Department of Chemistry & Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts, USA.
Anal Chem. 2012 Oct 2;84(19):8265-71. doi: 10.1021/ac3017407. Epub 2012 Sep 11.
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.
光谱细胞病理学（SCP）是一种强大且可重复的诊断技术，它采用红外光谱和多元统计方法（如主成分分析）来检测未染色的细胞样本，并在生化水平上区分变化。在过去的十年中，SCP 在疾病诊断中的应用取得了长足的进步。培养的细胞系已被证明是提供该领域详细生物学信息的有用模型系统；然而，在 SCP 中，细胞培养物的固定和储存对其的影响仍不完全清楚。传统的细胞病理学利用已经建立并广泛接受近一个世纪的固定和染色方法，专注于保持细胞的形态。相反，SCP 实践必须实施固定方案，以保留样本的生化组成并保持其光谱完整性，以免引入可能掩盖与疾病相关的差异的光谱变化。不仅需要评估对固定的脱落细胞的影响，还需要评估对固定培养细胞的影响，因为尽管它们是相似的系统，但它们表现出明显的差异。我们报告了研究传统细胞病理学和 SCP 中常用固定方法的影响的努力，包括应用于贴壁宫颈癌细胞系 HeLa 细胞的固定和未固定常规。数据表明与该系列第 1 部分中脱落细胞的发现平行，其中固定剂中的暴露时间和通过干燥储存样品的持续时间仅导致较小的光谱变化。这里呈现的结果加强了第 1 部分的观察结果，表明由疾病引起的变化远大于由于替代固定方法观察到的变化。通过与脱落细胞（第 1 部分）相同的条件和方案固定的 HeLa 细胞的主成分分析得出几乎相同的结果。更重要的是，总体结论是，必须对所有进行比较分析的样本进行相同的制备，因为尽管变化很小，但它们确实存在。
