Comparative Analysis of Sample Loop and Counting Bead-Based Methods for Size-Dependent Bias in Flow Cytometry

Reliable and accurate particle number concentration measurements are essential across various fields, including clinical diagnostics, environmental monitoring, and industrial applications. Flow cytometry is widely used for these measurements, where the use of counting beads is a common approach. However, this method can introduce size-dependent bias when the target particles differ in size from the counting beads. To evaluate size-dependent bias, this study systematically compares the conventional counting bead-based method with a sample loop-based method that relies on total counting with a defined sample volume. Experimental results show that while both methods yield similar concentrations for beads of comparable size, discrepancies arise when there are significant size differences between the counting beads and target particles. To investigate the cause of this bias, simulations based on force balance analysis were conducted, revealing that larger beads experience stronger forces that facilitate their movement toward the detection area, while smaller beads are more influenced by Brownian motion, which impedes their overall motion. These findings provide a mechanistic explanation for the observed size-dependent bias, confirming that differences in hydrodynamic behavior contribute to variations in bead distribution and motion. By using the sample loop method, which minimizes size-dependent bias, and employing an empirical equation derived from the results, this study offers a reliable approach for predicting and mitigating bias in particle concentration measurements. This work therefore contributes to the development of more precise and traceable methods for particle number concentration measurement, with implications for a wide range of biological and industrial applications.