Validation of a free phenytoin assay on Cobas c501 analyzer using calibrators from Cobas Integra free phenytoin assay by comparing its performance with fluorescence polarization immunoassay for free phenytoin on the TDx analyzer.

For many years, fluorescence polarization immunoassay (FPIA) on the TDx analyzer has been used for determination of free phenytoin concentration. Recently Abbott Laboratories decided to discontinue the TDx analyzer and related assays on this analyzer. Free phenytoin assay is also available from Roche Diagnostics for application on the Cobas Integra analyzer (fluorescence polarization assay) but not on Cobas c510 analyzer. Free phenytoin calibrators from the Cobas Integra free phenytoin assay and the reagents from the KIMSphenytoin assay were used for the determination of free phenytoin on the Cobas c501 analyzer. The intra-run and inter-run precisions were both <7.2%. The assay was linear from 0.2 to 4 µg/ml. The free phenytoin assay on the Cobas c501 was compared with the FPIAassay on the TDx analyzer using sera from 25 patients receiving phenytoin (phenytoin concentration between 0.3 and 3.7 µg/ml). The following regression equation was observed: y = 0.9899 x + 0.0408 (r = 0.98, n = 25). In conclusion, the free phenytoin assay on the Cobas c501 analyzer is a valid alternative to free phenytoin assay on the TDx analyzer.

Comparison of mycophenolic acid concentrations determined by a new PETINIA assay on the dimension EXL analyzer and a HPLC-UV method.

OBJECTIVE: Mycophenolic acid requires routine therapeutic drug monitoring. We evaluated the suitability of a new PETINIA (particle enhanced turbidimetric inhibition immunoassay) assay on the Dimension EXL analyzer for monitoring of mycophenolic acid by comparing values obtained by this assay with a HPLC-UV method. DESIGN AND METHODS: Mycophenolic acid concentrations determined in sera of 60 organ transplant recipients using high performance liquid chromatography combined with ultraviolet detection (HPLC-UV, reference method) and the new immunoassay on the Dimension RxL analyzer. RESULTS: The within and between run precision of the new PETINIA assay was <10%. The assay was linear for a mycophenolic acid concentration up to 30µg/mL. When mycophenolic acid concentrations in 60 transplant recipients obtained by the HPLC-UV (x-axis) method were compared with corresponding values obtained by the PETINIA assay (y-axis), the following regression equation was obtained: y=1.1204 x+0.0881 (r=0.983, n=60). CONCLUSIONS: If PETINIA assay is used for therapeutic drug monitoring of mycophenolic acid, caution must be exercised in interpreting serum mycophenolic acid level due to observed positive bias.

Evaluation of QMS everolimus assay using Hitachi 917 Analyzer: comparison with liquid chromatography/mass spectrometry.

Everolimus is an immunosuppressant requiring routine monitoring in whole blood. We evaluated the analytical performance of a new immunoassay for everolimus, Quantitative Microsphere System (QMS) everolimus (Thermo Fisher Scientific), which is CE marked and currently under review by Food and Drug Administration of the United States by comparing results with values obtained by using liquid chromatography/mass spectrometry. The total coefficient of variations (CVs) were 8.3% for low control (mean: 3.8 ng/mL), 6.1% for the medium control (mean: 8.0 ng/mL), and 7.5% for the high control (mean: 14.4 ng/mL) (n = 80 for each control, run over 20 nonconsecutive days). The respective total CVs for patients' pool were 13.3% (mean: 4.0 ng/mL), 7.5% (mean: 8.2 ng/mL), and 8.7% (mean: 11.7 ng/mL) (n = 80 for each patient pool). The assay was linear from a whole-blood everolimus level between 1.5 and 20 ng/mL, and the limit of quantitation was 1.3 ng/mL. Comparison was carried out using 90 renal transplant patient samples, and we observed the following Passing and Bablok linear regression plot: y = 1.11, slope = -0.005 (R = 0.92). This assay was not affected by commonly used 70 drugs, but sirolimus, a drug structurally similar to everolimus, showed 46% cross-reactivity. We conclude that QMS everolimus immunoassay has adequate sensitivity and specificity for the determination of whole-blood everolimus and can be used for routine therapeutic drug monitoring.

Naproxen metabolites interfere with certain bilirubin assays: elimination of interference by using a Roche bilirubin assay on the Hitachi 917 analyzer.

A recent report indicated significant interference of naproxen with total bilirubin measurement when using the Jendrassik and Grof method. We explored the possibility of avoiding this interference by using a different method. We observed that naproxen has no effect on total bilirubin measurement by any method, but O-desmethylnaproxen (the naproxen metabolite) interfered significantly with total bilirubin assays using the Jendrassik and Grof method (Beckman Coulter [Brea, CA] and Siemens [Tarrytown, NY] bilirubin method). The Roche (Indianapolis, IN) bilirubin assay based on a different method was not affected. When serum samples of 9 patients receiving naproxen were analyzed for total bilirubin level, we observed good correlation in total bilirubin values measured by all 3 methods in 7 of 9 patients with therapeutic naproxen concentrations. In contrast, in 2 patients with elevated naproxen levels, the total bilirubin values observed by using the Beckman and Siemens methods were significantly higher than the value obtained by the Roche assay. The naproxen metabolite interferes with assays based on the Jendrassik and Grof method but not with the Roche bilirubin assay.

Hydroxyzine and cetirizine interfere with the PENTINA carbamazepine assay but not with the ADVIA CENTEUR carbamazepine assay.

Because of a published report indicating significant interference of hydroxyzine with the particle-enhanced turbidimetric inhibition immunoassay (PENTINA) carbamazepine assay, we investigated whether such interference can be avoided by using the ADVIA Centaur carbamazepine assay. Both the Dimension Vista analyzer and ADVIA Centaur analyzer are available from Siemens Diagnostics. Aliquots of a drug-free serum pool were supplemented with various concentrations of hydroxyzine or cetirizine (0.05 microg/mL to 20 microg/mL covering therapeutic and toxic levels in serum) followed by analysis using both assays. We observed significant apparent carbamazepine concentrations using the PENTINA assay but no apparent carbamazepine level using the ADVIA Centaur assay. Because crossreactivity should be studied in the presence of the primary analyte, we also prepared a serum carbamazepine pool from patients receiving carbamazepine and then supplemented aliquots of this pool with various amounts of hydroxyzine or cetirizine followed by reanalyzing carbamazepine concentration using both assays. We observed falsely elevated carbamazepine values using the PENTINA assay but no interference was observed using the ADVIA Centaur assay. However, the falsely elevated carbamazepine values using the PENTINA assay were clinically significant at hydroxyzine or cetirizine concentrations expected in patients with severe overdoses with these drugs. We conclude that the ADVIA Centaur carbamazepine assay is free from interference of both hydroxyzine and cetirizine.

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

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 micro mol/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 micro mol/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 micro mol/L.