Cytopathology by optical methods: spectral cytopathology of the oral mucosa

Abstract

Spectral cytopathology (SCP) is a novel approach for diagnostic differentiation of disease in individual exfoliated cells. SCP is carried out by collecting information on each cell's biochemical composition through an infrared micro-spectral measurement, followed by multivariate data analysis. Deviations from a cell's natural composition produce specific spectral patterns that are exclusive to the cause of the deviation or disease. These unique spectral patterns are reproducible and can be identified and used through multivariate statistical methods to detect cells compromised at the molecular level by dysplasia, neoplasia, or viral infection. In this proof of concept study, a benchmark for the sensitivity of SCP is established by classifying healthy oral squamous cells according to their anatomical origin in the oral cavity. Classification is achieved by spectrally detecting cells with unique protein expressions: for example, the squamous cells of the tongue are the only cell type in the oral cavity that have significant amounts of intracytoplasmic keratin, which allows them to be spectrally differentiated from other oral mucosa cells. Furthermore, thousands of cells from a number of clinical specimens were examined, among them were squamous cell carcinoma, malignancy-associated changes including reactive atypia, and infection by the herpes simplex virus. Owing to its sensitivity to molecular changes, SCP often can detect the onset of disease earlier than is currently possible by cytopathology visualization. As SCP is based on automated instrumentation and unsupervised software, it constitutes a diagnostic workup of medical samples devoid of bias and inconsistency. Therefore, SCP shows potential as a complementary tool in medical cytopathology.

Figure 1

Spectral Heterogeneity between Anatomical Origins in the Oral Cavity (A) PCA scores plot of the significant spectral differences among normal oral cells from 5 distinct origins of the oral cavity: cheeks, gums, palate, tongue, and mouth floor. (B) Mean second derivative, vector normalized spectra representative of oral cells from the various anatomical origins and a reference spectrum of human keratin. The asterisks mark the spectral patterns contributed by keratin. The arrows signify an amide I shift to higher wavenumbers. (C-E) 40X visual images of epithelial cells from the cheek, mouth floor, and tongue, respectively.

Figure 2

Drug Metabolite Influence on Mouth Floor Cells (A) PCA scores plot of the significant spectral differences between normal epithelial mouth floor cells before and after Ibuprofen ingestion. (B) Mean second derivative, vector normalized spectra representative of mouth floor cells before (red) and after (blue) Ibuprofen influence. (C&D) 40X visual images of epithelial mouth floor cells before and after ingestion of Ibuprofen, respectively.

Figure 3

Malignancy Associated Changes in SCC of the Tongue and Palate PCA scores plot of the significant spectral differences between normal oral cells and oral cells of (A) reactive and (B) cancer biopsies. (C) Second derivative, vector normalized spectra representative of oral cells in successive states of abnormality. 40X visual images of epithelial palate cells from diagnosed (D) reactive and (E) carcinoma samples. (F&G) 40X visual images of morphologically normal epithelial tongue cells and morphologically cancerous epithelial tongue cancer cells, both from diagnosed cancer biopsies.

Figure 4

Herpes Simplex Viral Infection (A) PCA scores plot of the significant spectral differences between normal epithelial cheek cells and virally infected epithelial cheek cells. (B) Second derivative, vector normalized spectra of normal (blue) and Herpes infected (green) cheek cells. (C&D) 40X visual images of epithelial cheek cells diagnosed with Herpes viral infection.