EFFECTS OF CELL PURIFICATION METHODS ON CD11B AND L-SELECTIN EXPRESSION AS WELL AS THE ADHERENCE AND ACTIVATION OF LEUKOCYTES

This study investigated the effects of commonly used procedures for th e isolation of leucocytes from human blood in comparison with cells in whole blood on the surface expression of CD11b and L-selectin (adhesi on molecules which are known to be increased and decreased respectivel y by cell activation). Washing of granulocytes or monocytes with Hanks ' buffered salt solution after separation by either dextran sedimentat ion or density gradient centrifugation, increased surface expression o f CD11b (p < 0.05). The number of monocytes bearing CD11b was enhanced (p < 0.05) by dextran sedimentation and two layer density gradient ce ntrifugation (Histopaque). The increase in CD11b expression on granulo cytes was associated with enhanced binding of the cells to endothelial monolayers that were either untreated (r = 0.902; p < 0.001) or treat ed with tumour necrosis factor alpha (TNF-alpha) (r = 0.68; p = 0.004) . The expression of L-selectin was reduced on granulocytes that had be en isolated by dextran sedimentation followed by hypotonic lysis of co ntaminating erythrocytes. All isolates of granulocytes demonstrated a loss of L-selectin following activation with fMLP though this effect w as less marked with cells subjected to erythrocyte lysis. The various separation methods had little effect on expression or distribution of CD11b or L-selectin on lymphocytes. We conclude that isolation of lymp hocytes by density gradient centrifugation and of granulocytes and mon ocytes by dextran-sedimentation and centrifugation using Histopaque gr adients, but avoiding washing and the use of hypotonic erythrocyte lys is, are appropriate techniques for studying the expression and functio n of adhesion molecules.