Flow cytometric evaluation of material-induced platelet and complement activation

Flow cytometry is used to characterize the activation state of platelets and leukocytes within whole blood after contact for 4 h at 37 degreesC with various materials under conditions of low shear. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic(TM), PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles, P-selectin expression and platelet-leukocyte aggregates. Only contact with PVA hydrogel surfaces led to dramatic increases in CD11b up-regulation on monocytes and neutrophils that was inhibited by complement inhibition (sCR1). Flow cytometry was also used to evaluate the effectiveness of various agents to inhibit material-induced complement activation. The assay involved incubating 10 mum polystyrene beads for 1 h with serum at 37 degreesC before isolating the beads so as to label them with a monoclonal antibody against a neoantigen on SC5b-9. The beads were then identified by flow cytometry and the fluorescence associated with their SC5b-9 level recorded. The ability of C1-INH, pentamidine and benzamidine to moderately inhibit SC5b-9 levels suggests a role for classical complement activation in material-induced complement activation.