Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

拉曼光谱可以提供样品化学指纹的快速、无标记、无损测量，并显示出用于癌症筛查和诊断的潜力。在这里，我们报告了对不同脱落细胞学样本（宫颈、口腔和肺）进行拉曼显微光谱分析的协议，涵盖样本制备、光谱采集、预处理和数据分析。该协议需要 2 小时 20 分钟进行样品制备、测量和数据预处理，最多需要 8 小时进行完整分析。该协议的一个关键特征是它使用与诊断细胞学实验室（即载玻片上的液基细胞学）相同的样品制备程序，确保与临床工作流程的兼容性。我们的协议还涵盖了校正玻璃光谱贡献的方法和样品预处理方法，以去除可能掩盖脱落细胞光谱特征并导致变异的污染物（如血液和粘液）。该协议建立了一个标准化的临床程序，允许收集高度可重复的拉曼光谱细胞病理学数据，用于宫颈癌和肺癌的癌症诊断应用以及监测口腔癌的可疑病变。