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INTRODUCTION
Tacrolimus is a calcineurin inhibitor immunosuppressive agent discovered in 1984 [1]. Since its discovery, tacrolimus has been widely prescribed for organ transplant recipients and other patients in need of immunosuppression. However, this drug has a relatively narrow therapeutic index and concentration-related side effects [2]. Tacrolimus at inappropriate levels may cause nephrotoxicity, neurotoxicity, and diabetogenicity [2] or increase the risk of infection due to over-immunosuppression. Conversely, insufficient concentration of tacrolimus increases the risk of allograft rejection [3]. Careful monitoring of tacrolimus concentration in transplant recipients is essential to prevent side effects and graft rejection.
Tacrolimus concentration is measured using various methods, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay [4]. Tacrolimus measurement values may differ depending on the test method and pre-treatment employed. Although the reference measurement method for tacrolimus is based on isotope dilution LC-MS/MS [5], immunoassays for tacrolimus are often used in clinical laboratories because they offer the advantage of automation through the use of modular analyzers.
Previous multicenter studies evaluated the performance of tacrolimus electrochemiluminescence immunoassay (ECLIA) [6]. However, the launch of a new diagnostic platform requires additional validation, including comparison with existing measurement procedures. Cobas pure integrated solutions (Roche Diagnostics International AG, Rotkreuz, Switzerland) is a new modular diagnostic system combining clinical chemistry, immunoassay, and ion-selective electrode (ISE) diagnostic testing. In the present study, we evaluated the analytical performance of tacrolimus ECLIA on cobas pure platform and compared it with that of LC-MS/MS and affinity chrome-mediated immunoassay (ACMIA).
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MATERIALS AND METHODS
This study was waived from deliberation by the Institutional Review Board of Asan Medical Center (S2020-1899).
1. Laboratory measurements
Before measurement, pretreatment including cell lysis, tacrolimus extraction, and precipitation of blood protein was conducted using Elecsys ISD sample pretreatment reagent (Roche Diagnostics GmbH, Mannheim, Germany). The pretreated samples were centrifuged, and the supernatants containing tacrolimus were assayed. Blood tacrolimus concentrations were measured using Elecsys Tacrolimus assay (Roche Diagnostics GmbH), which is based on the principle of ECLIA, and cobas pure integrated solutions (Roche Diagnostics International Ltd., Rotkreuz, Switzerland) in accordance with the manufacturer’s instructions. For comparison, Dimension TAC Flex assay (Siemens Healthineers, Tarrytown, NY, USA), which is based on the principle of affinity chrome-mediated immunoassay (ACMIA), was conducted on a Dimension ExL 200 analyzer (Siemens Healthineers). LC-MS/MS was performed on an ACQUITY UPLC®zHSS SB C18, 2.1 mm×30 mm, 1.8 fim and Xevo TQ-S spectrometer (Waters Corporation, Milford, MA, USA).
2. Precision
The assay precision was evaluated in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline EP5-A3 [7]. Quality control (QC) materials (PreciControl ISD, Roche Diagnostics GmbH) at low, medium, and high concentrations were used. Two replicates of each QC sample were analyzed twice a day for 20 days. The acceptance criterion for precision was less than 6% analytical coefficient of variation (CV), as recommended by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee (IATDMCT) [8].
3. Linearity
Linearity was evaluated in accordance with CLSI guideline EP6-A [9]. Patient samples of high (27.43 ng/mL) and low (0.91 ng/mL) tacrolimus concentrations were diluted to produce five equally spaced concentrations. Samples at each concentration were measured in four replicates. The means of the measured values of each concentration were compared with assigned values.
4. Comparison
Method comparison was performed in accordance with CLSI guideline EP9-A3 [10]. In January 2021, we collected residual whole blood samples from patients receiving tacrolimus. Values measured using Elecsys Tacrolimus assay on cobas pure integrated solutions, Dimension TAC Flex assay on a Dimension ExL 200 analyzer, and LC-MS/MS on a Xevo TQ-S spectrometer were compared. Eight samples per day were measured on each measurement system.
5. Sample carryover
Samples of high and low concentrations were prepared for carryover evaluation. After measuring high-concentration samples four times (H1, H2, H3, and H4) consecutively, low-concentration samples (L1, L2, L3, and L4) were measured four times consecutively. The carryover between samples (%) was calculated according to the following equation:
6. Statistical analysis
Statistical analysis was conducted using Microsoft Excel (Microsoft Corp., Redmond, WA, USA), EP Evaluator Release 12 software (David G. Rhoads Associates, Kennett Square, PA, USA), and GraphPad Prism version 9 for Windows (GraphPad Software, San Diego, CA, USA).
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RESULTS
1. Precision
The results of the assay precision are shown in Table 1. The total CVs of the QC materials at low, medium, and high concentrations were 4.0%, 3.1%, and 3.2%, respectively, which were higher than the within-run CVs of 1.9%, 1.0%, and 1.0%, respectively.
