Influence of neutrophil separation on the expression of adhesion molecules

In many assays of polymorphonuclear neutrophil (PMN) function the first step is separation of PMN from whole blood. In the present investigation it was examined if PMN separation leads to an altered expression of neutrophil surface membrane adhesion molecules. Samples have been taken from 20 healthy volunteers (10 male, 10 female; 39.7 +/- 11.8 years of age). PMN activation was measured cytometrically using the following antibodies against PMN surface membrane receptors: L-selectin (CD 62 L), beta-2-integrin Mac-1 (CD 11b) and Intercellular Adhesion Molecule 1 (CD54). PMN activation was determined in whole blood and after separation of PMN using density gradients. After PMN separation all three adhesion molecules appeared increased but the effect was only statistically significant for CD 54 (Wilcoxon test). Data (mean fluorescence intensity in arbitrary units) were: CD 62 L: 62 +/- 37 in whole blood, 82 +/- 28 after separation; p = 0.0674, CD 11b: 94 +/- 55 in whole blood, 111 +/- 47 after separation; p = 0.1454, CD 54; 13 +/- 12 in whole blood, 81 +/- 35 after separation; p < 0.0001. With the present data available it can be assumed that separation of PMN from whole blood can influence the results of flow cytometric assays.