Inertial Microfluidics Enables Functional Analysis of Neutrophils Isolated from Ultralow Blood Volume Samples

Monitoring immune cell function is increasingly being recognized as a more relevant biomarker than traditional white blood cell counts, yet the need for repeated relatively large blood volumes still continues to pose a significant challenge. To overcome this, we developed a sample-sparing platform using inertial microfluidics that can process as little as 10 μL of blood to isolate leukocytes for downstream functional analysis. Our platform isolates leukocytes with ∼80% purity, >90% in-device recovery, and >95% viability. Neutrophils were our primary focus due to their sensitivity to external stimuli and their critical role in immune responses. Neutrophils isolated through our new method did not show inadvertent activation, as evidenced by unchanged expression of activation markers CD62L and CD11b, with phenotypes comparable to control cells in whole blood. We conducted a range of functional assays, including phagocytosis, ROS production, and NETosis with all tests confirming that neutrophils maintained their functionality after isolation. These assays were performed using standard laboratory workflows, demonstrating the platform's compatibility with techniques such as flow cytometry and cell culture assays. Furthermore, we showed the versatility of our platform by successfully isolating leukocytes from challenging samples, including mouse blood from the vena cava or tail vein, as well as human capillary blood obtained by fingerstick. This adaptability highlights the potential of this platform for clinical and research applications, particularly in frequent immune monitoring or cases where sample volume is limited.