Platelet-monocyte aggregates are increasingly being used to quantify platelet activation. The variables that influence platelet-monocyte aggregates have not been well defined. We sought to determine the effect of blood collection, handling and processing techniques on detected levels of platelet-monocyte aggregates using a flow cytometric assay. Whole blood was labelled with anti-CD14-PE and anti-CD42a-FITC. Thereafter, samples were fixed and red cells lysed. Analysis was performed with the flow cytometer initially triggering on light scatter and then on FL-2 to identify CD14-PE positive monocytes. Platelet-monocyte aggregates were defined as monocytes positive for CD42a. The effect of collection, handling and processing techniques on this assay were assessed. Anticoagulation with heparin (20.1 +/- 2.0%), PPACK (16.8 +/- 1.9%), sodium citrate (12.3 +/- 1.6%) and EDTA (9.5 +/- 1.0%) resulted in markedly different levels of platelet-monocyte aggregation (P < 0.0001). Platelet-monocyte aggregation was higher in samples obtained from intravenous cannulae compared to those obtained by venepuncture (20.9 +/- 3.9% vs.13.8 +/- 2.4%, P = 0.03). For every 10 minutes of delay prior to processing platelet-monocyte aggregates increased by 2.8% (P = 0.0001) in PPACK anticoagulated blood and 1.7% (P = 0.01) in citrate anticoagulated blood. Erythrocyte lysis together with fixation does not affect platelet-monocyte aggregation. Platelet-monocyte aggregates remained stable over 24 hours when fixed and stored at 4 degrees C. Multiple handling and processing factors may affect platelet-monocyte aggregation. We recommend the measurement of platelet-monocyte aggregates on samples collected by direct venepuncture, using a direct thrombin inhibitor as the anticoagulant and minimising the time delay before sample fixation.