Patient-independent variables affecting the assessment of aspirin responsiveness by serum thromboxane measurement

Abstract

The serum TXB (sTXB) assay reflects the pharmacodynamics of platelet inhibition by low-dose aspirin. However, different studies reported variable sTXB values. sTXB assay requires whole blood incubation at 37 °C as a condition for optimal thrombin generation, arachidonic acid release and its metabolism by platelet cyclooxygenase-1 to form TXA. Access to 37 °C incubation may be variably delayed, and different methods to quantitate sTXB may contribute to variable results between different Centers. We investigated whether delaying 37 °C incubation and/or analytical issues affect sTXB concentrations, biasing the assessment of aspirin responsiveness. Sixty-eight samples from 54 volunteers, on- and off-aspirin, were incubated at 37 °C immediately after sampling (reference sample) or after 5, 10, 15, 20, 30 or 60 minutes at room temperature (RT); 8 samples remained at RT 60 minutes, without subsequent incubation; 314 sera were measured by enzyme immunoassay (EIA) and liquid chromatography-tandem mass-spectrometry (LC/MS-MS) methods. sTXB concentrations decreased exponentially as a function of the delay before 37 °C incubation, ranging from 94 ± 11 % at 5 minutes to 23 ± 22 % of the reference sample after 60 minutes at RT. There was high agreement between EIA and LC/MS-MS. Moreover, we simulated the influence of a 15- or 30-minute delayed incubation on 300 sTXB2 measurements from previously-studied, aspirin-treated patients. Delayed incubation reduced the percentage of aspirin 'non-responders' by 22 % to 52 %, depending on the response threshold. In conclusion, a variable delay in the 37 °C incubation of blood samples may affect the assessment of platelet cyclooxygenase-1 inhibition by aspirin and confound the characterization of the determinants of aspirin responsiveness.

The Aspirin Regimens in Essential Thrombocythemia (ARES) phase II randomized trial design: Implementation of the serum thromboxane B2 assay as an evaluation tool of different aspirin dosing regimens in the clinical setting

“…Consistent with this expectation, a comparison of serum TXB 2 values in two large, multicenter cohorts of aspirin-treated patients 45 , 46 showed up to 10-fold difference in median TXB 2 levels (7 and 0.6 ng/ml in the ADRIE 46 and BOSTON 45 studies, respectively) that could not be explained by patient characteristics or analytical biases 47 . Two recent in vitro studies showed that even a minor delay in starting 37 °C incubation can time-dependently underestimate serum TXB 2 levels 17 , 48 , and thus potentially account for variable aspirin responsiveness across studies and centers. Thus, we assessed the feasibility of obtaining reliable serum TXB 2 measurements across the ARES study centers.…”

“…All samples were shipped frozen to a Core Lab, where centralized measurements were performed. Serum TXB 2 was measured by enzyme immunoassay (EIA) as previously described 17 , 23 . This EIA method has a limit of detection calculated as 80% B / B 0 of 3 ± 2 pg/ml, an inter-assay coefficient of variation of 6% ( n = 75 determinations), and has been validated against gas chromatography/mass spectrometry 17 .…”

“…Thus, data in ET suggest a pathogenetic link between persistently enhanced platelet activation and thrombotic complications, requiring an effective antiplatelet therapy. Low-dose aspirin (75–100 mg once daily [od] 17 ) is currently recommended for both secondary and primary CV prevention in the majority of ET patients 1 , 9 , with the exception of young patients without traditional CV risk factors, defined at “low risk”, in whom aspirin in primary prophylaxis remains controversial 18 and possibly dependent on the mutation profile 19 .…”

Development and validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of serotonin and thromboxane B2 from activated platelets

“…It is synthesized from arachidonic acid by activated platelets and after binding to its receptor on platelets causes vasoconstriction, activation, amplification, and irreversible platelet aggregation . TxA2 is rapidly hydrolyzed non‐enzymatically to the biologically inactive form TxB2 …”

“…7,8 TxA2 is rapidly hydrolyzed non-enzymatically to the biologically inactive form TxB2. 8,9 We validated our UHPLC-MS/MS method according to the European Medicines Agency (EMA) guideline for bioanalytical method validation. 10 The method was then used in combination with light transmission aggregometry (LTA) to identify good platelet responders for the diagnosis of HIT.…”