Evaluation of microparticles in whole blood by multicolour flow cytometry assay.

OBJECTIVE: To develop and evaluate a multicolour flow cytometry method for analysis of microparticles (MPs) in fresh whole blood without any centrifugation steps or freezing/thawing procedure.

MATERIALS AND METHODS: Flow cytometry was performed using a FC500 MPL cytometer. The compensation in the protocol was performed based on the platelet population. Polystyrene microspheres 0.50-1.27 μm were used for size position, and the MP gate was set as particles 0.5-1.0 μm. Whole blood was incubated with annexin V and antibodies to tissue factor (TF), platelets (CD41 and CD62P), monocyte (CD14) and endothelial cells (CD144). For comparison, MPs from platelet free supernatant was used. The TF activity was evaluated by Calibrated Automated Thrombogram.

RESULTS: Annexin V was used to distinguish true events from background noise. For standardization, each analysis included 10,000 events in the gate of platelets. There were 622(462-1001) MP(annV+)/10,000 platelets and of these, 66 (49-82)/10,000 platelets expressed TF. After correction for the individual platelet counts, the amount of circulating MP(annV+) was 17.1 (12.1-24.9) × 10(9)/L in whole blood, and of these, 10% (6-12%) expressed TF. The majority of the MPs expressed CD41, and 5.6% (2.2-6.9%) of these co-expressed TF. The amount of CD41 + MP(annV+) tended to correlate to the TF activity in whole blood. There was no correlation between the MP(annV+) in whole blood and MPs derived from platelet free supernatant. Patients with pulmonary arterial hypertension and stable coronary artery disease had increased concentrations of CD41 + MP(annV+) in whole blood.

CONCLUSION: This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.