Evaluating different fixation protocols for spectral cytopathology, part 1

Abstract

Spectral cytopathology (SCP) is a novel approach for disease diagnosis that utilizes infrared spectroscopy to interrogate the biochemical components of cellular samples and multivariate statistical methods, such as principal component analysis, to analyze and diagnose spectra. SCP has taken vast strides in its application for disease diagnosis over the past decade; however, fixation-induced changes and sample handling methods are still not systematically understood. Conversely, fixation and staining methods in conventional cytopathology, typically involving protocols to maintain the morphology of cells, have been documented and widely accepted for nearly a century. For SCP, fixation procedures must preserve the biochemical composition of samples so that spectral changes significant to disease diagnosis are not masked. We report efforts to study the effects of fixation protocols commonly used in traditional cytopathology and SCP, including fixed and unfixed methods applied to exfoliated oral (buccal) mucosa cells. Data suggest that the length of time in fixative and duration of sample storage via desiccation contribute to minor spectral changes where spectra are nearly superimposable. These findings illustrate that changes influenced by fixation are negligible in comparison to changes induced by disease.

Figure 1

(A) Stacked mean absorbance spectra (overlay shown in insert), (B) overlay mean 2^nd derivative spectra (9 Point Savitzky-Golay smoothing window) and (C) stacked 2^nd derivative spectra of unfixed (dried) cells at 24 hours (red), 1 week (green), and 4 weeks (blue) after sample preparation.

Figure 2

(A) PCA scores plot of unfixed (dried) cells at 24 hours (red), 1 week (green), and 4 weeks (blue) after sample preparation. Representative loading vectors (B) PC1 and (C) PC2.

Figure 3

Photomicrographs of (bottom) unfixed (dried) cells and (middle) Surepath® and (top) formalin fixed cells at 24 hours (red), 1 week (green), and 4 weeks (blue) after sample preparation.

Figure 4

Mean 2^nd derivative spectra (9 Point Savitzky-Golay smoothing window) of cells fixed and unfixed for 24 hours at 24 hours (red), 1 week (green), and 4 weeks (blue) after sample preparation.

Figure 5

(A) PCA Scores plot of cells fixed by three different methods: unfixed (dried), Surepath® and formalin, 24 hours post deposition. (B) and (C): corresponding loading vectors PC1 and PC2 respecitvely.

Figure 6

(A) PCA scores plot of exfoliated oral (buccal) mucosa cells fixed for 24 hours in Surepath® and desiccated between each time-point (24 hours, 1 week and 4 weeks). (B) and (C): corresponding loading vectors PC1 and PC2. (D) Photomicrographs of one cell monitored at each time-point chronologically from top to bottom.

Figure 7

Mean (A) absorbance (overlay shown in insert) and (B) 2^nd derivative spectra of exfoliated oral mucosa cells fixed in Surepath® for 24 hours (red), 1 week (green), or 4 weeks (blue).

Figure 8

Mean (A) absorbance (overlay shown in insert) and (B) 2^nd derivative spectra of exfoliated oral mucosa cells fixed in buffered formalin for 24 hours (red), 1 week (green), or 4 weeks (blue).

Figure 9

(A) PCA scores plot and its representative loading vectors (B) PC1 and (C) PC2 of fixative data presented thus far (red, green, and blue) and diseased, yet morphologically normal looking, oral mucosa (buccal) cells (black)