Simultaneous measurement of biopolymer-mediated Mac-1 up-regulation and adherence of neutrophils: a novel flow cytometric approach for predicting initial inflammatory interaction with foreign materials

Implantation of any medical device normally causes an inflammatory cell int eraction with the foreign material. In vitro cell activation of human neutr ophils (Mac-1 upregulation) has been taken as one measure to assess the att ributable risk of inflammation due to biopolymers before their clinical app lication. Mac-1 expression has generally been measured by flow cytometric a ssays, whereas quantification of neutrophil adhesion to the biopolymer surf aces has been performed by separate and time-consuming assays, e.g. microsc opically by differential cell counting. However, due to an increasing numbe r of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate the se analyses, we have developed a novel flow cytometric assay pennitting sim ultaneous measurement of biopolymer-mediated neutrophil activation and adhe sion. The biopolymers were used as beads (diameter 25 +/- 10 mum), and were demon strated to be non-phagocytosable and non-fluorescent before being co-incuba ted with whole human blood (range of ratio granulocytes/beads from 5:1 to 1 :1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface. After establishing this assay, we evaluated it by comparing six different b iopolymers. We observed high levels of Mac-1 expression (> 70% of positive control) accompanied by increased adhesiveness (> 60% of neutrophils) for p olyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA ) beads. Low Mac-1 expression levels (< 10%) accompanied by a low percentag e of adhering neutrophils ( < 10%) were observed for polyethylene (PE), pol yisoprene (PI), and silicone (SI) beads. (C) 2001 Elsevier Science B.V. All rights reserved.