Advancing magnetic flow cytometry to quantitative epitope analysis in high hematocrit conditions for point-of-care testing

Quantitative cell function measurements are essential for many clinical decisions but are primarily tied to centralized laboratories. Limited access to these laboratories in low-resource settings or for immobile patients highlights the urgent need for Point-of-Care testing (POCT) infrastructure. Magnetic flow cytometers (MFC) offer a solution, albeit phenotyping is limited, and sample processing steps like cell lysis or washing increase MFC's workflow complexity. Here, we investigate conditions for novel phenotyping and direct cell concentration quantification in a streamlined workflow suitable for POCT in high hematocrit environments. We characterize magnetic nanoparticles (MNP) by their size, magnetic moment, and opportunities for high signal-to-noise ratios. With adapted theoretical models, we provide the framework for quantifying bound MNPs per cell. This reveals labeling quality and gives insight into system requirements for reliable cell detection and rational cell phenotyping. We investigate temporal labeling dynamics, which show suboptimal MNP binding kinetics in whole blood (WB), leading to long incubation periods and only 50% recovery concentrations. With our streamlined workflow favoring small (<50 nm) MNPs, we quantify CD14(+) monocytes in WB and achieve coefficients of variation of <11%. By simultaneously assessing quantitative epitope expression, we extend MFC's capabilities to clinical subtyping for POCT.