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

Abstract

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.

Circulating tissue factor‐positive extracellular vesicles and their association with thrombosis in different diseases

“…We recently evaluated a new bead‐based assay called the MACSPlex Exosome kit to measure levels of human TF antigen on EVs in plasma. Unfortunately, in contrast to our in‐house activity assay, we found that the kit was not sensitive enough to detect the low levels of TF + EVs in plasma from LPS stimulated whole blood and plasma from patients with ovarian cancer 23 …”

“…Unfortunately, in contrast to our inhouse activity assay, we found that the kit was not sensitive enough to detect the low levels of TF + EVs in plasma from LPS stimulated whole blood and plasma from patients with ovarian cancer. 23 We and others developed similar in-house FXa generation assays that measure EVTF activity kinetically 9 or as an endpoint. 14 Briefly, EVs in plasma are pelleted by centrifugation and washed twice with buffer.…”

Detection of tissue factor–positive extracellular vesicles using the ExoView R100 system

“…Blood samples were collected from patients with ovarian cancer using a protocol approved by the University of North Carolina at Chapel Hill Institutional Review Board (11-1201). Some of the ovarian cancer samples were used in a previous study [ 39 ]. Some of the trauma samples were used in previous studies [ 40 ].…”

Measurement of tissue factor-positive extracellular vesicles in plasma: strengths and weaknesses of current methods

“…It should be noted that the study used serum instead of plasma and added the labeled beads directly to the serum rather than isolating the EVs as recommended by the manufacturer. We evaluated the ability of the MACSPlex Exosome Kit to detect TFþ EVs in plasma [94]. We found that the kit was unable to distinguish between samples with or without TFþ EVs.…”