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

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

Keywords

• counting CD4⁺ T cells
• magnetic activated cells sorting (MACS)
• Countess™
• fluorescence activated cell sorting (FACS)
• anti-CD4
• magnetic nanoparticles