Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood

Enzyme inhibitors have been compared with conventional anticoagulants for the flow cytometric analysis of live leucocytes in whole blood. At 18 to 20 degrees C in vitro, PPACK (60 microM), hirudin (10 units ml-1), leupeptin (20 microg ml-1) and aprotinin (50 microg ml-1) inhibited blood clotting for 4 h or more, whereas PMSF (8 mg ml-1) or AEBSF (5 mg ml-1) inhibited clotting for only 20 or 40 min, respectively. When labelled with CD11b antibodies and analysed immediately ex vivo at 4 degrees C, the percentages of lymphocytes, monocytes, and polymorphs which stained positively and their mean fluorescence intensities were similar, irrespective into which anticoagulant blood was collected. Less than 1.5-fold increases in expression occurred on monocytes and polymorphs when blood anticoagulated with enzyme inhibitors or conventional anticoagulants was kept at 4 degrees C, or when blood anticoagulated with citrate, heparin, or hirudin was kept at 18 to 20 degrees C for 1 h before labelling and analysis, whereas approximately 2-fold increases in expression occurred in blood kept with K3EDTA, leupeptin, or aprotinin and more than 3-fold increases in blood kept with AEBSF or PPACK at 18 to 20 degrees C for 1 h. Further studies showed that leupeptin could be used effectively as the anticoagulant when investigating functional responses of live leucocytes in whole blood samples by flow cytometry and for the isolation of leucocytes with minimal modulation of adhesion molecules.