# Raman Spectroscopy of Individual Cervical Exfoliated Cells in Premalignant and Malignant Lesions
## 3 Results
When carrying out the discriminant analysis (CAP, Figure 3), similar predictive power or correct classification of the samples in healthy and CIN patients was obtained for both the cytoplasm (76.5%, \(\delta\) = 0.567, \(p\) = 0.0002) and the nucleus (82.7%, \(\delta\) = 0.767, \(p\) = 0.0001). For the cytoplasmic samples, of the total number of patients who were true negatives (29 healthy from the histopathology) in the healthy group, Raman spectroscopy identified 23, 79.3% of correct classification from the CAP analysis. In contrast, 13 patients of 52 from the CIN were classified by CAP analysis as (false) negatives (incorrectly classified as healthy). From the histopathology results, some samples had some degree of lesion. Overall, the percentage of incorrect classification was 23.5%. Conversely, the analysis of the nuclei showed successes and errors in both healthy and CIN patients in their derived taxonomy. Of the 29 healthy patients, 6 were classified as affected (79.3% correct); as for the patients with CIN, 8 were incorrectly classified as healthy (15.4%), while 44 of 52 patients with CIN (results according to histopathology) were correctly classified with the presence of a cervical lesion (84.6%). The results are shown in Table 2. We include a comparative analysis with other reports in Supplemental Material. Figure 2: Metric Multidimensional Scaling (MDS) plots and bootstrap averages of biochemical patterns. Samples of healthy patients (H, blue) and CIN patients (CIN, red). (**A**) Cytoplasmic analysis and (**B**) nuclear analysis. CIN = cervical lesion. Dots represent centroids and areas 95% confidence level. If the patients with the disease are classified into subgroups based on the different degrees of the cervical lesion (CIN1, CIN2, and CIN3/CIS) and the healthy patients are subdivided into two subgroups: no HPV detected and those with viral latency (presence of HPV but no abnormalities in cervical tissue), higher intensity is observed in bands corresponding to proteins, lipids, and nucleic acids in patient samples presenting cervical lesions (CIN) compared to bands of the Raman spectra of healthy patients and those with viral latency. Similar results were obtained for both the Raman spectra obtained from the cytoplasm and the nucleus, Figure 4A,B. \begin{table}
\begin{tabular}{c c c c c c} \hline  & **Group** & **Healthy** & **CIN** & **Total** & **Raman Classification Score \%** \\ \hline \multirow{3}{*}{Cytoplasm} & Healthy & 23 & 6 & 29 & 79.3 \\ \cline{2-6}  & CIN & 13 & 39 & 52 & 75.0 \\ \cline{2-6}  & \multicolumn{4}{c}{Overall classification efficiency 62/81 76.5\%} \\ \cline{2-6}  & \multicolumn{4}{c}{Incorrect classification 23.5\%} \\ \hline \multirow{3}{*}{Nucleus} & Healthy & 23 & 6 & 29 & 79.3 \\ \cline{2-6}  & CIN & 8 & 44 & 52 & 84.6 \\ \cline{2-6}  & \multicolumn{4}{c}{Overall classification efficiency 67/81 82.7\%} \\ \cline{2-6}  & \multicolumn{4}{c}{Incorrect classification 17.3\%} \\ \hline \end{tabular}
\end{table} 
Table 2: Model classification and cross-validation of Healthy and CIN samples from CAP analysis. Figure 3: Canonical analysis of principal coordinates (CAP) of biochemical patterns. Samples of healthy patients (H, blue triangles) and CIN patients (CIN, red triangles). (**A**) Cytoplasmic analysis (\(\delta\) = 0.567, \(p\) = 0.0002) and (**B**) nuclear analysis (\(\delta\) = 0.767, \(p\) = 0.0001). HC = Health condition. CIN= cervical lesion. Euclidean distance-based analysis. The bands with differences in intensity between groups correspond to protein and lipid components, like collagen and amide (Raman bands at 1320, 1396, 1440, and 1546 cm\({}^{-1}\)). Other essential differences are presented for DNA/RNA components such as cytosine, guanine, adenine, and thymine in the Raman bands 1320 and 1603 cm\({}^{-1}\). Therefore, it is necessary to increase the number of samples for each study group to obtain a better comparison and thus to be able to establish a fingerprint for healthy patients and another for different degrees of cervical lesions.