Flow-Stabilized Microfluidic Sensing of Thermally Induced Changes in Red Blood Cell Aggregation During Closed-Loop...

Red blood cell (RBC) aggregation is a sensitive indicator of hemorheological alterations, but conventional methods generally rely on intermittent or end-point measurements. This study presents a flow-stabilized closed-loop microfluidic platform for continuous measurement of the RBC aggregation index (AI) during controlled circulation and thermal exposure. The system integrates a peristaltic pump, hydraulic flow stabilizer (FS), microfluidic aggregation sensor, and thermal mixer to suppress flow pulsation and RBC sedimentation. The FS increases the characteristic flow-response time by approximately 17.6-fold, markedly attenuating pump-induced fluctuations. Under stabilized conditions, flow rates in the main channel and test chamber remain highly linear with the imposed flow rate, while stable AI measurements are maintained for at least 30 min. The platform also resolves dextran-dependent changes in RBC aggregation during continuous circulation. During thermal exposure from 25 to 47 °C, main-channel image intensity (I_mc) and AI remain nearly constant up to 43 °C, whereas exposure at ≥45 °C causes a progressive increase in I_mc and a marked decrease in AI. These results demonstrate continuous detection of thermally induced changes in RBC aggregation at a fixed measurement temperature of 25 °C without interrupting circulation and provide a versatile framework for investigating progressive changes in aggregation behavior under controlled and reproducible in vitro conditions.