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

The serum TXB2 (sTXB2) assay reflects the pharmacodynamics of plateletinhibition by low-dose aspirin. However, different studies reportedvariable sTXB2 values. sTXB2 assay requires whole blood incubationat 37 °C as a condition for optimal thrombin generation, arachidonicacid release and its metabolism by platelet cyclooxygenase-1to form TXA2. Access to 37 °C incubation may be variably delayed, anddifferent methods to quantitate sTXB2 may contribute to variable resultsbetween different Centers. We investigated whether delaying37 °C incubation and/or analytical issues affect sTXB2 concentrations,biasing the assessment of aspirin responsiveness. Sixty-eight samplesfrom 54 volunteers, on- and off-aspirin, were incubated at 37 °C immediatelyafter sampling (reference sample) or after 5, 10, 15, 20, 30or 60 minutes at room temperature (RT); 8 samples remained at RT 60minutes, without subsequent incubation; 314 sera were measured byenzyme immunoassay (EIA) and liquid chromatography-tandem massspectrometry(LC/MS-MS) methods. sTXB2 concentrations decreasedexponentially as a function of the delay before 37 °C incubation, rangingfrom 94±11 % at 5 minutes to 23±22 % of the reference sampleafter 60 minutes at RT. There was high agreement between EIA andLC/MS-MS. Moreover, we simulated the influence of a 15– or 30-minutedelayed incubation on 300 sTXB2 measurements from previouslystudied,aspirin-treated patients. Delayed incubation reduced the percentageof aspirin ‘non-responders’ by 22 % to 52 %, depending onthe response threshold. In conclusion, a variable delay in the 37 °C incubationof blood samples may affect the assessment of plateletcyclooxygenase-1 inhibition by aspirin and confound the characterizationof the determinants of aspirin responsiveness.