Publications by authors named "Valérie Untereiner"

38 Publications

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

Biomolecules 2021 01 30;11(2). Epub 2021 Jan 30.

Université de Reims Champagne-Ardenne, Laboratoire de Biochimie Médicale et Biologie Moléculaire, 51097 Reims, France.

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.
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http://dx.doi.org/10.3390/biom11020192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912031PMC
January 2021

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

Endosc Int Open 2021 Feb 3;9(2):E203-E209. Epub 2021 Feb 3.

Department of Hepato-Gastroenterology and Digestive Oncology, Reims University Hospital, Reims, France.

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.  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).  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 ± 5.9 µmol/L vs 991 ± 1039 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). White bile in patients with inoperable malignant biliary obstruction is an independent factor of poor survival.
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http://dx.doi.org/10.1055/a-1324-2721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857955PMC
February 2021

New insights into hidradenitis suppurativa diagnosis via salivary infrared biosignatures: A pilot study.

J Biophotonics 2021 Mar 2;14(3):e202000327. Epub 2020 Dec 2.

Université de Reims Champagne-Ardenne, UFR Odontologie, Département de Biologie Orale, Reims, France.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease which can lead to a prolonged physical disability. HS diagnosis is exclusively clinical with the absence of biomarkers. Our study aims at assessing the HS-diagnostic potential of infrared spectroscopy from saliva, as a biofluid reflecting the body's pathophysiological state. Infrared spectra from 127 patients (57 HS and 70 non-HS) were processed by multivariate methods: principal component analysis coupled with Kruskal-Wallis or Mann-Whitney tests to identify discriminant spectral wavenumbers and linear discriminant analysis to evaluate the performances of HS-diagnostic approach. Infrared features, mainly in the 1300 cm -1600 cm region, were identified as discriminant for HS and prediction models revealed diagnostic performances of about 80%. Tobacco and obesity, two main HS risk factors, do not seem to alter the infrared diagnosis. This pilot study shows the potential of salivary "liquid biopsy" associated to vibrational spectroscopy to develop a personalized medical approach for HS patients' management.
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http://dx.doi.org/10.1002/jbio.202000327DOI Listing
March 2021

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

Anal Chem 2020 12 21;92(24):15745-15756. Epub 2020 Nov 21.

Mass Spectrometry Laboratory, MolSys Research Unit, University of Liege, Place du 20 Aoǔt 7, 4000 Liège, Belgium.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.
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http://dx.doi.org/10.1021/acs.analchem.0c02696DOI Listing
December 2020

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

Molecules 2020 Sep 19;25(18). Epub 2020 Sep 19.

Laboratoire de Biochimie Médicale et Biologie Moléculaire, Université de Reims Champagne-Ardenne, 51097 Reims, France.

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. 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.
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http://dx.doi.org/10.3390/molecules25184300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570935PMC
September 2020

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

Molecules 2020 Sep 10;25(18). Epub 2020 Sep 10.

Département de Biologie Orale, UFR Odontologie, Université de Reims Champagne-Ardenne, 2 rue du Général Koenig, 51100 Reims, France.

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 ( = 16/18) and more specifically surface-enhanced Raman spectroscopy ( = 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.
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http://dx.doi.org/10.3390/molecules25184142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570680PMC
September 2020

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

Molecules 2020 Sep 7;25(18). Epub 2020 Sep 7.

Université de Reims Champagne-Ardenne, BioSpecT-EA7506, UFR de Pharmacie, 51097 Reims, France.

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.
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http://dx.doi.org/10.3390/molecules25184092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570752PMC
September 2020

Vibrational spectroscopy of liquid biopsies for prostate cancer diagnosis.

Ther Adv Med Oncol 2020 30;12:1758835920918499. Epub 2020 Jul 30.

Radiation and Environmental Science Centre, Focas Research Institute, Technological University Dublin, Dublin, Dublin D08 NF82, Ireland.

