A SIMPLE FLOW CYTOMETRIC PROCEDURE FOR THE DETERMINATION OF SURFACE-ANTIGENS ON UNFIXED LEUKOCYTES IN WHOLE-BLOOD

A novel procedure has been developed for the quantitation by flow cyto metry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood antico agulated with the serine esterase inhibitor, phenylmethylsulphonyl flu oride, is mixed with the vital nucleic acid stain, LDS-751, labelled w ith monoclonal antibodies for 5 min at 4-degrees-C, diluted and analys ed in a five-parameter flow cytometer. The three major leucocyte subpo pulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining int ensity with LDS-751 in the FL3 channel (erythrocytes and platelets sta in very weakly), whilst the fluorescence intensity due to bound fluore scein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives , erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitat ion of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of t hose leucocyte surface antigens whose expression is not subject to rap id modulation.