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Sahu A.,Chilakapati Laboratory | Dalal K.,All India Institute of Medical Sciences | Naglot S.,All India Institute of Medical Sciences | Aggarwal P.,All India Institute of Medical Sciences | Krishna C.M.,Chilakapati Laboratory
PLoS ONE | Year: 2013

The currently prescribed tests for asthma diagnosis require compulsory patient compliance, and are usually not sensitive to mild asthma. Development of an objective test using minimally invasive samples for diagnosing and monitoring of the response of asthma may help better management of the disease. Raman spectroscopy (RS) has previously shown potential in several biomedical applications, including pharmacology and forensics. In this study, we have explored the feasibility of detecting asthma and determining treatment response in asthma patients, through RS of serum. Serum samples from 44 asthma subjects of different grades (mild, moderate, treated severe and untreated severe) and from 15 reference subjects were subjected to Raman spectroscopic analysis and YKL-40 measurements. The force expiratory volume in 1 second (FEV1) values were used as gold standard and the serum YKL-40 levels were used as an additional parameter for diagnosing the different grades of asthma. For spectral acquisition, serum was placed on a calcium fluoride (CaF2) window and spectra were recorded using Raman microprobe. Mean and difference spectra comparisons indicated significant differences between asthma and reference spectra. Differences like changes in protein structure, increase in DNA specific bands and increased glycosaminoglycans-like features were more prominent with increase in asthma severity. Multivariate tools using Principal-component-analysis (PCA) and Principal-component based-linear-discriminant analysis (PC-LDA) followed by Leave-one-out-cross-validation (LOOCV), were employed for data analyses. PCA and PC-LDA results indicate separation of all asthma groups from the reference group, with minor overlap (19.4%) between reference and mild groups. No overlap was observed between the treated severe and untreated severe groups, indicating that patient response to treatment could be determined. Overall promising results were obtained, and a large scale validation study on random subjects is warranted before the routine clinical usage of this technique. © 2013 Sahu et al.

Deshmukh A.,Chilakapati Laboratory | Singh S.P.,Chilakapati Laboratory | Chaturvedi P.,Tata Memorial Hospital | Krishna C.M.,Chilakapati Laboratory
Journal of Biomedical Optics | Year: 2011

Oral squamous cell carcinoma is one of among the top 10 malignancies. Optical spectroscopy, including Raman, is being actively pursued as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex vivo tissues. Spectral features showed predominance of lipids and proteins in normal and cancer conditions, respectively, which were attributed to membrane lipids and surface proteins. In view of recent developments in deep tissue Raman spectroscopy, we have recorded Raman spectra from superior and inferior surfaces of 10 normal oral tissues on intact, as well as incised, biopsies after separation of epithelium from connective tissue. Spectral variations and similarities among different groups were explored by unsupervised (principal component analysis) and supervised (linear discriminant analysis, factorial discriminant analysis) methodologies. Clusters of spectra from superior and inferior surfaces of intact tissues show a high overlap; whereas spectra from separated epithelium and connective tissue sections yielded clear clusters, though they also overlap on clusters of intact tissues. Spectra of all four groups of normal tissues gave exclusive clusters when tested against malignant spectra. Thus, this study demonstrates that spectra recorded from the superior surface of an intact tissue may have contributions from deeper layers but has no bearing from the classification of a malignant tissues point of view. © 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

Bhattacharjee T.,Chilakapati Laboratory | Kumar P.,Chilakapati Laboratory | Maru G.,Maru Laboratory | Ingle A.,Laboratory Animal Facility | And 2 more authors.
Lasers in Medical Science | Year: 2014

