Raman Spectroscopic Evaluation of Efficacy of Current Paraffin Wax Section Dewaxing Agents

Abstract

During a spectroscopic study to identify biochemical changes in cervical tissue with the onset of carcinogenesis, residual paraffin wax contributions were observed on almost all dewaxed formalin-fixed paraffin-processed (FFPP) tissue sections examined. Subsequently, the present study was formulated to evaluate the efficacy of current dewaxing agents using Raman spectroscopy. Three cervical FFPP sections were subjected to each of the protocols. Sections were dewaxed using four common dewaxing protocols, namely, xylene, Histoclear, heat-mediated antigen retrieval (HMAR) using xylene and citrate buffer, and Trilogy (combined deparaffinization and unmasking of antigens). The potential for hexane as a dewaxing agent was also evaluated. Sections were dewaxed in multiple dewaxing cycles using xylene, Histoclear, and hexane. Residual paraffin wax contributions remained at 1062 cm⁻¹, 1296 cm⁻¹, and 1441 cm⁻¹. HMAR using xylene and citrate buffer, and HMAR using Trilogy, showed a similar efficacy, resulting in incomplete removal of wax. Hexane was shown to be the most effective dewaxing agent, resulting in almost complete removal of wax. Immunohistochemistry was carried out on dewaxed slides, and those dewaxed with hexane displayed a stronger positivity (≍28%). Implications for histopathology and immunohistochemistry are considered, as well as problems that residual wax poses for spectroscopic evaluation of dewaxed FFPP sections with a view to disease diagnosis.