Stone N, Stavroulaki P, Kendall C, Birchall M, Barr H
Cranfield Postgraduate Medical School, Gloucestershire Royal Hospital, Gloucester, United Kingdom.
Laryngoscope. 2000 Oct;110(10 Pt 1):1756-63. doi: 10.1097/00005537-200010000-00037.
Raman spectroscopy, the analysis of scattered photons after monochromatic laser excitation, is well established in nonbiological sciences. Recently this method has been used to differentiate premalignant and malignant lesions from normal tissue. Its application for early diagnosis has been explored in a variety of sites (e.g., esophagus, cervix), but not, to date, in laryngeal cancer. The objective of this study was to perform a feasibility study of the use of Raman spectroscopy for early diagnosis of laryngeal malignancy.
Biopsy specimens were snap-frozen, and top sections were sent for histopathological analysis. Only homogenous samples with clearly defined pathological findings were used in this study: seven histologically normal samples, four exhibiting dysplasia, and four with carcinoma. Samples were defrosted and placed under a Renishaw (Wotton-Under-Edge, UK) System 1000 Raman microspectrometer for analysis. Between 5 and 12 spectra were acquired from each sample, with an excitation wavelength of 830 nm. Average characteristic spectra for each disease or condition were compared. Further multivariate statistical analysis of the data was carried out to evaluate and maximize the differences in the spectra for each disease or condition.
The most visible differences in the spectra occur between 850 and 950 cm(-1) and 1,200 and 1,350 cm(-1). The later peaks are directly related to protein conformation and C-H bond stretch in nucleic acid bases. The relative intensity of the nucleic acid peak increases with progression to malignancy. Use of linear discriminant analysis made it possible to separate the spectra with disease to a greater degree of accuracy than using empirical peak ratio methods alone. Classification results obtained from cross-validation of the discriminant model showed prediction sensitivities of 83%, 76%, and 92% and specificities of 94%, 91%, and 90% for normal, dysplastic, and squamous cell carcinoma of the larynx, respectively.
There was strong evidence to support spectral identification of malignancy and earlier abnormal changes. More substantive studies of the spectral differences between malignant and non-neoplastic tissue are warranted. Raman spectroscopy may become a useful adjunct to pathological diagnosis allowing directed or guided biopsies and assessment of adequacy of resection margins.
拉曼光谱法是一种在单色激光激发后对散射光子进行分析的技术，在非生物科学领域已得到广泛应用。近年来，该方法已被用于区分癌前病变和恶性病变与正常组织。其在早期诊断中的应用已在多种部位（如食管、宫颈）进行了探索，但迄今为止尚未应用于喉癌。本研究的目的是对拉曼光谱法用于喉恶性肿瘤早期诊断进行可行性研究。
活检标本速冻后，取顶部切片进行组织病理学分析。本研究仅使用病理结果明确的均匀样本：7个组织学正常样本、4个显示发育异常的样本和4个癌样本。样本解冻后置于雷尼绍（英国沃顿-埃奇）1000型拉曼显微光谱仪下进行分析。每个样本采集5至12个光谱，激发波长为830nm。比较每种疾病或状况的平均特征光谱。对数据进行进一步的多变量统计分析，以评估并最大化每种疾病或状况光谱之间的差异。
光谱中最明显的差异出现在850至950cm⁻¹以及1200至1350cm⁻¹之间。后一组峰与蛋白质构象以及核酸碱基中的C-H键伸展直接相关。核酸峰的相对强度随着恶性程度的进展而增加。使用线性判别分析比单独使用经验峰比方法能更准确地将不同疾病的光谱区分开来。判别模型交叉验证得到的分类结果显示，对于喉的正常组织、发育异常组织和鳞状细胞癌，预测敏感性分别为83%、76%和92%，特异性分别为94%、91%和90%。
有充分证据支持通过光谱识别恶性肿瘤及早期异常变化。有必要对恶性组织与非肿瘤组织之间的光谱差异进行更深入的研究。拉曼光谱法可能成为病理诊断的有用辅助手段，有助于进行定向或引导活检以及评估切除边缘的充分性。
