Evaluating Different Fixation Protocols for Spectral Cytopathology, Part 2: Cultured Cells

Abstract

Spectral cytopathology (SCP) is a robust and reproducible diagnostic technique that employs infrared spectroscopy and multivariate statistical methods, such as principal component analysis to interrogate unstained cellular samples and discriminate changes on the biochemical level. In the past decade, SCP has taken considerable strides in its application for disease diagnosis. Cultured cell lines have proven to be useful model systems to provide detailed biological information to this field; however, the effects of sample fixation and storage of cultured cells are still not entirely understood in SCP. Conventional cytopathology utilizes fixation and staining methods that have been established and widely accepted for nearly a century and are focused on maintaining the morphology of a cell. Conversely, SCP practices must implement fixation protocols that preserve the sample’s biochemical composition and maintain its spectral integrity so not to introduce spectral changes that may mask variance significant to disease. It is not only necessary to evaluate the effects on fixed exfoliated cells but also fixed cultured cells because although they are similar systems, they exhibit distinct differences. We report efforts to study the effects of fixation methodologies commonly used in traditional cytopathology and SCP including both fixed and unfixed routines applied to cultured HeLa cells, an adherent cervical cancer cell line. Data suggest parallel results to findings in Part 1 of this series for exfoliated cells, where the exposure time in fixative and duration of sample storage via desiccation contribute to minor spectral changes only. The results presented here reinforce observations from Part 1 indicating that changes induced by disease are much greater than changes observed as a result of alternate fixation methodologies. Principal component analysis of HeLa cells fixed via the same conditions and protocols as exfoliated cells (Part 1) yield nearly identical results. More importantly, the overall conclusion is that it is necessary that all samples subjected to comparative analysis should be prepared identically because although changes are minute, they are present.

Molecular pathology via IR and Raman spectral imaging

“…After mounting tissue sections on appropriate substrates, the samples are air dried, which can be considered an effective way of fixation because the samples are resistant toward further degradation and remain virtually unaffected for days or weeks [48,49]. On the other hand, dehydration causes denaturation of biomolecules such as lipids, proteins and nucleic acids.…”

Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells

“…Without live-cell or well-fixed reference spectra the IR spectra of MeOH and 8% PFA fixed cells would not immediately be identifiable as erroneous despite the cells under investigation being significantly damaged by the fixation. Previously it has been argued that fixation induced effects are inherently reproduced sample-wide and are not significant enough to confound diseasestate changes or other differences being examined 43,44 . However, we have shown here that differences between experiments arise even when the same optimized fixation protocol is carried 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 out by the same individual, on the same day, in the same lab, thus demonstrating the irreproducibility of fixation due to the sheer number of variables at play.…”

Can mid-infrared biomedical spectroscopy of cells, fluids and tissue aid improvements in cancer survival? A patient paradigm

“…[27][28][29] Now, however, high definition (HD) IR imaging is more routinely possible on a bench-top FTIR spectrometer, achieving a pixel resolution of ∼1.1 μm using a standard thermal source. 30,31 For some clinical applications, a pixel resolution of ∼5.5 μm would be appropriate for cytological screening when isolating single cell spectra from cultured cells as demonstrated by Mazur et al 32 and Filik et al 33 However, for instances where there may be a clinical requirement to isolate the IR signature of single cells condensed within a tissue cross-section where cells therefore appear smaller in magnitude, increasing the effective pixel size is of use to more accurately resolve the cells spatially and to reduce 'pixel mixing' effects of neighbouring tissue structures.…”