Breast cancer is the most common cancer affecting females worldwide. As early detection results in better prognosis, screening tools for breast cancer are being explored. Raman spectroscopy, a rapid, objective, and noninvasive tool, has shown promising results in the diagnosis of several cancers including breast cancer. For development as a screening tool, a study of spectral signatures associated with breast cancer progression is imperative. However, such studies are not possible in human subjects. Hence, there is a need for a suitable animal model, which is conducive to transcutaneous in vivo Raman spectroscopic measurements of breast with minimal interference from skin and hair and has contribution from functional mammary epithelium of breast. In this study, rodent models like C57, Swiss albino, Swiss bare, agouti mice, and Sprague-Dawley rats were evaluated. Among these models, transcutaneous breast spectra of hairless Swiss bare mice have the best signal-to-noise ratio and were closest to reported ex vivo as well as intraoperative in vivo human breast spectra. Principal component-linear discriminant analysis of several anatomical sites confirms minimal skin interference and suggests contribution from functional mammary epithelium of breast. Moreover, transcutaneous spectra from normal breast and breast tumors of Swiss bare mice could be classified with 99 % efficiency, which is better than the previous reports. Thus, Swiss bare mice model may be better suited for transcutaneous in vivo Raman spectroscopic studies of breast physiology and pathology, especially breast cancer. Prospectively, in addition to cancer progression, breast-to-bone metastasis can also be studied, since these anatomical sites can be uniquely classified. © 2013 Springer-Verlag London.

Sahu A.,Chilakapati Laboratory | Deshmukh A.,Chilakapati Laboratory | Ghanate A.D.,Chilakapati Laboratory | Singh S.P.,Chilakapati Laboratory | And 2 more authors.
Technology in Cancer Research and Treatment | Year: 2012

Biophysical techniques play an important role in detecting physiological alterations during pathogenesis. Raman spectroscopy has shown immense potential in identifying several diseased conditions, including oral cancers. Classification of normal, inflammatory, premalignant and malignant conditions has been demonstrated using ex vivo Raman spectroscopy. Feasibility of recording in vivo spectra in clinically implementable time has also been shown. Translation of this technology to clinics requires extensive validation of methodologies, building of robust models and testing the same under stringent conditions as well as on diverse populations. In this context, the ability of Raman spectroscopy in identifying subtle changes in oral mucosa with increasing age, and the influence of these aging related changes on classification with tobacco-related pathological changes was evaluated. A total of 451 spectra from 62 subjects were recorded from buccal mucosa of healthy subjects of 4 different age groups (aged 20-60 years). Also, 478 spectra from 85 subjects belonging to 4 different categories, tobacco exposed mucosa, contralateral normal (opposite side of tumor), premalignant patches and tumors on buccal mucosa were recorded using fiber optic probe-coupled commercial Raman spectrometer. Differences in spectra were explored by unsupervised Principal Component Analysis (PCA) and supervised Linear Discriminant Analysis (LDA), followed by Leave one out cross validation. Results indicate feasibility of classifying early and late age groups. Also, clear classification is observed between healthy and pathological groups, thus inherent heterogeneity in healthy groups seems to have no bearing on classification of normal with abnormal conditions. Findings of the study indicate high sensitivity of Raman spectroscopy in detecting subtle mucosal changes, further supporting efficacy of Raman spectroscopic approaches in oral cancer applications. Prospectively, more vigorous validation studies of Raman methodology would enable routine clinical applications. © Adenine Press (2012).

Gohulkumar M.,Annamalai University | Kumar P.,Chilakapati Laboratory | Murali Krishna C.,Chilakapati Laboratory | Krishnakumar N.,Annamalai University
Journal of Raman Spectroscopy | Year: 2016

Raman spectroscopy is a vibrational spectroscopic technique that can be used to monitor the therapeutic efficacy of anticancer drugs during carcinogenesis in a non-invasive and label-free manner. The present study aims to investigate the biochemical changes exerted upon free silibinin (SIL) and its nanoparticulate (SILNPs) treatment against 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in the fingerprint region of 1800-500cm-1 using HE-785 Raman spectrometer. Raman spectra differed significantly between the control and tumor tissues, with tumor tissues characterized by increased intensities of vibrational bands such as nucleic acids, phenylalanine and tryptophan and a lower percentage of lipids when compared to the control tissues. Further, oral administration of free SIL and SILNPs significantly increased lipids and decreased the levels of tryptophan, phenylalanine and nucleic acid contents. Overall, the treatment of nanoparticulate SIL was found to be a more potent antitumor effect than free SIL in preventing the formation of tumor and also brought back the several Raman bands to a normal range in the buccal mucosa of hamsters during DMBA-induced oral carcinogenesis. In addition, the detailed secondary structure of proteins in the control and experimental groups is also presented. Furthermore, the diagnostic algorithms based on principal component linear discriminant analysis (PC-LDA) achieved an overall sensitivity of 94-100% and specificity of 76-100%. These results further demonstrate that Raman spectroscopy associated with PC-LDA diagnostic algorithms could be a valuable tool for rapid and sensitive detection of specific biomolecular changes at the molecular level in response to anticancer drug. © 2016 John Wiley & Sons, Ltd.

