A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids.

BACKGROUND: The procoagulant activity of tissue factor-bearing microvesicles (MV-TF) has been associated with the risk of developing venous thrombosis in cancer patients. However, MV-TF assays are limited either by i) a lack of specificity, ii) a low sensitivity, or iii) a lack of repeatability when high-speed centrifugation (HS-C) is used to isolate MV. Therefore, our objective was to develop a new hybrid "capture-bioassay" with improved reproducibility combining MV immunocapture from biofluids and measurement of their TF activity. MATERIALS AND METHODS: Factor Xa generation and flow cytometry assays were used to evaluate IMS beads performance, and to select the most effective capture antibodies. The analytical performance between IMS-based and HS-C-based assays was evaluated with various models of plasma samples (from LPS-activated blood, spiked with tumoral MV, or with saliva MV) and different biofluids (buffer, plasma, saliva, and pleural fluid). RESULTS: Combining both CD29 and CD59 antibodies on IMS beads was as efficient as HS-C to isolate plasmatic PS+ MV. The IMS-based strategy gave significantly higher levels of MV-TF activity than HS-C in tumor MV spiked buffer, and both pleural fluids and saliva samples. Surprisingly, lower TF values were measured in plasma due to TFPI (TF pathway inhibitor) non-specifically adsorbed onto beads. This was overcome by adding a TFPI-blocking antibody. After optimization, the new IMS-based assay significantly improved reproducibility of MV-TF bioassay versus the HS-C-based assay without losing specificity and sensitivity. In addition, this approach could identify the cellular origin of MV-TF in various biological fluids. CONCLUSION: Compared to HS-C, the IMS-based measurement of MV-TF activity in body fluids improves reproducibility and makes the assay compatible with clinical practice. It can facilitate future automation.

Increasing the sensitivity of the human microvesicle tissue factor activity assay.

INTRODUCTION: The TF-FVIIa complex is the primary activator of coagulation. Elevated levels of microvesicle (MV) bearing tissue factor (TF)-dependent procoagulant activity are detectable in patients with an increased risk of thrombosis. Several methods have been described to measure MV TF activity but they are hampered by limited sensitivity and specificity. The aim of this work was to increase the sensitivity of the MV TF activity assay (called Chapel Hill assay). MATERIAL AND METHODS: Improvements of the MV TF activity assay included i/ speed and time of centrifugation, ii/ use of a more potent inhibitory anti-TF antibody iii/ use of FVII and a fluorogenic substrate to increase specificity. RESULTS: The specificity of the MV TF activity assay was demonstrated by the absence of activity on MV derived from a knock-out-TF cell line using an anti-human TF monoclonal antibody called SBTF-1, which shows a higher TF inhibitory effect than the anti-human TF monoclonal antibody called HTF-1. Experiments using blood from healthy individuals, stimulated or not by LPS, or plasma spiked with 3 different levels of MV, demonstrated that the new assay was more sensitive and this allowed detection of MV TF activity in platelet free plasma (PFP) samples from healthy individuals. However, the assay was limited by an inter-assay variability, mainly due to the centrifugation step. CONCLUSIONS: We have improved the sensitivity of the MV TF activity assay without losing specificity. This new assay could be used to evaluate levels of TF-positive MV as a potential biomarker of thrombotic risk in patients.