Improved Open-Well Stability of Affinity Column–Mediated Immunoassay Reagent for Determining Blood Tacrolimus
Tacrolimus, a calcineurin inhibitor, is the primary immunosuppressive agent used in allograft transplantation1 and autoimmune diseases such as rheumatoid arthritis.2 Because the pharmacokinetics of tacrolimus exhibit large individual variation and the drug shows a narrow therapeutic window for optimal blood concentration, therapeutic drug monitoring is required to optimize the dosage regimen for individual patients.3 The current target blood tacrolimus concentration range has become low (2–6 ng/mL) for maintenance dose for patients with rheumatoid arthritis and those under multiple adjuvant immunosuppressive therapy, including calcineurin inhibitor and everolimus.4,5 This situation necessitates improving the assay quality and precision at lower blood concentrations of tacrolimus.
We previously reported that an affinity column–mediated immunoassay (ACMIA) using the Dimension TACR Flex reagent cartridge (Catalog No. DF107; Siemens Healthcare Diagnostics, Tokyo, Japan) showed time-dependent reductions in measured concentrations for quality control (QC) and patient samples, especially at lower blood concentrations.6 It was suggested that tacrolimus immobilized on chromium dioxide particles (CrO2-TACR) becomes unstable 24–48 hours after opening the reagent cartridge.6 This may be one of the reasons for the low reproducibility in determining blood tacrolimus concentrations. Recently, a novel reagent cartridge (Catalog No. DF207) was developed, which consists of improved CrO2-TACR. Bargnoux et al7 confirmed the reliable and reproducible performance of the novel reagent by comparing the measured concentrations using ACMIA and liquid chromatography–tandem mass spectrometry at lower tacrolimus concentrations. In this study, we compared the blood tacrolimus concentration in QC and patient samples using the novel (lot numbers DA5301, FB6062, and GB6160) and original reagents to assess the assay stability and precision on the Dimension Xpand Plus analyzer (Siemens Healthcare Diagnostics). This study was approved by the Ethical Committee of the University of Tsukuba Hospital (Tsukuba, Japan), and written informed consent was obtained from the patients.
Tac/CsA immunosuppressant controls (More Diagnostics; lot number 33081 for low, 33082 for medium, and 33083 for high concentration; Los Osos, CA) were used as the QC samples, and 22 whole blood samples from renal transplant recipients treated with tacrolimus were used to evaluate the performance of ACMIA. QC and patient samples were analyzed using both original and novel reagents on the same day. Blood tacrolimus concentrations were determined in triplicate. Reagent cartridges were stored at 6°C after opening. The between-day coefficients of variation (CVs) were evaluated by measuring QC samples on 5 different days. The between-day CVs for the assay at low, medium, and high levels of QC samples were 2.6%, 1.4%, and 2.2% for the novel cartridges, and were 6.6%, 4.2%, and 2.9% for the original cartridges, respectively. Time-dependent reduction in the measured concentration was assessed by comparing between just after (0 hours) and 47 hours after opening the cartridges in original and novel reagents, for both of which expiration is 48 hours after the opening according to their package insert. No time-dependent reductions in the measured concentrations of QC samples were observed in the novel cartridges after automatically opening the reagent well (Table 1). Reduction in the measured QC concentrations (low, medium, and high) at 47 hours after opening the reagent well were 37.2%, 17.6%, and 14.0%, respectively, for the original cartridges (Table 1). Similar results were observed with the patients' whole blood samples (Table 1). The time-dependent reductions were 13.9% and 9.9% at a concentration of <10 and ≥10 ng/mL, respectively, when the original cartridges were used. This reduction disappeared when the novel cartridges were applied for both samples of <10 and ≥10 ng/mL. These results suggest that the reagents in the novel cartridges are stable for 47 hours, even after opening the reagent cartridge.