QUANTITATION OF ADHESION MOLECULES AND OTHER FUNCTION-ASSOCIATED ANTIGENS ON HUMAN PERIPHERAL-BLOOD LEUKOCYTES

Quantitating satisfactorily the expression of adhesion molecules and o ther functional antigens on the surface of human peripheral blood gran ulocytes by flow cytometry is difficult. Artefactual (in vitro) change s, which occur in the expression of these molecules during conventiona l cell preparation and labelling procedures, led to the development of a leucocyte preparation technique that prevents upregulation of the l eucocyte integrins and other antigens. The technique, as originally ev aluated, involves fixing freshly drawn blood with formaldehyde (final concentration 0.2%) for 4 min at 37-degrees-C before adding ammonium c hloride to lyse the erythrocytes; procedures which are inconvenient wh en 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 formaldehyd e for a similar period before processing was continued. The results sh owed that when quantitating those granulocyte surface antigens which c an be rapidly upregulated: i) that it is preferable to mix freshly dra wn unanticoagulated blood directly with formaldehyde; ii) that if bloo d must be collected into anticoagulants, it should be kept for only th e briefest possible time before processing; and iii) that the time for which cells are exposed to 0.2% formaldehyde must be carefully contro lled and limited to 4 min. A corollary of the results with anticoagula nts is that it is probably impossible to isolate live granulocytes by currently available procedures without artefactually altering the expr ession of these antigens in vitro. (C) 1993 Wiley-Liss, Inc.