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The diagnostic usefulness of capture assays for measuring global/specific extracellular micro-particles in plasma
Jean Amiral1, Jerard Seghatchian2
1HYPHEN BioMed Research, Neuville sur Oise, France.
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.
Insights
New capture assays measure micro-particle (MP) pro-coagulant activity, offering complementary insights beyond flow cytometry. These validated assays detect thrombin and Factor Xa generation, aiding in disease assessment and thrombo-embolic event prediction.
Area of Science:
- Coagulation Science
- Biomarker Discovery
- Hemostasis Research
Background:
- Micro-particles (MPs), particularly those originating from platelets, erythrocytes, leukocytes, monocytes, and endothelial cells, play a significant role in hemostasis and thrombosis.
- MPs exposing Tissue Factor (MP-TF) are associated with certain cancers and contribute to pathological processes and thrombo-embolic events.
- Existing flow cytometry methods may not fully capture the global pro-coagulant activity of MPs, especially smaller ones (<0.40 µ) with high Phosphatidyl Serine (PS) exposure.
Purpose of the Study:
- To develop and validate capture assays for measuring global pro-coagulant activity (MP-Activity) and specific MP-TF activity.
- To compare the information provided by capture assays with traditional flow cytometry methods.
- To present the performance characteristics and applications of these optimized capture-based assays.
Main Methods:
- Development and validation of capture assays to measure thrombin generation (MP-Activity) and Factor Xa generation (MP-TF).
- Assays utilize ELISA equipment or incubators, making them accessible to skilled hemostasis laboratories.
- Quantification of MP-Activity from <0.1 nM to >2.5 nM Phospholipids (PS equivalent) and MP-TF from <0.10 pg/ml to >5.00 pg/ml (TF equivalent).
Main Results:
- Capture assays provide complementary information to flow cytometry, specifically measuring the coagulant activity associated with MPs.
- Smaller MPs (<0.40 µ) exposing PS exhibit greater pro-coagulant surface than larger MPs measured by flow cytometry.
- No measurable MP-TF was found in normal individuals, but significant levels were generated in whole blood treated with Lipo-Poly-Saccharides.
Conclusions:
- Capture-based assays are highly useful for measuring pro-coagulant or specific cellular MP activities in a laboratory setting.
- These assays offer a more specific measure of pro-coagulant activity associated with MPs compared to flow cytometry.
- The validated assays can be readily implemented in hemostasis laboratories for research and clinical applications.