Background: Screening for prostate cancer with prostate specific antigen and digital rectal examination allows early diagnosis of prostate malignancy but has been associated with poor sensitivity and specificity. There is also a considerable risk of over-diagnosis and over-treatment, which highlights the need for better tools for diagnosis of prostate cancer. This study investigates the potential of high throughput Raman and Fourier Transform Infrared (FTIR) spectroscopy of liquid biopsies for rapid and accurate diagnosis of prostate cancer.

Methods: Blood samples (plasma and lymphocytes) were obtained from healthy control subjects and prostate cancer patients. FTIR and Raman spectra were recorded from plasma samples, while Raman spectra were recorded from the lymphocytes. The acquired spectral data was analysed with various multivariate statistical methods, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and classical least squares (CLS) fitting analysis.

Results: Discrimination was observed between the infrared and Raman spectra of plasma and lymphocytes from healthy donors and prostate cancer patients using PCA. In addition, plasma and lymphocytes displayed differentiating signatures in patients exhibiting different Gleason scores. A PLS-DA model was able to discriminate these groups with sensitivity and specificity rates ranging from 90% to 99%. CLS fitting analysis identified key analytes that are involved in the development and progression of prostate cancer.

Conclusions: This technology may have potential as an alternative first stage diagnostic triage for prostate cancer. This technology can be easily adaptable to many other bodily fluids and could be useful for translation of liquid biopsy-based diagnostics into the clinic.
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http://dx.doi.org/10.1177/1758835920918499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412923PMC
July 2020

Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods.

Sci Rep 2020 06 26;10(1):10471. Epub 2020 Jun 26.

Vectorology and Anticancer Therapies, UMR 8203, CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 114 rue Edouard Vaillant, 94805, Villejuif, France.

The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes.
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http://dx.doi.org/10.1038/s41598-020-67402-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319994PMC
June 2020

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

Cancer Sci 2020 Aug 9;111(8):2907-2922. Epub 2020 Jul 9.

Department of Gynecology and Obstetrics, Münster University Hospital, Münster, Germany.

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.
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http://dx.doi.org/10.1111/cas.14539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419026PMC
August 2020

Label-Free Infrared Spectral Histology of Skin Tissue Part II: Impact of a Lumican-Derived Peptide on Melanoma Growth.

Front Cell Dev Biol 2020 29;8:377. Epub 2020 May 29.

Université de Reims Champagne-Ardenne, BioSpecT-EA7506, Reims, France.

Melanoma is the most aggressive type of cutaneous malignancies. In addition to its role as a regulator of extracellular matrix (ECM) integrity, lumican, a small leucine-rich proteoglycan, also exhibits anti-tumor properties in melanoma. This work focuses on the use of infrared spectral imaging (IRSI) and histopathology (IRSH) to study the effect of lumican-derived peptide (L9Mc) on B16F1 melanoma primary tumor growth. Female C57BL/6 mice were injected with B16F1 cells treated with L9Mc ( = 10) or its scrambled peptide ( = 8), and without peptide (control, = 9). The melanoma primary tumors were subjected to histological and IR imaging analysis. In addition, immunohistochemical staining was performed using anti-Ki-67 and anti-cleaved caspase-3 antibodies. The IR images were analyzed by common K-means clustering to obtain high-contrast IRSH that allowed identifying different ECM tissue regions from the epidermis to the tumor area, which correlated well with H&E staining. Furthermore, IRSH showed good correlation with immunostaining data obtained with anti-Ki-67 and anti-cleaved caspase-3 antibodies, whereby the L9Mc peptide inhibited cell proliferation and increased strongly apoptosis of B16F1 cells in this mouse model of melanoma primary tumors.
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http://dx.doi.org/10.3389/fcell.2020.00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273845PMC
May 2020

Label-Free Infrared Spectral Histology of Skin Tissue Part I: Impact of Lumican on Extracellular Matrix Integrity.

