Digital Dewaxing of Raman Signals: Discrimination between Nevi and Melanoma Spectra Obtained from Paraffin-Embedded Skin Biopsies

Abstract

Malignant melanoma (MM) is the most severe tumor affecting the skin and accounts for three quarters of all skin cancer deaths. Raman spectroscopy is a promising nondestructive tool that has been increasingly used for characterization of the molecular features of cancerous tissues. Different multivariate statistical analysis techniques are used in order to extract relevant information that can be considered as functional spectroscopic descriptors of a particular pathology. Paraffin embedding (waxing) is a highly efficient process used to conserve biopsies in tumor banks for several years. However, the use of non-dewaxed formalin-fixed paraffin-embedded tissues for Raman spectroscopic investigations remains very restricted, limiting the development of the technique as a routine analytical tool for biomedical purposes. This is due to the highly intense signal of paraffin, which masks important vibrations of the biological tissues. In addition to being time consuming and chemical intensive, chemical dewaxing methods are not efficient and they leave traces of the paraffin in tissues, which affects the Raman signal. In the present study, we use independent component analysis (ICA) on Raman spectral images collected on melanoma and nevus samples. The sources obtained from these images are then used to eliminate, using non-negativity constrained least squares (NCLS), the paraffin contribution from each individual spectrum of the spectral images of nevi and melanomas. Corrected spectra of both types of lesion are then compared and classified into dendrograms using hierarchical cluster analysis (HCA).

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells

“…Accordingly, it is common to record and subtract "dark" and substrate spectra from the sample spectra before removing any arbitrary background. As the substrate spectrum is an independent spectral component, it can effectively be removed by ICA sand NCLS, in a similar process to the so-called "digital dewaxing" of tissue spectra [28]. Further artifacts in Raman Spectroscopy include nonlinearity of axes and dark noise.…”

“…Notably, analyses of archived tissue libraries may add much to understanding disease progression and patient prognosis. It has been demonstrated that it is not necessary to remove the paraffin to obtain usable spectral information, particularly in the case of FTIR spectroscopy [28]. Standard tissue microarray protocols involve paraffin embedded tissue.…”

“…Leaving the paraffin in place reduces scattering artefacts and effects of further variable removal of aromatic solvent soluble components [28][29][30][31]. The spectrally well defined paraffin contribution can be digitally Please do not adjust margins Please do not adjust margins removed in a preprocessing step, involving Independent Component Analysis (ICA) and Non-Negatively Constrained Least Squares Analysis (NCLS), so called digital dewaxing [28]. However, it may be argued that greater consistency of spectral information is achieved when sections are deparaffinised.…”

Raman spectroscopy for screening and diagnosis of cervical cancer

“…Notably, analyses of archived tissue libraries may add much to understanding disease progression and patient prognosis. It has been demonstrated that it is not necessary to remove the paraffin to obtain usable spectral information [53]. Leaving the paraffin in place reduces scattering artefacts and effects of further variable removal of aromatic solvent soluble components.…”

Spectropathology for the next generation: Quo vadis?

“…16 Standard tissue microarray protocols involve paraffin-embedded tissue. Leaving the paraffin in place reduces scattering artefacts and the effects of further variable removal of aromatic solvent-soluble components.…”