A Lysis- and Sonication-Based Method for the Quantification of Extracellular Vesicle-Bound Cardiac Troponin T Using a High-Sensitivity Immunoassay.

Background/Objectives: High-sensitivity cardiac troponin (hs-cTn) assays are used in routine diagnostics to detect myocardial injury. However, a fraction of circulating cardiac troponin T (cTnT) enclosed within extracellular vesicles (EVs) goes widely undetected. This study introduces a combined lysis- and sonication-based protocol to release and quantify EV-bound cTnT in a time-efficient manner using a state-of-the-art hs-cTnT immunoassay. Methods: Plasma samples from patients with non-ST-segment elevation myocardial infarction (NSTEMI), unstable angina, pulmonary embolism, decompensated aortic stenosis, atrial fibrillation, myocarditis, and healthy controls were treated with a lysis buffer and subsequently sonicated. Treated and untreated samples were assessed and compared to a conventional EV isolation method. Results: Following combined lysis and sonication, cTnT levels were significantly higher compared to native, unprocessed samples across all cohorts. The median increase post-processing ranged from ~10% in decompensated aortic stenosis to ~34% in young healthy controls. In NSTEMI, the EV-bound cTnT accounted for ~15% of plasma cTnT and remained stable over 72 h. The EV cTnT/plasma cTnT ratios were comparable between the combined lysis and sonication approach and the EV isolation method. Processing time prior to cTnT measurement was reduced from ~2.5 h to ~10 min using the lysis and sonication protocol. Conclusions: Our method allows for the rapid liberation of a previously inaccessible EV-bound fraction of cTnT without the need for time-consuming and resource-intensive EV isolation workflows and is therefore readily implementable alongside standard hs-cTnT testing. The observed EV-cTnT patterns suggest differential compartmentation of cTnT, potentially reflecting the myocardial pathophysiology underlying troponin elevation.