Analytical validation of the PRO-Trac II ELISA for the determination of tacrolimus (FK506) in whole blood

Abstract

Background: The analytical validation of multiple lots of the PRO- Trac(TM) II ELISA (DiaSorin) for the determination of tacrolimus in whole blood is described. Methods: The analytical parameters assessed included analytical sensitivity, dilution linearity, functional sensitivity, values in samples containing no tacrolimus, intra-and interassay precision, supplementation and recovery, metabolite cross-reactivity, interference studies and method comparisons HPLC-tandem mass spectrometry (HPLC/MS/MS) and the IMx® Tacrolimus II multiparticle enzyme immunoassay. Where appropriate, assessments were performed according to NCCLS guidelines. Results: The mean analytical detection limit was <0.25 μg/L for all lots, whereas the functional sensitivity was 1.0 μg/L. Excellent linear correlation (r = 0.985) was observed for dilution linearity. The intraassay imprecision was <7%, and the total imprecision by ANOVA was <10%. Recovery was 109% ± 11%. Metabolite cross-reactivity was consistent with previous reports for this antibody. No interference was observed for 35 tested drugs. Method comparison with HPLC/MS/MS showed no statistically significant differences. Samples exhibited stability through four freeze/thaw cycles and for 1 week at room temperature. Conclusion: These data demonstrate that the PRO-Trac II ELISA is a robust, accurate, and precise tool for the assessment and management of tacrolimus blood concentrations in transplant patients.