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


OBJECTIVE
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.


CONCLUSIONS
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.
Department of Pathology, the Johns Hopkins School of Medicine, Baltimore, MD, USA.
M T
MT
Olson
T S
TS
Kickler
J A
JA
Lawson
R C
RC
McLean
J
J
Jani
G A
GA
FitzGerald
J J
JJ
Rade
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002318:Cardiovascular Diseases / Q000453:epidemiology* / Q000652:urine; D002853:Chromatography, Liquid; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D012307:Risk Factors; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D013929:Thromboxane B2 / Q000031:analogs & derivatives* / Q000652:urine
D000911:Antibodies, Monoclonal; D013929:Thromboxane B2; C049235:11-dehydro-thromboxane B2