The PF4-TRA is highly sensitive and specific for the detection of platelet-activating HIT and VITT antibodies. (A) PEA and SRA results for the cohort of HIT-suspected patient samples tested in the PF4-TRA are shown on the x-axis and y-axis, respectively. Red dotted lines represent positive cutoff values (19% P-selectin expression for the PEA and 20% serotonin release for the SRA). (B) PF4-TRA results for 34 HIT-suspected patient samples and 4 VITT patient samples are shown. ELISA-positive/PEA-positive samples, ELISA-positive/PEA-negative samples, and ELISA-negative/PEA negative samples are indicated by closed circles, closed triangles, and open triangles, respectively. Each data point represents the mean of technical duplicates. An ordinary 1-way analysis of variance was used for comparisons, and means are shown as dotted lines. (C) TRA results for OD-matched HIT ELISA-positive/PEA-positive patient samples (closed circles) vs HIT ELISA-positive/PEA-negative patient samples (closed triangles) are shown. Each data point represents the mean of technical duplicates. A 2-tailed, unpaired Student t test was used to compare OD values and TSP1 fold changes. (D) PEA and (E) SRA results were correlated with TSP1 release for the HIT-suspected patient samples tested in the PF4-TRA. Linear regression analysis (black solid line) with 95% CI (dashed lines) are presented. The fold increase in TSP1 released from platelets incubated with the HIT/VITT cohort samples relative to release seen with a normal serum control is depicted on the y-axis for (B), (C), (D), and (E). ∗∗∗∗P < .0001. IgG, immunoglobulin G; SpHIT, spontaneous HIT.