Quantitation of adhesion molecules and other function-associated antigens on human peripheral blood leucocytes

Quantitating satisfactorily the expression of adhesion molecules and other functional antigens on the surface of human peripheral blood granulocytes by flow cytometry is difficult. Artefactual (in vitro) changes, which occur in the expression of these molecules during conventional cell preparation and labelling procedures, led to the development of a leucocyte preparation technique that prevents upregulation of the leucocyte integrins and other antigens. The technique, as originally evaluated, involves fixing freshly drawn blood with formaldehyde (final concentration 0.2%) for 4 min at 37 degrees C before adding ammonium chloride to lyse the erythrocytes; procedures which are inconvenient when blood samples are taken in wards or clinics that are some distance from a laboratory. We therefore investigated i) whether blood could be kept with commonly used anticoagulants for up to 1 h before fixation, and ii) whether unanticoagulated blood could be kept with formaldehyde for a similar period before processing was continued. The results showed that when quantitating those granulocyte surface antigens which can be rapidly upregulated: i) that it is preferable to mix freshly drawn unanticoagulated blood directly with formaldehyde; ii) that if blood must be collected into anticoagulants, it should be kept for only the briefest possible time before processing; and iii) that the time for which cells are exposed to 0.2% formaldehyde must be carefully controlled and limited to 4 min. A corollary of the results with anticoagulants is that it is probably impossible to isolate live granulocytes by currently available procedures without artefactually altering the expression of these antigens in vitro.