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Application of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 high volume clinical chemistry analyzer
Clinical Biochemistry 39 (2006) 1041 – 1043
Application of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 high volume clinical chemistry analyzer Klas Boer ⁎, Thomas Deufel, Dirk Schmidt, Sibylle Streck, Michael Kiehntopf Institut fur Klinische Chemie und Labordiagnostik FSU Jena, Germany Received 4 April 2006; received in revised form 6 June 2006; accepted 21 June 2006 Available online 21 July 2006
Abstract Objectives: Evaluation of the performance of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 analyzer. Design and Methods: Imprecision studies were performed and patient samples were assayed by EMIT assay and by LC-MS/MS. Results: Limit of quantification was established at 2.8 μg/L. A positive bias of 17.5% between results measured on EMIT and on LC-MS/MS was detected. Conclusions: EMIT 2000 Tacrolimus assay is suitable for automated analyses of Tacrolimus on the Architect c8000. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: EMIT; Tacrolimus; Architect; TDM; c8000
Introduction Tacrolimus (tac) is a calcineurin inhibitor widely used to prevent allograft rejection in organ transplant patients. The oral bioavailability of the drug is low [1]; following oral administration, maximum blood concentrations are reached after about 1 to 2 h [1]. Whole blood trough levels are used for therapeutic drug monitoring [2] (TDM). With 30% more requests in 2005 compared to 2004 in our laboratory, monitoring blood levels of post-transplant patients is an increasing challenge for clinical laboratories. HPLC and HPLC-MS based methods have become the gold standard for the measurement of tac [3] due to an unsurpassed accuracy and economical advantages. However, due to turnaround-time (TAT) requirements in a routine diagnostic environment, immunoassays such as the PRO-TRAC II (Diasorin) assay, the Abbott MEIA II assay and the EMIT 2000 Tacrolimus assay (Dade Behring) are widely used; for the latter, adaptations both to the Bayer Advia 1650 and the Beckmann Coulter LX20 PRO analyzer have been described [4,5]. We adapted this assay
to the Architect c8000 clinical chemistry analyzer and report the results of our evaluation of analytical performance of this application and compare it with LC-MS/MS analysis. Methods EMIT 2000 Tacrolimus assay reagents, pre-treatment reagent, assay calibrators were purchased from Dade Behring, Schwalbach; reference material was purchased from More Diagnostic, Los Osos. For sample preparation 400 μL of whole blood were mixed with 100 μL of pre-treatment reagent and 400 μL of methanol (JT Baker, Netherlands). Vortexed samples were incubated for 2 min and centrifuged at 12000 × g for 3 min. Sample analysis on the Viva Twin (Viva Scientific) Samples were pre-treated as described above. The analyzer was set according to the standard assay manufacturer's instructions [6]. Sample analysis on the Architect c8000
⁎ Corresponding author. E-mail address: [email protected] (K. Boer).
Antibody and buffer reagent (reagent A) were mixed 2:1 prior to use; the enzyme reagent (reagent B) was directly placed
0009-9120/$ - see front matter © 2006 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2006.06.011
1042
K. Boer et al. / Clinical Biochemistry 39 (2006) 1041–1043
into the analyzer. To ensure rapid processing pre-treated samples were positioned into the emergency slots of the analyzer. We used volumes of 142 μL mixed reagent A, B, 42 μL of reagent C and 25 μL of sample. Absorbance was followed bichromatically (340/412) for 198 seconds after addition of reagent B. The assay was recalibrated daily using the 2-point alignment calibration. Linearity of the assay on the Architect system was assessed measuring duplicates of a 2-fold serial dilution of control material in analyte-free calibrator.
Table 1 Comparison between tac blood levels assayed on Architect c8000 and on LCMS/MS
Sample analysis on the LC-MS/MS
Mean and median of the tac blood levels assayed on Architect c8000, absolute bias and relative bias between the methods depending on the type of transplantation received.
