## 3 Results
### Comparison between MACS-Countes to FACS Methods
#### 3.3.2 MACS-Countess Compared with FACS  
After determining the STCs and operation scheme of the MACS-Countess system, we performed CD4+ T cell counting using the MACS-Countess system on 15 samples, including 9 samples with CD4+ T of \(>\)200 cells/ uL (belonging to groups I + II) and the other six samples of 100-200 cells/ uL (belonging to group III). The original CD4+ T cell counts in the blood of each group were deduced from the experimental values using the corresponding STCs, as mentioned above. CD4+ T cell counts by MACS-Coutness and their deduced values, CD4+ T cell counts by FACS, and the bias of the deduced values compared with the FACS values in each sample group are listed in Table 1. We found that the biases ranged from \(-\)34.8% to 32.7% in group I + II and from \(-\)25.1% to 29.3% in group III. By adding 33 samples used for generating STCs (38 samples excluding 5 samples with blood concentrations of \(<\)100 cells/ uL) to these 15 samples, a total of 48 samples was used for deducing the CD4+ T cell counts in blood based on the MACS-Countess method (in brief, deduced MACS-Countess) to evaluate the reliability of this method compared with FACS (in brief, FACS). Linear regression showed that the deduced MACS-Countess had a good correlation with FACS, with an R2 equals to 0.86, slope of 0.96, and y-intercept of 22.3 (Figure 5a). We also compared the agreement of the deduced MACS-Countess with the PIMA analyzer, which is a commercially launched product for CD4+ T cell counting, using FACS as a reference. It has been reported that PIMA is comparable to conventional FACS with a mean bias of 0.2% and LoA from \(-\)42% to 42% in the field setting evaluation [13]. Meanwhile, the mean bias of the deduced MACS-Countess was 3.5% and LoA ranged from \(-\)36.4% to 43.3% (Figure 5b). The mean percentage similarity (MPS) of MACS-Countess was 102.9% with a coefficient of variance (CV) equaling 10.6% (Figure 5c), which is slightly different from that of PIMA with an MPS of 96.4% and CV of 13.1% [14]. The mean bias at cut-off \(<\)350 cells/ uL of MACS-Countess was 6.4%, which was not significantly different from that at the cut-off of \(<\)500 cells/ uL (5.4%, \(p<0.05\)), suggesting that a goodcell count performance is ensured at both low and high cell concentrations. The MPS of MACS-Countess at the two thresholds were 104.3% and 103.9%, respectively, and were not significantly different (\(p<0.05\)). Sensitivity, specificity, PPV, NPV, and misclassification rates were calculated to better understand the diagnostic results (Table 2). MACS-Countess deduced a good specificity of 94.7% at a cut-off of 350 cells/\(\mu\)L; however, the sensitivity was only 89.7%. This led to  
Figure 4: MACS-Countess operation and analysis scheme for CD4\({}^{+}\) T cell counting. The 50 \(\mu\)L of blood to be tested is transferred to a new 1.5 mL microcentrifuge tube together with the addition of 25 \(\mu\)L NP-CD4 and 450 \(\mu\)L PBS\({}^{+}\). After 30 min of incubation at room temperature, the sample is then washed twice each with 1 mL PBS on the DynamagTM-2 magnetic stand and is finally resuspended in 50 \(\mu\)L PBS. Then, 10 \(\mu\)L of the separated sample is introduced in the CountlessTM slide and counted on the cell counter machine (Countess). For deducing the original CD4\({}^{+}\) T cell count value: a Countess count of \(>\)200 cells/\(\mu\)L is applied to STC (I + II): y = 1.15x + 35.87, Countess count of \(\leq\)200 cells/\(\mu\)L is applied to STC (III): y = 0.4x + 90.27. Figure 5: CD4\({}^{+}\) T cell counts by the deduced MACS-Countess and FACS methods. (**a**) Deduced MACS-Countess (\(x\) axis) correlates with FACS (\(y\) axis) at R\({}^{2}\) = 0.86. (**b**) Bland–Altman plot with a mean bias of 3.5%, LoA ranging from \(-\)36.4% (Mean bias \(-\)1.96 SD) to 43.3% (Mean bias +1.96 SD). (**c**) Percentage similarity with MPS of 102.9% and CV of 10.6%. an upward misclassification rate of 10.3% and a downward misclassification rate of 5.3%.