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 degrees C 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, 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 (sCRI). Flow cytometry was also used to evaluate the effectiveness of various agents to inhibit material-induced complement activation. The assay involved incubating 10 microm polystyrene beads for 1 h with serum at 37 degrees C 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.
University Health Network, Toronto, Ontario, Canada. cynthia.gemmell@utoronto.ca
C H
CH
Gemmell
D001672:Biocompatible Materials / Q000737:chemistry / Q000494:pharmacology* / Q000592:standards; D001769:Blood; D003167:Complement Activation / Q000187:drug effects*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D006023:Glycoproteins / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology / Q000592:standards; D013314:Stress, Mechanical
D001672:Biocompatible Materials; D015938:Complement Membrane Attack Complex; D006023:Glycoproteins; D011108:Polymers; C047610:SC5b-9 protein complex; D003165:Complement System Proteins