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Updated: Mar 9, 2026
Author Spotlight: High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis
Published on: December 29, 2023
Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy
Yohei Hisada1, Alyson C Auriemma2, Wyeth Alexander2
1Department of Medicine, Division of Hematology and Oncology, Thrombosis and Hemostasis Program, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; K.G. Jensen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø, Norway.
Introduction:
Increased levels of tissue factor-positive extracellular vesicles (TF+EVs) have been detected in the plasma of patients with various diseases, including cancer and endotoxemia. Levels of TF+EVs in plasma samples can be measured by antigen and activity assays. The aim of the present study was to visualize TF+EVs by laser scanning confocal microscopy (LSCM).
Methods:
EVs were isolated from the supernatant of two cultured human pancreatic cancer cell lines (Panc-1 and BxPc-3), from untreated or lipopolysaccharide (LPS) treated whole blood, and from plasma of pancreatic cancer patients. EV-TF activity was determined using an in-house assay. The EVs were labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, which is converted to the impermeant green fluorescent molecule carboxyfluorescein inside the EVs. EVs were either captured using annexin V and detected using a fluorescent-labeled anti-TF antibody, or captured using an anti-TF antibody and detected using fluorescent-labeled annexin V. EVs were visualized by LSCM.
Results:
TF+EVs were easily detected from high TF-expressing BxPc-3 cells using annexin V capture, whereas the addition of tyramide amplification was required to detect TF+EVs from low TF-expressing Panc-1 cells. Visualization of TF+EVs in plasma from LPS treated whole human blood and in plasma from pancreatic cancer patients required either capture with annexin V and detection with a fluorescent-labeled anti-TF antibody with tyramide signal amplification, or capture with an anti-TF antibody and detection with a fluorescent-labeled annexin V.
Conclusion:
LSCM enables visualization of TF+EVs in the supernatant from cultured cells and in clinical samples.