2. Linearity
The results of linearity evaluation are shown in Fig. 1. The linear range, showing a coefficient of determination (R2) greater than 0.99, was 0.91–27.43 ng/mL (Conversion factors: ng/mL×1.2438=nmol/L). The linearity error % was 2.6% when the assigned value and the values from the linear fit were compared. The linearity regression slope and intercept were as follows:
Y (observed)=1.014x (expected)–0.0267
3. Comparison
A total of 80 samples ranging from 1.33 ng/mL to 27.63 ng/mL were analyzed for method comparison. Among the 80 patients, 35 received kidney transplantation, 30 liver transplantation, 13 lung transplantation, and 2 heart transplantation. The results of comparisons between the Elecsys Tacrolimus assay on cobas pure, Dimension TAC Flex assay on Dimension ExL 200 analyzer, and LC-MS/MS on Xevo TQ-S spectrometer are shown in Fig. 2. The results obtained using cobas pure were significantly correlated with those obtained using Dimension TAC Flex and LC-MS/MS (R=0.9771 and R=0.9941, respectively). Tacrolimus levels measured by the cobas pure method showed positive mean differences compared to those measured by the Dimension TAC Flex and LC-MS/MS methods, 1.55 and 1.42 ng/mL, respectively, as demonstrated by Bland-Altman plots.
 | Fig. 2Scatter plots and Bland-Altman plots between Elecsys Tacrolimus assay on Cobas Pure and different tacrolimus assays. (A) Comparison between Elecsys Tacrolimus assay on Cobas pure and LC-MS/MS on Xevo TQ-S spectrometer. (B) Comparison between Elecsys Tacrolimus assay and Dimension TAC Flex assay on Dimension ExL 200. In the scatter plots, the dotted line represents 1:1 line and the red line represents Deming regression. In the Bland-Altman plots, the dotted line represents the mean bias. (Conversion factors: ng/mL ×1.2438=nmol/L).
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4. Sample carryover
Four samples of high concentrations (18.4, 18.1, 18.2, and 18.2 ng/mL, respectively) and four samples of low concentrations (2.83, 2.82, 2.81, and 2.84 ng/mL, respectively) were analyzed for sample carryover evaluation. The carryover between samples was acceptable below 1.0% and was 0.03%.
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DISCUSSION
In terms of precision, we observed within-run CVs between 1.0% and 1.9%, and total CVs between 3.1% and 4.0%, depending on the concentrations of the QC materials. Within-run CVs of 2.0–4.5% and total CVs of 2.7–8.1% were observed in previous multicenter studies that evaluated Elecsys Tacrolimus assay [6, 13]. In addition, Dimension TAC Flex, another immunoassay used for comparison, showed a total CV of 5.7–7.3% in a previous evaluation study [14]. The results of the present study on the tacrolimus measurement of cobas pure showed a similar or better precision than those of previous studies. In 2016, an assay precision performance of less than 6% analytical CV was presented by IATDMCT as a goal in the case of immunosuppressive drugs [8]. In this manner, Elecsys Tacrolimus assay on cobas pure was deemed sufficient for clinical implementation. The linearity and sample carryover of the assay were also acceptable.
Comparison with LC-MS/MS and ACMIA also showed acceptable correlation coefficient values of R>0.975. However, compared with LC-MS/MS and ACMIA, tacrolimus ECLIA on cobas pure showed average bias values of 1.42 and 1.55 ng/mL, respectively, indicating a slightly higher value of tacrolimus. A previous study that evaluated the same tacrolimus ECLIA on other platforms also observed an average of 4.4% positive bias compared with LC-MS/MS [13]. Immunoassay results can be affected by various factors, such as matrix effect [15], different calibration [16], non-specific binding [17], cross-reactivity, and autoantibodies and heterophile antibodies [18-20]. Additionally, tacrolimus is extracted from whole blood samples, thus it is influenced by the whole blood extraction method and post-extraction stability [21, 22]. Furthermore, tacrolimus undergoes extensive metabolism in the body and exists as various types of metabolites [23], and different tacrolimus metabolites can be sources of cross-reactivity. Using different calibrators from different manufacturers can also affect tacrolimus measurements. With the availability of certified reference materials (CRMs), using calibrators with assigned values traceable to higher CRMs can be helpful for tacrolimus standardization [24]. Further efforts are needed to standardize tacrolimus assays and reduce the inter-assay variations in tacrolimus measurement.
Cobas pure is a novel modular automatic analyzer developed for use in limited spaces, such as small- and medium-sized laboratories or satellite laboratories. Combining clinical chemistry, ISE, and immunoassay modules, this analyzer can be used to test a large number of analytes. Although we did not evaluate all the analytes available on cobas pure in this study, we observed no significant difference between the results of tacrolimus assay and those of Dimension TAC assay and LC-MS/MS conducted on other platforms.
In conclusion, the performance of tacrolimus ECLIA on cobas pure integrated solutions was acceptable. Until now, tacrolimus concentrations vary according to the measurement method, especially among immunoassays. Improving the reliability of measurements of immunosuppressants, including tacrolimus, is crucial to improve the prognosis of organ transplant recipients.
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