S Jaychandran1, PK Meenapriya1* and S Ganesan1
1Department of Oral Medicine and Radiology, Tamil Nadu Government Dental College and Hospital, Chennai - 600 003, Tamil nadu, India
2Department of Oral Medicine and Radiology, J.K.K.Nattraja Dental College and hospital, Komarapalayam, Tamilnadu -638183, India
3Department of Medical Physics, Anna University, Chennai, Tamilnadu, India
Received: 22 January, 2016; Accepted: 08 March, 2016; Published: 09 March, 2016
Dr. PK Meenapriya, M.D.S., Senior Lecturer, Department of Oral Medicine and Radiology, J.K.K.Nattraja Dental College and hospital, Komarapalayam, Tamilnadu - 638183, India, Tel: 9865248844; E-mail:
Jaychandran S, Meenapriya PK, Ganesan S (2016) Raman Spectroscopic Analysis of Blood, Urine, Saliva and Tissue of Oral Potentially Malignant Disorders and Malignancy-A Diagnostic Study. Int J Oral Craniofac Sci 2(1): 011-014. DOI: 10.17352/2455-4634.000013
© 2016 Jaychandran S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Raman spectroscopy; Oral potentially malignant disorders (OPMDs); Oral cancer
Background: Oral cancers are mostly being preceded by oral potentially malignant disorders (OPMD). Early diagnosis of oral cancer or OPMDs speed up treatment and improve prognosis. Raman spectroscopy of blood, urine, saliva and tissue samples collected from OPMD and oral cancer patients were compared with similar samples from normal controls.
Context and purpose: Raman spectroscopy is a noninvasive inelastic light scattering technique in which the wavelength of the incident laser light shifts depending on the vibrations of the molecules. The specific biochemical, structural and conformational changes occurring in tissues is reflected by their Raman spectra well before the clinical manifestations start. This helps in early diagnosis and speedy treatment planning.
Results: Raman spectroscopy gave an accuracy of 78%, 90.5%, 93.1%, and 97.4% respectively for blood, urine, saliva and tissue samples in discriminating oral pre malignancy and malignancy from normal control.
Conclusion: Results of the study validate that Raman spectroscopy has the potential to be a diagnostically useful tool for the in vitro detection of OPMDs and oral cancers at the molecular level.
Brief Summary: Early diagnosis of OPMD and oral cancer by analyzing Raman spectroscopic changes improves the prognosis. Total sample size was 205. Spectra recorded on a confocal Micro Raman System (LABRAM HR 800) were statistically analyzed using the computer software SPSS/PC +19 under one of the multivariant technique analysis - Principal Component analysis followed with the Linear Discriminant Analysis (PC-LDA). The statistical analysis using PC-LDA combination of normal vs malignant vs premalignant group accuracy are shown.
Oral cancer is ranked as the sixth most common cancers in the world . Oral potentially malignant disorders (OPMDs) which are clinically evident precede most of the oral squamous cell carcinomas . Most cancers of the oral cavity are oral squamous cell carcinomas (OSCC), and tobacco, alcohol and betel use are the main risk factors for these and many OPMDs [3,4]. The high risk group is older adult males who use tobacco and alcohol. It is expected that early diagnosis of OPMDs can reduce mortality [5,6]. Early diagnosis of OSCC can speed proceeding early intervention to treatment and can improve the prognosis . Conventional oral examination (COE) is the standard method of revealing OPMDs and OSCC, confirming the clinical suspicion by biopsy. It is subject to interpretation of pathologists, and although it can detect cellular changes, it can only detect molecular changes if special techniques are employed. Currently available diagnostic technologies are histopathological examination, vital staining, biomarkers, DNA analysis, brush biopsy and optical techniques 8. Early diagnosis of OPMDs and oral cancers play an important role in reducing the mortality rate [5-7]. Such early diagnosis is made possible with optical spectroscopy which will contain information about histological and biochemical characteristics . The study is done to assess the diagnostic utility of Raman spectroscopy in metabolic fingerprinting of biologic fluids and tissues in OPMDs and oral cancer. Raman Effect , is based on interaction of light with matter. Raman or inelastic scattering is produced by about 1 in 106 to 1 in 108 of incident photons. The scattered photon has a wavelength different from the incident photon. This wavelength shift is recorded in Raman spectroscopy, which produces a detailed biochemical ‘fingerprint’ of the sample characteristic for the constituent chemical bonds . In neoplastic cells nuclear material, nuclear to cytoplasmic ratio and mitotic activity are increased. There is a progressive loss of cell maturation, abnormal chromatin distribution, decreased differentiation, cellular crowding and disorganization. Rapid angiogenesis with leaky vessels are present due to increased metabolic activity. So neoplastic cells show specific changes in quantities and/or conformations of protein, nucleic acid, carbohydrate and lipid , which is reflected as change in spectral characteristics of these cells from their normal counterparts.
Aim and objective
The study is to analyze Raman spectrum of blood, urine, saliva and tissue samples in oral premalignancy and malignancy and to analyze, correlate the diagnostic predictability.
Materials and Methods
After approval from the institutional ethical committee and obtaining written consent from the study and control groups, samples were collected in the Department of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospitals, Chennai, with laboratory and technical support from Department of Medical Physics, Anna University, Chennai. Total sample size was 205 (male 152(74%), female 53(26%), of age group 18 to 80 years) which includes 94(46%) cases of premalignancy (53(26%) cases of oral sub mucous fibrosis and 41(20%) cases of leukoplakia), 63(31%) cases of oral cancer (oral squamous cell carcinoma) and 48(23%) cases of healthy controls. From these groups 158 samples of blood, 158 samples of urine, 158 samples of saliva (50(32%) oral cancer, 87(55%) OPMDs, 21(13%) control), and 89 tissue samples, 29(32%) oral cancer, 22(25%) premalignancy, 38(43%) control were collected.
Patients clinically diagnosed with oral leukoplakia (Warnakulasuriya 2007), oral submucous fibrosis (WHO Bulletin OMS. Vol 72 1994) and clinically and histopathologically (Anneroth et al.) diagnosed with oral squamous cell carcinoma were included in the study. Willing patients who visited the hospital for other treatments were enrolled as normal controls.
Subjects with history of systemic diseases, those under any medications and those with regional or distant metastasis or a history of recurrences of any of the lesions/ conditions under study were excluded from the study.
Urine, saliva and blood samples (random) were collected at a standardized time of the day (9 am to 11 am) and transported in ice box to the laboratory. 3ml of unstimulated saliva is collected in a sterile container by drooling method.3 ml of blood is collected under aseptic conditions from ante-cubital vein in sterile EDTA coated tube. Patient is asked to collect 5 ml of urine in a sterile plastic container. Tissue sample is obtained from the lesion site and stored in normal saline for analysis. The samples were refrigerated and analyzed the same day in laboratory. Tissue samples for biopsy and for Raman spectroscopic analysis were taken at the same appointment and were of similar size and range.
Spectra were recorded on a confocal Micro Raman System (LABRAM HR 800), equipped with Peltier cooled CCD detector, with 600 gm/mm grating edge filter. Excitation was with a 785 nm diode laser (SDL 8530) with laser power of 100Mw
The statistical analysis was done using the computer software SPSS/PC +19.Principal Component analysis followed with the Linear Discriminant Analysis (PC-LDA) of normal vs. malignant vs. premalignant group accuracy are shown.
From the results we observe intensity variations and peak shift among the three groups.
Blood samples (Figure 1)
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