## 3 Results and Discussion
[17]; in particular, they found that three types of cells (two from cancer cell lines and one from peripheral blood) deposited on coverslip can be discriminated by PC1 component of PCA technique. Instead, we have shown that such a discrimination is also possible for different types of cells grown on glass slides, even with a larger separation grade with respect to coverslip. In addition, the spectra in Reference [17] were obtained with Synchrotron based FTIR, which involves an infrared beamline as synchrotron light source. Such beamlines are large systems mainly located away from hospitals, where diagnostic analyses are performed. In our case, FTIR measurements were carried out by means \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Substrate** & \begin{tabular}{c} **Average PC1** \\ **(HUKE)** \\ \end{tabular} & \begin{tabular}{c} **Average PC1** \\ **(SH-SYSY)** \\ \end{tabular} & \
\begin{tabular}{c} **Statistical** \\ **Significance** \\ \end{tabular} &
\begin{tabular}{c} **Separation** \\ **Grade** \\ \end{tabular} \\ \hline CaF\({}_{2}\) & \(0.06\pm 0.03\) & \(0.26\pm 0.08\) & \(p<0.001\) & \(0.32\pm 0.11\) \\ low-e & \(0.12\pm 0.04\) & \(-0.11\pm 0.03\) & \(p<0.001\) & \(0.23\pm 0.07\) \\ glass & \(-0.12\pm 0.08\) & \(0.06\pm 0.04\) & \(p<0.001\) & \(0.18\pm 0.12\) \\ coverslip & \(0.07\pm 0.11\) & \(-0.03\pm 0.09\) & \(p<0.001\) & \(0.10\pm 0.20\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Average values of PC1 for HUKE (second column) and SH-SYSY (third column) cells grown on different substrates (first column). Statistical significance of separation of PC1 values for the two types of cells is reported in the fourth column, whereas the fifth column shows the difference of average PC1 values. Figure 2: PCA score plot for the 2750–3000 cm\({}^{-1}\) spectral range for HUKE (red circles) and SH-SYSY (black circles) cells grown on CaF\({}_{2}\) (**a**), low-e (**b**), glass (**c**), and coverslip (**d**) substrates. of an apparatus consisting of an IR microscope coupled to a FTIR interferometer; it can be easily located as a facility in a hospital setting. It is important to investigate whether univariate analysis can give information similar to those obtained by PCA about differences in the spectra of the two types of cells. Since the main differences between the HUKE and SH-SYSY spectra occur for the relative intensity of peaks at 2956 and 2924 cm\({}^{-1}\), we examined the intensity ratio I\({}_{2956/2924}\) of these two peaks for the two types of cells. Because of the attribution of such peak, I\({}_{2956/2924}\) is proportional to the relative content of proteins with respect to lipids in the investigated sample. Figure 3 shows the average values of I\({}_{2956/2924}\) for the two cell lines grown on the investigated substrates; for each substrate, such a value results in being larger for SH-SYSY cells than for HUKE cells. The difference of average values is statistically significant in all cases, although it is larger for cells grown on low-e slides with respect to the other substrates. In addition, the distribution of I\({}_{2956/2924}\) values results quite broad in the case of SH-SYSY cells grown on CaF\({}_{2}\) substrate. The differences between the average values of I\({}_{2956/2924}\) for the two cell lines are reported in Table 2. It is evident that the separation between HUKE and SH-SYSY cells was statistically significant, regardless of growth substrate, although low-e and glass substrates are able to provide large separation capability. These results shown in Figure 3 suggest that the lipid content, assessed with respect to the protein content, is larger in HUKE cells than in SH-SYSY cells. It is not in contrast with the well-known fact that lipid metabolism and lipogenesis increases in cancerous cells with respect to normal ones [24]. In fact, I\({}_{2956/2924}\) is proportional to the protein/lipid ratio content in two different cells type, which are