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 signal for phagocytic clearance of apoptotic cells. In order to measure PS exposure in blood cells, we developed a flow-cytometric procedure to measure annexin V binding to leukocytes and platelets in whole-blood samples. Leukocytes were identified by CD45 and side-scatter gating, and platelets by CD61 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 equal 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-fold 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 markers of platelet activation, CD62P and the activation-dependent epitope of glycoprotein IIb/IIIa detected by the PAC1 antibody, indicated that platelets from normal donors showed the least amount of activation with the annexin V marker. Whole-blood flow cytometry with annexin V can reliably measure the state of PS exposure in platelets and leukocytes, and the results confirm that these cell types normally circulate with extremely low levels of exposed PS.