Samples were analyzed on LC-MS/MS as recently described [7] with a slight modification. In short, 100 μL whole blood were deproteinized with 200 μL methanol/water solution (v/v = 80/ 20) containing 10 g/L ZnSO4, ascomycin and cyclosporin D as internal standard. The supernatant (10 μL) was analyzed by LCMS/MS using a HPLC Agilent 1100-System and an API-4000 tandem mass spectrometer (Applied Biosystems, Darmstadt). Mobile phase with a flow rate of 600 μL/min consisted of a methanol: water-solution (v/v = 60:40) containing 10 mmol/L ammonium acetate and 0.1% acetic acid. Chromatography was performed using a Phenomenex Luna Phenylhexyl column
Type of Number Mean Median Bias in % transplantation of in in μg/L (c8000 versus patients μg/L LC-MS/MS)
Abs. bias in μg/L (c8000 versus LC-MS/MS)
Bone marrow Kidney Liver Heart Lung All
1.1 1.1 1.7 1.1 1.1 1.4
24 59 87 17 8 195
6.9 9.5 12.3 9.0 11.8 10.5
4.0 8.8 11.3 8.9 10.3 9.5
28.1 16.2 17.3 16.8 10.0 17.5
(Phenomenex, Aschaffenburg) with a security guard cartridge as pre-column. The measurement time was 4.5 min for each run. Statistical analysis Passing–Bablok regression and statistical analysis were performed using MedCalc (MedCalc Software, Belgium) and Microsoft Excel (Microsoft, United States). Results Reference material was assayed twice daily in duplicates for 20 days; between-run imprecision was 6.3% (mean = 3.8 μg/L) for Q1, 2.4% (mean = 9.8 μg/L) for Q2 and 1.6% (mean = 16.3 μg/L) for Q3. Inter-day imprecision was 9.0% for Q1, 4.7% for Q2 and 5.4% for Q3 with mean recoveries of 96.8% for Q1 (SD = 14.1%), 90.7% for Q2 (SD = 6.7%) and 93.4% for Q3 (SD = 6.6%) respectively. Limit of quantification was established at 2.8 μg/L (coefficient of variation > 20% for repeated analysis (n = 5) of a sample with a known concentration). Linearity was analyzed between 1.44 μg/L and 23.86 μg/L with a regression line calculated as y = 0.996 x + 0.258, with y = mean measured value and x = expected value. Comparison of tac concentrations measured on the Architect c8000 and the Viva Twin (Fig. 1A) were assessed using Passing–Bablok regression analysis (y = 1.0658 x − 1.3875, Intercept: 95% CI: − 1.9966, − 0.7318, Slope: 95% CI: 0.9997, 1.1346) with no deviation from linearity (p < 0.05) and a mean bias of − 7% (− 0.5 μg/L) for the Architect c8000 (Fig. 1). Corresponding comparison between Architect c8000 and LC-MS/MS (y = 0.8821x − 0.0953, Intercept: 95% CI: − 0.3753, 0.0985, Slope: 95% CI: 0.8496, 0.9131) resulted in no deviation from linearity (p < 0.05) (Fig. 1B). Overall bias between methods was 17.5% (+ 1.38 μg/L) with higher results measured on the Architect c8000 (Table 1).
Discussion Fig. 1. Passing–Bablok regression analysis of patient whole blood tac concentrations assayed on Abbott Architect c8000 versus Viva Twin (A, n = 166) and versus LC-MS/ MS (B, n = 195).
Imprecision of the EMIT assay on the Architect c8000 is comparable to results previously published for the LX20 PRO analyzer [4]; the limit of quantification at 2.8 μg/L is lower than
K. Boer et al. / Clinical Biochemistry 39 (2006) 1041–1043
the range estimated by Stephen et al. [5] for the ADVIA 1650 and below the limit of detection reported by LeGatt et al. [4] for the LX20 Pro. With between-day imprecision ranging from 9.0% to 4.7% the method is also suitable for the longitudinal monitoring of patients. Comparing the Viva Twin and Architect c8000 systems we observed a bias of 7% between both analyzers. LeGatt et al. [4] have noted previously a difference between the Abbott MEIA II assay and the EMIT 2000 assay on the Viva Twin that was larger than the difference between the MEIA II assay and the EMIT assay on the LX20 PRO, suggesting that the application of the same assay to different analyzers may still result in a bias between the analyzers. Tac concentrations measured on LCMS/MS by us and by other groups [4,8] were significantly lower as compared to the immunochemical assay; this is readily explained by the fact that the antibodies used in the EMIT and MEIA assays also bind pharmacologically inactive metabolites of tac [9] and by the possible impact of a low serum albumin and a low hematocrit [8]. In our study, absolute bias was comparable in all transplantation groups (Table 1); it is more significant in patients receiving a low dose of tac such as patients after BMT as described recently [8]. Therefore, the method used has to be reported to the clinician to avoid over- or under-dosing. LC-MS/MS based methods are labor-intensive and require expensive instrumentation. Due to restraints in TAT requirements immunochemical methods are still widely used. On the Architect c8000, high numbers of clinical samples can be processed in a relatively short time (10 min using the emergency slots provided by the analyzer). The Architect reagent cartridges hold up to 90 mL for both reagents (dead volume 2.5 mL) which is sufficient to load up to 600 tests onto the analyzer. In summary, applying EMIT 2000 Tacrolimus assay to the Architect c8000 we were able to consolidate standard routine
