Label-free microfluidic small-volume blood processing for analysis of platelet activation and neutrophil–platelet aggregates

Platelets and leukocytes are central regulators of thrombo-inflammatory processes, and their activation states and interactions provide important insights into disease progression and therapeutic response. However, conventional blood processing relies on centrifugation-based workflows that are labor-intensive, require large sample volumes, and can induce artefactual platelet activation or disrupt native cell-cell interactions. These limitations reduce translational relevance, particularly for applications requiring small blood volumes or longitudinal sampling. Here, we present a two-stage, label-free microfluidic workflow based on Dean Flow Fractionation (DFF) for isolating and reconcentrating functionally intact platelets and leukocytes from whole blood. The platform integrates spiral inertial fractionation with a downstream serpentine channel for on-chip leukocyte reconcentration without off-chip processing. The device is fabricated in poly(methyl methacrylate) (PMMA) to enhance robustness and scalability for translational use. Performance was validated using human blood samples from healthy donors and fever patients, as well as a streptozotocin-induced diabetic mouse model. The workflow achieved approximately 70-80% leukocyte recovery and 45% platelet recovery from 100 μL of whole blood within five minutes. Importantly, platelet quiescence was preserved relative to centrifugation, and platelet-leukocyte aggregates were maintained. The platform supports downstream functional analyses including flow cytometry and digital holographic microscopy. In mouse studies, small-volume sampling enabled longitudinal monitoring of platelet activation and neutrophil-platelet interactions without terminal blood collection. Together, this work establishes a practical microfluidic workflow for functional platelet and leukocyte analysis from limited blood volumes.