Comparison of the Abbott IMx Tacrolimus I and Tacrolimus II Assays

Tacrolimus (FK-506, Prograf, Fujisawa Pharmaceutical Co.), first approved in the United States for liver transplants, is now widely used in other organ transplants (1). Its immunosuppressive potency is 10- to 100-fold greater than cyclosporine A. Because subtherapeutic concentrations are associated with organ rejection and high concentrations are associated with nephrotoxicity, neurotoxicity, and opportunistic infections, therapeutic drug monitoring of FK-506 is well-accepted and is a common laboratory practice (2). Currently, there is no consensus on the therapeutic range for FK-506. Substantial amounts of the drug are present intracellularly, with a red blood cells-to-plasma ratio >4:1. In addition, clinical effects correlate better with whole blood concentrations than with serum or plasma concentrations (3). Therefore, routine laboratory assays determine concentrations on whole blood. The whole blood therapeutic range was originally thought to be 15–25 μg/L, but has subsequently been modified at most US centers to 5–20 μg/L (1). Several methods are available for monitoring the concentration of FK-506. These include bioassays, radioreceptor assays, HPLC, ELISA (Incstar Corp.), and microparticle enhancement immunoassay (MEIA;Abbott Diagnostics) (4). HPLC, the reference method, is generally used for research purposes only. The ELISA, although more sensitive than MEIA (detection limit, 0.2 vs 1.5 μg/L), is a time-consuming method, taking >4 h of a technologist’s time per batch. Furthermore, the manufacturer of the ELISA method recommends running the calibration curve with every batch. These factors make ELISA inconvenient for routine use and expensive for small hospital laboratories, which run only a few samples at a time. The MEIA (Abbott IMx) is the most commonly used assay for FK-506 (5). This is in part because of the ease of instrument handling and the stability of the calibration curve over time. In the Abbott Tacrolimus I assay, the analytical detection limit is 5.0 μg/L. In light of the downward shift in the therapeutic ranges, Abbott has developed a new assay (Tacrolimus II) for FK-506. The Tacrolimus II assay is more sensitive than the Tacrolimus I assay, with an analytical detection limit of 1.5 μg/L. In the process of switching from the Tacrolimus I assay to the Tacrolimus II assay, we compared these assays. To avoid the questions of sample stability and drug metabolism, FK-506 was assayed the same day by both methods. Each sample was done in duplicate by each method. Both methods use a similar protocol of organic extraction and assay by MEIA except for the ratios of sample to extraction buffer. The Tacrolimus I assay uses 100 μL of whole blood and 200 μL of extraction buffer. The Tacrolimus II assay uses 150 μL of whole blood and 150 μL of extraction buffer. We found that the two assays yielded significantly different values for FK-506. In our laboratory, the Tacrolimus II assay gave significantly lower values (P <0.001) than the Tacrolimus I assay. Fig. 1 shows the comparison of the two assays, with a slope of 0.80 and intercept of −1.7. We do not believe that this bias is because of laboratory error, but think that this is a real bias based on these facts: (a) In each assay, the samples were run in duplicate and the duplicates agreed within an average of 5.5% and 6.8% for Tacrolimus I and II assays, respectively; (b) in all the assays, the controls were within manufacturer-stated ranges; and (c) CVs for the controls of the two assays were comparable. The CVs for the Tacrolimus I assay at 14, 25, and 41 μg/L were 11.0%, 9.0%, and 6.6%, respectively, and the CVs for the Tacrolimus II assay at 5, 10, and 23 μg/L were 11.2%, 8.2%, and 10.1%, respectively. Comparison between the Abbott IMx Tacrolimus I and II assays (n = 36), using least-squares analysis. Each point represents the mean of duplicate estimations from different patients. The coefficient of correlation (r) was 0.96. The regression data was: slope = 0.799 (95% confidence intervals, 0.71 to 0.88), intercept = −1.7 (95% confidence interval, −2.98 to −0.33); and Sy‖x = 1.661. To discover if the lower values given by the Tacrolimus II assay are because of lower recoveries, we added 60 μg/L calibrator from the Tacrolimus I assay to four negative blood samples, to get a drug concentration of 20 μg/L. The average recovery on these four samples by the Tacrolimus II assay was 80%, compared with 102% by the Tacrolimus I assay. These lower recoveries in the Tacrolimus II assay seem to be the cause of lower values in the newer assay. We think that the change in sample-to-extraction buffer ratio in the newer assay produces incomplete drug extraction from red blood cells. When this report was under review, similar differences between the Tacrolimus I and II assays were reported (6)(7). In the first report, the bias was throughout the range studied; in the second report, the Tacrolimus I assay gave higher values at the lower concentrations because of a significant intercept value. In conclusion, compared to the Tacrolimus I assay, there is a significant negative bias with the Abbott Tacrolimus II assay. At least part of this bias seems to be because of poor drug extraction. For better therapeutic drug monitoring, physicians should be made aware of this bias.