1043
workflow and increase productivity by concentrating more assays on one analyzer system. Acknowledgment The authors would like to thank Mrs. Haeuser for the technical support. References [1] Wallemacq PE, Verbeeck RK. Comparative clinical pharmacokinetics of tacrolimus in paediatric and adult patients. Clin Pharmacokinet 2001;40:283–95. [2] Wallemacq PE. Therapeutic monitoring of immunosuppressant drugs. Where are we? Clin Chem Lab Med 2004;42:1204–11. [3] Taylor PJ. Therapeutic drug monitoring of immunosuppressant drugs by high-performance liquid chromatography-mass spectrometry. Ther Drug Monit 2004;26:215–9. [4] LeGatt DF, Shalapay CE, Cheng SB. The EMIT 2000 Tacrolimus assay: an application protocol for the Beckman Synchron LX20 PRO analyzer. Clin Biochem 2004;37:1022–30. [5] Stephen DW, Rooke P, Clark S, Coutts M, Crowe L, Docherty D. Evaluation of the Dade Behring Syva EMIT 2000 Tacrolimus assay on the Bayer Advia 1650. Ann Clin Biochem 2003;40:697–700. [6] Dade Behring. EMIT® 2000 Tacrolimus Application Sheet. For the VIVA®, Viva-E™ and V-Twin™ Analyzers. 2004. [7] Koal T, Deters M, Casetta B, Kaever V. Simultaneous determination of four immunosuppressants by means of high speed and robust on-line solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. J Chromatogr, B: Anal Technol Biomed Life Sci 2004;805: 215–22. [8] Brown NW, Gonde CE, Adams JE, Tredger JM. Low hematocrit and serum albumin concentrations underlie the overestimation of Tacrolimus concentrations by microparticle enzyme immunoassay versus liquid chromatography-tandem mass spectrometry. Clin Chem 2005;51:586–92. [9] Iwasaki K, Shiraga T, Matsuda H, Nagase K, Tokuma Y, Hata T, et al. Further metabolism of FK506 (Tacrolimus). Identification and biological activities of the metabolites oxidized at multiple sites of FK506. Drug Metab Dispos 1995;23:28–34.
Application of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 high volume clinical chemistry analyzer Klas Boer ⁎, Thomas Deufel, Dirk Schmidt, Sibylle Streck, Michael Kiehntopf Institut fur Klinische Chemie und Labordiagnostik FSU Jena, Germany Received 4 April 2006; received in revised form 6 June 2006; accepted 21 June 2006 Available online 21 July 2006
Abstract Objectives: Evaluation of the performance of the EMIT 2000 Tacrolimus assay on the Abbott Architect c8000 analyzer. Design and Methods: Imprecision studies were performed and patient samples were assayed by EMIT assay and by LC-MS/MS. Results: Limit of quantification was established at 2.8 μg/L. A positive bias of 17.5% between results measured on EMIT and on LC-MS/MS was detected. Conclusions: EMIT 2000 Tacrolimus assay is suitable for automated analyses of Tacrolimus on the Architect c8000. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: EMIT; Tacrolimus; Architect; TDM; c8000
Introduction Tacrolimus (tac) is a calcineurin inhibitor widely used to prevent allograft rejection in organ transplant patients. The oral bioavailability of the drug is low [1]; following oral administration, maximum blood concentrations are reached after about 1 to 2 h [1]. Whole blood trough levels are used for therapeutic drug monitoring [2] (TDM). With 30% more requests in 2005 compared to 2004 in our laboratory, monitoring blood levels of post-transplant patients is an increasing challenge for clinical laboratories. HPLC and HPLC-MS based methods have become the gold standard for the measurement of tac [3] due to an unsurpassed accuracy and economical advantages. However, due to turnaround-time (TAT) requirements in a routine diagnostic environment, immunoassays such as the PRO-TRAC II (Diasorin) assay, the Abbott MEIA II assay and the EMIT 2000 Tacrolimus assay (Dade Behring) are widely used; for the latter, adaptations both to the Bayer Advia 1650 and the Beckmann Coulter LX20 PRO analyzer have been described [4,5]. We adapted this assay
to the Architect c8000 clinical chemistry analyzer and report the results of our evaluation of analytical performance of this application and compare it with LC-MS/MS analysis. Methods EMIT 2000 Tacrolimus assay reagents, pre-treatment reagent, assay calibrators were purchased from Dade Behring, Schwalbach; reference material was purchased from More Diagnostic, Los Osos. For sample preparation 400 μL of whole blood were mixed with 100 μL of pre-treatment reagent and 400 μL of methanol (JT Baker, Netherlands). Vortexed samples were incubated for 2 min and centrifuged at 12000 × g for 3 min. Sample analysis on the Viva Twin (Viva Scientific) Samples were pre-treated as described above. The analyzer was set according to the standard assay manufacturer's instructions [6]. Sample analysis on the Architect c8000
⁎ Corresponding author. E-mail address: [email protected] (K. Boer).
