Detection and identification of plasma extracellular vesicles in patients with systemic lupus erythematosus using different methodologies

INTRODUCTION: The evaluation of extracellular vesicles (EVs) is crucial for understanding their role as monitoring biomarkers in systemic lupus erythematosus (SLE), a chronic, autoimmune disease. This study aimed to evaluate plasma extracellular vesicles, derived from leukocytes and platelets, in systemic lupus erythematosus patients using different methods for EV identification and quantification. METHODS: A cross-sectional study (IRB#3383573/UFF) was performed with 49 plasma samples obtained from SLE patients and 13 samples from healthy individuals (nSLE). Disease activity was determined by the SLE Disease Activity Index (SLEDAI-2K: 0, 1-4, ≥ 5). After thawing, plasma EV isolation was performed using differential centrifugation, 12,000xg 2 minutes 4°C to obtain supernatant with a subsequent 20,000xg 20 minutes 4°C centrifugation. The pellet was resuspended in PBS for nanoparticle tracking assay and ELISA, or annexin buffer for flow cytometry. For the EV detection, we used nanoparticle tracking analysis (ZetaView® PMX-220 TWIN), nanoscale flow cytometry (CytoFLEX S, Beckman Coulter), and immunoenzymatic assay (in-house ELISA). For nanoparticle tracking assay different dilutions of EVs were prepared to a final volume of 1.2 mL. In flow cytometry, EV identification was based on size after calibration with polystyrene beads (~100-900 nm, Gigamix) and positivity for the Annexin V-FITC, anti-CD41-APC or anti-CD45-APC monoclonal antibodies. Anti-Annexin V, Anti-CD45, and Anti-CD41 antibodies monoclonal were used in the in-house ELISA. RESULTS: Of the 49 patients, 44 (89.8%) were female, with a median age of 40 (28-53) years-old. The nanoparticle tracking assay exhibited similar EV diameter and particle count between the SLE and nSLE groups. In-house ELISA and flow cytometry showed increased platelets-derived EVs levels in SLE patients compared to nSLE (p < 0.001). However, flow cytometry methods showed increased leukocytes-derived EVs in SLE (p = 0.03), while lower counts were found in the in-house ELISA (p = 0.03). ROC curve analysis showed that both flow cytometry and in-house ELISA (AUC ≥ 0.7) were able to differentiate groups. There was no correlation between SLEDAI-2K and leukocytes- and platelets-derived EVs for flow cytometry or ELISA. Importantly, we identified that patients under immunosuppressive therapy presented significantly higher leukocytes-derived EVs (p = 0.02). CONCLUSION: Our study suggests that plasma EVs may be early markers of inflammation in SLE, but are not associated with SLEDAI-2K. Moreover, in SLE patients, immunosuppression should be considered when evaluating leukocytes-derived EVs.