Background: Therapeutic drug monitoring of tacrolimus is necessary for appropriate dose adjustment for a successful immunosuppressive therapy. Several commercial immunoassays are available for tacrolimus measurements. This study aimed at simultaneously evaluating the analytical performances of 4 such immunoassays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a standard. For the first time, cross-reactivity to tacrolimus metabolites was assessed at concentrations frequently observed in clinical settings, as opposed to the higher concentrations tested by assay manufacturers.
Methods: An affinity column-mediated immunoassay (ACMIA), using upgraded flex reagents; released in 2015, a chemiluminescence immunoassay (CLIA), an electrochemiluminescence immunoassay (ECLIA), and a latex agglutination turbidimetric immunoassay (LTIA) were evaluated using frozen whole blood samples collected from transplantation patients. Cross-reactivities to 3 major tacrolimus metabolites (13-O-demethyl-tacrolimus [M-I], 31-O-demethyl-tacrolimus [M-II], and 15-O-demethyl-tacrolimus [M-III]) were evaluated.
Results: Each immunoassay correlated well with LC-MS/MS, and the Pearson's correlation coefficients (R) were 0.974, 0.977, 0.978, and 0.902 for ACMIA, CLIA, ECLIA, and LTIA, respectively. Using Bland-Altman difference plots to compare the immunoassays with LC-MS/MS, the calculated average biases were -6.73%, 6.07%, 7.46%, and 12.27% for ACMIA, CLIA, ECLIA, and LTIA, respectively. The cross-reactivities of ACMIA to the tacrolimus metabolites M-II and M-III were 81% and 78%, respectively, when blood was spiked at 2 ng/mL, and 94% and 68%, respectively, when it was spiked at 5 ng/mL.
Conclusions: Each immunoassay was useful, but had its own characteristics. ACMIA cross-reactivities to M-II and M-III were much higher than the respective 18% and 15% reported on its package insert, suggesting that cross-reactivity should be examined at clinically relevant concentrations.