Use of fluorescence polarization immunoassay for salicylate to avoid positive/negative interference by bilirubin in the Trinder salicylate assay

Abstract

Background: Significant positive bias of bilirubin in the Trinder salicylate method on automated analysers has been reported. Because the fluorescence polarization immunoassay (FPIA) for salicylate is also widely used in the clinical laboratory, we studied the potential interference of bilirubin in the salicylate FPIA. Methods: Salicylate serum pools (three different pools) were prepared from patients receiving salicylate. We also prepared a normal serum pool containing no salicylate and serum pools containing no salicylate but elevated bilirubin. Aliquots of one salicylate pool were supplemented with various concentrations of bilirubin (42.8- 427.5 µmol/L) and salicylate concentrations were measured by the salicylate FPIA (TDxFLx and AxSYM analysers). We also assayed these specimens with the Trinder salicylate method, using both Synchron LX and Hitachi 917 analysers for comparison with the results obtained by the FPIA method. In another experiment, aliquots of the two other salicylate pools were supplemented with various concentrations of bilirubin (42.8-684.0 µmol/L) in order to further study the effect of very high bilirubin concentrations on the salicylate FPIA. We also added known amounts of salicylate to serum pools containing elevated bilirubin but no salicylate and measured salicylate using the FPIA in order to study the recovery of salicylate in the presence of elevated bilirubin concentrations. Results: The FPIA showed minimal interference from bilirubin. We also observed good recovery of salicylate when specimens high in bilirubin but containing no salicylate were supplemented with known amounts of salicylate and the FPIA was used for the measurement of salicylate concentration. However, we observed falsely low salicylate concentrations with the Trinder method using the Synchron LX (primary wavelength 560 nm, secondary wavelength 700 nm) analyser and falsely increased salicylate concentrations using the same reagent but the Hitachi 917 (primary wavelength 546 nm, no secondary wavelength) analyser in the presence of elevated bilirubin levels compared with the FPIA results. Conclusion: We conclude that the FPIA for salicylate is not affected by high bilirubin concentrations up to 427.5 µmol/L.