Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry

Platelet aggregation and microaggregate formation were measured in samples of stirred whole blood by flow cytometry. Blood samples were stirred in a multi-sample agitator with ADP, fixed and labelled with a platelet-specific CD42a-FITC fluorescent antibody. The blood was then diluted and applied directly to a flow cytometer. Platelets were identified using a gating procedure based on their expression of CD42a and then quantified. Aggregation was monitored as a fall in the number of single platelets. Both reversible and irreversible aggregation responses to ADP were determined and these were found to correlate directly with aggregation responses determined using a well-established single platelet counting technique using the Ultra-Flo 100 Whole Blood Platelet Counter. We found from flow cytometry that ADP-induced aggregation was coupled with a transient formation of platelet microaggregates over the initial 60 s following ADP addition. Assessment of single platelet loss by flow cytometry was found to be a reliable way of monitoring aggregation responses and provided new information on rapid microaggregate formation in ADP-stimulated blood.