Introduction
Immunosuppressive drugs (ISD), such as the calcineurin inhibitors (cyclosporine (CSA) and tacrolimus (TAC)) and mammalian target of rapamycin (mTOR) inhibitors (sirolimus (SRL) and everolimus), are critical to the maintenance of solid organ transplantation 1. CSA is a cyclic undecapeptide and TAC (also known as FK-506) is a macrolide lactone. CSA binds to cyclophilin A/B and inhibits calcineurin. TAC binds to FK506-binding protein 12 (FKBP-12) to form the calcineurin inhibitory complex. Inhibition of calcineurin, a serine/threonine phosphatase, leads to altered calcium-dependent signal transduction, and decreases T-cell activation and downregulates anti-inflammatory response-related genes 2, 3. SRL (also known as rapamycin) is a 31-membered macrolide antibiotic that binds to FKBP-12 and allosterically targets the mTOR pathway, inhibiting cell cycle progression, T-cell proliferation and differentiation 2. SRL has structural similarities to TAC and competes with TAC for FKBP-12 binding 1, 2. All three ISDs are characterized by having variable absorption, poor bioavailability, strong affinity to blood proteins, leukocytes, and/or erythrocytes, and metabolism via cytochrome CYP3A4/5 and efflux transport by P-glycoprotein 2.
Therapeutic drug monitoring is a mainstay in immunosuppressant therapy. ISDs have narrow therapeutic ranges 2, high interindividual variability in pharmacokinetics and pharmacogenetics 4, 5, susceptibility to food- and drug-drug interactions 6, and adverse consequences if plasma drug levels are not maintained 5, 7. Similar toxic effects have been described for CSA and TAC, due to their overlapping mechanism of action, and includes nephrotoxicity, hypertension, and neurotoxicity 2. TAC is a more potent calcineurin inhibitor than CSA, due to increased affinity for FKBP-12 and the advantage of decreased nephrotoxicity, risk of hyperlipidemia and hypertension 1, 2, 8. TAC, however, is more likely to cause post-transplantation diabetes 1, 9, 10. SRL does not cause renal toxicities; however, long-term SRL use can induce leukopenia, thrombocytopenia, and dyslipidemia 11, 12. The target therapeutic range for each ISD may vary depending on type of organ transplanted, time from transplantation, co-administered drugs, and method of analysis.
Recently, semi-automated electrochemiluminescence immunoassays (ECLIA) for the quantification of CSA, TAC, and SRL in whole blood were developed and made available by Roche Diagnostics (GmbH, Mannheim, Germany) 13, 14. In this study, we evaluated the analytical performance of ECLIA method for CSA, TAC, and SRL on the Roche cobas e411 analyzer and compared to the commonly used chemiluminescent microparticle immunoassay (CMIA) method on the Abbott ARCHITECT i2000 analyzer (Abbott Laboratories, Abbott Park, IL, USA). This is the first report on the evaluation of ECLIA SRL, and compares the performance of all three ISDs together.
Methods
Specimen source and handling
Ethical approval for this study was waived by the Research Ethics Board at the University Health Network in Toronto, Ontario, Canada (16-6312) for use of routine collected specimens for the evaluation of method performance. Residual EDTA whole blood specimens from 300 patients undergoing immunosuppressant therapy (either cyclosporine, tacrolimus, or sirolimus) at the University Health Network, and CAP proficiency testing samples were used in this evaluation. Samples were collected and either analyzed within the same day or stored as per manufacturer recommendations. Samples were thawed and equilibrated to room temperature for 30 minutes and mixed well prior to analysis. In accordance with stability studies on whole blood ISD specimens, samples were analyzed within three months of collection and did not undergo more than two freeze-thaw cycles 15– 18.
