The EMIT® Cyclosporine Assay: Development of Application Protocols for the Boehringer Mannheim Hitachi 911 and 917 Analyzers

Objective: The purpose of this work was to develop applications for the EMIT® Cyclosporine (CsA) Assay on the Hitachi 911 and 917 analyzers. Methods and Results: Instrument settings were optimized to arrive at the following assay characteristics on the Hitachi 917. Limit of sensitivity was 50 μg/L. Intra-assay coefficients of variation (CV) were 8.1% (n = 20; = 62 μg/L) and 4.2% (n = 20; = 315 μg/L), while interassay CVs were 13.0% (n = = 73 μg/L) and 5.7% (n = 43; = 391 μg/L). Recoveries of 95-104% were obtained by spiking aliquots of 3 whole blood patient pools of known CsA concentrations with CsA. Serial dilutions-of 3 patient specimens demonstrated linear relationships between expected and actual CsA concentrations (r = 0.99, 0.99, 0.98; regression lines: y = 1.19x −17.1; y = 0.75x + 18.0; y = 1.01x + 3.7). Specimen carryover was not evident. Calibration stability is at least 10 days. Comparable assay characteristics were found for the Hitachi 911. Sequentially-collected trough whole blood specimens from renal (n = 3), liver (n = 3) and heart (n = 4) transplant patients prescribed CsA were collected up to 78 days post-transplant and analyzed by EMIT® on the Hitachi 917 and also by fluorescence polarization immunoassay (FPIA) and high performance liquid chromatography (HPLC). The following linear regression equations were produced for the renal [EMIT® = 0.801 (TDx®) + 4.98, r = 0.91, Sy/x = 32, n = 37; EMIT® = 0.877 (HPLC) + 56, r = 0.87, Sy/x = 38, n = 37]; liver [EMIT® = 0.808 (TDx®) − 27, r = 0.94, Sy/x = 42, n = 37; EMIT® = 0.953 (HPLC) + 44, r = 0.89, Sy/x = 57, n = 37] and heart [EMIT® = 0.820 (TDx®) − 24, r = 0.94, Sy/x = 31, n = 45; EMIT® = 0.956 (HPLC) + 54, r = 0.91, Sy/x = 38, n = 45] patient samples. FPIA values average 32% more than EMIT®-derived CsA concentrations on the Hitachi 917, which in turn averaged 15% more than HPLC values. In addition, these levels were compared intra-individually. CsA concentrations within all patients were significantly higher (p< 0.05, paired t-test) by FPIA compared to EMIT® and by FPIA compared to HPLC. Although CsA concentrations within most patients were significantly higher (p< 0.05) by EMIT® compared to HPLC, levels determined in 4 transplant patients (1 renal, 1 liver, 2 heart) were not different. Conclusion: Development of applications for the EMIT® CsA Assay on two highly automated, random access instruments, the Hitachi 911 and Hitachi 917, enhances the versatility of the immunoassay for routine therapeutic drug monitoring of this immunosuppressant in the clinical setting