Neutrophil integrin assay for clinical studies

The level of expression of neutrophil adhesion molecules may be a useful marker for neutrophil activation in clinical studies. We therefore determined neutrophil integrin expression under various experimental conditions using a Fluorescence Activated Cell Sorter (FACS) after the cells had been labelled with fluorescent conjugated antibodies to the integrin subunits CD11a, CD11b and CD18. Levels of labelled CD11b and CD18 increased after activation with the chemotactic peptide formyl-methionyl-leucyl phenylalanine (fMLP) in a dose- and time-dependent manner, but CD11a did not, indicating that CD11a would not be a useful marker of neutrophil activation. The baseline expression of CD11b and CD18 on unstimulated neutrophils was similar in heparin and EDTA anti-coagulated blood but the response to activation with fMLP was significantly less for the EDTA anti-coagulated samples (p < 0.01 in paired t-test). The labelling of integrins was significantly higher in unfixed whole blood samples compared to samples fixed with 1 per cent paraformaldehyde. However, the increase in labelling induced by fMLP was similar whether or not the samples were fixed after activation. Labelling of CD11b and CD18 was greater for preparations of isolated neutrophils than for neutrophils in whole blood, and the response to fMLP stimulation tended to be lower for the isolated cells. Our results indicate that heparin should be used as anti-coagulant in clinical studies utilizing whole blood if subsequent activation of neutrophils is planned (e.g. to detect in vivo priming), although EDTA may be used if baseline expression alone is to be measured. Fixation of blood samples should not affect the ability to detect neutrophil activation.