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High-Dimensional Flow Cytometry Identifies Immune Signatures Associated with Septic Shock Severity in Critically Ill
Miguel Bardají-Carrillo1, Rosa Dolores Prieto-Utrera2, Rosa Cobo-Zubia2
1BioCritic, Group for Biomedical Research in Critical Care Medicine, 47003, Valladolid, Spain; Anesthesiology and Critical Care, Clinical University Hospital of Valladolid, Av. Ramón y Cajal, 3, 47003, Valladolid, Spain; Department of Surgery, University of Valladolid, 47003, Valladolid, Spain; Health Research Institute of Valladolid (IBioVALL), Valladolid, Spain; Biomedical Research Networking Center in Infectious Diseases (CIBERINFEC), Carlos III Health Institute, Madrid, Spain.
Background:
Sepsis and septic shock are associated with severe immune dysregulation and high mortality, while current biomarkers provide limited accuracy for early risk stratification. This study aimed to characterize immune cell alterations using high-dimensional spectral flow cytometry in postoperative ICU patients and to explore their association with disease severity in combination with clinical biomarkers.
Methods:
We conducted a prospective multicentre study including 219 postoperative ICU patients classified as non-septic ICU (n = 77), sepsis (n = 62), and septic shock (n = 80). PBMCs were analysed by spectral flow cytometry. Immune populations were defined using supervised gating. Associations with clinical severity were assessed using logistic regression and ROC analysis. External validation was performed using single-cell RNA sequencing.
Results:
Patients with septic shock showed a significant reduction in CD2-/CD8+ NK-like cells and dendritic cells compared with sepsis and non-septic ICU patients. In multivariable models, lactate, neutrophil-to-lymphocyte ratio, dendritic cells, and NK-like cells were associated with septic shock. The combined model showed superior performance (AUC = 0.870). Single-cell RNA sequencing confirmed reduced dendritic and NK-like populations and revealed transcriptional alterations consistent with immune and metabolic dysregulation.
Conclusions:
Septic shock is associated with a distinct immune signature characterized by reduced dendritic cells and NK-like cells. Integration of immune profiling with routine biomarkers is associated with improved discrimination of disease severity in this setting and supports further investigation of immune phenotyping approaches in sepsis.
