Modelling innate and adaptive immune responses in whole blood: A modified ex vivo assay without anticoagulants and synthetic media

Background: Blood is central to immune defense, rendering accurate assessment of its immunoreactivity vital for medical and biotechnological applications. Objective: This study presented a novel whole blood immunoreactivity assay (WBIA) designed to mimic natural physiological conditions, preserving essential cell–cell and cell–cytokine interactions for ex vivo immunological analysis. Methods: Fresh whole blood (with or without heparin) was stimulated with lipopolysaccharide (LPS), concanavalin A (Con A), or both, activating innate and adaptive immunity. Cytokine levels were measured through enzyme-linked immunosorbent assay after incubation. Results: Coagulation enhanced secretion of interleukin (IL)-2 and vascular endothelial growth factor (VEGF) in mitogen-stimulated samples. LPS induced tumor necrosis factor (TNF)-α, IL-6, and VEGF, while LPS + Con A co-stimulation produced the highest levels of interferon (IFN)-γ, IL-2, and IL-10. Peak cytokine concentrations were reached at 18 h, declining by 48–72 h. In 18 h LPS + Con A-stimulated serum blood samples from 30 healthy donors (19 women, 11 men, aged 30–55), cytokine levels (pg/mL, mean ± standard error of the mean) were as follows: IL-1β at (521 ± 62), IL-2 (24 ± 4), IL-6 (569 ± 43), IL-8 (277 ± 28), IL-10 (198 ± 35), IL-18 (293 ± 19), IFN-γ (227 ± 108), TNF-α (930 ± 126), and VEGF (655 ± 55). Conclusion: The WBIA provides a reliable, physiologically relevant model for evaluating immune responses to stimuli. Its high fidelity to in vivo conditions makes it a valuable tool for testing immunomodulatory drugs and monitoring immune status in clinical settings.