Spectral cytopathology of cervical samples: detecting cellular abnormalities in cytologically normal cells

Abstract

Spectral cytopathology (SCP) is a novel spectroscopic method for objective and unsupervised classification of individual exfoliated cells. The limitations of conventional cytopathology are well recognized within the pathology community. In SCP, cellular differentiation is made by observing molecular changes in the nucleus and the cytoplasm, which may or may not produce morphological changes detectable by conventional cytopathology. This proof of concept study shows SCP's potential as an enhancing tool for cytopathologists by aiding in the accurate and reproducible diagnosis of cells in all states of disease. Infrared spectra are collected from cervical cells deposited onto reflectively coated glass slides. Each cell has a corresponding infrared spectrum that describes its unique biochemical composition. Spectral data are processed and analyzed by an unsupervised chemometric algorithm, principal component analysis. In this blind study, cervical samples are classified by analyzing the spectra of morphologically normal looking squamous cells from normal samples and samples diagnosed by conventional cytopathology with low-grade squamous intraepithelial lesions. SCP discriminated cytopathological diagnoses amongst 12 different cervical samples with a high degree of specificity and sensitivity. SCP also correlated two samples with abnormal spectral changes: these samples had a normal cytopathological diagnosis but had a history of abnormal cervical cytology. The spectral changes observed in the morphologically normal looking cells are most likely because of an infection with human papillomavirus (HPV). HPV DNA testing was conducted on five additional samples, and SCP accurately differentiated these samples by their HPV status. SCP tracks biochemical variations in cells that are consistent with the onset of disease. HPV has been implicated as the cause of these changes detected spectroscopically. SCP does not depend on identifying the sparse number of morphologically abnormal cells within a large sample to make an accurate classification, as does conventional cytopathology. These findings suggest that the detection of cellular biochemical variations by SCP can serve as a new enhancing screening method that can identify earlier stages of disease.

Figure 1

(Left) Visual image of a 2 mm × 2 mm area on a sample spot on a low-e slide collected on the PE Spotlight microscope. (Right) Binary masked image, constructed through PapMap, of the image shown on the Left. The white spots constitute the area occupied by a cell. The cellular diameter ranges from 3 to 10 pixels, or 19 μm to 63 μm (1 pixel equals 6.25 μm).

Figure 2

(A) PCA scores plot of cervical cells from five normal samples (blue), five abnormal samples (red), and two samples from patients with a history of an abnormal Pap test (yellow). The number of cells included from each sample is listed in Table 1. Each symbol represents the spectrum of an individual cell. There is a separation of the blue and red classes along PC2. The cellular spectra from samples of patients with a history of abnormal cervical cytology (yellow) co-cluster with the abnormal samples (red). All cells were morphologically normal as shown in (B–D), representative high resolution 40x images of cells from each classification. (E) Mean second derivative vector normalized spectra of the normal samples and abnormal samples for the spectral range 3100-2800 and 1700-1200 cm⁻¹.

Figure 3

(A) PCA scores plot of eight cells that exhibit morphological abnormalities (green) and the morphologically normal looking cells (red) from the same two samples. (B–C) Representative high resolution 40x images of a morphologically normal looking cell and a morphologically abnormal cell, respectively.

Figure 4

(A) PCA scores plot of 5 samples which were tested for hrHPV DNA: 3 samples with normal cytology were hrHPV negative (blue circles), 1 sample with normal cytology was hrHPV positive (pink triangles), and 1 sample was diagnosed by cytology with low grade dysplasia and was hrHPV positive (red squares). The samples differentiate along PC2 based on their HPV status. (B) Mean second derivative vector normalized spectra for each class for the spectral range 3100-2800 and 1700-1200 cm⁻¹.