Whole Blood Deep Immunophenotyping and Platelet-Leukocyte Aggregate Quantification by Spectral Flow Cytometry

Platelet-leukocyte aggregates (PLAs) are a proposed interface between coagulation and immune regulation, but their distribution across leukocyte subsets beyond monocytes and neutrophils, and their behavior in acute disorders of immune regulation, such as sepsis and severe trauma, remain largely undefined. The study of thromboimmune mechanisms in these acute conditions has relied heavily on murine sepsis models, whose species differences in platelet and leukocyte biology limit translation to human disease. Direct measurement in human whole blood is needed yet reported PLA frequencies vary widely because quantification is highly sensitive to sample handling. Here we present a robust, minimally manipulative 27-color spectral flow cytometry protocol that allows simultaneous deep immunophenotyping and classification of PLAs across >40 leukocyte subpopulations. In healthy donors, PLA frequency ranged from 1.7% on memory CD4 T cells to over 50% on monocyte subsets, with monocytes contributing 25% of all PLAs. We also found novel platelet-binding leukocyte populations, including Th9 and Th22 CD4+ T cells. Optimization identified anticoagulant choice, centrifugation conditions, dilution, and sample processing time as key determinants of reproducible measurement; PLA quantification was validated by imaging flow cytometry. In a small validation cohort of sepsis and trauma patients, the assay recapitulated established immunophenotypic features of critical illness, including neutrophilia and myeloid-derived suppressor cells (MDSC) expansion, and quantified PLAs within each subset. This protocol provides a reference framework for studying thromboinflammation and other dysregulated immune response diseases directly in human blood, and a foundation for further studies, defining the mechanisms and clinical relevance of platelet-leukocyte interactions in disease.