Abstract Number: PB2136
Meeting: ISTH 2020 Congress
Background: Standardized and ultra-sensitive measurement of MV-associated TF-dependent activity is mandatory to evaluate thrombotic risk. Although several versions of FXa generation assays have been described, those may not be sensitive and specific enough. Our highly sensitive new method (Vallier, Thromb. Res. 2019) has been adapted into a kit, switching detection technology from fluorescence to absorbance for wider applicability.
Aims: The aim of this study was to compare chromogenic versus fluorogenic assay versions and illustrate performance of this new MV-TF Activity kit.
Methods: MVs were purified from platelet-free plasma by high-speed centrifugation (HS-C – 24,000 g – 1h). Robustness was ensured by easy pipeting volumes, a lyophilized mix of FVII + FX and a wide recTF calibration range over 2 decades in log scales. The FXa substrate was switched from fluorogenic to chromogenic wo losing sensitivity. Precision measurements were realized using the kit controls and kit’s performance tested using 1) TF liposomes, 2) MV-free plasma spiked with tumoral TF+ vesicles and 3) normal and pathological plasma samples.
Results: Noteworthy, for samples with detectable but low values (< 30 fM) in optimal pre-analytical conditions, increased TF activities were observed when one of paired blood tubes was purposely agitated during transportation. The FXa generation assay itself is robust with CV< 5% for repeatability and < 15% for inter-operator reproducibility. However, the pre-test HS-C step still induces significant inter-operator variability (CV ~25%). An alternative approach for MV extraction using magnetic beads is considered.
Conclusions: This new kit allowing robust and sensitive measurement on purified MVs may help clarifying the biomarker value of MV-TF activity in future studies. It advocates for the presence of active MV-TF in normal plasma and for taking care of transportation conditions.
To cite this abstract in AMA style:Bouriche T, Judicone C, Franco C, Vallier L, Cucini V, Mathieu O, Dignat-George F, Lacroix R, Poncelet P. A Newly Developed Kit for High-Sensitivity Measurement of TF-Dependent Procoagulant Activity on Microvesicles Purified by High-Speed Centrifugation [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-newly-developed-kit-for-high-sensitivity-measurement-of-tf-dependent-procoagulant-activity-on-microvesicles-purified-by-high-speed-centrifugation/. Accessed January 26, 2022.
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