Raman Spectroscopy: New Perspectives for Its Clinical Application in Diagnosis

R aman spectroscopy is an analytical tool that has, undoubtedly, demonstrated itself to be a powerful technique for biochemical characterization and clinical diagnosis. ¹ Recent studies have demonstrated that Raman and optical reflectance spectroscopy are able to distinguish benign and malignant renal tumors in ex vivo environments. ² Raman spectroscopy can be used for the identification of different cells and tissues, such as breast cancer; atherosclerosis and other vascular processes as, for example, thrombosis following rupture of the plaque; ³ inflammatory diseases; ⁴ individual neoplastic and normal hematopoietic cells; bladder cancer melanoma cancer; uterine cervix cancer; squamous cell carcinoma; basal cell carcinoma; skin cancer; and lung cancer. ⁵ Evolving diagnostic applications of Raman spectroscopy, with special emphasis on transplant allograft rejection and cancer detection, has received special attention. ⁶ Raman spectra are sensitive to biochemical changes caused by proliferation in mammalian cell cultures, and they exhibit small differences between normal and malignant cells. ⁵ A Raman spectroscopic database with potential to diagnose cancer has been created. Auner and coworkers collected and classified 1352 spectra from 55 specimens from fresh or frozen normal brain, kidney, and adrenal glands and their malignancies. ⁷ Raman spectroscopy can also be employed in association with other diagnostic procedures, particularly in complex cases. The spectral profile associated with characteristic biomolecules inherent to the neoplastic tissue, demonstrates, unequivocally, different peculiarities when compared with the tissue without pathological modifications. Therefore, these molecular signatures form the basis for the identification of complex biomolecules, and can be used to differentiate normal from neoplastic tissue. ⁷

Similarly, in pediatric studies, the number of applications of Raman spectroscopy is increasing in recent years. The technique has been applied in the diagnosis of several types of neoplastic, inflammatory, allergic, and other pathologies. ⁸ Diseases difficult to diagnose, such as Alzheimer`s disease, ⁹ diabetes, ¹⁰ dengue, ¹¹ and tuberculosis, ¹² have also been analyzed with significant success with Raman spectroscopy. Hematological diseases, such as thalassemia, have also been detected with a great number of spectral details by Raman spectroscopy. Different kinds of thalassemia (alpha and beta) and normal erythrocytes (at acid or alkaline pH) present dissimilar Raman spectra in the acidic environment. ¹³ This tool has been used both in vivo, mostly using optical fibers for tissue illumination, as well as on ex vivo tissue sections in a microscopic imaging approach defined as “spectral histopathology.” ⁸ In this context, it is important to note that a great number of catheters of optical fibers and other instrumental strategies have been developed, allowing several clinical applications of this technique. ^14,15 Raman spectroscopy has a huge potential for minimally invasive or noninvasive real-time, bedside and intraoperatory diagnosis. In addition, it has great use as an ex vivo imaging tool for pathologists. ⁸

This great potential to identify spectral profiles of several diseases is strongly associated with the significant ability to discriminate biochemical compounds. Raman spectroscopy is described as a new and potentially powerful diagnostic tool in comparison with routine biochemical tests, as Raman spectroscopy can provide rapid and simultaneous identification of several “biochemical fingerprints,” such as glucose, acetone, creatinine, urea, lipid profile, uric acid, total protein, among others, which are directly related to several pathologies. ¹⁶ It is important to notice that some of these compounds, such as carbohydrates and proteins, are also relevant as “fingerprint compounds” in food analysis. ¹⁷ This area of interest constitutes a promising focus for the application of Raman spectroscopy.

The versatility of Raman spectroscopy as an instrumental analytical method is evolving continuously. This technique is a vibrational spectroscopy, which implies in great capability for structural characterization of compounds of great biochemical relevance. Interestingly, the water molecules do not cause a significant negative impact in the analysis of biochemical mixtures by Raman spectroscopy, which occurs in other vibrational spectroscopies, such as Fourier transform infrared (FTIR). Ali and coworkers, ¹⁸ in a comparative study between Raman, FTIR, and attenuated total reflectance (ATR)-FTIR microspectroscopy for imaging human skin tissue sections, observed that at similar conditions, Raman scattering produced a more detailed spectra, which, for example, could differentiate the stratum corneum from the underlying epithelial layer, and, in the absence of melanin in an artificial skin model, could further differentiate the basal layer from the overlying epithelium. Discussing the technological and spectroscopic differences between the respective tools, ¹⁸ the authors concluded that the differences in the performance of the techniques were not only specific instrumentation characteristics, which denotes the extraordinary potential of Raman spectroscopy as a technique to analyze biological tissues.

The development of new devices has improved the perspectives of applications of Raman spectroscopy in vivo, mainly the studies of endoluminal organs. Several spectroscopy techniques, with special emphasis on Raman spectroscopy, have been considered as the basis for minimally invasive and nondestructive measuring systems, ¹⁹ as these instrumental analytical methodologies are able to detect a great number of components that are present in low physiological concentration in body fluids (e.g., mmol L⁻¹ to nmol L⁻¹), ²⁰ in a noninvasive or, at least, minimally invasive way. Among the applications of optical spectroscopy in medicine, its employment in gastrointestinal endoscopy, which is likely to be one of the most important areas of impact of biomedical engineering, can be mentioned, ^21,22 It is interesting to cite the work of Matousek and Stone, ²³ which reviews the called “deep non-invasive medical diagnosis,” mainly associated with bone diseases, cancer, and diabetes, which allows for inferring of the great development that Raman spectroscopy will generate in the diagnostic procedures in the next few years, with the greater use of this tool in small clinics and hospitals. We believe that Raman spectroscopy can be an interesting alternative in terms of diagnosis when employed as a unique diagnostic tool, as well as when used as a coadjuvant device for the confirmation of clinical analyses obtained by other methods, especially in severe diseases.