FTIR Microspectroscopy Coupled with Two-Class Discrimination Segregates Markers Responsible for Inter- and Intra-Category Variance in Exfoliative Cervical Cytology

Abstract

Infrared (IR) absorbance of cellular biomolecules generates a vibrational spectrum, which can be exploited as a “biochemical fingerprint” of a particular cell type. Biomolecules absorb in the mid-IR (2–20 μm) and Fourier-transform infrared (FTIR) microspectroscopy applied to discriminate different cell types (exfoliative cervical cytology collected into buffered fixative solution) was evaluated. This consisted of cervical cytology free of atypia (i.e. normal; n = 60), specimens categorised as containing low-grade changes (i.e. CIN1 or LSIL; n = 60) and a further cohort designated as high-grade (CIN2/3 or HSIL; n = 60). IR spectral analysis was coupled with principal component analysis (PCA), with or without subsequent linear discriminant analysis (LDA), to determine if normal versus low-grade versus high-grade exfoliative cytology could be segregated. With increasing severity of atypia, decreases in absorbance intensity were observable throughout the 1,500 cm–1 to 1,100 cm–1 spectral region; this included proteins (1,460 cm–1), glycoproteins (1,380 cm–1), amide III (1,260 cm–1), asymmetric (νas) PO2– (1,225 cm–1) and carbohydrates (1,155 cm–1). In contrast, symmetric (νs) PO2–(1,080 cm–1) appeared to have an elevated intensity in high-grade cytology. Inter-category variance was associated with protein and DNA conformational changes whereas glycogen status strongly influenced intra-category. Multivariate data reduction of IR spectra using PCA with LDA maximises inter-category variance whilst reducing the influence of intra-class variation towards an objective approach to class cervical cytology based on a biochemical profile.