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Quantitative Infrared Spectroscopy of Formalin-Fixed, Paraffin-Embedded Tissue Specimens: Paraffin Wax Removal with Organic Solvents



Curtis W. Meuse, Peter E. Barker


Formalin-fixed, paraffin embedded tissue specimens for histopathology are cut to 3-20 μm thickness to allow transmission of light, and optimize tissue morphology. Tissue is stabilized by formalin fixation followed by impregnation with wax in nonpolar solvents to provide an appropriate density for mictotome sectioning. After sectioning, the paraffin must be removed and tissue rehydrated prior to morphological staining, or molecular analysis with DNA probes or antibodies. Wax removal methods have been adequate for morphological evaluation, but, inconsistent quantitative molecular biomarker results suggest preanalytical processing might better address quantitation and reproducibility. For example, interlaboratory comparisons reveal the presence of residual wax. We have developed an infrared spectroscopy method to quantitate wax left by processing procedures. We estimate that all processing methods remove over 97 % of the wax based on changes in spectral intensities before and after washing. Measurements directly through glass microscope slides are limited to the spectral region above 2000 cm-1 where the absorption bands of the wax and tissue, overlap. To compare the efficacy of several washing methods, we calculate the correlation coefficients of the spectra of the wax and the washed tissue sections over the region 2840 cm-1 to 2965 cm-1. The correlation coefficients describe the similarity of the spectra of the tissue and the wax and provide a quantitative method to determine what fraction of the dewaxed tissue could still be paraffin. Our correlation analysis indicates that over 3 different tissue sample types, xylene washing leaves only about half as much wax as washing with hexane or limonene, in agreement with other reports.
Applied Immunohistochemistry & Molecular Morphology
Report Number


formalin fixed, paraffin embeded, FTIR, tissue specimens, correlation coefficient


Meuse, C. and Barker, P. (2009), Quantitative Infrared Spectroscopy of Formalin-Fixed, Paraffin-Embedded Tissue Specimens: Paraffin Wax Removal with Organic Solvents, Applied Immunohistochemistry & Molecular Morphology (Accessed June 24, 2024)


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Created December 1, 2009, Updated February 19, 2017