Antibody and buffer reagent (reagent A) were mixed 2:1 prior to use; the enzyme reagent (reagent B) was directly placed
0009-9120/$ - see front matter © 2006 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2006.06.011
1042
K. Boer et al. / Clinical Biochemistry 39 (2006) 1041–1043
into the analyzer. To ensure rapid processing pre-treated samples were positioned into the emergency slots of the analyzer. We used volumes of 142 μL mixed reagent A, B, 42 μL of reagent C and 25 μL of sample. Absorbance was followed bichromatically (340/412) for 198 seconds after addition of reagent B. The assay was recalibrated daily using the 2-point alignment calibration. Linearity of the assay on the Architect system was assessed measuring duplicates of a 2-fold serial dilution of control material in analyte-free calibrator.
Table 1 Comparison between tac blood levels assayed on Architect c8000 and on LCMS/MS
Sample analysis on the LC-MS/MS
Mean and median of the tac blood levels assayed on Architect c8000, absolute bias and relative bias between the methods depending on the type of transplantation received.
Samples were analyzed on LC-MS/MS as recently described [7] with a slight modification. In short, 100 μL whole blood were deproteinized with 200 μL methanol/water solution (v/v = 80/ 20) containing 10 g/L ZnSO4, ascomycin and cyclosporin D as internal standard. The supernatant (10 μL) was analyzed by LCMS/MS using a HPLC Agilent 1100-System and an API-4000 tandem mass spectrometer (Applied Biosystems, Darmstadt). Mobile phase with a flow rate of 600 μL/min consisted of a methanol: water-solution (v/v = 60:40) containing 10 mmol/L ammonium acetate and 0.1% acetic acid. Chromatography was performed using a Phenomenex Luna Phenylhexyl column
Type of Number Mean Median Bias in % transplantation of in in μg/L (c8000 versus patients μg/L LC-MS/MS)
Abs. bias in μg/L (c8000 versus LC-MS/MS)
Bone marrow Kidney Liver Heart Lung All
1.1 1.1 1.7 1.1 1.1 1.4
24 59 87 17 8 195
6.9 9.5 12.3 9.0 11.8 10.5
4.0 8.8 11.3 8.9 10.3 9.5
28.1 16.2 17.3 16.8 10.0 17.5
(Phenomenex, Aschaffenburg) with a security guard cartridge as pre-column. The measurement time was 4.5 min for each run. Statistical analysis Passing–Bablok regression and statistical analysis were performed using MedCalc (MedCalc Software, Belgium) and Microsoft Excel (Microsoft, United States). Results Reference material was assayed twice daily in duplicates for 20 days; between-run imprecision was 6.3% (mean = 3.8 μg/L) for Q1, 2.4% (mean = 9.8 μg/L) for Q2 and 1.6% (mean = 16.3 μg/L) for Q3. Inter-day imprecision was 9.0% for Q1, 4.7% for Q2 and 5.4% for Q3 with mean recoveries of 96.8% for Q1 (SD = 14.1%), 90.7% for Q2 (SD = 6.7%) and 93.4% for Q3 (SD = 6.6%) respectively. Limit of quantification was established at 2.8 μg/L (coefficient of variation > 20% for repeated analysis (n = 5) of a sample with a known concentration). Linearity was analyzed between 1.44 μg/L and 23.86 μg/L with a regression line calculated as y = 0.996 x + 0.258, with y = mean measured value and x = expected value. Comparison of tac concentrations measured on the Architect c8000 and the Viva Twin (Fig. 1A) were assessed using Passing–Bablok regression analysis (y = 1.0658 x − 1.3875, Intercept: 95% CI: − 1.9966, − 0.7318, Slope: 95% CI: 0.9997, 1.1346) with no deviation from linearity (p < 0.05) and a mean bias of − 7% (− 0.5 μg/L) for the Architect c8000 (Fig. 1). Corresponding comparison between Architect c8000 and LC-MS/MS (y = 0.8821x − 0.0953, Intercept: 95% CI: − 0.3753, 0.0985, Slope: 95% CI: 0.8496, 0.9131) resulted in no deviation from linearity (p < 0.05) (Fig. 1B). Overall bias between methods was 17.5% (+ 1.38 μg/L) with higher results measured on the Architect c8000 (Table 1).
