Methods for platelet microaggregation test and platelet reactivity assay. (A) Platelets were gated based on FSC/SSC properties. (B) Platelet microaggregation test. Test platelets, derived from either a patient or healthy control were stained with PKH and mixed in a 1:9 ratio with healthy control platelets, stained with CFSE. Platelets were analyzed in the phycoerythrin (PE; PKH) and fluorescein isothiocyanate (CFSE) channels; platelets positive in both channels were identified as aggregates. The left panel shows unstimulated platelets after 0 minutes, the middle panel unstimulated platelets after 10 minutes, and the right panel PMA-stimulated platelets after 10 minutes. (C) Platelet reactivity assay. The MFI of RPE-labeled mouse anti-human P-selectin antibodies and Alexa Fluor 488–labeled fibrinogen on platelets was measured by FACS analysis, representing the quantity of P-selectin and open GPIIbIIIa receptor per platelet. The left panel shows unstimulated platelets; the right panel shows platelets stimulated with 625 μM TRAP.