Anticoagulant selection influences flow cytometric determination of CD11b upregulation in vivo and ex vivo

Abstract

We recently devised three-colour flow cytometic assay for evaluating expression of CD11b on neutrophils and monocytes in circulation. Artefactual upregualtion of CD11b ex vivo was minimized by cooling blood samples on ice. In this communication we further characterize the method in terms of differet anticoagulants. EDTA was less optimal than ACD or heparin because (i) saturating concentrations of CD11b antibody (clone D12) were not achieved with resting cells; (ii) CD11b fluorescence intensity of synovial fluid cells, i.e., in vivo activated cells expressing CD11b at high levels, was significantly lower in EDTA plasma, and (iii) EDTA mediated more cell damage at 37°C, as determined by PI staining. The fluorescence data suggested that D12 antibody binding was dependent on divalent cations. Saturating concentrations in the presence of EDTA in medium were easily obtained with synovial fluid cells and peripheral blood phagocytes activated with chemotactic peptide FMLP, suggesting that cell activation decreased cation concentrations required for D12 antibody binding. Using another CD11b antibody (2MPL19c), whose binding proved to be cation independent, it was shown that CD11b upregulation was not affected by EDTA. ACD was superior to heparin and phenylalanylprolylarginyl chloromethyl ketone (PPACK), a thrombin inhibitor, because cell counts were significantly lower in heparinized samples in cold, and in PPACK-anticoagulated samples treated with LPS at 37°C. Kinetics of l-selectin shedding was similar in heparin and ACD, suggesting that cell loss did not derive from differences in cell activation. In comparison of buffy coat cell assay and whole blood assay, neutrophil CD11b expression was similar but background fluorescence was significantly higher in whole blood preparations. This implies that nonspecific antibody binding may occur more in whole blood assay, whereas in the buffy coat cell assay, sample manipulation procedures may slightly increase CD11b antibody binding, but not control antibody binding. Finally, it was confirmed that warming from 0°C, but not from room temperature, to 37°C increased CD11b expression significantly on neutrophils, and it was further shown that monocytes undergo similar changes. Cooling did not upregulate CD11b, and completely prevented LPS-induced upregulation. In conclusion, the results support use of ACD in evaluating CD11b expression; if EDTA is used, it is important to make sure that binding of CD11b antibody selected does not require presence of divalent cations in medium.