A Simple Approach for Counting CD4(+) T Cells Based on a Combination of Magnetic Activated Cell Sorting and Automated Cell Counting Methods

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(TM), an automated cell-counting system. Using the cell counting protocol, 25 mu L anti-CD4 conjugated magnetic nanoparticles (NP-CD4, BD Bioscience) were optimized for separating CD4(+) T cells from 50 mu L of blood in PBS using a Dynamag(TM)-2 magnet, followed by the introduction of 10 mu L separated cells into a Countess(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/mu 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.