Sahu A.,Chilakapati Laboratory | Sawant S.,Tata Memorial Center | Mamgain H.,WITec GmbH | Krishna C.M.,Chilakapati Laboratory
Analyst | Year: 2013

Early diagnosis of oral cancers, one of the major cancers, is of utmost importance as 5-year disease-free survival rates are some of the lowest, despite advances in treatment and surgical modalities. In vivo Raman spectroscopy has shown efficacy in the detection of normal, premalignant and malignant lesions and even of early changes such as cancer-field-effects/malignancy-associated- changes. However, the need for a dedicated instrument and stringent laboratory conditions, at all diagnostic centers, limits wide screening applications of this method. In light of this, it is pertinent to explore ex vivo samples like serum due to its ease of collection, storage, transport and analysis at a centralized facility. Hence, Raman studies were carried out on serum from 14 buccal mucosa and 40 tongue cancers as well as 16 healthy control samples. Spectral features indicate differential contributions of proteins, DNA, and amino acids like Phe, Trp and Tyr and p-carotene in the analyzed groups. Highly intense Raman bands assigned to p-carotene could be due to resonance Raman, and were observed in all sera with the highest relative intensity in normal samples. Higher DNA and protein content were observed in the mean cancer spectra. Principal component-linear discriminant analysis (PC-LDA) followed by cross-validation using leave-one-out cross-validation (LOOCV) were employed for data analysis which was carried out both spectra- and patient-wise. Findings indicate the possibility of classifying normal and oral cancer sera in both these approaches; however, the patient-wise approach could be the preferred mode for prospective studies. Besides, a tendency of classification for buccal mucosa and tongue cancers was also observed. Prospective validation of these results on a large sample size may help in the translation of this methodology to clinics. © The Royal Society of Chemistry 2013.

Gurushankar K.,Annamalai University | Gohulkumar M.,Annamalai University | Kumar P.,Chilakapati Laboratory | Murali Krishna C.,Chilakapati Laboratory | Krishnakumar N.,Annamalai University
Laser Physics Letters | Year: 2016

Recently it has been shown that Raman spectroscopy possesses great potential in the investigation of biomolecular changes of tumor tissues with therapeutic drug response in a non-invasive and label-free manner. The present study is designed to investigate the antitumor effect of hespertin-loaded nanoparticles (HETNPs) relative to the efficacy of native hesperetin (HET) in modifying the biomolecular changes during 7,12-dimethyl benz(a)anthracene (DMBA)-induced oral carcinogenesis using a Raman spectroscopic technique. Significant differences in the intensity and shape of the Raman spectra between the control and the experimental tissues at 1800-500 cm-1 were observed. Tumor tissues are characterized by an increase in the relative amount of proteins, nucleic acids, tryptophan and phenylalanine and a decrease in the percentage of lipids when compared to the control tissues. Further, oral administration of HET and its nanoparticulates restored the status of the lipids and significantly decreased the levels of protein and nucleic acid content. Treatment with HETNPs showed a more potent antitumor effect than treatment with native HET, which resulted in an overall reduction in the intensity of several biochemical Raman bands in DMBA-induced oral carcinogenesis being observed. Principal component and linear discriminant analysis (PC-LDA), together with leave-one-out cross validation (LOOCV) on Raman spectra yielded diagnostic sensitivities of 100%, 80%, 91.6% and 65% and specificities of 100%, 65%, 60% and 55% for classification of control versus DMBA, DMBA versus DMBA + HET, DMBA versus DMBA + HETNPs and DMBA + HET versus DMBA + HETNPs treated tissue groups, respectively. These results further demonstrate that Raman spectroscopy associated with multivariate statistical algorithms could be a valuable tool for developing a comprehensive understanding of the process of biomolecular changes, and could reveal the signatures of the antitumor response of drugs. © 2016 Astro Ltd.

