Effect of assay specificity on the association of urine 11-dehydro thromboxane B2 determination with cardiovascular risk

Abstract

Background: Elevated urine 11-dehydro TXB(2), 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 TXB(2) 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 TXB(2) 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 TXB(2) to clinical outcome was assessed for each assay using multivariate modeling.

Results: Median 11-dehydro TXB(2) 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 TXB(2), a thromboxane metabolite present in a similar concentration but with a poor direct correlation with 11-dehydro TXB(2). In contrast to the first-generation ELISA, 11-dehydro TXB(2) measured by the monoclonal antibody-based ELISA failed to associate with the risk of vein graft occlusion.

Conclusion: Quantification of urine 11-dehydro TXB(2) by monoclonal antibody-based ELISA was confounded by interference from 11-dehydro-2,3-dinor TXB(2) which reduced the accuracy and clinical utility of this second-generation assay.

Conflict of interest statement

Disclosure of Conflict of Interest

J.J. Rade and T.S. Kickler received royalty payments as part of a licensing agreement between Siemens Healthcare Diagnostics and Johns Hopkins University; J.J. Rade has received honoraria from Corgenix Medical Corporation for speaking at sponsored CME-approved symposia. All other authors report no conflicts.

© 2012 International Society on Thrombosis and Haemostasis.

Figures

Fig. 1

Inter-assay correlation of 11-dehydro TXB2 results in 451 Reduction in Graft Occlusion Rates (RIGOR) urine samples measured by polyclonal and monoclonal antibody-based ELISAs. (A) Distribution of result values. Dashed lines denote respective median values. (B) Scatter plot of the result values. Solid line denotes the regression trend line and the dashed line has a slope = 1. (C) Bland–Altman plot of percent bias vs. mean.

Fig. 2

Correlation of ELISA results with mass spectrometry. Gray and black solid lines denote regression lines for 11-dehydro TXB2 results measured by the monoclonal and polyclonal antibody-based ELISAs, respectively. Dashed line has a slop of one.

Fig. 3

Differences in primary antibody specificity between assays. (A) Pooled urine sample with low concentration of 11-dehydro TXB2 were spiked with an increasing concentration of the indicated thromboxane metabolites and apparent 11-dehydro TXB2 levels determined by polyclonal (left panel) and monoclonal antibody-based ELISA (right panel). (B) Correlation between urine 11-dehydro 2,3-dinor TXB2 and 11-dehydro TXB2 levels determined by performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS).

Fig. 4

Percentage of occluded vein grafts in Reduction in Graft Occlusion Rates (RIGOR) subjects stratified by quartile of urine 11-dehydro TXB2. *P = 0.004; **P = 0.01.