MICROPARTICLES PROCOAGULANT ACTIVITY IN CITRATED PLASMA
Abstract number: P-S-513
Vissac1 A.M., Peyrafitte1 M., Amiral1 J.J.
1RESEARCH, HYPHEN BIOMED SAS, Neuville sur Oise, France
How-to-cite Vissac AM, Peyrafitte M, Amiral JJ. MICROPARTICLES PROCOAGULANT ACTIVITY IN CITRATED PLASMA. J Thromb Haemost 2007; 5 Supplement 2: P-S-513
Introduction: A method, based on the original principle reported by Aupeix K et Al, for measuring the procoagulant activity of circulating microparticles (MPs), in citrated plasma, has been optimized and adapted to routine clinical laboratory practice. It allows measuring these MPs in plasma, as the consequence or the cause of cardiovascular complications.
Methods: The assay is performed on human plasma, prepared from citrated blood, centrifuged at room temperature for 15 min at 2,000 g, and the supernatant, centrifuged again for 2 min at 13,000 g. Plasma diluted 1:20 in a buffer containing Ca2+ and protease inhibitors, is incubated in a micro-Elisa plate coated with streptavidin and biotinylated Annexin V, then incubated for 1 hour at 37 °C. MPs are captured by Annexin V. Following a washing step factors Va, Xa, then prothrombin are introduced and incubated for 10 min at 37 °C. There is a microparticle concentration dose dependent thrombin generation, which is tested with a specific chromogenic substrate. A linear relationship between MPs concentration (in nM equivalent Phosphatidyl Serine, PS) and A405 is obtained. Calibration is performed with washed, activated and lysed platelets, highly diluted, which MPs concentration is evaluated against a PS standard.
Results: A working range from 0 to 50 nM in plasma is obtained. MPs are present at a low concentration in normal individuals (< 10 nM, and frequently < 5 nM), and are importantly increased in many pathological situations (inflammation, infections, cardiovascular diseases, ...) with concentrations > 10 nM and up to 100 nM or more. The detection threshold is of 0.1 nM in the tested dilution.
Conclusions: This method offers a new and standardized tool for measuring MPs coagulant activity, for diagnosis or prognosis applications, or for monitoring some therapies for circulatory diseases.
References: Aupeix K et Al. J. Clin. Invest. 1997, 99, 1546-54
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Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
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