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 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 </= 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 PAC I 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
Tait
J F
JF
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110, USA. tait@u.washington.edu
Smith
C
C
Wood
B L
BL
D017304:Annexin A5 / Q000032:analysis; D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000097:blood; D001792:Blood Platelets / Q000737:chemistry* / Q000648:ultrastructure; D002138:Calibration; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D019963:Clinical Chemistry Tests / Q000379:methods / Q000592:standards; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007962:Leukocytes / Q000737:chemistry* / Q000648:ultrastructure; D019007:P-Selectin; D010718:Phosphatidylserines / Q000097:blood*; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology; D012016:Reference Values
D017304:Annexin A5; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D019007:P-Selectin; D010718:Phosphatidylserines; D019039:Platelet Glycoprotein GPIIb-IIIa Complex