EFFECT OF FIXATION ON QUANTIFICATION OF THE EXPRESSION OF LEUKOCYTE FUNCTION-ASSOCIATED SURFACE-ANTIGENS

The surface expression of adhesion molecules and other function-associ ated antigens on peripheral blood leucocytes may be measured by flow c ytometry. However, quantification of these antigens is difficult, beca use their expression can be rapidly and artefactually modulated if the cells are activated in vitro, Consequently, it is common, when analyz ing these antigens either: (1) to label leucocytes in whole blood at 4 degrees C, to lyse erythrocytes and then fix the leucocytes, or (2) t o fix the leucocytes in whole blood, to lyse the erythrocytes, and the n label the leucocytes. We have compared the mean fluorescence intensi ty (MFI) values for CD11b, CD18, and L-selectin (Leu-8 and TQ1 epitope s) on human peripheral blood leucocytes, using these two approaches. I n addition, we have simultaneously evaluated how anticoagulants (acid citrate, K(3)EDTA, and heparin) and the presence or absence of divalen t metal ions (Ca2+ and Mg2+) affect the expression levels of these ant igens. The results for all four epitopes varied markedly depending on the preparation procedure used but were less affected by the choice of anticoagulant and whether divalent cations were or were not present i n the media used for cell preparation and labelling. Comparison of the results obtained using these procedures, which involve fixation with formaldehyde, with those obtained by a recently developed procedure in which unfixed leucocytes were labelled with the vital nuclear dye LDS -751 and antibodies together, then analysed in unlysed whole blood at 4 degrees C, showed that formaldehyde-based preparation techniques und erestimated the expression (MFI) of CD18, Leu-8, and TQ1. It is recomm ended that, whenever practicable, measurements are made on unfixed cel ls stained using the newer procedure. (C) 1994 Wiley-Liss, Inc.