Electrochemiluminescence immunoassay (ECLIA) method on Roche cobas e 411
The cobas ECLIAs (Roche Diagnostics GmbH, Mannheim, Germany) for CSA, TAC, and SRL are based on the competition of analyte in sample with a ruthenium-labeled analogue. A voltage is applied and electrochemiluminescence signal is detected. Testing was performed according to the manufacturer’s instructions. Briefly, the samples (calibrators, QC, whole blood samples) were manually pretreated by combining 300 µL of whole blood with 300 µL of Universal ISD Sample Pretreatment Reagent (containing zinc sulfate and methanol) and vortexed for 10 seconds to lyse the red blood cells, precipitate proteins and extract the analyte. The samples were centrifuged for 4 minutes at 15,000 x g, and the supernatant was decanted for analysis. Analysis was performed within 30 minutes of preparation to prevent evaporation of the extracted samples. The ECLIA assays were calibrated five times during the evaluation by a 2-point calibration using calibrators traceable to pure standard materials reconstituted in whole blood matrix by gravimetrical methods.
Chemiluminescent microparticle immunoassay (CMIA) method on Abbott ARCHITECT i2000
The ARCHITECT CMIAs (Abbott Laboratories, Abbott Park, IL, USA) for CSA, TAC, and SRL are based on competition of analyte in sample with acridinium-labeled analogue. The samples were manually pretreated according to the manufacturer’s instructions and site-specific standard operating procedures. For CSA, 100 µL of Cyclosporine Solubilizing Reagent (4% saponin) and 400 µL of Cyclosporine Precipitation Reagent (zinc sulfate in methanol and ethylene glycol) was added to 200 µL of sample 19, 20. For TAC, 200 µL of sample was mixed with 200 µL of Tacrolimus Precipitation Reagent (zinc sulfate in methanol) 21, 22. For SRL, 150 µL of sample was mixed with 300 µL of Sirolimus Precipitation Reagent (zinc sulfate in >50% v/v DMSO and ethylene glycol), vortexed and heated at 42 oC for 10 minutes 23, 24. All ISD samples were then vortexed for 10 seconds and centrifuged for 4 minutes at 15,000 x g. The supernatants were decanted into labelled tubes and assayed within 30 minutes of sample preparation. The ARCHITECT CMIAs are calibrated according to the site-specific standard operation procedures, with a 6-point 4-parameter logistic curve fit (4PLC, y-weighted) that is traceable to pure standard materials in a whole blood matrix by gravimetrical methods. Internal QC was evaluated with Bio-Rad Lyphochek Whole Blood ISD Controls levels 1, 3, and 4.
Imprecision
Three levels of manufacturer multi-analyte QC materials (Roche Diagnostics PreciControl ISD levels 1, 2, and 3) and third-party multi-analyte QC materials (Bio-Rad Lyphochek Whole Blood ISD Controls levels 1, 3, and 4) were analyzed. QC samples were prepared and measured in duplicate, one run per day over 10 days. The acceptance criterion for total imprecision was based on the recommendation of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) expert consensus group of ≤ 10% 25.
Functional sensitivity
Residual patient sample with levels 2–3 times the claimed limit of quantification (LoQ) was used to generate a series of dilutions with blank whole blood. The neat sample and dilutions were measured in triplicates within one day. The precision profile curve was used to calculate the LoQ concentration corresponding to a CV of 20% with the upper 95% confidence limit.
Linearity
Since there is a lack of elevated CSA and TAC patient specimen, CSA and TAC linearity were assessed using CAP EQA linearity materials (6 concentrations measured in duplicate). SRL linearity was assessed using a patient sample above the upper measuring range diluted with blank whole blood to 6 concentrations and measured in duplicate. The acceptance criterion was defined as slope of 1.00 ± 0.05 and deviation <10%.
Method comparison
Method comparison experiments were assessed where anonymized residual patient samples spanning the analytical measuring range for each analyte were measured one replicate per method. CSA samples concentrations ranged from 41.0 to 1808.0 μg/L, TAC ranged from 2.1 to 30.0 μg/L, and SRL ranged from 1.8 to 34.6 μg/L as determined by ARCHITECT CMIA. Roche ECLIA measurements were compared to ARCHITECT CMIA (n=100). To further elucidate the accuracy between immunoassays, a subset of samples was also analyzed by LC-MS/MS (n=20) ( Supplementary File 1). Lot-to-lot assessment was also performed between two lots of reagents for each ISD (n=20). The slope, intercept, correlation coefficient r were calculated by Deming regression analysis. The acceptance criteria for method comparison were defined as a slope of 1.00 ± 0.15 and r of ≥ 0.95, meanwhile for lot-to-lot comparison were defined as a slope of 1.00 ± 0.05 and r of ≥ 0.95.
