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Performance characteristics of the Architect cortisol immunoassay
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Clinica Chimica Acta 388 (2008) 219 – 221 www.elsevier.com/locate/clinchim
Letter to the Editor Performance characteristics of the Architect cortisol immunoassay
Keywords: Automated immunoassay; Cortisol; Imprecision; Linearity; Method comparison; Reference interval; Serum; Urine
Dear Editor, Cortisol is the primary glucocorticoid produced by the adrenal gland and is produced after stimulation of adrenocorticotrophic hormone from the pituitary gland. Regulation of cortisol is maintained by a negative feedback loop. The measurement of cortisol is the first step in the diagnostic work up of adrenal excess or deficiency. Follow-up with additional suppression or stimulation testing can further aid in diagnosis of Cushing's syndrome and Addison's disease. In practice, immunoassays are the easiest and most commonly used methods for cortisol measurements in serum and urine and are widely available in most clinical laboratories on different analyzers [1]. Over the years, random-access automated immunoassays have provided many benefits. Most cortisol immunoassays performed in clinical laboratories are direct assays and require no initial extraction of steroids from the specimen [1]. Such extraction techniques are not always suitable for large numbers of samples and require significant experience to yield useful results [2]. Although highly specific antibodies are available for immunoassays, lack of specificity and cross-reactivity with both endogenous and exogenous compounds are common concerns. Limit of detection, linearity, imprecision, and reference interval were determined for the cortisol immunoassay on the Architect i2000SR (Abbott Diagnostics, Abbott Park, IL). Method comparison testing was performed using the Advia® Centaur (Siemens Medical Solutions Diagnostics, Tarrytown, NY), AxSYM® (Abbott Diagnostics, Abbott Park, IL), Immulite® 2000 (Siemens Medical Solutions Diagnostics, Los Angeles, CA), Modular® E170 (Roche Diagnostics, Indianapolis, IN), and UniCel® DxI 800 (Beckman Coulter, Fullterton, CA). All testing was performed according to manufacturers' instructions. Urine samples were tested without organic solvent extraction. A urine cortisol method was not available on the Immulite 2000 analyzer. Limit of detection was determined by averaging 3 separate runs with each run consisting of 20 replicates of the 0 calibrator (Calibrator A, 0 nmol/l) and 15 replicates of non-zero material (3 serum pools ranging between 6.9 to 20.7 nmol/l). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.11.005
Specimens used for linearity testing were collected following completion of clinical testing. A high patient serum pool with an analyte concentration near the upper reportable limit was diluted with a low pool of cortisol-free stripped serum to yield concentrations of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0% of the original high pool. Five replicates of each dilution were tested. Imprecision was assessed using 3 levels of manufacturer's quality control material. Controls were run twice a day, for 5 days, in replicates of 2, using a fresh aliquot for each run. A minimum of 2 h separated each run for a total of 20 replicates for each control level. Interference studies were performed as previously described [4]. Briefly, a serum pool with a cortisol concentration of 822 nmol/l was supplemented with the following: (1) RBC hemolysate to a final hemoglobin concentration of 37.0 g/l, (2) bilirubin to a final concentration of 1157 μmol/l (677 mg/l), and (3) Intralipid to a final triglycerides concentration of 46.7 mmol/l (41.3 g/l). A deviation of N15% from the target cortisol concentration was considered a clinically significant degree of interference. Serum specimens for a reference interval study were collected from apparently healthy subjects, not taking any prescription medications, drawn between 8:00 AM and 10:00 AM from 75 males and 75 females. All studies using samples obtained from human subjects were approved by the Institutional Review Board of the University of Utah. Samples were retrieved from − 70 °C storage, thawed, mixed thoroughly, and analyzed on the Architect. Method comparison testing was performed with 80 residual serum and 104 residual urine specimens that had been submitted for clinical testing. Some of the serum samples were obtained from hospitalized subjects. The albumin and total protein concentrations of each serum sample were determined using a Modular P analyzer (Roche Diagnostics) with Roche reagents. All serum and urine specimens were tested for cortisol by all analyzers except urine specimens were not tested by the Immulite analyzer because this sample type is not listed as an acceptable sample type in the package insert for this method. A previously described LC-MS/MS was used as the comparison method for urine samples [5]. Prior to testing, specimens were thawed, mixed thoroughly, subjected to centrifugation at 3000 ×g for 10 min, and serum samples were checked for clots. EP Evaluator Release 5 software (David G. Rhoads Associates, Kennett Square, PA) was used to calculate limit of detection, linearity, imprecision, and reference intervals. Passing-Bablok and linear regression analysis was performed using Analyse-It, ver. 1.71 (Analyse-It Software, Leeds, England).
220
Letter to the Editor
Fig. 1. Method comparison using serum samples. The Architect method was compared to all methods using Passing-Bablok analysis. For graphs B and D, arrow points to repeated outliers included in regression analysis.
The limit of detection was calculated and compared with the manufacturer's claimed value. The average limit of detection was 3.6 nmol/l with a manufacturer's claim of ≤22.1 nmol/l. The target value for each linearity sample was calculated based
on the samples with the lowest and highest concentrations within the analytic measurement range. The Architect had a maximum deviation from the target recovery of 14.9% from a high patient serum pool diluted with cortisol-free stripped
Fig. 2. Method comparison using urine samples. Urinary free cortisol by LC-MS/MS was compared to all methods using Passing-Bablok analysis.
