Human platelet aggregation test with whole blood. Platelets were labeled with CD31-Pacific Blue or CD31-FITC and mixed in 1:1 proportion prior to stimulation. (A) Representative dot plots before and after platelet aggregation upon stimulation with ristocetin. All populations (unstained, single-stained platelets and double-colored aggregates) are plotted against the forward and side scatter and indicated by colored arrowheads. PLT, platelet; RBC, red blood cell. (B) Aggregation was measured by flow cytometry in time after stimulation with PMA, collagen, Aggretin A, and ristocetin. Average and standard error of the mean are depicted, n > 3. Nonstimulated labeled platelets are shown as baseline for spontaneous aggregation. Colored asterisks at each data point indicate statistical significance with the unstimulated control (P < .05). (C) Whole blood platelet aggregation analysis performed with blood from Glanzmann thrombasthenia and LAD-III patients upon collagen, PMA, or ristocetin stimulation. Note that Glanzmann platelets respond about half normally to collagen but not to PMA, whereas in LAD-III patients, collagen and PMA-induced aggregation is equally reduced. Control platelets were incubated with tirofiban (β3 integrin inhibitor) or tirofiban + LIA 1/2.1 (integrin β1 blocking moAb) to mimic the phenotype of Glanzmann thrombasthenia and LAD-III variant patients, respectively.