemolytic plasmas did not show spectral differences in the range of hemoglobin concentrations observed in spurious or pathological hemolysis. By contrast, spectra from lipidemic plasmas had different spectral profiles compared with control plasma, exhibiting increased absorbance in lipid bands. Differences in the same spectral regions were observed in spectra from icteric plasma, which may be explained by the hyperlipidemia associated with cholestasis. PCA did not discriminate between control and hemolytic plasmas up to 1 g/L hemoglobin but confirmed the interference of bilirubin and triglycerides concentrations on spectral classification. Our results show that hemolysis does not have an impact on the plasma spectral profile except for high concentrations of hemoglobin rarely observed in clinical practice, whereas icterus and hyperlipidemia constitute significant confounding factors. Graphical abstract.

Vibrational Spectroscopy Saliva Profiling as Biometric Tool for Disease Diagnostics: A Systematic Literature.

Saliva is a biofluid that can be considered as a "mirror" reflecting our body's health status. Vibrational spectroscopy, Raman and infrared, can provide a detailed salivary fingerprint that can be used for disease biomarker discovery. We propose a systematic literature review based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines to evaluate the potential of vibrational spectroscopy to diagnose oral and general diseases using saliva as a biological specimen. Literature searches were recently conducted in May 2020 through MEDLINE-PubMed and Scopus databases, without date limitation. Finally, over a period of 10 years, 18 publications were included reporting on 10 diseases (three oral and seven general diseases), with very high diagnostic performance rates in terms of sensitivity, specificity, and accuracy. Thirteen articles were related to six different cancers of the following anatomical sites: mouth, nasopharynx, lung, esophagus, stomach, and breast. The other diseases investigated and included in this review were periodontitis, Sjögren's syndrome, diabetes, and myocardial infarction. Moreover, most articles focused on Raman spectroscopy (n = 16/18) and more specifically surface-enhanced Raman spectroscopy (n = 12/18). Interestingly, vibrational spectroscopy appears promising as a rapid, label-free, and non-invasive diagnostic salivary biometric tool. Furthermore, it could be adapted to investigate subclinical diseases-even if developmental studies are required.

Analysis of Hepatic Fibrosis Characteristics in Cirrhotic Patients with and without Hepatocellular Carcinoma by FTIR Spectral Imaging.

The evolution of cirrhosis is marked by quantitative and qualitative modifications of the fibrosis tissue and an increasing risk of complications such as hepatocellular carcinoma (HCC). Our purpose was to identify by FTIR imaging the spectral characteristics of hepatic fibrosis in cirrhotic patients with and without HCC. FTIR images were collected at projected pixel sizes of 25 and 2.7 µm from paraffinized hepatic tissues of five patients with uncomplicated cirrhosis and five cirrhotic patients with HCC and analyzed by k-means clustering. When compared to the adjacent histological section, the spectral clusters corresponding to hepatic fibrosis and regeneration nodules were easily identified. The fibrosis area estimated by FTIR imaging was correlated to that evaluated by digital image analysis of histological sections and was higher in patients with HCC compared to those without complications. Qualitative differences were also observed when fibrosis areas were specifically targeted at higher resolution. The partition in two clusters of the fibrosis tissue highlighted subtle differences in the spectral characteristics of the two groups of patients. These data show that the quantitative and qualitative changes of fibrosis tissue occurring during the course of cirrhosis are detectable by FTIR imaging, suggesting the possibility of subclassifying cirrhosis into different steps of severity.

Infrared Microspectroscopy and Imaging Analysis of Inflammatory and Non-Inflammatory Breast Cancer Cells and Their GAG Secretome.

Glycosaminoglycans (GAGs)/proteoglycans (PGs) play a pivotal role in the metastasis of inflammatory breast cancer (IBC). They represent biomarkers and targets in diagnosis and treatment of different cancers including breast cancer. Thus, GAGs/PGs could represent potential prognostic/diagnostic biomarkers for IBC. In the present study, non-IBC MDA-MB-231, MCF7, SKBR3 cells and IBC SUM149 cells, as well as their GAG secretome were analyzed. The latter was measured in toto as dried drops with high-throughput (HT) Fourier Transform InfraRed (FTIR) spectroscopy and imaging. FTIR imaging was also employed to investigate single whole breast cancer cells while synchrotron-FTIR microspectroscopy was used to specifically target their cytoplasms. Data were analyzed by hierarchical cluster analysis and principal components analysis. Results obtained from HT-FTIR analysis of GAG drops showed that the inter-group variability enabled us to delineate between cell types in the GAG absorption range 1350-800 cm-1. Similar results were obtained for FTIR imaging of GAG extracts and fixed single whole cells. Synchrotron-FTIR data from cytoplasms allowed discrimination between non-IBC and IBC. Thus, by using GAG specific region, not only different breast cancer cell lines could be differentiated, but also non-IBC from IBC cells. This could be a potential diagnostic spectral marker for IBC detection useful for patient management.

