PF4 enhanced PP flow cytometry. Plasma samples from 6 VITT-positive patients and 8 VITT-negative patients previously tested on the standard flow cytometry assay (A) or 15 individuals classified as negative, inconclusive, or equivocal on the standard assay were retested on the PF4 enhanced assay (B). Green line represents a patient with heparin-enhancing response. Donor whole blood was treated with platelet agonist 5 µM SFLLRN and patient plasma in the presence of 25 µg/mL purified native human PF4, unfractionated heparin (0.5 U/mL or 100 U/mL), and/or FcγRIIa-blocking antibody IV.3 (10 µg/mL). Dotted horizontal line represents 1.7-fold increase above no plasma baseline determined in Figure 3 as the optimal cutoff for VITT. The PP response induced by VITT-positive plasma was correlated with anti-PF4 antibody titer represented by ELISA optical density (OD) values (n = 87) (C), patient platelet count at the time of testing (n = 98) (D), and D-dimer levels represented by fold-change above upper limit of normal (n = 96) (E). All patients recorded a platelet count nadir of <150 × 10⁹/L. Spearman correlation analysis was performed.