Statistical analysis
Microsoft Excel (version 1708, Microsoft Office) and/or EP Evaluator (version 7.0.0.307, Data Innovations) were used for statistical analysis.
Results and discussion
To assess imprecision, three levels of manufacturer (Roche PreciControl) and third-party (Bio-Rad Lyphochek) multi-analyte QC materials were analyzed using one lot of reagents in duplicate, one run per day over 10 days ( Table 1). For the PreciControl, the total imprecision was <7.1% for CSA, <9.4% for TAC, and <5.6% for SRL. Imprecision for CSA and TAC were comparable to other studies 13, 14. Our study additionally evaluated third-party QC performance on ECLIA ISD assays, a total imprecision of <4.7% for CSA, <6.3% for TAC, and <8.2% for SRL were determined. The imprecision goal of ≤10%, based on the recommendation of the IATDMCT expert consensus group, was achieved for all QC samples 25. Overall, the ECLIA methods demonstrate acceptable precision.
Table 1.
Total imprecision for cyclosporine, tacrolimus, and sirolimus determined by the ECLIA method (duplicate per run, 1 run per day for 10 days).
| Samples | Mean Conc.
| Total CV
| |
|---|---|---|---|
| Cyclosporine
| Roche ISD L1 | 63.1 | 7.1 |
| Roche ISD L2 | 271.1 | 5.1 | |
| Roche ISD L3 | 976.9 | 3.8 | |
| Bio-Rad WB ISD L1 | 67.3 | 4.7 | |
| Bio-Rad WB ISD L3 | 346.5 | 4.5 | |
| Bio-Rad WB ISD L4 | 740.1 | 3.3 | |
| Tacrolimus
| Roche ISD L1 | 2.5 | 9.4 |
| Roche ISD L2 | 9.2 | 6.9 | |
| Roche ISD L3 | 16.9 | 4.1 | |
| Bio-Rad WB ISD L1 | 4.1 | 6.3 | |
| Bio-Rad WB ISD L3 | 7.8 | 5.3 | |
| Bio-Rad WB ISD L4 | 15.2 | 3.9 | |
| Sirolimus
| Roche ISD L1 | 3.4 | 4.6 |
| Roche ISD L2 | 8.7 | 5.6 | |
| Roche ISD L3 | 15.4 | 4.7 | |
| Bio-Rad WB ISD L1 | 5.9 | 4.7 | |
| Bio-Rad WB ISD L3 | 9.5 | 5.7 | |
| Bio-Rad WB ISD L4 | 14.2 | 8.2 |
*Conventional unit: 1 µg/L = 1 ng/mL
The ECLIA methods offer a wider linear analytical measuring range for CSA and TAC than CMIA methods. ECLIA CSA, TAC, and SRL were linear up to 960.9 μg/L, 27.1 μg/L, and 32.3 μg/L, respectively. The higher upper limit allows TDM and pharmacokinetic analysis of ISD at different time points and peak concentrations offering additional flexibility 26, 27.
The claimed functional sensitivity of the ECLIA ISD methods are improved for TAC and SRL compared to CMIA ISD methods. The functional sensitivities were assessed and the precision profile was used to calculate the LoQ corresponding to a CV of 20% with upper 95% confidence limit. The functional sensitivities were determined to be <6.5 μg/L for CSA, 1.1 μg/L for TAC, and <0.1 μg/L for SRL, which meets the 2007 European consensus guideline and IATDMCT expert consensus group recommended LoQ of 20.0 μg/L for CSA and a LoQ of 1.0 μg/L for both TAC and SRL 25, 28.