Shedding light on confounding factors likely to affect salivary infrared biosignatures.

Total human saliva is a biofluid which can be considered as a "mirror" reflecting the state of the body's health. The "spectral mid-infrared fingerprint" represents a snapshot of the intrinsic biomolecular composition of a saliva sample translating multiple information about the patient, and likely to be related not only to his physiopathological status but also to his behavioral habits or even current medical treatments. These different patient-related characteristics are "confounding factors," which may strongly affect the infrared data of salivary samples and disrupt the search for specific salivary biomarkers in the detection of diseases, especially in the case of complex pathologies influenced by multiple risk factors such as genetic factors and behavioral factors, and also other comorbidities. In this study, dealing with the processing of infrared saliva spectra from 56 patients, our aim was to highlight spectral features associated with some patient characteristics, namely tobacco smoking, periodontal diseases, and gender. By using multivariate statistical methods of feature selection (principal component analysis coupled with Kruskal-Wallis test, linear discriminant analysis coupled with randfeatures function), we were able to identify the discriminant vibrations associated with a specific factor and to assess the related spectral variability. Based on the methodology demonstrated here, it could be very valuable in the future to develop processing aimed at neutralizing these variabilities, in order to determine specific spectroscopic markers related to a multifactorial disease for diagnostic or follow-up purposes.

Characterization of inflammatory breast cancer: a vibrational microspectroscopy and imaging approach at the cellular and tissue level.

Inflammatory breast cancer (IBC) has a poor prognosis because of the lack of specific biomarkers and its late diagnosis. An accurate and rapid diagnosis implemented early enough can significantly improve the disease outcome. Vibrational spectroscopy has proven to be useful for cell and tissue characterization based on the intrinsic molecular information. Here, we have applied infrared and Raman microspectroscopy and imaging to differentiate between non-IBC and IBC at both cell and tissue levels. Two human breast cancer cell lines (MDA-MB-231 and SUM-149), 20 breast cancer patients (10 non-IBC and 10 IBC), and 4 healthy volunteer biopsies were investigated. Fixed cells and tissues were analyzed by FTIR microspectroscopy and imaging, while live cells were studied by Raman microspectroscopy. Spectra were analyzed by hierarchical cluster analysis (HCA) and images by common k-means clustering algorithms. For both cell suspensions and single cells, FTIR spectroscopy showed sufficient high inter-group variability to delineate MDA-MB-231 and SUM-149 cell lines. Most significant differences were observed in the spectral regions of 1096-1108 and 1672-1692 cm-1. Analysis of live cells by Raman microspectroscopy gave also a good discrimination of these cell types. The most discriminant regions were 688-992, 1019-1114, 1217-1375 and 1516-1625 cm-1. Finally, k-means cluster analysis of FTIR images allowed delineating non-IBC from IBC tissues. This study demonstrates the potential of vibrational spectroscopy and imaging to discriminate between non-IBC and IBC at both cell and tissue levels.

Comprehensive Characterization of the Interaction between Pulsed Electric Fields and Live Cells by Confocal Raman Microspectroscopy.

This study reports a comprehensive analysis of the effect of 100 µs electric pulses on the biochemical composition of live cells using a label-free approach, confocal Raman microspectroscopy. We investigated different regions of interest around the nucleus of the cells and the dose-effect relationship related to different electric pulse parameters. We also extended the study to another cell type. Membrane resealing was monitored by pulsing the cells in reversible or irreversible electropermeabilization condition at different temperatures. Our results confirmed a previous publication showing that proteins and lipids were highly impacted by the delivery of electric pulses. These chemical changes were similar in different locations around the cell nucleus. By sweeping the field magnitude, the number of electric pulses, or their repetition rate, the Raman signatures of live cells appeared to be related to the electropermeabilization state, verified by Yo-Pro-1 uptake. We also demonstrated that the chemical changes in the Raman signatures were cell-dependent even if common features were noticed between the two cell types used.

Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems.

