Quantitative Magnetic Flow Cytometry in High Hematocrit Conditions for Point-of-Care Testing

Abstract

Quantitative cell analysis in liquid biopsies is essential for many clinical decisions, but it is primarily tied to centralized laboratories. However, access to these laboratories is limited in low-resource settings or for immobile patients, highlighting the urgent need for Point-of-Care (POC) testing infrastructure. Magnetic flow cytometers (MFC) offer a solution, albeit sample processing steps like cell lysis or washing crucially disrupt POC-capable MFC workflows. Here, we investigate conditions for immunomagnetic labeling and direct cell quantification in a streamlined workflow suitable for high hematocrit environments. Magnetic nanoparticles (MNP) are characterized by their size, magnetic moment, and potential to generate signal noise, favoring small (< 50 nm) MNPs. Theoretical models provide the framework for quantifying bound MNPs per cell, revealing labeling quality and giving insight into system requirements for reliable cell detection. Temporal labeling dynamics show suboptimal binding kinetics in whole blood (WB), leading to long incubation periods and only 50% recovery of optically determined concentrations. Besides showing quantitative MFC in WB with biomimetic microbeads, we finally quantify CD14+monocytes in WB with our streamlined workflow, achieving an intra-assay coefficient of variation (CV) of 0.11 and a CV across multiple donors of 0.10, demonstrating reliable POC flow cytometry close to regulatory standards.