Discussion Fig. 1. Passing–Bablok regression analysis of patient whole blood tac concentrations assayed on Abbott Architect c8000 versus Viva Twin (A, n = 166) and versus LC-MS/ MS (B, n = 195).
Imprecision of the EMIT assay on the Architect c8000 is comparable to results previously published for the LX20 PRO analyzer [4]; the limit of quantification at 2.8 μg/L is lower than
K. Boer et al. / Clinical Biochemistry 39 (2006) 1041–1043
the range estimated by Stephen et al. [5] for the ADVIA 1650 and below the limit of detection reported by LeGatt et al. [4] for the LX20 Pro. With between-day imprecision ranging from 9.0% to 4.7% the method is also suitable for the longitudinal monitoring of patients. Comparing the Viva Twin and Architect c8000 systems we observed a bias of 7% between both analyzers. LeGatt et al. [4] have noted previously a difference between the Abbott MEIA II assay and the EMIT 2000 assay on the Viva Twin that was larger than the difference between the MEIA II assay and the EMIT assay on the LX20 PRO, suggesting that the application of the same assay to different analyzers may still result in a bias between the analyzers. Tac concentrations measured on LCMS/MS by us and by other groups [4,8] were significantly lower as compared to the immunochemical assay; this is readily explained by the fact that the antibodies used in the EMIT and MEIA assays also bind pharmacologically inactive metabolites of tac [9] and by the possible impact of a low serum albumin and a low hematocrit [8]. In our study, absolute bias was comparable in all transplantation groups (Table 1); it is more significant in patients receiving a low dose of tac such as patients after BMT as described recently [8]. Therefore, the method used has to be reported to the clinician to avoid over- or under-dosing. LC-MS/MS based methods are labor-intensive and require expensive instrumentation. Due to restraints in TAT requirements immunochemical methods are still widely used. On the Architect c8000, high numbers of clinical samples can be processed in a relatively short time (10 min using the emergency slots provided by the analyzer). The Architect reagent cartridges hold up to 90 mL for both reagents (dead volume 2.5 mL) which is sufficient to load up to 600 tests onto the analyzer. In summary, applying EMIT 2000 Tacrolimus assay to the Architect c8000 we were able to consolidate standard routine
1043
workflow and increase productivity by concentrating more assays on one analyzer system. Acknowledgment The authors would like to thank Mrs. Haeuser for the technical support. References [1] Wallemacq PE, Verbeeck RK. Comparative clinical pharmacokinetics of tacrolimus in paediatric and adult patients. Clin Pharmacokinet 2001;40:283–95. [2] Wallemacq PE. Therapeutic monitoring of immunosuppressant drugs. Where are we? Clin Chem Lab Med 2004;42:1204–11. [3] Taylor PJ. Therapeutic drug monitoring of immunosuppressant drugs by high-performance liquid chromatography-mass spectrometry. Ther Drug Monit 2004;26:215–9. [4] LeGatt DF, Shalapay CE, Cheng SB. The EMIT 2000 Tacrolimus assay: an application protocol for the Beckman Synchron LX20 PRO analyzer. Clin Biochem 2004;37:1022–30. [5] Stephen DW, Rooke P, Clark S, Coutts M, Crowe L, Docherty D. Evaluation of the Dade Behring Syva EMIT 2000 Tacrolimus assay on the Bayer Advia 1650. Ann Clin Biochem 2003;40:697–700. [6] Dade Behring. EMIT® 2000 Tacrolimus Application Sheet. For the VIVA®, Viva-E™ and V-Twin™ Analyzers. 2004. [7] Koal T, Deters M, Casetta B, Kaever V. Simultaneous determination of four immunosuppressants by means of high speed and robust on-line solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. J Chromatogr, B: Anal Technol Biomed Life Sci 2004;805: 215–22. [8] Brown NW, Gonde CE, Adams JE, Tredger JM. Low hematocrit and serum albumin concentrations underlie the overestimation of Tacrolimus concentrations by microparticle enzyme immunoassay versus liquid chromatography-tandem mass spectrometry. Clin Chem 2005;51:586–92. [9] Iwasaki K, Shiraga T, Matsuda H, Nagase K, Tokuma Y, Hata T, et al. Further metabolism of FK506 (Tacrolimus). Identification and biological activities of the metabolites oxidized at multiple sites of FK506. Drug Metab Dispos 1995;23:28–34.