V. B. Kartha

Tissue Raman Spectroscopy for the Study of Radiation Damage: Brain Irradiation of Mice

Abstract Jyothi Lakshmi, R., Kartha, V. B., Murali Krishna, C., Solomon, J. G. R., Ullas, G. and Uma Devi, P. Tissue Raman Spectroscopy for the Study of Radiation Damage: Brain Irradiation of Mice. Radiat. Res. 157, 175–182 (2002). Radiotherapy is routinely employed in the treatment of head and neck cancers. Acute cell death, radiation-activated chemical cascades, and the induction of genes coding for protective factors like cytokines are considered to be the major processes involved in radiation damage and repair. It should be possible to follow these processes by monitoring the biochemical interactions initiated by radiation. We have carried out Raman spectroscopy studies on tissue from mice subjected to brain irradiation to identify the biochemical changes occurring in tissue and brain as a result of radiation injury. These studies show that brain irradiation produces drastic spectral changes even in tissue far removed from the irradiation site. The changes are very similar to those produced by the stress of inoculation and restraint and the administration of an anesthetic drug. While the changes produced by stress or anesthetics last for only a short time (a few hours to 1 or 2 days), radiation-induced changes persist even after 1 week. The spectral changes can be interpreted in terms of the observation of new spectra that are dominated by bands due to proteins. The results thus support the hypothesis that various protective factors are released throughout the body when the central nervous system (CNS) is exposed to radiation.

Micro-Raman Spectroscopy for Optical Pathology of Oral Squamous Cell Carcinoma

Micro-Raman spectra of formalin-fixed oral squamous normal and carcinoma tissues, stored at room temperature for 2 months, have been recorded. Spectra were recorded both in the epithelial and subepithelial regions of the tissues. No noticeable spectral contamination due to formalin was observed. Very significant differences between spectra of normal epithelial and malignant epithelial samples were found. No such differences in spectra of subepithelial malignant and subepithelial normal samples could be observed. This study shows that spectra from the epithelial region changes drastically because of malignancy-induced biochemical changes in this region. Major differences between normal and malignant spectra seem to arise from the protein composition, conformational/structural changes, and possible increase in protein content in malignant epithelia. The differences between normal epithelial and subepithelial spectra, as expected, arise mainly from the collagen in subepithelial tissue. Principal component analysis of the combined sets of spectra—epithelial and subepithelial, normal and malignant— showed that very good discrimination can be achieved by Raman microspectroscopy. This study thus validates the suitability of formalin-fixed tissues for optical pathology in oral malignancy.

Osteoradionecrosis (ORN) of the Mandible: A Laser Raman Spectroscopic Study

Laser Raman spectroscopy has been used in this study to characterize mandibular bone samples from patients who had undergone radiation therapy for oral cancer. The paper discusses spectral changes resulting in osteoradionecrosis (ORN) of the mandibular bone, a serious complication that may occur after radiation therapy. Histopathological studies normally reveal the radiation damage on vascular canals and loss in bone cells, but will not reveal any structural or biochemical changes. All radiation-induced side effects are attributed to this hypovascularity and hypocellularity caused by early- and/or late-delayed effects. Our Raman studies on normal and ORN bone and on bone exposed to radiation, but not in the ORN state, show that irradiation produces immediate structural changes in the inorganic bone matrix with a slight loss in cells. ORN bone, in addition to the structural changes that had already occurred on radiation exposure, shows almost complete loss of cellular components. Since bone tissue is continuously being remodeled (dissolved and rebuilt) under normal conditions, our results suggest that the immediate structural changes in the calcium hydroxy apatite mineral part is not repaired in ORN, due to loss of the highly transient osteoblasts and osteoclasts resulting from destruction of stem cells. The spectral studies also show changes in the organic matrix, which is mostly type I collagen.

Prediction of radiotherapy response in cervix cancer by Raman spectroscopy: A pilot study

Radiotherapy is the choice of treatment for locally advanced stages of the cervical cancers, one of the leading female cancers. Because of intrinsic factors, tumors of same clinical stage and histological type often exhibit differential radioresponse. Radiotherapy regimen, from first fraction of treatment to clinical evaluation of response, spans more than 4 months. Clinical assessment by degree of tumor shrinkage is the only routinely practiced method to evaluate the tumor response. Hence, a need is created for development new methodologies that can predict the tumor response to radiotherapy at an early stage of the treatment which can lead to tailor‐made protocols. To explore the feasibility of prediction of tumor radioresponse, Raman spectra of cervix cancer tissues that were collected before (malignant) and 24 h after patient was treated with 2nd fraction of radiotherapy (RT) were recorded. Data were analyzed by Principal Components Analysis (PCA) and results were correlated with clinical evaluation of radioresponse. Mean Raman spectra of RT tissues corresponding to different levels of tumor response, complete, partial, and no response, showed minute but significant variations. The unsupervised PCA of malignant tissues failed to provide any classification whereas RT spectra gave clear classification between responding (complete and partial response) and nonresponding conditions as well as a tendency of separation among responding conditions. These results were corroborated by supervised classification, by means of discrimination parameters: Mahalanobis distance and spectral residuals. Thus, findings of the study suggest the feasibility of Raman spectroscopic prediction of tumor radioresponse in cervical cancers.

