Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

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.

A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance.

Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8-3.0] vs 3.1 [1.7-18] A405 × 10-3/min, p = 0.02; 1.4 [1-1.6] vs 5.2 [2.2-16] A405 × 10-3/min, p = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance.

Tips and tricks for flow cytometry-based analysis and counting of microparticles.

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties.

High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles--brief report.

OBJECTIVE: Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels. METHODS AND RESULTS: Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 µm) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8- to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information. CONCLUSIONS: Recent improvements in FCM provide access to previously undetectable MP and represent a new opportunity to enhance their impact as biomarkers in clinical practice.