FTIR spectroscopy is a widely used technique that provides insights into disease processes at the molecular level. Due to its numerous advantages it is becoming an increasingly powerful tool for the study of biologi- cal materials and has the potential to become an excellent diagnostic method, especially considering the low cost of trans fl ection substrates. However, questions about the usefulness of the trans fl ection measure- ment mode due to the complicated nature of physical processes occurring during the measurement and in particular the Electric Field Standing Wave (EFSW) e ff ect have been raised. In this paper we present a com- parison of the two most common FT-IR measurement modes: transmission and transfection using healthy and pathologically altered tissue (histiocytic sarcoma). We found that the major di ff erences between normal and cancerous tissue were associated with changes DNA and carbohydrate content. In particular we ident- i fi ed a band at 964 cm − 1 assigned to a nucleic acid phosphodiester backbone mode, which appeared more pronounced in cancerous tissue irrespective of the substrate. We applied Principal Component Analysis, Unsupervised Hierarchical Cluster Analysis and k -means clustering to transmission and trans fl ection sub- strates and found that both measurement modes were equally capable of discrimination normal form can- cerous tissue. Moreover, the di ff erences between spectra from cancerous and normal tissue were signi fi cantly more important than the ones arising from the measurement modes.