Bhattacharjee T.,Chilakapati Laboratory | Maru G.,Maru Laboratory | Ingle A.,Laboratory Animal Facility | Krishna C.M.,Chilakapati Laboratory
Vibrational Spectroscopy | Year: 2013

The risk of female breast cancer increases as age progresses. This can be explained by Pike's model, which suggests that the process of aging in breast is not uniform. 'Breast ageing' is most rapid during menarche, slows with each pregnancy, slows further during perimenopause, and is least after the menopause. In this study, the feasibility of using transcutaneous in vivo Raman spectroscopy to detect age-related changes in mouse breast and its effect on tumor detection were explored. Spectra acquired transcutaneously from breast of 2 (menarche), 4-6 (mid reproductive phase), 10-12 (perimenopause), 13-15 month (menopause) old mice and frank breast tumors were analyzed using principal component-linear discriminant analysis (PC-LDA). A classification efficiency of ~80% was achieved for different age groups. Further, it was observed that the number of misclassifications among age groups increase as age progresses. For example, 3% spectra from menarche misclassify with other age groups, while 19-28% from mid-reproductive, perimenopause and menopause misclassify with each other. Misclassifications between groups indicate homogeneity in tissues. Thus, results suggest that menarche breast is biochemically distinct while breast during mid-reproductive, perimenopause and menopause is relatively homogenous. This probably indicates that the rate of aging is rapid during menarche, but slows down during other phases. Thus, spectroscopic data correlate with Pike's model. Although sensitive to age-related changes, RS could classify tumors with 95% efficiency. Overall, results suggest possibility of distinguishing age-related changes using RS without affecting ability to classify tumors. © 2013 Elsevier B.V.

PubMed | Vaidya Laboratory, D Y Patil Dental College, Indian Institute of Technology Bombay, The Surgical Center and Chilakapati Laboratory
Type: Journal Article | Journal: Journal of biomedical optics | Year: 2015

Serum Raman spectroscopy (RS) has previously shown potential in oral cancer diagnosis and recurrence prediction. To evaluate the potential of serum RS in oral cancer screening, premalignant and cancer-specific detection was explored in the present study using 328 subjects belonging to healthy controls, premalignant, disease controls, and oral cancer groups. Spectra were acquired using a Raman microprobe. Spectral findings suggest changes in amino acids, lipids, protein, DNA, and -carotene across the groups. A patient-wise approach was employed for data analysis using principal component linear discriminant analysis. In the first step, the classification among premalignant, disease control (nonoral cancer), oral cancer, and normal samples was evaluated in binary classification models. Thereafter, two screening-friendly classification approaches were explored to further evaluate the clinical utility of serum RS: a single four-group model and normal versus abnormal followed by determining the type of abnormality model. Results demonstrate the feasibility of premalignant and specific cancer detection. The normal versus abnormal model yields better sensitivity and specificity rates of 64 and 80%; these rates are comparable to standard screening approaches. Prospectively, as the current screening procedure of visual inspection is useful mainly for high-risk populations, serum RS may serve as a useful adjunct for early and specific detection of oral precancers and cancer.

PubMed | Tata Memorial Center, Chilakapati Laboratory and Tata Memorial Hospital
Type: Journal Article | Journal: Journal of biomedical optics | Year: 2014

In vivo Raman spectroscopy is being projected as a new, noninvasive method for cervical cancer diagnosis. In most of the reported studies, normal areas in the cancerous cervix were used as control. However, in the Indian subcontinent, the majority of cervical cancers are detected at advanced stages, leaving no normal sites for acquiring control spectra. Moreover, vagina and ectocervix are reported to have similar biochemical composition. Thus, in the present study, we have evaluated the feasibility of classifying normal and cancerous conditions in the Indian population and we have also explored the utility of the vagina as an internal control. A total of 228 normal and 181 tumor in vivo Raman spectra were acquired from 93 subjects under clinical supervision. The spectral features in normal conditions suggest the presence of collagen, while DNA and noncollagenous proteins were abundant in tumors. Principal-component linear discriminant analysis (PC-LDA) yielded 97% classification efficiency between normal and tumor groups. An analysis of a normal cervix and vaginal controls of cancerous and noncancerous subjects suggests similar spectral features between these groups. PC-LDA of tumor, normal cervix, and vaginal controls further support the utility of the vagina as an internal control. Overall, findings of the study corroborate with earlier studies and facilitate objective, noninvasive, and rapid Raman spectroscopic-based screening/diagnosis of cervical cancers.

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