A multi-gradient microfluidic chip-based neutrophil chemotaxis analysis for sepsis auxiliary diagnosis and prognostic monitoring.

Sepsis involves life-threatening immune dysregulation where impaired neutrophil chemotaxis is a critical indicator. We developed a multi-gradient neutrophil chemotaxis analysis microfluidic chip (NCA chip) capable of simultaneously generating triple-chemotactic factor gradients (fMLP, IL-8, and LTB4). Chemotaxis trajectories of neutrophils from healthy individuals (NH = 25) and patients with sepsis (NS1 = 25) were detected, and three parameters, chemotaxis speed (V), chemotaxis index (CI), and number of chemotaxis stops (S) were manually extracted and analyzed. The three parameters were integrated into a multi-gradient-based neutrophil function analysis index (NFA Index). The NFA Index strongly correlated with sepsis severity scores (Sequential Organ Failure Assessment, Acute Physiology and Chronic Health Evaluation Ⅱ), inflammatory markers (Procalcitonin and Brain Natriuretic Peptide). In the NS1 group, the NFA Index showed robust predictive performance for 8-day mortality (AUC = 0.904; cut-off = 9.5; specificity = 92.4%; sensitivity = 75%). In an independent cohort (NS2 = 10), all patients with an NFA Index >9.5 survived, confirmed its clinical prognostic value. Furthermore, the NFA Index exhibited excellent diagnostic efficacy in distinguishing patients with sepsis from healthy individuals (AUC = 0.985; cut-off = 12.5; sensitivity = 92%; specificity = 94.3%), and achieved 100% classification accuracy in a small-sample double-blind validation (NB = 10). Additionally, a deep learning method directly analyzing chemotaxis trajectories diagnosed sepsis with 90% accuracy in the NB, supporting AI-driven immune assessment. The NCA chip and NFA Index provide a precise platform for monitoring neutrophil chemotaxis, demonstrating significant potential for sepsis diagnosis, prognosis, and immune function evaluation.