A study examining the effects of tissue processing on human tissue sections using vibrational spectroscopy

The use of vibrational spectroscopy in the detection of cancer is a newly emerging diagnostic field, which has shown great potential to date. Many investigations have been carried out on frozen tissue samples, which by their very nature are hard to obtain. However, histology departments have archives of thousands of tissue samples, preserved and mounted in wax blocks. If this archival material can be shown to yield good Raman and IR spectra capable of differentiating between normal and cancerous tissue, it would improve the diagnostic capabilities of spectroscopy even further. Results from these formalin-fixed paraffin processed (FFPP) tissue sections, will give a better understanding of the effects of processing and could unlock the potential diagnostic capabilities of FFPP sections. This study investigated the effect of freezing, formalin fixation, wax embedding and de-waxing. Spectra were recorded from parallel tissue sections of placenta to examine biochemical changes before, during and after processing with both Raman and IR spectroscopy. FFPP sections were shown to provide good quality Raman and IR spectra but new peaks due to freezing and formalin fixation as well as shifts in the amide bands resulting from changes in protein conformation and possible cross-links were found. Residual wax peaks were observed clearly in the Raman spectra. In the FT-IR spectra a single wax contribution was seen which may contaminate the characteristic CH₃ deformation band in biological tissue. This study has therefore confirmed that FFPP sections have diagnostic potential provided that researchers are aware of the biochemical changes due to tissue processing highlighted by this study.