Evaluation of a new bead‐based assay to measure levels of human tissue factor antigen in extracellular vesicles in plasma

BACKGROUND: Circulating tissue factor (TF)‐expressing extracellular vesicles (EVs) are associated with thrombosis in several diseases, such as coronavirus disease 2019 (COVID‐19). Activity assays have higher sensitivity and specificity compared to antigen assays for measuring TF+ EVs in plasma. The MACSPlex Exosome Kit is designed to detect 37 exosomal surface epitopes, including TF, on EVs in plasma using various fluorescently labeled beads. The different EV‐bead complexes are detected by flow cytometry. A recent study used the MACSPlex Exosome Kit to measure levels of TF+ EVs in serum from patients with COVID‐19. OBJECTIVES: To evaluate the ability of the MACSPlex Exosome Kit to detect TF on EVs in plasma. METHODS: We measured levels of TF+ EVs isolated from plasma with or without TF detected using our in‐house EVTF activity assay and the MACSPlex Exosome Kit. RESULTS: The MACSPlex Exosome Kit gave a very low TF antigen signal (TF bead signal) compared to platelet‐derived CD41b+ EVs, which was used as a control. Lipopolysaccharide (LPS) increased levels of EVTF activity but not TF bead signal in four donors. Inhibition of TF reduced levels of EVTF activity but did not affect the TF bead signal in EVs isolated from plasma from LPS‐treated blood. Finally, we found no correlation between levels of EVTF activity and TF bead signal in EVs isolated from plasma from ovarian cancer patients (r = .16, P = .62). CONCLUSION: Our data suggest that the MACSPlex Exosome Kit gives a nonspecific signal for TF and does not have the sensitivity to detect TF+ EVs in plasma.