Establishment of Reference Values for Platelet Activation Markers by Flow Cytometry

Abstract

Background: This study was purposed to establish reference values for platelet activation markers and leukocyte-platelet aggregates in the evaluation of the platelet function tests using flow cytometry.

Methods: Whole blood samples were obtained from 30 volunteers of healthy adults. Diluted blood samples, either in the resting state or in activated state by the addition of agonist, 20 M ADP or 100 M TRAP, were stained with fluorescent conjugated monoclonal antibody of PAC1 or CD62P. Then, the percentages of expression for each marker were analyzed by flow cytometry. For leukocyte-platelet aggregates, monoclonal antibodies of CD41a, CD14 and CD45 were added simultaneously to undiluted whole blood.

Results: Reference values for the percentages of the expression of PAC1 and CD62P, respectively,were 0.1-12.5% and 0.0-4.7% at the resting state, 65.3-92.4% and 39.0-75.7% with the addition of 20 M ADP, and 68.1-93.1% and 60.5-91.2% with the addition of 100 M TRAP. Reference values for leukocyte-platelet aggregates, granulocyte-platelet aggregates, and lymphocyte-platelets aggregates were 2.8-23.6%, 5.3-34.2%, and 4.9-21.6%, respectively.

Conclusion: The platelet activation markers at the resting or an activated state with agonists and leukocyte-platelet aggregates could be analyzed using flow cytometry. These reference values should be helpful in interpreting platelet function tests by flow cytometry.