CD9+ and CD82+ extracellular vesicles in synovial fluid differentiate aseptic from septic endoprosthesis loosening

Aim: Extracellular vesicles (EVs) hold great promise as emerging biomarkers for a variety of diseases. However, their clinical application is still hindered by complex and time-consuming isolation procedures. A clinically relevant scenario where improved biomarker-based diagnostics are urgently needed is prosthetic joint loosening, which may result from either aseptic inflammation or periprosthetic joint infection (PJI) - two conditions requiring fundamentally different therapeutic approaches. This study investigated whether imaging flow cytometry (IFCM) enables the discrimination between aseptic and septic loosening by profiling EVs directly in minimally processed synovial fluid. Methods: We analyzed synovial fluid from 35 aseptic and 13 septic cases using IFCM to detect surface marker-defined EV subpopulations without prior isolation. Samples were classified based on clinical and microbiological findings. Marker abundance was quantified and analyzed using logistic regression. Results: Septic loosening was associated with significantly increased levels of CD82⁺ EVs and decreased levels of CD9⁺ EVs. CD82⁺ EVs showed a sensitivity of 83.3% and specificity of 90.0%, while CD9⁺ EVs demonstrated 100% specificity but lower sensitivity (58.8%) for aseptic loosening. CD82⁺ EV abundance was identified as an independent predictor of septic loosening. Conclusion: IFCM enables rapid and direct detection of diagnostically relevant EVs in native synovial fluid. CD9⁺ and CD82⁺ EVs serve as promising biomarkers for distinguishing aseptic from septic endoprosthesis loosening, offering a fast and robust diagnostic tool that may complement current clinical diagnostics and support timely treatment decisions.