Glycosaminoglycans (GAGs) are natural, linear and negatively charged heteropolysaccharides which are incident in every mammalian tissue. They consist of repeating disaccharide units, which are composed of either sulfated or non-sulfated monosaccharides. Depending on tissue types, GAGs exhibit structural heterogeneity such as the position and degree of sulfation or within their disaccharide units composition being heparin, heparan sulfate, chondroitine sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. They are covalently linked to a core protein (proteoglycans) or as free chains (hyaluronan). GAGs affect cell properties and functions either by direct interaction with cell receptors or by sequestration of growth factors. These evidences of divert biological roles of GAGs make their characterization at cell and tissue levels of importance. Thus, non-invasive techniques are interesting to investigate, to qualitatively and quantitatively characterize GAGs in vitro in order to use them as diagnostic biomarkers and/or as therapeutic targets in several human diseases including cancer. Infrared and Raman microspectroscopies and imaging are sensitive enough to differentiate and classify GAG types and subtypes in spite of their close molecular structures. Spectroscopic markers characteristic of reference GAG molecules were identified. Beyond these investigations of the standard GAG spectral signature, infrared and Raman spectral signatures of GAG were searched in complex biological systems like cells. The aim of the present review is to describe the implementation of these complementary vibrational spectroscopy techniques, and to discuss their potentials, advantages and disadvantages for GAG analysis. In addition, this review presents new data as we show for the first time GAG infrared and Raman spectral signatures from conditioned media and live cells, respectively.

Digital de-waxing on FTIR images.

This paper presents a procedure that digitally neutralizes the contribution of paraffin to FTIR hyperspectral images. A brief mathematical derivation of the procedure is demonstrated and applied on one normal human colon sample to exemplify the de-waxing procedure. The proposed method includes construction of a paraffin model based on PCA, EMSC normalization and application of two techniques for spectral quality control. We discuss every step in which the researcher needs to take a subjective decision during the de-waxing procedure, and we explain how to make an adequate choice of parameters involved. Application of this procedure to 71 hyperspectral images collected from 55 human colon biopsies (20 normal, 17 ulcerative colitis, and 18 adenocarcinoma) showed that paraffin was appropriately neutralized, which made the de-waxed images adequate for analysis by pattern-recognition techniques such as k-means clustering or PCA-LDA.

Developing and understanding biofluid vibrational spectroscopy: a critical review.

Vibrational spectroscopy can provide rapid, label-free, and objective analysis for the clinical domain. Spectroscopic analysis of biofluids such as blood components (e.g. serum and plasma) and others in the proximity of the diseased tissue or cell (e.g. bile, urine, and sputum) offers non-invasive diagnostic/monitoring possibilities for future healthcare that are capable of rapid diagnosis of diseases via specific spectral markers or signatures. Biofluids offer an ideal diagnostic medium due to their ease and low cost of collection and daily use in clinical biology. Due to the low risk and invasiveness of their collection they are widely welcomed by patients as a diagnostic medium. This review underscores recent research within the field of biofluid spectroscopy and its use in myriad pathologies such as cancer and infectious diseases. It highlights current progresses, advents, and pitfalls within the field and discusses future spectroscopic clinical potentials for diagnostics. The requirements and issues surrounding clinical translation are also considered.

Rapid screening of classic galactosemia patients: a proof-of-concept study using high-throughput FTIR analysis of plasma.

Classic galactosemia is an autosomal recessive metabolic disease involving the galactose pathway, caused by the deficiency of galactose-1-phosphate uridyltransferase. Galactose accumulation induces in newborns many symptoms, such as liver disease, cataracts, and sepsis leading to death if untreated. Neonatal screening is developed and applied in many countries using several methods to detect galactose or its derived product accumulation in blood or urine. High-throughput FTIR spectroscopy was investigated as a potential tool in the current screening methods. IR spectra were obtained from blood plasma of healthy, diabetic, and galactosemic patients. The major spectral differences were in the carbohydrate region, which was first analysed in an exploratory manner using principal component analysis (PCA). PCA score plots showed a clear discrimination between diabetic and galactosemic patients and this was more marked as a function of the glucose and galactose increased concentration in these patients' plasma respectively. Then, a support vector machine leave-one-out cross-validation (SVM-LOOCV) classifier was built with the PCA scores as the input and the model was tested on median, mean and all spectra from the three population groups. This classifier was able to discriminate healthy/diabetic, healthy/galactosemic, and diabetic/galactosemic patients with sensitivity and specificity rates ranging from 80% to 94%. The total accuracy rate ranged from 87% to 96%. High-throughput FTIR spectroscopy combined with the SVM-LOOCV classification procedure appears to be a promising tool in the screening of galactosemia patients, with good sensitivity and specificity. Furthermore, this approach presents the advantages of being cost-effective, fast, and straightforward in the screening of galactosemic patients.

Diagnosis approach of chronic lymphocytic leukemia on unstained blood smears using Raman microspectroscopy and supervised classification.

