Platelet activation in the PIFPA test. Platelet activation induced by (heat-inactivated; 56°C, 30 minutes) sera and PF4 is shown by CD62P expression and given by the MFI multiplied by percentage of gated events. Data are shown for 16 samples from patients with VITT (14 sera, 2 plasmas), 10 sera from patients after vaccination with positive EIA but negative PIPA result, 10 sera from patients after vaccination with negative EIA and PIPA, and 4 sera from patients with typical HIT. One data point for each serum represents median activation from 4 different whole-blood samples. Cutoff was determined with sera of 13 unvaccinated healthy controls incubated with 5 µg/mL PF4 as the mean plus 2 standard deviations (SD). With the addition of 5 µg/mL PF4, sera from patients with VITT can be readily discriminated from vaccinated donors with no functionally relevant antibodies (P < .0003). Statistical significance was calculated by the unpaired Student t test. *P < .05; **P < .01; ***P < .001. HIPA, heparin-induced platelet aggregation; UFH, unfractionated heparin.