For method comparison, anonymized residual patient samples spanning the analytical measuring range for each analyte were measured. CSA samples concentrations ranged from 41.0 to 1808.0 μg/L, TAC ranged from 2.1 to 30.0 μg/L, and SRL ranged from 1.8 to 34.6 μg/L as determined by CMIA. ECLIA ISDs measurements were compared to CMIA ISDs (n=100). The acceptance criteria were defined as a slope of 1.00 ± 0.15 and r of ≥ 0.95. Figure 1 shows the Deming regression and Bland-Altman analysis for CSA, TAC, and SRL. ECLIA and CMIA CSA ( Figure 1A) showed good agreement with a slope of 0.917 (95% CI: 0.885-0.949), intercept of -15.2 (95% CI: -39.4-9.0), and r of 0.985. For TAC, the ECLIA TAC also showed good agreement with CMIA TAC ( Figure 1B) with a slope of 0.938 (95% CI: 0.895-0.981), intercept of 0.2 (95% CI -0.4-0.8), and r of 0.974. Similar trends were observed by others (slopes of 0.87 for CSA, and 0.96-0.98 for TAC) 14, 29. Reported for the first time, method comparison of ECLIA and CMIA SRL ( Figure 1C) showed a slope of 0.842 (95% CI: 0.810-1.110), intercept of 0.9 (95% CI: 0.4-1.4), and r of 0.982. Overall, all three ECLIA ISDs met our acceptance criteria, with SRL slightly exceeding the limit for the slope.
Figure 1.
Method comparisons and Bland-Altman plots for ( A) cyclosporine (CSA), ( B) tacrolimus (TAC), and ( C) sirolimus (SRL) between cobas e411 ECLIA and ARCHITECT i2000 CMIA.
To further examine ECLIA performance with CMIA, a subset of samples was also analyzed by LC-MS/MS (n=20). For both CSA immunoassays, a positive bias was observed when compared with LC-MS/MS, similarly observed by others 13, 29, 30 ( Figure S1). For TAC, both the ECLIA and CMIA TAC had good agreement with LC-MS/MS, also similarly observed by others for different cohorts of solid organ transplant 14, 21, 29 ( Figure S2). Reported for the first time, the ECLIA SRL compared to LC-MS/MS showed a slope of 0.971 (95% CI: 0.913-1.030), intercept of 2.4 (95% CI: 1.6-3.3), and r of 0.993 ( Figure 2). Meanwhile, CMIA SRL compared to LC-MS/MS showed a slope 1.119 (95% CI: 1.051-1.187), intercept of 1.4 (95% CI: 0.5-2.4), and r of 0.993. Based on our small sample size, both ECLIA and CMIA SRL generally showed good correlation with LC-MS/MS, with ECLIA SRL with better agreement to LC-MS/MS.
Figure 2.
Method comparisons and Bland-Altman plots for sirolimus (SRL) between cobas e411 ECLIA and LC-MS/MS.
For lot-to-lot comparisons, Deming regression analysis of 20 residual patient samples tested by 2 lots of reagent for CSA shows a slope of 0.998 (95% CI: 0.967-1.029), intercept of 8.5 (95% CI: -10.2-27.2), and r of 0.998. For TAC, a slope of 0.972 (95% CI: 0.936-1.008), intercept of -0.4 (95% CI: -0.8-0.0), and r of 0.997. And for SRL, a slope of 0.913 (95% CI: 0.841-0.985), intercept of 0.1 (95% CI: -0.9-1.2), and r of 0.988. All three ISDs had good correlation between 2 different lots of reagents.
Evaluation on practical considerations included ease-of-use, throughput, and workflow of the method. The sample pretreatment for the ECLIA method is faster, simpler, and more convenient than CMIA method due to the use of a single universal sample pretreatment reagent and protocol for all three ISDs. Additionally, there is no heating step for the SRL ECLIA method, which leads to a simpler workflow. The ECLIA universal sample pretreatment reagent and protocol would enable better workflow, simpler sample handling and inventory control.
Conclusion
In conclusion, the overall analytical evaluation of the ECLIA method for CSA, TAC, and SRL met acceptable performance. ECLIA CSA showed better precision than our current CMIA CSA. ECLIA CSA and TAC showed better linearity range, and ECLIA TAC and SRL showed good functional sensitivity than CMIA methods. Method comparisons showed good correlations and agreement between ECLIA ISDs and CMIA ISDs.
Data availability
The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Fung AWS et al.
Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
Dataset 1: File containing the raw data and a table of contents for the data file. doi, 10.5256/f1000research.12775.d180033 31
Copyright: © 2017 Fung AWS et al. This work is licensed under the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/3.0/ ) (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.