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A sensitive flow cytometric assay for circulating platelet–leucocyte aggregates

A whole blood flow cytometric method for studies of platelet–leucocyte aggregates (PLAs) in vivo, involving neither fixation nor centrifugation, is described. With this method, PLAs in the total leucocyte population (PLA/L) were 15.3 ± 8.5% in 36 healthy volunteers. Blocking antibodies had little effect on PLAs in the absence of in vitro stimulation, suggesting that the aggregates were preformed in vivo. Fixation with formaldehyde or paraformaldehyde increased PLA/L significantly. Similarly, prefixation of the blood or red blood cell lysis, with repeated washing and centrifugation, caused artefactual 3–5‐fold increases in PLAs (P < 0.05). ADP, thrombin, platelet activating factor (PAF) or N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) each increased PLA formation in unfixed whole blood dose‐dependently; additive effects were found when they were combined. Experiments with blocking MAbs suggested that different ligand–receptor systems mediate PLA formation by different agonists. PLA formation by the platelet agonist ADP was inhibited by P‐selectin blockade, but enhanced by GPIIb/IIIa blockade (which inhibits platelet–platelet interactions). PLA formation by the leucocyte agonist fMLP was inhibited by GPIIb/IIIa blockade, suggesting linking via fibrinogen. Platelet–leucocyte aggregate analysis by this whole blood method appears to reflect in vivo conditions, and enables investigations of the mechanisms involved in their formation.