Evaluation of high-performance liquid chromatography laser-induced fluorescence for serum protein profiling for early diagnosis of oral cancer

The present work deals with the evaluation of a high-performance liquid chromatography laser-induced fluorescence (HPLC-LIF) technique developed in our laboratory for early detection of oral cancer from protein profiles of body fluids. The results show that protein profiles of serum samples from a given class of samples, say, normal, premalignant, or malignant, are statistically very close to each other, while profiles of members of any class are significantly different from other classes. The performance of the technique is evaluated by the use of sensitivity and specificity pairs, receiver operating characteristic (ROC) analysis, and Youdens Index. The technique uses protein profile differences in serum samples, registered by the HPLC-LIF technique. The study is carried out using serum samples from volunteers diagnosed as normal or premalignant clinically, and as malignant by histopathology. The specificities and sensitivities of the HPLC-LIF method at an ideal threshold (M-distance = 2) for normal, malignant, and premalignant classes are 100, 69.5, and 61.5%, and 86.5, 87.5, and 87.5% respectively.

Protein profile study of clinical samples of ovarian cancer using High Performance Liquid Chromatography-Laser Induced Fluorescence (HPLC-LIF)

New techniques for the early detection of cancer are fast emerging. This is essential for more effective diagnosis and control of the disease. We have used a High Performance Liquid Chromatography-Laser Induced Fluorescence (HPLCLIF) technique to record chromatograms of proteins in serum and ovarian tissue samples. The recorded chromatograms of normal, benign and malignant samples were analyzed using statistical (Principal Component Analysis) methods. It is shown that chromatograms of the samples can be classified into sets, and a model based on such a classification can be used to analyze protein profiles of test samples of serum and ovarian tissue for the detection of malignancies.

Protein Profile Analysis of Cellular Samples from the Cervix for the Objective Diagnosis of Cervical Cancer using HPLC-LIF

Protein profiles of cytologic samples from the cervix were studied using High Performance Liquid Chromatographic (HPLC) separation combined with ultra-sensitive laser induced fluorescence (LIF) detection. HPLC-LIF protein profiles of samples from clinically normal subjects, individuals suffering from cervical cancer (different stages), and subjects who had other gynecological problems related to cervix, like erosion of cervix and Nabothian cyst, but no malignancy, were subjected to Principal Component Analysis (PCA). The application of HPLC-LIF protein profiling combined with PCA was found to be a highly efficient method for discrimination of different classes of samples with high sensitivity and specificity. Diagnostic accuracy and optimal threshold - decision criterion - for objective discrimination were estimated using sensitivity-specificity pairs and Youdens index (J) plots.

A hybrid LIBS–Raman system combined with chemometrics: an efficient tool for plastic identification and sorting

AbstractClassification of plastics is of great importance in the recycling industry as the littering of plastic wastes increases day by day as a result of its extensive use. In this paper, we demonstrate the efficacy of a combined laser-induced breakdown spectroscopy (LIBS)–Raman system for the rapid identification and classification of post-consumer plastics. The atomic information and molecular information of polyethylene terephthalate, polyethylene, polypropylene, and polystyrene were studied using plasma emission spectra and scattered signal obtained in the LIBS and Raman technique, respectively. The collected spectral features of the samples were analyzed using statistical tools (principal component analysis, Mahalanobis distance) to categorize the plastics. The analyses of the data clearly show that elemental information and molecular information obtained from these techniques are efficient for classification of plastics. In addition, the molecular information collected via Raman spectroscopy exhibits clearly distinct features for the transparent plastics (100% discrimination), whereas the LIBS technique shows better spectral feature differences for the colored samples. The study shows that the information obtained from these complementary techniques allows the complete classification of the plastic samples, irrespective of the color or additives. This work further throws some light on the fact that the potential limitations of any of these techniques for sample identification can be overcome by the complementarity of these two techniques. Graphical Abstractᅟ

Development of a Stand-off Laser Induced Breakdown Spectroscopy (ST-LIBS) system for the analysis of complex matrices

In the present work, we discuss the evaluation and optimization of a stand-off laser induced breakdown spectroscopy (ST-LIBS) system, developed indigenously for remote analysis of heavy elements in soil. A compact Q-switched Nd:YAG laser operating at fundamental wavelength 1064 nm was used for plasma generation at distances up to 6 meters. Techniques for optimal experimental results were evaluated for detection of Cd, Cr, Pb, Mo and Ni in soil. The system was evaluated with two NIST certified soil samples. The effect of working distance on the LIBS signal is also discussed briefly. Results confirm the capabilities of the system for remote monitoring.

Salivary protein markers: a noninvasive protein profile-based method for the early diagnosis of oral premalignancy and malignancy

Abstract. An ultra-sensitive hyphenated technique, high-performance liquid chromatography-laser-induced fluorescence detection protein profiling of saliva, is evaluated for early detection and diagnosis of oral premalignancy and malignancy. Calibration sets of protein profiles of unstimulated whole saliva are collected from clinically/pathologically normal, premalignant, and malignant subjects and used as standards. Three parameters—scores of factors, sum of squared residuals, and Mahalanobis distance—derived from principal component analysis of protein profiles of the standard calibration sets, and blind samples are used for “match/no-match” diagnosis of the blind samples. Analyses of the results show that the method is capable of differentiating normal, premalignant, and malignant conditions with the sensitivity and specificity of 79% and 78%, respectively. The technique provides a fast, highly objective (free from personal judgment and statistically defined), and noninvasive diagnostic method for screening and early detection of oral cancer.