D101-12 High-throughput Imaging Captures Neutrophil Dysfunction in Critical Illness

Rationale Sepsis and acute respiratory distress syndrome (ARDS) remain major causes of ICU mortality, yet clinicians lack tools to identify patients at risk of deterioration before organ failure occurs1. Neutrophils, central to inflammatory injury, exhibit transcriptional and functional heterogeneity that correlates with disease severity2. This study aims to determine whether neutrophil morphology, captured by high-throughput imaging, can serve as a rapid, interpretable surrogate for immune dysregulation. Methods We adapted a Dean flow-based spiral microfluidic device to focus and image neutrophils in suspension under brightfield microscopy. Morphometric features (n = 249) were extracted using MATLAB-based image analysis and combined with nonlinear dimensionality reduction (PCA, UMAP) and trajectory inference (Slingshot) to capture continuous morphological transitions. Human neutrophils were isolated using the StemCell immunomagnetic isolation protocol and activated ex vivo using phorbol myristate acetate (PMA), and morphological dynamics were then compared to healthy controls. Results From >4,000 human neutrophil images, control cells showed uniform circularity (mean 0.97 ± 0.02) and consistent diameter (∼9.4 µm). PMA-stimulated neutrophils, on the other hand, demonstrated marked morphological dispersion, with increased diameter, irregularity, and fractal complexity. Unsupervised clustering and UMAP visualization revealed clear separation between control and PMA-activated cells, supported by a significant pseudotime shift (p < 2.2 × 10-16, η² = 0.38). Quantitative shape features such as cell radius and perimeter increased gradually with activation, mapping a continuous morphological trajectory that mirrors known functional transformations that underlie neutrophil activation. Conclusions and Ongoing Work High-throughput imaging combined with computational morphology resolves dynamic states of neutrophil activation, identifying quantitative features that reflect immune dysregulation. These morphological signatures provide a scalable framework for characterizing neutrophil phenotypes in critical illness. Ongoing experiments include (1) murine sepsis models using cecal slurry induction versus wild-type controls and (2) prospectively collected blood samples from patients with sepsis and/or ARDS. Together, these datasets will expand a growing image library linking morphology with multi-omics data and clinical outcomes to establish a high-throughput, microscopy-based platform for immune risk stratification in critical illness. Citations:1. Rhee, C. et al. “Incidence and trends of sepsis in US hospitals using clinical vs claims data, 2009-2014”, JAMA 318, 1241-1249 (2017).2. Moore, A.R. et al. “A consensus immune dysregulation framework for sepsis and critical illnesses.” Nat Med (2025). This abstract is funded by: None