###### Abstract  
Frequent tests for CD4\({}^{+}\) T cell counting are important for the treatment of patients with immune deficiency; however, the routinely used fluorescence-activated cell-sorting (FACS) gold standard is costly and the equipment is only available in central hospitals. In this study, we developed an alternative simple approach (shortly named as the MACS-Countess system) for CD4\({}^{+}\) T cell counting by coupling magnetic activated cell sorting (MACS) to separate CD4\({}^{+}\) T cells from blood, followed by counting the separated cells using Countess\({}^{\text{TM}}\), an automated cell-counting system. Using the cell counting protocol, 25 \(\upmu\)L anti-CD4 conjugated magnetic nanoparticles (NP-CD4, BD Bioscience) were optimized for separating CD4\({}^{+}\) T cells from 50 \(\upmu\)L of blood in PBS using a Dynamag\({}^{\text{TM}}\)-2 magnet, followed by the introduction of 10 \(\upmu\)L separated cells into a Countess\({}^{\text{TM}}\) chamber slide for automated counting of CD4\({}^{+}\) T cells. To evaluate the reliability of the developed method, 48 blood samples with CD4\({}^{+}\) T cell concentrations ranging from 105 to 980 cells/\(\upmu\)L were analyzed using both MACS-Countess and FACS. Compared with FACS, MACS-Countess had a mean bias of 3.5% with a limit of agreement (LoA) ranging from \(-36.4\%\) to 43.3%, which is close to the reliability of the commercial product, PIMA analyzer (Alere), reported previously (mean bias 0.2%; LoA ranging from \(-42\%\) to \(42\%\), FACS as reference). Further, the MACS-Countess system requires very simple instruments, including only a magnet and an automated cell counter, which are affordable for almost every lab located in a limited resource region. c 2021 11 9786 2021  
by which CD4\({}^{+}\) T cells are lost during SARS-CoV infection are direct invasion [6], T cell exhaustion [7; 8] or apoptosis [9], etc. Usually, CD4\({}^{+}\) T cell counting is performed on a FACS system, also known as the gold standard method for cell sorting and enumeration. The key mechanism of cell sorting by FACS is that fluorescence-labeled antibodies recognize and bind specifically to markers expressed on cells; the cells are then passed through single-cell flow and fluorescence signals are detected using a laser beam. To count CD4\({}^{+}\) T cells, fluorescence-labeled anti CD4 antibodies are used to recognize the CD4 markers on targeted cells. Though the CD4 marker is typical for CD4\({}^{+}\) T cells, other cell types such as dendritic cells and monocytes also express CD4 but at lower levels [10]. Therefore, to confirm whether the separated cells are actually CD4\({}^{+}\) T cells, a mixture of distinctive fluorescent dyes conjugated with CD4 and CD3 monoclonal antibodies is usually used for targeting the corresponding markers on CD4\({}^{+}\) T cells, which do not co-appear on other cells expressing CD4 alone. For diagnostic purposes, many factors are of great concern. The first and utmost important is the result reliability, followed by a short time for obtaining results as well as low cost, and availability. The FACS method has excellent reliability [11], but is rather complicated and thus requires technicians who are properly trained. Further, the high cost of reagents and maintenance is an additional cause leading to the unavailability of FACS in limited resource regions. Thus, a system that can perform CD4\({}^{+}\) T cell counts, which is fast, cheap, reliable, and easy to handle, is urgently needed. Several commercial point-of-care (POC) products for counting CD4 are already available including the CyFlow miniPOC (Partec), Pima analyzer (Alere), and CD4 Counter (Daktari). The frequently utilized technology for cell counting in these systems is the fluorescence-based method; for example, PIMA uses disposable cartridges which enable fluorescence labeling of CD4\({}^{+}\) T cells, followed by signal collection to discriminate labeled cells from others [12].