Front Cell Dev Biol 2020 12;8:320. Epub 2020 May 12.

Université de Reims Champagne-Ardenne, Laboratoire de Biochimie Médicale et Biologie Moléculaire, Reims, France.

Proteoglycans (PG) play an important role in maintaining the extracellular matrix (ECM) integrity. Lumican, a small leucine rich PG, is one such actor capable of regulating such properties. In this study, the integrity of the dermis of lumican-deleted vs. wild-type mice was investigated by conventional histology and by infrared spectral histology (IRSH). Infrared spectroscopy is a non-invasive, rapid, label-free and sensitive technique that allows to probe molecular vibrations of biomolecules present in a tissue. Our IRSH results obtained on control (WT, = 3) and ( = 3) mice showed that different histological structures were identified by using K-means clustering and validated by hematoxylin eosin saffron (HES) staining. Furthermore, an important increase of the dermis thickness was observed in compared to WT mice. In terms of structural information, analysis of the spectral images also revealed an intra-group homogeneity and inter-group heterogeneity. In addition, type I collagen contribution was evaluated by HES and picrosirius red staining as well as with IRSH. Both techniques showed a strong remodeling of the ECM in mice due to the looseness of collagen fibers in the increased dermis space. These results confirmed the impact of lumican on the ECM integrity. The loss of collagen fibers organization due to the absence of lumican can potentially increase the accessibility of anti-cancer drugs to the tumor. These results are qualitatively interesting and would need further structural characterization of type I collagen fibers in terms of size, organization, and orientation.
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http://dx.doi.org/10.3389/fcell.2020.00320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235349PMC
May 2020

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma.

J Biophotonics 2020 07 7;13(7):e201960173. Epub 2020 Apr 7.

Radiation and Environmental Science Centre, Focas Research Institute, Technological University Dublin, Dublin, Ireland.

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.
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http://dx.doi.org/10.1002/jbio.201960173DOI Listing
July 2020

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

Anal Chem 2020 03 21;92(5):4053-4064. Epub 2020 Feb 21.

Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom, L69 7ZB.

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.
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http://dx.doi.org/10.1021/acs.analchem.9b05658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997108PMC
March 2020

Transmission Fourier Transform Infrared Spectroscopic Imaging, Mapping, and Synchrotron Scanning Microscopy with Zinc Sulfide Hemispheres on Living Mammalian Cells at Sub-Cellular Resolution.

Appl Spectrosc 2020 May 30;74(5):544-552. Epub 2020 Mar 30.

University of Reims Champagne-Ardenne, Pharmacy, Reims, France.

Fourier transform infrared (FT-IR) spectroscopic imaging and microscopy of single living cells are established label-free technique for the study of cell biology. The constant driver to improve the spatial resolution of the technique is due to the diffraction limit given by infrared (IR) wavelength making subcellular study challenging. Recently, we have reported, with the use of a prototype zinc sulfide (ZnS) transmission cell made of two hemispheres, that the spatial resolution is improved by the factor of the refractive index of ZnS, achieving a λ/2.7 spatial resolution using the synchrotron-IR microscopy with a 36× objective with numerical aperture of 0.5. To refine and to demonstrate that the ZnS hemisphere transmission device can be translated to standard bench-top FT-IR imaging systems, we have, in this work, modified the device to achieve a more precise path length, which has improved the spectral quality of the living cells, and showed for the first time that the device can be applied to study live cells with three different bench-top FT-IR imaging systems. We applied focal plane array (FPA) imaging, linear array, and a synchrotron radiation single-point scanning method and demonstrated that in all cases, subcellular details of individual living cells can be obtained. Results have shown that imaging with the FPA detector can measure the largest area in a given time, while measurements from the scanning methods produced a smoother image. Synchrotron radiation single-point mapping produced the best quality image and has the flexibility to introduce over sampling to produce images of cells with great details, but it is time consuming in scanning mode. In summary, this work has demonstrated that the ZnS hemispheres can be applied in all three spectroscopic approaches to improve the spatial resolution without any modification to the existing microscopes.
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http://dx.doi.org/10.1177/0003702819898275DOI Listing
May 2020

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

Anal Bioanal Chem 2020 Feb 19;412(4):805-810. Epub 2019 Dec 19.

