## 4 Discussion  
In this research, we used Anti-Human CD4 Particles--DM (NP-CD4, BD Biosciences) for capturing CD4\({}^{+}\) T cells with a specificity higher than 95% from PBMCs sample (Figure 1a,b). Specificity of cell separation from whole blood sample as high as >95% was also confirmed (Figure 2a,b). This high selectivity could be attributed to a higher level of CD4\({}^{+}\) biomarker expression on the surface of CD4\({}^{+}\) T cells than on the surface of other cells, resulting in a stronger interaction affinity of the NP-CD4 with CD4\({}^{+}\) T cells than with monocytes and dendritic cells. The optimal number of anti-CD4 molecules per nanoparticle of NP-CD4 could thus be a major factor in its high specificity. Compared with the conventional method requiring 4 mL blood input, the optimized MACS procedure enables the separation of CD4\({}^{+}\) T cells directly from a small blood volume of 50 \(\mu\)L, making the method friendly to patients. Further, the optimized procedure does not require a centrifugation process, resulting in a considerably shorter time and less labor for performing, while greatly reducing the risk of sample contamination to the environment. The simplicity and small scale of the optimized MACS procedure are also available for further development for other applications, such as integration into microfluidic systems for automated operation. Microfluidic devices specifically for bio-separation have been developed for fractionation of CD34\({}^{+}\) cells [33], rare cancer cells [34], and for capturing bacteria [35]. It is thus obvious that automation could reduce manual work and, therefore, decrease the errors originating from manual operation while increasing batch analysis. This study also provides a reproducible model of applying MACS to diagnosis with a simple cell isolation process, whereas a minimum of complicated instruments is required. Using a similar format, many other diagnostic tests can be developed by simply changing the antibody conjugated to the magnetic nanoparticles. The limit of detection of this assay is estimated to be 100 cells/\(\mu\)L and the assay accuracy deteriorates below this threshold. HIV-infected individuals with cell counts below 200 cells/\(\mu\)L have severe immunodeficiency and are usually susceptible to opportunistic infections. The treatment regimens for these patients in such circumstances are based on the type of opportunistic disease and/or CD4\({}^{+}\) T cell counts mostly at a threshold of 100 cells/\(\mu\)L, as in yalaromycosis, Toxoplasma gondii encephalitis, cryptosporidiumosis, and so on, a threshold of 150 cells/\(\mu\)L such as in histoplasmosis, or higher [36]. This LOD threshold is also sufficient enough to aid to assess the immune status of patient with severe SARS-CoV-2 infection because they usually have significantly lower CD4\({}^{+}\) T cell counts (<200 cells/\(\mu\)L) than for mild-to-moderate group \begin{table}
\begin{tabular}{l c c c c c c c} \hline \hline \multirow{2}{*}{**Method**} & \multirow{2}{*}{**Cut-Off**} & \multirow{2}{*}{**Sensitivity**} & \multirow{2}{*}{**Specificity**} & \multirow{2}{*}{**PPV**} & \multicolumn{2}{c}{**Upward**} & \multirow{2}{*}{
\begin{tabular}{c} **Downward** \\ **Misclassification** \\ **Rate** \\ \end{tabular} } \\  & & & & & & \\ \hline MACS-Countess & 350 cells/\(\mu\)L & 89.7\% & 94.7\% & 96.3\% & 85.7\% & 10.3\% & 5.3\% \\  & 500 cells/\(\mu\)L & 89.7\% & 88.9\% & 97.2\% & 66.7\% & 10.3\% & 11.1\% \\ PIMA [29; 30; 31; 32] & 350 cells/\(\mu\)L & 89.6-100\% & 85.7–86.7\% & 91.2\% & 92.3\% & 0–10.4\% & 133.1-14.3\% \\  & 500 cells/\(\mu\)L & 95.5\% & 84.2\% & 76.0–80.2\% & 94.9–96.4\% & 4.5\% & 15.8\% \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Sensitivity, specificity, PPV, NPV, and misclassification rates of MACS-Countess to the reference FASC at cut-off 350 and 500 cells/\(\mu\)L.  
(\(<\)400 cells/\(\mu\)L) [4; 5; 37]. Lower CD4\({}^{+}\) T cell counts also associated with higher mortality during disease treatment [5; 38]. Furthermore, [PERSON] et al.