Simultaneous assay of activated platelet count and platelet-activating capacity by P-selectin detection using K2-EDTA-treated whole blood for antiplatelet agents

• Published in: International journal of laboratory hematology Vol. 34; no. 6; pp. 621 - 629
• Main Authors: Okano, K., Araki, M., Mimura, Y., Nogaki, H., Ichihara, K.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.12.2012
• Subjects: adenosine diphosphate; Adenosine Diphosphate - pharmacology; Anticoagulants - pharmacology; Aspirin - therapeutic use; aspirin monitoring; assay optimization; Blood Platelets - drug effects; Blood Platelets - metabolism; Citrates - pharmacology; collagen; Collagen - pharmacology; Drug Monitoring - methods; Edetic Acid - pharmacology; Female; Flow Cytometry; Heparin - pharmacology; Humans; Male; P-Selectin - blood; Platelet Activation - drug effects; Platelet Aggregation Inhibitors - therapeutic use; Platelet Count; Reproducibility of Results; Thrombosis - blood; Thrombosis - diagnosis; Thrombosis - prevention & control; Time Factors; Young Adult; collagen; Flow cytometry; assay optimization; aspirin monitoring; adenosine diphosphate
• ISSN: 1751-5521; 1751-553X
• DOI: 10.1111/j.1751-553X.2012.01447.x

Summary Introduction It is well recognized that examinations of activated platelets (aPLTs) and platelet‐activating capacity are very important to observe and prevent embolic diseases (events) such as ischemic stroke and myocardial infarction. Previously, we reported an appropriate measurement technique of aPLT for clinical assay. In this paper, we investigated stable conditions for measurement of activating capacity of platelets. Methods Blood samples were taken from healthy volunteers using anticoagulants of 2K‐EDTA, sodium citrate and heparin, and platelets were stimulated with adenosine diphosphate (ADP) or collagen. We demonstrated platelet‐activating capacity by detection of scattering light, absorbance, microscopic observation, and P‐selectin (CD62P) expression. We also performed basic experiments in seven healthy volunteers to test the clinical application of these assays with monitoring aspirin therapy. Results We judged that samples of whole blood with 2K‐EDTA were suitable for CD62P expression assay as functional assessments of platelet activity, because platelets treated with anticoagulants such as sodium citrate and heparin were extremely damaged after stimulation, and it was difficult to measure the CD62P expression by flow cytometry. For optimal results, samples should be tested within 1 h after the drawing of blood and stimulated with ADP or collagen for 10 min. The CD62P‐positive platelet value of blood from volunteers who had taken aspirin was decreased, and platelet activation was inhibited as well. Conclusion The simultaneous assay of aPLT and platelet‐activating capacity by CD62P detection using whole blood treated with the K2‐EDTA anticoagulant was useful for the monitoring of antiplatelet drugs.