Université de Reims Champagne-Ardenne, BioSpecT EA7506, UFR de Pharmacie, 51097, Reims, France.

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.
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http://dx.doi.org/10.1007/s00216-019-02312-0DOI Listing
February 2020

CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth.

J Cyst Fibros 2020 05 29;19(3):466-475. Epub 2019 Nov 29.

Université de Reims Champagne Ardenne, BIOS EA 4691, Biomatériaux et Inflammation en site osseux, SFR CAP-Santé (FED 4231), 1, Avenue du Maréchal Juin, 51097 Reims, France. Electronic address:

Background: The lack of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing to severe lung disease, reduced growth and osteopenia. Both reduced bone content and strength are increasingly recognized in infants with CF before the onset of significant lung disease, suggesting a developmental origin and a possible role in bone disease pathogenesis. The role of CFTR in bone metabolism is unclear and studies on humans are not feasible. Deletion of CFTR in pigs (CFTR pigs) displays at birth severe malformations similar to humans in the intestine, respiratory tract, pancreas, liver, and male reproductive tract.

Methods: We compared bone parameters of CFTR male and female pigs with those of their wild-type (WT) littermates at birth. Morphological and microstructural properties of femoral cortical and trabecular bone were evaluated using micro-computed tomography (μCT), and their chemical compositions were examined using Raman microspectroscopy.

Results: The integrity of the CFTR bone was altered due to changes in its microstructure and chemical composition in both sexes. Low cortical thickness and high cortical porosity were found in CFTR pigs compared to sex-matched WT littermates. Moreover, an increased chemical composition heterogeneity associated with higher carbonate/phosphate ratio and higher mineral crystallinity was found in CFTR trabecular bone, but not in CFTR cortical bone.

Conclusions: The loss of CFTR directly alters the bone composition and metabolism of newborn pigs. Based on these findings, we speculate that bone defects in patients with CF could be a primary, rather than a secondary consequence of inflammation and infection.
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http://dx.doi.org/10.1016/j.jcf.2019.10.023DOI Listing
May 2020

Investigating pre-analytical requirements for serum and plasma based infrared spectro-diagnostic.

J Biophotonics 2019 12 13;12(12):e201900177. Epub 2019 Aug 13.

Université de Reims Champagne-Ardenne, BioSpecT EA7506, UFR de Pharmacie, Reims, France.

Infrared spectroscopy is a rapid, easy-to-operate, label-free and therefore cost-effective technique. Many studies performed on biofluids (eg, serum, plasma, urine, sputum, bile and cerebrospinal fluid) have demonstrated its promising application as a clinical diagnostic tool. Given all these characteristics, infrared spectroscopy appears to be an ideal candidate to be implemented into the clinics. However, before considering its translation, a clear effort is needed to standardise protocols for biofluid spectroscopic analysis. To reach this goal, careful investigations to identify and track errors that can occur during the pre-analytical phase is a crucial step. Here, we report for the first time, results of investigations into pre-analytical factors that can affect the quality of the spectral data acquired on serum and plasma, such as the impact of long-term freezing time storage of samples as well as the month-to-month reproducibility of the spectroscopic analysis. The spectral data discrimination has revealed to be majorly impacted by a residual water content variation in serum and plasma dried samples.
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http://dx.doi.org/10.1002/jbio.201900177DOI Listing
December 2019

Monitoring Radiotherapeutic Response in Prostate Cancer Patients Using High Throughput FTIR Spectroscopy of Liquid Biopsies.

Cancers (Basel) 2019 Jul 2;11(7). Epub 2019 Jul 2.

