## 4 Discussion
[37] has reported that among severe patients, the improved subgroup has a T lymphocyte count gradually back to a normal range within 15-25 days, while that of the dead subgroup keeps falling until death. These highlighted the need of monitoring CD4\({}^{+}\) T cell counts in the treatment of COVID-19 patients having severe symptoms. Therefore, we consider that doctors can still refer to test results using the MACS-Countess method to make treatment decisions, or use this method for screening purposes. In terms of accuracy, MACS-Countess showed high agreement with the gold standard FACS method with a mean bias of 3.5% and LoA from \(-\)36.4% to 43.3%. This is comparable to the commercially launched product, PIMA analyzer, which has a mean bias of 0.2% and LoA from \(-\)42% to 42% in the field setting evaluation (with FACS as the reference method). Compared with the PIMA analyzer, MACS-Countess has slightly poorer sensitivity, resulting in a higher misclassification rate. This is reasonable as PIMA tends to underestimate the cell count with an MPS of 96.37% compared with 102.3% for MACS-Countess. Therefore, the upward misclassification rate of MACS-Countess can be reduced once the MPS is less than 100%. In practice, many other non-flow cytometric platforms tend to underestimate the CD4\({}^{+}\) T cell counts. Underestimation would decrease the upward misclassification while increasing the downward misclassification, which will benefit the earlier initiation of ART. [PERSON] et al. reported that Dynabeads (Dynal T4 Count, Dynal Biotech, Oslo, Norway) have a good sensitivity of 94% at a cut-off of \(<\)350 cells/\(\mu\)L. The specificity, however, is poor at 69% as Dynabeads highly underestimate the cell count, as illustrated by a low slope of 0.85 and the absolute mean difference (FACS--Dyna) of 8.8 cells/\(\mu\)L [39]. In the same report, the FDA-approved Cytospheres (CD4 Manual Count, Beckman Coulter, CA, USA) also repeat the same pattern with a high sensitivity yet low specificity, very low slope, and high absolute difference compared with the reference method at 99%, 32%, 0.58, and 58.6 cells/\(\mu\)L, respectively [39]. Conversely, a high positive bias leads to low sensitiity, typically illustrated by PointCare NOW (PointCare), also an FDA-approved platform [40], with a mean relative bias of 35% and a mean sensitivity of only 53% [41]. Based on these analyzed data, the MACS-Countess can be considered an alternative simple method for diagnostic purposes and is affordable for limited resource areas where expensive conventional FACS methods are unavailable. To enable cell count by FACS, a center has to invest at least USD 27000 to buy a cytometer. In addition, the complexity of the fluidic system and optical system of the cytometer restricts its use to situations where the support of well-qualified technicians is present. In contrast, the MACS-Countess is very easy to access as the system requirements simply include: (i) a strong magnet for placing 1.5 mL microcentrifuge tubes that cost only several hundred US dollars, and (ii) an automated cell counter of only about USD 5000-6000, both of which are cost-effective and affordable for hospitals with limited resources. It is estimated that a CD4\({}^{+}\) T cell counting test with MACS-Countess would cost less than USD 10 (the cost of 25 \(\mu\)L NP-CD4 accounts for USD 5). Meanwhile, the cost of a test based on the FACS method includes fluorescent-labeled antibodies, Trucount tubes, the fluid needed to run the cytometer machine (shutdown solution, clean solution, etc.) and cytometer maintenance and calibration, which is estimated to range roughly from USD 18--on the FACSCount system--to USD 30-40 on the FACSCalibur/FACSCanto system (these costs were estimated based on the import prices of reagents and consumables into Vietnam, which may not be universal).