# Raman Spectroscopy of Individual Cervical Exfoliated Cells in Premalignant and Malignant Lesions
## 4 Discussion
The percentage of incorrect classification provided by the SIMPER analysis in this study was attributed to \"healthy patients\" with low-grade cervical lesions or CIN1, a stage where there are still no marked differences at the molecular/cellular level compared with the present non-affected cell. Nonetheless, good classification power was achieved for samples from patients with CIN, with good sensitivity (84.6%, nucleus). It is important to note that Raman spectroscopy included in the multivariate analysis (PERMANOVA, SIMPER, and CAP analysis) of the nuclear spectra observed from healthy and CIN patients resulted in a specificity of 79.3% and a sensitivity of 84.6% (Table 2). In addition, the predictive power obtained from the Raman measurements of both the cytoplasm and the nucleus (76.5% and 82.7%, respectively) is similar to those previously reported in studies conducted on cell pellets (~79%). Comparing the Raman spectra by separating the samples into five groups, healthy, viral latency, CIN1, CIN2, and CIN3/CIS, there is a corresponding increase in the intensity in bands of samples from cervical lesion compared to the spectra of healthy patients and with viral latency. Significant differences were found in the intensity of some bands in the spectra from samples with cervical lesions compared to the spectra of healthy samples and those with viral latency. Furthermore, this increase in the intensity of some bands seems to be proportional to the degree of a cervical lesion, being more intense in CIN2 and CIN3. The spectra corresponding to viral latency in Figure 4A,B are very similar to that of healthy samples. The objective of separating the spectra of healthy samples from those of viral latency was to determine if in the viral latency state there would be cellular changes due to the viral action that could be expressed in the Raman spectrum even when no morphological cellular modifications were observed through cytological diagnostic methods. However, even when the virus is present, it does not necessarily cause visible damage to cervical tissue. As we show in these figures, no significant spectral differences were observed when comparing the spectra of samples from healthy patients with those of viral latency (Figure 4A,B). In this study, we observe differences in band intensities in Raman spectra of patients with a cervical lesion, particularly in high-grade lesion samples, compared to healthy patients. Additionally, the potential of the Raman spectroscopy technique to correctly classify cytology or cervical exfoliation samples into healthy patients and CIN patients, that is, with some degree of the cervical lesion, was evaluated. The collection and analysis of the Raman spectra used to assess the cytoplasm and nucleus of single cervical cell preparations were performed on 81 samples. The sensitivity and the classification power were 75% and 76.5%; respectively, according to the PERMANOVA and SIMPER analysis of the Raman spectrum bands obtained from the cytoplasm, and 84.6% and 82.7%, respectively, according to the examination of the spectra of the cell nucleus. These results were similar to the sensitivity reported for the Pap test (74-96%) and the classification efficiency of Raman studies on cell pellets (~79%). In addition, Raman spectroscopy showed good sensitivity (84.6%), which would encourage its application in the future as a support method to existing diagnostic approaches for the detection of cervical lesions, thus reducing the false-negative rate and avoiding delays in diagnosis and therefore in treatment and prognosis. The application of this technique in the detection of CINs and cervical cancer would have the advantage of being carried out on cervical exfoliation, which is a type of sample that is obtained routinely in the initial screening in the diagnosis; therefore, it is possible to perform the two studies, Papanicolaou and Raman measurements, from a single sample.