Radiation and Environmental Science Centre, Focas Research Institute, Technological University Dublin, D08 NF82 Dublin, Ireland.

Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, RT causes a wide range of adverse late effects that can affect a patient's quality of life. There are currently no predictive assays in clinical use to identify patients at risk of normal tissue radiation toxicity. This study aimed to investigate the potential of Fourier transform infrared (FTIR) spectroscopy for monitoring radiotherapeutic response. Blood plasma was acquired from 53 prostate cancer patients at five different time points: prior to treatment, after hormone treatment, at the end of radiotherapy, two months post radiotherapy and eight months post radiotherapy. FTIR spectra were recorded from plasma samples at all time points and the data was analysed using MATLAB software. Discrimination was observed between spectra recorded at baseline versus follow up time points, as well as between spectra from patients showing minimal and severe acute and late toxicity using principal component analysis. A partial least squares discriminant analysis model achieved sensitivity and specificity rates ranging from 80% to 99%. This technology may have potential to monitor radiotherapeutic response in prostate cancer patients using non-invasive blood plasma samples and could lead to individualised patient radiotherapy.
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http://dx.doi.org/10.3390/cancers11070925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679106PMC
July 2019

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

Anal Bioanal Chem 2019 Apr 16;411(11):2283-2290. Epub 2019 Feb 16.

BioSpecT EA7506, Faculty of Pharmacy, University of Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096, Reims Cedex, France.

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.
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http://dx.doi.org/10.1007/s00216-019-01669-6DOI Listing
April 2019

Raman spectroscopy-based insight into lipid droplets presence and contents in liver sinusoidal endothelial cells and hepatocytes.

J Biophotonics 2019 04 21;12(4):e201800290. Epub 2019 Jan 21.

Faculty of Chemistry, Jagiellonian University, Krakow, Poland.

Liver sinusoidal endothelial cells (LSECs), a type of endothelial cells with unique morphology and function, play an important role in the liver hemostasis, and LSECs dysfunction is involved in the development of nonalcoholic fatty liver disease (NAFLD). Here, we employed Raman imaging and chemometric data analysis in order to characterize the presence of lipid droplets (LDs) and their lipid content in primary murine LSECs, in comparison with hepatocytes, isolated from mice on high-fat diet. On NAFLD development, LDs content in LSECs changed toward more unsaturated lipids, and this response was associated with an increased expression of stearylo-CoA desaturase-1. To the best of our knowledge, this is a first report characterizing LDs in LSECs, where their chemical composition is analyzed along the progression of NAFLD at the level of single LD using Raman imaging.
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http://dx.doi.org/10.1002/jbio.201800290DOI Listing
April 2019

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

Analyst 2018 Dec;143(24):6103-6112

Université de Reims Champagne-Ardenne, Laboratoire de Biochimie Médicale et de Biologie Moléculaire, UFR de Médecine, Reims, France.

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.
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http://dx.doi.org/10.1039/c8an01292jDOI Listing
December 2018

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

Anal Chem 2017 10 5;89(20):10790-10797. Epub 2017 Oct 5.

Vectorology and Anticancer Therapies, UMR 8203, CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay , 114 rue Edouard Vaillant, 94805 Villejuif, France.

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.
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http://dx.doi.org/10.1021/acs.analchem.7b02079DOI Listing
October 2017

Lumican delays melanoma growth in mice and drives tumor molecular assembly as well as response to matrix-targeted TAX2 therapeutic peptide.

Sci Rep 2017 08 9;7(1):7700. Epub 2017 Aug 9.

Université de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Campus Moulin de la Housse, 51100, Reims, France.

