Ticagrelor Interference in the Serotonin-Release Assay

Introduction:

Ticagrelor is a widely used reversible platelet adenosine 5’-diphosphate (ADP) P2Y12 receptor inhibitor used in patients with acute coronary syndrome and coronary interventions. Ticagrelor treated patients may be concomitantly exposed to heparin treatment for prophylaxis/treatment of thrombosis, and heparin induced thrombocytopenia (HIT), a thrombotic thrombocytopenic adverse event may be suspected in such patients.

Objective:

Here we describe the serological profile of a patient on ticagrelor treatment and strong clinical suspicion of HIT who presented with negative serotonin release assay (SRA) results. A follow up systematic study on the impact of ticagrelor on this gold standard HIT assay is presented.

Methods:

Remnants from 23 patient samples submitted for clinical SRA testing (5- SRA negative; 6- “low positive” [20-50%]; 5- “moderate positive” [>50 to <90%]; 7- “strong positive” [>90%]) were spiked with ticagrelor in concentrations of 100, 250, 500, and 1000 ng/mL before SRA re-testing. Testing was performed using heat inactivated serum, with low (0.1 units/mL) or high (100 units/mL) heparin and washed platelets (Leger, THSNA 2022). A positive SRA result was defined as ≥20% serotonin release with the low heparin condition and >50% inhibition of serotonin release with high concentration of heparin.

A subset of SRA positive patient samples that tested falsely negative upon ticagrelor addition were subsequently incubated with activated charcoal and retested in the SRA to determine if ticagrelor interference was reversible using this methodology.

Results:

The index patient on ticagrelor had a high 4Ts score, ELISA optical density >1.0 but tested negative in the SRA. Re-testing of this patient with an activated charcoal treated sample demonstrated a striking change in SRA to a strong positive result (91% serotonin release).

Upon testing of ticagrelor-spiked samples in the SRA negative patient cohort, as expected, the drug had no effect on results, with all 5 samples persistently staying SRA negative. Of the 6 patients that had low positive SRA results, all converted to SRA negative with ticagrelor concentrations >500 ng/mL. One of these patients became SRA negative at 100 ng/mL and 3 at 250 ng/mL. Of the 5 patients with moderately positive SRA results, 2 converted to SRA negative with ticagrelor concentrations >100 ng/mL, and 4 of 5 patients became negative with spiking at 500 ng/mL. Finally, of the 7 patients with strong positive SRA results, 1 became falsely negative in the SRA at a ticagrelor concentration of 1000 ng/mL.

Activated charcoal was incubated with 6 samples that demonstrated negative SRA results with drug concentration of 500 ng/mL. Two samples reverted to SRA positive, while 4 samples remained persistently negative.

Conclusion:

In preliminary studies, ticagrelor has been shown to interfere with the heparin induced platelet activation (HIPA) assay but its impact on the SRA has been relatively unexplored. Here, we demonstrate that patients on ticagrelor may present with spuriously false HIT functional assay testing by the SRA, particularly if the HIT antibody is not strongly platelet activating. Activated charcoal treatment was only able to reverse drug interference in a subset of samples, even at drug concentrations below peak levels recorded in patients (approx. 800ng/mL). Given these findings, we recommend de-emphasizing the utility of the SRA in ticagrelor treated patients with reliance more on criteria such as pre-test probability of HIT (e.g. 4Ts score), and ELISA results in order to facilitate an accurate diagnosis of HIT.