Flow Cytometric Characterization of Synovial Fluid Extracellular Vesicles for the Diagnostic Stratification of Inflammatory Arthritis

Background and aim of study Synovial fluid (SF) directly reflects the joint microenvironment and may provide disease-relevant information in inflammatory arthritis. Extracellular vesicles (EVs) are emerging mediators and biomarkers of intercellular communication, but their phenotypic composition in SF across different forms of arthritis remains poorly defined. We aimed to characterize SF EV profiles in psoriatic arthritis (PsA), rheumatoid arthritis (RA), and crystal-induced arthritis (CIA), using osteoarthritis (OA) as a degenerative joint disease comparator. Methods Adult patients undergoing ultrasound-guided knee arthrocentesis were included: PsA (n=11), RA (n=5), CIA (n=6), and OA (n=8). SF was assessed for white blood cell (WBC) count, differential cell composition, and monosodium urate/calcium pyrophosphate crystals. EV-enriched pellets were analyzed by high-sensitivity flow cytometry. EVs were identified within a size-calibrated 0.1–0.9 μm gate and by Calcein-AM positivity, then phenotyped using CD14/CD66b and CD4/CD8 antibody panels. Non-parametric tests with false discovery rate correction were used. Results Inflammatory arthritis showed the expected inflammatory SF profile, with higher WBC counts than OA (median 8200 vs 450 cells/µL, q< 0.001) and higher PMN percentages (82% vs 0%, q< 0.001). EV size distribution was markedly different: inflammatory arthritis was enriched in small EVs (79% vs 11%, q< 0.001), while medium/large EVs predominated in OA (84% vs 18%, q< 0.001). Immune-associated EVs were also higher in inflammatory arthritis, including CD14+ EVs (83% vs 10%, q= 0.002), CD14+CD66b+ EVs (2% vs 1%, q=0.012), CD8+ EVs (58% vs 0%, q< 0.001), and CD4+CD8+ EVs (31% vs 0%, q< 0.001). Among inflammatory subtypes, CD4+CD8+ EVs also differed significantly (q=0.013), mainly reflecting lower values in RA (1%) compared with both PsA (33%) and CIA (41%). CD8+ EVs differed significantly across all three diseases (overall q=0.009), with the highest proportions in CIA (74%), intermediate values in PsA (58%), and markedly lower values in RA (1%). CIA showed numerically higher CD66b+ and CD14+CD66b+ EVs than PsA, suggesting a more neutrophil-associated profile. Conclusion SF EV phenotyping by high-sensitivity flow cytometry identifies distinct vesicular signatures in inflammatory arthritis. Compared with OA, inflammatory arthritis is enriched in small and immune-cell-associated EVs. Within inflammatory arthritis, CD8-associated EVs differentiated PsA, RA, and CIA, while neutrophil-associated EVs may help further characterize CIA. These findings support SF EV profiling as a candidate tool for synovial disease stratification in diagnostically complex inflammatory effusions.