## 3 Results
### Reliability of the qFCM for Viral Titration
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline Samples & \multicolumn{8}{c}{**AFN**} & \multicolumn{8}{c}{**MSEF**} \\ \hline
**Banks** & **AFI** & **MESF** & **AFI** & **MESF** & **AFI** & **MESF** & **Mean** & **SD** & **CV** & **Mean** & **SD** & **CV** \\ \hline B1 & 15.2 & 1929.0 & 17.7 & 1929.0 & 16.1 & 1929.0 & 16.33 & 1.3 & 7.8 & 1929.00 & 0.00 & 0.00 \\ B2 & 36.6 & 8750.0 & 37.8 & 8750.0 & 39.2 & 8750.0 & 37.87 & 1.3 & 3.4 & 8750.00 & 0.00 & 0.00 \\ B3 & 130.0 & 34,864.0 & 133.0 & 34,864.0 & 135.0 & 34,864.0 & 132.67 & 2.5 & 1.9 & 34,860.00 & 0.00 & 0.00 \\ B4 & 432.0 & 127,311.0 & 465.0 & 127,311.0 & 489.0 & 127,311.0 & 4620.00 & 28.8 & 6.2 & 127,311.00 & 0.00 & 0.00 \\ B5 & 1227.4 & 421,992.0 & 1390.0 & 421,992.0 & 1473.0 & 421,992.0 & 1370.23 & 114.2 & 8.3 & 421,992.00 & 0.00 & 0.00 \\ Blank & 107.00 & 24,818.8 & 94.3 & 194,803.0 & 133.0 & 28,911.68 & 111.45 & 19.7 & 17.7 & 24,403.69 & 4729.0 & 19.4 \\ C-MOCK & 139.03 & 33,818.8 & 151.86 & 34,341.5 & 180.3 & 41,054.13 & 156.97 & 21.0 & 13.4 & 34,215.67 & 4017.3 & 11.7 \\ FITC & 1699.80 & 66,255.1 & 1852.74 & 668,078.5 & 1901.67 & 62,937.2 & 1818.07 & 105.3 & 5.8 & 635,525.29 & 20,6147.4 & 3.2 \\ (MFI) & (24,028,067.6) & (25,327,328.0) & (29,859,019.5) & & & & & & & & \\ \hline AFI, arbitrary fluorescence intensity units (geometric mean values): MESF, Molecules of Equivalent Soluble Fluorochrome units; SD, Standard deviation; CV, Coefficient of variation (SD/average \(\times\) 100); C-MOCK, Negative control (Mock infected cells); MFI, Mean fluorescence Intensity of the infected cells. & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: _Cont._ \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline  & \multicolumn{4}{c}{**Inoculated Virus 1**} & \multicolumn{4}{c}{**Assay 1**} & \multicolumn{4}{c}{**Assay 2**} \\ \hline
**MOI** & **TCID\_/m1** & **pfu/mL** & **\%’ Cells 2** & **AFI 3** & **MESF 4** & **MFI 5** & **\%’ Cells 2** & **AFI** & **MESF** & **MFI** \\ \hline
0.1 & \(1\times 10^{6}\) & \(2.6\times 10^{6}\) & 66.4 & 7901 & 206,112 & 13,685,836.8 & 82.6 & 5761 & 241,276 & 19,929,423.9 \\
0.01 & \(1\times 10^{5}\) & \(2.6\times 10^{5}\) & 54.1 & 6641 & 173,314 & 9,376,287.4 & 67.8 & 4154 & 166,502 & 11,288,862.9 \\
0.001 & \(1\times 10^{4}\) & \(2.6\times 10^{4}\) & 13.3 & 6967 & 181,801 & 2,417,953.3 & 23.7 & 1655 & 58,628.7 & 1,389,500.3 \\
0.0001 & \(1\times 10^{3}\) & \(2.6\times 10^{4}\) & 17.6 & 6344 & 165,581 & 2,914,225.6 & 18.6 & 1395 & 48,297.5 & 893,333.3 \\
0.00001 & \(1\times 10^{2}\) & \(2.6\times 10^{2}\) & 18.7 & 136,165 & 3347.12 & 67,211.4 & 2.63 & 874 & 28,418.9 & 74,741.8 \\
0.00001 & \(1\times 10^{1}\) & \(2.6\times 10^{1}\) & 19.7 & 11,188 & 291,618 & 574,487.5 & 2.76 & 963 & 31,723 & 87,555.6 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Reliability of the qFCM for viral titration. On the other hand, a significant correlation (R\({}^{2}\) = 0.9559, assay 1; R\({}^{2}\) = 0.9577, assay 2) was observed between the viral titre and the percentage of infected cells, by a second-degree polynomial regression (y = 2.8979x\({}^{2}\) - 6.7267x + 5466, assay 1; y = 3.3423x\({}^{2}\) - 6.2588x + 4.229, assay 2) (data not shown). More importantly, a clear correlation between viral titres and the MFI values was observed, also by a second-degree polynomial regression (Figure 3). Finally, to compare the limit of detection of qFCM with that of the traditional method involving virus isolation, the same viral dilutions employed in qFCM were subjected to re-isolation in BF-2, observing that re-isolation was not possible from the last dilution (corresponding to 10\({}^{1}\) TCID\({}_{50}\)/mL or 2.6 \(\times\) 10\({}^{1}\) pfu/mL). Figure 3: Reliability of qFCM for viral titration. Second degree polynomial regression between the viral titre (in pfu and TCID\({}_{50}\)) and fluorescence (in MFI). Each data corresponds to a single data point.