Lumican is a small leucine-rich proteoglycan (SLRP) being known as a key regulator of collagen fibrillogenesis. However, little attention has been given so far in studying its influence on tumor-associated matrix architecture. Here, we investigate the role of host lumican on tumor matrix organization as well as on disease progression considering an immunocompetent model of melanoma implanted in Lum vs. wild type syngeneic mice. Conjointly, lumican impact on tumor response to matrix-targeted therapy was evaluated considering a previously validated peptide, namely TAX2, that targets matricellular thrombospondin-1. Analysis of available genomics and proteomics databases for melanoma first established a correlation between lumican expression and patient outcome. In the B16 melanoma allograft model, endogenous lumican inhibits tumor growth and modulates response to TAX2 peptide. Indeed, IHC analyses revealed that lumican deficiency impacts intratumoral distribution of matricellular proteins, growth factor and stromal cells. Besides, innovative imaging approaches helped demonstrating that lumican host expression drives biochemical heterogeneity of s.c. tumors, while modulating intratumoral collagen deposition as well as organization. Altogether, the results obtained present lumican as a strong endogenous inhibitor of tumor growth, while identifying for the first time this proteoglycan as a major driver of tumor matrix coherent assembly.
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http://dx.doi.org/10.1038/s41598-017-07043-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550434PMC
August 2017

Demonstration of the Protein Involvement in Cell Electropermeabilization using Confocal Raman Microspectroscopy.

Sci Rep 2017 01 19;7:40448. Epub 2017 Jan 19.

Vectorology and Anticancer Therapies, UMR 8203, CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 114 rue Edouard Vaillant, 94805 Villejuif, France.

Confocal Raman microspectroscopy was used to study the interaction between pulsed electric fields and live cells from a molecular point of view in a non-invasive and label-free manner. Raman signatures of live human adipose-derived mesenchymal stem cells exposed or not to pulsed electric fields (8 pulses, 1 000 V/cm, 100 μs, 1 Hz) were acquired at two cellular locations (nucleus and cytoplasm) and two spectral bands (600-1 800 cm and 2 800-3 100 cm). Vibrational modes of proteins (phenylalanine and amide I) and lipids were found to be modified by the electropermeabilization process with a statistically significant difference. The relative magnitude of four phenylalanine peaks decreased in the spectra of the pulsed group. On the contrary, the relative magnitude of the amide I band at 1658 cm increased by 40% when comparing pulsed and control group. No difference was found between the control and the pulsed group in the high wavenumber spectral band. Our results reveal the modification of proteins in living cells exposed to pulsed electric fields by means of confocal Raman microspectroscopy.
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http://dx.doi.org/10.1038/srep40448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5244372PMC
January 2017

Digital de-waxing on FTIR images.

Analyst 2017 Apr;142(8):1358-1370

Universidade de São Paulo, Avenida dos Bandeirantes, 3900, Ribeirão Preto, SP, Brazil.

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.
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http://dx.doi.org/10.1039/c6an01975gDOI Listing
April 2017

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

Glycoconj J 2017 06 7;34(3):309-323. Epub 2016 Dec 7.

CNRS UMR7369, Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Reims, France.

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.
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http://dx.doi.org/10.1007/s10719-016-9743-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487820PMC
June 2017

Developing and understanding biofluid vibrational spectroscopy: a critical review.

Chem Soc Rev 2016 Apr 27;45(7):1803-18. Epub 2015 Nov 27.

WESTChem, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, 99 George Street, Glasgow, G1 1RD, UK.

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.
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http://dx.doi.org/10.1039/c5cs00585jDOI Listing
April 2016

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

Analyst 2015 Jul;140(13):4465-72

Université de Reims Champagne-Ardenne, Équipe MéDIAN Biophotonique et Technologies pour la Santé, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096, Reims Cedex, France.

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.
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http://dx.doi.org/10.1039/c4an02085eDOI Listing
July 2015

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

Analyst 2015 Apr;140(7):2280-6

Université de Reims Champagne-Ardenne, Equipe MéDIAN, Biophotonique et Technologies pour la Santé, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims, France.

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.
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http://dx.doi.org/10.1039/c4an01942cDOI Listing
April 2015