We have investigated the potential of Raman microspectroscopy combined with supervised classification algorithms to diagnose a blood lymphoproliferative disease, namely chronic lymphocytic leukemia (CLL). This study was conducted directly on human blood smears (27 volunteers and 49 CLL patients) spread on standard glass slides according to a cytological protocol before the staining step. Visible excitation at 532 nm was chosen, instead of near infrared, in order to minimize the glass contribution in the Raman spectra. After Raman measurements, blood smears were stained using the May-Grünwald Giemsa procedure to correlate spectroscopic data classifications with cytological analysis. A first prediction model was built using support vector machines to discriminate between the two main leukocyte subpopulations (lymphocytes and polymorphonuclears) with sensitivity and specificity over 98.5%. The spectral differences between these two classes were associated to higher nucleic acid content in lymphocytes compared to polymorphonuclears. Then, we developed a classification model to discriminate between neoplastic and healthy lymphocyte spectra, with a mean sensitivity and specificity of 88% and 91% respectively. The main molecular differences between healthy and CLL cells were associated with DNA and protein changes. These spectroscopic markers could lead, in the future, to the development of a helpful medical tool for CLL diagnosis.

Probing non-enzymatic glycation of type I collagen: a novel approach using Raman and infrared biophotonic methods.

BACKGROUND: Non-enzymatic glycation is the main post-translational modification of long-life proteins observed during aging and physiopathological processes such as diabetes and atherosclerosis. Type I collagen, the major component in matrices and tissues, represents a key target of this spontaneous reaction which leads to changes in collagen biomechanical properties and by this way to tissue damages. METHODS: The current study was performed on in vitro glycated type I collagens using vibrational microspectroscopies, FT-IR and Raman, to highlight spectral features related to glycation effect. RESULTS AND CONCLUSIONS: We report a conservation of the triple-helical structure of type I collagen and noticeable variations in the exposure of proline upon glycation. Our data also show that the carbohydrate band can be a good spectroscopic marker of the glycation level, correlating well with the fluorescent AGEs formation with sugar addition. GENERAL SIGNIFICANCE: These non-invasive and label-free methods can shed new light on the spectral features of glycated collagens and represent an effective tool to study changes in the extracellular matrix observed in vivo during aging or on the advent of a pathological situation.

Glycosaminoglycan profiling in different cell types using infrared spectroscopy and imaging.

We recently identified vibrational spectroscopic markers characteristic of standard glycosaminoglycan (GAG) molecules. The aims of the present work were to further this investigation to more complex biological systems and to characterize, via their spectral profiles, cell types with different capacities for GAG synthesis. After recording spectral information from individual GAG standards (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate) and GAG-GAG mixtures, GAG-defective mutant Chinese hamster ovary (CHO)-745 cells, wild-type CHO cells, and chondrocytes were analyzed as suspensions by high-throughput infrared spectroscopy and as single isolated cells by infrared imaging. Spectral data were processed and interpreted by exploratory unsupervised chemometric methods based on hierarchical cluster analysis and principal component analysis. Our results showed that the spectral information obtained was discriminant enough to clearly delineate between the different cell types both at the cell suspension and single-cell levels. The abilities of the technique are to perform spectral profiling and to identify single cells with different potentials to synthesize GAGs. Infrared microspectroscopy/imaging could therefore be developed for cell screening purposes and further for identifying GAG molecules in normal tissues during physiological conditions (aging, healing process) and numerous pathological states (arthritis, cancer).

White bile in patients with malignant biliary obstruction is an independent factor of poor survival.

Background and study aims White bile is defined as a colorless fluid occasionally found in the biliary tract of patients with bile duct obstruction. Its significance is not clearly established. Our objective was to analyze the prognostic value of white bile in a series of patients with biliary obstruction due to biliary or pancreatic cancer. Patients and methods The study was conducted on a series of consecutive patients with malignant obstructive jaundice. They all underwent endoscopic retrograde cholangiopancreatography with collection of bile and biliary stent insertion. White bile was defined as bile duct fluid with bilirubin level < 20 µmol/L. Univariate and multivariate analyses were performed to identify variables associated with overall survival (OS). Results Seventy-three patients were included (32 pancreatic cancers, 41 bile duct cancers). Thirty-nine (53.4 %) had white bile. The mean bile duct bilirubin level in this group was 4.2 ±â€Š5.9 µmol/L vs 991 ±â€Š1039 µmol/L in patients with colored bile (P < 0.0001). In the group of 54 patients not eligible for surgery, the multivariate analysis demonstrated an association between the presence of white bile and reduced OS (HR 2.3, 95 %CI 1.1-4.7; P = 0.02). Other factors independently associated with OS were metastatic extension (HR 2.8, 95 %CI 1.4-5.7) and serum total bilirubin (HR 1.003, 95 %CI 1.001-1.006). There was a significant inverse correlation between serum and bile duct bilirubin levels (r = -0.43, P = 0.0001). Conclusion White bile in patients with inoperable malignant biliary obstruction is an independent factor of poor survival.

Hair Histology and Glycosaminoglycans Distribution Probed by Infrared Spectral Imaging: Focus on Heparan Sulfate Proteoglycan and Glypican-1 during Hair Growth Cycle.

The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia.