UNRAVELING MONOCYTE AND PLATELET MORPHO-FUNCTIONAL ALTERATIONS IN SEPSIS: EMERGING BIOMARKERS FOR EARLY DIAGNOSIS

Sepsis remains a leading cause of morbidity and mortality worldwide. It is characterized by a complex immune dysregulation and an intricate interplay between inflammation and thrombosis in the host. Identifying early and reliable biomarkers is crucial for timely diagnosis and risk stratification. This PhD thesis investigated the role of Damage-Associated Molecular Patterns, Pathogen-Associated Molecular Patterns and live bacteria in morpho-functional changes in monocytes and platelets, which are key players in thrombo-inflammation. Through an integrated approach combining ex vivo whole blood model and in vivo clinical data, this work examined how Monocyte Distribution Width (MDW) and platelet indices, such as Platelet Distribution Width (PDW) and Mean Platelet Volume (MPV), mirror qualitative alterations in monocytes and platelets, evaluating their diagnostic and prognostic value. Whole blood from healthy donors (EDTAK2/K3) was treated with a mixture of histones (0-200 μg/mL), Lipopolysaccharide (LPS) from E. coli (0-10 μg/mL), and live E. coli (106-1010 CFU/mL) to simulate a septic condition. Complete blood counts were performed using DxH690T/900 (Beckman Coulter) up to 3h after treatment. Peripheral blood smears were prepared for digital cell morphology analyses using CellaVision. Plasma samples were used to assay a panel of 27 cytokines using the Bio-Plex 200 system. Our results demonstrated that free histones, LPS and live E. coli induce a significant, early, dose- and time- dependent increase in MDW, as confirmed by enlarged volume, cytoplasmic vacuolization, increased granularity, nuclear deformation and phagocytosis, only in the case of E. coli. Comparing MDW kinetics across stimuli, considering appropriate anticoagulant-specific cut-off, emerged differences in both timing and intensity of increase. E. coli and LPS induced an earlier and higher increase in MDW than histone, according to the different activated signalling. Moreover, MDW values obtained in our ex vivo model with E. coli and LPS overlap those found in septic patients with a positive blood culture for E. coli, confirming the reliability of the whole blood model. This evidence suggested that bacterial components start the early inflammatory signalling, recognised by increased MDW and cytokine release. Meanwhile, extracellular histones amplify the host response by causing the release of further histones and inflammatory mediators. In this vicious cycle, histones promote platelet activation and aggregation, constituting a key target for both biomarkers and potential therapeutic approaches. Our experimental results confirmed that histones induced a very significant and early decrease in platelet count, due to aggregation by consumption, and a significant increase in MPV and PDW. This thesis sustains that assessing MDW and platelet indices, and possibly quantifying circulating histones, may provide an integrated panel of early biomarkers for identifying patients at risk of sepsis.