Measurement of phosphatidylserine exposure in leukocytes and platelets by whole-blood flow cytometry with annexin V

Phosphatidylserine (PS) exposure serves as a procoagulant stimulus and a si gnal for phagocytic clearance of apoptotic cells. In order to measure PS ex posure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leuko cytes were identified by CD45 and side-scatter gating, and platelets by CD6 1 and side-scatter gating, The absolute number of annexin V molecules bound per cell was determined from an independent calibration procedure, Normal populations had the following levels of annexin V binding (in molecules per cell): lymphocytes, 0.53 x 10(3); neutrophils, 1.75 x 10(3); monocytes, 2. 45 x 10(3); platelets, 0.14 x 10(3). These levels represent less than or eq ual to 0.1% of the values obtained after maximal stimulation of PS exposure with calcium ionophore, confirming that virtually all PS is intracellular in normal circulating leukocytes and platelets. Pretreatment of whole-blood samples with ammonium chloride to lyse erythrocytes caused a 9- to 300-fol d increase in annexin V binding to leukocytes, indicating that analysis of unlysed whole-blood samples is essential to avoid artifactual increases in annexin V binding to leukocytes. Comparison of annexin V with two other mar kers of platelet activation, CD62P and the activation-dependent epitope of glycoprotein IIb/IIIa detected by the PAC1 antibody, indicated that platele ts from normal donors showed the least amount of activation with the annexi n V marker. Whole-blood flow cytometry with annexin V can reliably measure the state of PS exposure in platelets and leukocytes, and the results confi rm that these cell types normally circulate with extremely low levels of ex posed PS.