The diagnostic usefulness of capture assays for measuring global/specific extracellular micro-particles in plasma

Abstract

Capture assays were developed and validated for measuring the global pro-coagulant activity of micro-particles (MPs, mainly originated from platelets), or specific extravascular cellular MPs (released from erythrocytes, leukocytes, monocytes, endothelial cells) as those exposing TF (MP-TF, mainly observed in patients with some cancers). Conversely to Flow Cytometry methods, these capture assays measure all coagulant activity associated with MPs, through thrombin generation (MP-Activity) or Factor Xa generation (MP-TF), and therefore they bring a complementary information, as they are more specific for the pro-coagulant activity associated with MPs. Small particles (<0.40 µ) exposing Phosphatidyl Serine (PS) exhibit a greater pro-coagulant surface than larger MPs (0.40 to >1.00 µ), those preferentially measured with flow cytometry. Activity associated with MPs is a consequence of disease but can also be a cause contributing to pathological processes and development of thrombo-embolic events. In many diseases, flow cytometry and capture assays do not totally correlate, and have different associations with disease evolution. Optimized capture based assays are presented and discussed, along with their performance characteristics and some applications. They can be performed in any technically skillful hemostasis laboratory, using a thermostated ELISA equipment, or an incubator. Dynamic ranges for MP-Activity assay is from <0.1 nM to >2.5 nM Phospholipids, expressed as Phosphatidyl Serine (PS) equivalent, in the tested dilution. For MP-TF the very sensitive bio-immunoassay reported allows measuring concentrations from <0.10 pg/ml (TF equivalent) to >5.00 pg/ml, in the assayed dilution. No measurable MP-TF was found in normals, although an important concentration was generated from whole blood treated with Lipo-Poly-Saccharides. Capture based assays are then highly useful in the laboratory setting for measuring the activities associated with pro-coagulant, or specific cellular MPs.