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Summary. Background: Elevated urine 11-dehydro TXB2, an indicator of persistent thromboxane generation in aspirin-treated patients, correlates with adverse cardiovascular outcome and has recently been identified as an independent risk factor for vein graft thrombosis after cardiac bypass surgery in the Reduction in Graft Occlusion Rates (RIGOR) study. The polyclonal antibody-based ELISA used to measure 11-dehydro TXB2 in these previous studies is no longer clinically available and has been supplanted by a Food and Drug Administration (FDA)-cleared second-generation monoclonal antibody-based ELISA. Objectives: To compare the laboratory and clinical performance of the first- and second-generation assays in a well-defined study population. Methods: 11-dehydro TXB2 was quantified in 451 urine samples from 229 Reduction in Graft Occlusion Rates (RIGOR) subjects using both ELISA. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and spiking studies were used to investigate discordant assay results. The association of 11-dehydro TXB2 to clinical outcome was assessed for each assay using multivariate modeling. Results: Median 11-dehydro TXB2 levels were higher by monoclonal antibody- compared with polyclonal antibody-based ELISA (856 vs. 399 pg mg−1 creatinine, P < 0.000001), with the latter providing values similar to UPLC-MS/MS. This discrepancy was predominantly as a result of cross-reactivity of the monoclonal antibody with 11-dehydro-2,3-dinor TXB2, a thromboxane metabolite present in a similar concentration but with a poor direct correlation with 11-dehydro TXB2. In contrast to the first-generation ELISA, 11-dehydro TXB2 measured by the monoclonal antibody-based ELISA failed to associate with the risk of vein graft occlusion. Conclusion: Quantification of urine 11-dehydro TXB2 by monoclonal antibody-based ELISA was confounded by interference from 11-dehydro-2,3-dinor TXB2 which reduced the accuracy and clinical utility of this second-generation assay.