Material-induced up-regulation of leukocyte CD11b during whole blood contact: material differences and a role for...

Material-induced thrombogenicity is in part a consequence of leukocyte activation. To evaluate and compare material-induced platelet damage, we have expanded our in vitro flow cytometric immunoassay to include assessment of leukocyte activation. We have used a very simple system whereby fresh, heparinized whole blood contacts materials for 1 h at 37 degrees C under low shear. Unlike other tests that focus on adherent leukocytes, this assay evaluates the leukocytes in the whole blood drained from the tube (1.57 mm internal diameter, 25 cm length) after material contact. We demonstrate that whole blood contact with a polyvinyl alcohol (PVA) hydrogel surface leads to a twofold up-regulation in CD11b surface expression of all monocytes and neutrophils. The activation is metal-ion dependent and highly material dependent in that blood contact with polyethylene and Silastic surface leads to minimal activation. The shedding of L-selection as a marker of leukocyte activation was found to be unsuitable in our assay given it ease of shedding in resting heparinized whole blood. Further, plasma levels of complement components Bb and sC5b-9 (ELISA assays) were significantly elevated only after blood contact with PVA hydrogel surfaces (9.4 micrograms/mL sC5b-9 and 9.6 micrograms/mL Bb). Use of recombinant soluble human CR1 (sCR1) to inhibit the action of the C3 and C5 convertases completely inhibited sC5b-9 levels in whole blood after contact with PVA hydrogel surfaces and inhibited CD11b up-regulation by over 70%, suggesting that material-induced leukocyte activation is partially mediated by C5a production.