Letter to the Editor
serum. To answer the question of a possible matrix effect, linearity studies were also performed with a low patient serum pool rather than cortisol-free stripped serum and the maximum deviation from the target recovery did not improve. All levels of quality control material demonstrated total imprecision of ≤ 10%. Interestingly, the highest level of control gave the highest CV of 10.4% at a mean cortisol concentration of 649 nmol/l. The lowest level of control had a CV of 6.0% and the medium control level had the lowest CV at 3.4%. Total imprecision for all control levels on the Architect fall within the range of previously observed CVs for other automated immunoassays [3]. Interference studies for hemolysis, icterus, and lipemia demonstrated b15% interference detected by the maximum concentration of each substance tested. Trending shows a likely negative effect on cortisol concentrations above the maximum concentrations tested for all interferents. The nonparametric reference interval for serum samples drawn from healthy volunteers between 8:00 AM and 10:00 AM was 110 to 613 nmol/l. This confirms the manufacturer's serum AM reference interval of 101 to 536 nmol/l. Many specimen types can be used for cortisol testing such as serum, plasma, urine, and saliva; however, not all specimen types are approved for every method. The Architect was compared against other methods for serum and urine specimens. For serum samples used for the method comparison study, the median albumin concentration was 45 g/l with a range of albumin concentrations of 31–52 g/l. The median total protein concentration for these samples was 70 g/l with a range of total protein concentrations of 48–86 g/l. Furthermore, the range of serum cortisol concentrations was 75–1570 nmol/l, indicating varying degrees of activation of the adrenal glands. These data indicate that in addition to relatively healthy patients, some of the serum samples were obtained from patients with significant illness. Serum specimens demonstrated favorable agreement with correlation coefficients of 0.98 or better and slopes ranging from 0.92 to 1.11 (Fig. 1). Method comparison results for serum are comparable to what has been observed previously for other automated immunoassays [3]. It is noteworthy that for the method comparison with serum specimens there was one sample with an Architect result of 676 nmol/l and AxSYM and Modular E170 results of 1212 and 1256 nmol/l, respectively (Fig. 1). Repeat testing of this sample confirmed the initial results of each of the three methods. Interestingly, this sample did not appear to be an outlier with any of the other cortisol methods. No clinical history was available on the subject from whom this sample was collected. The possibility of an interfering substance, either endogenous or exogenous, cannot be excluded. Urine specimens demonstrated varying degrees of agreement with correlation coefficients of 0.57 to 0.81 and slopes ranging from 1.77 to 18.5 using LC-MS/MS as the comparison method (Fig. 2). One explanation may be the limited range of urine cortisol concentrations tested. Also, due to the lack of clinical data associated with collected specimens, we are unable to identify the possibility of biologic factors causing interference. Observed method comparison results for urine specimens may reflect matrix effects and/or differing specificities towards endogenous
221
steroids, which have been concerns for other automated immunoassays [6,7]. In conclusion, the Architect cortisol immunoassay shows excellent sensitivity, acceptable imprecision, and compares favorably against all methods, particularly for serum specimens. Differences in correlation of urine specimens are unlikely to be improved by calibration standardization but are likely due to variable cross-reactivities of endogenous substances. Variation between lots of reagents and calibrators reagent lots and variability in individual calibrations presents obstacles to assay harmonization in the field. Although most methods claim standardization to the Institute for Reference Material and Measurements/International Federation for Clinical Chemistry 451 serum reference panel, continuing standardization efforts have proven beneficial since an earlier study was conducted [3]. Acknowledgements Support for this study was provided by Abbott Diagnostics and the ARUP Institute for Clinical and Experimental Pathology. We gratefully acknowledge Abbott Diagnostics and Roche Diagnostics for providing instrumentation to perform testing using their methods. We thank Mamak Vedadi for her assistance with sample collection and de-identification. References [1] Demers LM. The adrenal cortex. 4th ed. St Louis, MO: Elsevier Saunders; 2005 [2034–7pp.]. [2] Holder G. Measurement of glucocorticoids in biological fluids. Methods Mol Biol 2006;324:141–57. [3] Roberts RF, Roberts WL. Performance characteristics of five automated serum cortisol immunoassays. Clin Biochem 2004;37:489–93. [4] Owen WE, Roberts WL. Performance characteristics of the IMMUNLITE 2000 erythropoietin assay. Clin Chim Acta 2004;340:213–7. [5] Kushnir MM, Rockwood AL, Nelson GJ, Terry AH, Meikle AW. Liquid chromatography-tandem mass spectrometry analysis of urinary free cortisol. Clin Chem 2003;49:965–7. [6] Gray G, Shakerdi L, Wallace AM. Poor specificity and recovery of urinary free cortisol as determined by the Bayer Advia Centaur extraction method. Ann Clin Biochem 2003;40:563–5. [7] Horie H, Kidowaki T, Koyama Y, et al. Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations. Clin Chim Acta 2007;378:66–70.
Sonia L. La'ulu ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States William L. Roberts Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT, United States E-mail address: [email protected]. Corresponding author. c/o 500 Chipeta Way, Salt Lake City, UT 84108, United States. Tel.: +1 800 242 2787x2086; fax: +1 801 584 5207. 24 August 2007
Clinica Chimica Acta 388 (2008) 219 – 221 www.elsevier.com/locate/clinchim
Letter to the Editor Performance characteristics of the Architect cortisol immunoassay
Keywords: Automated immunoassay; Cortisol; Imprecision; Linearity; Method comparison; Reference interval; Serum; Urine
Dear Editor, Cortisol is the primary glucocorticoid produced by the adrenal gland and is produced after stimulation of adrenocorticotrophic hormone from the pituitary gland. Regulation of cortisol is maintained by a negative feedback loop. The measurement of cortisol is the first step in the diagnostic work up of adrenal excess or deficiency. Follow-up with additional suppression or stimulation testing can further aid in diagnosis of Cushing's syndrome and Addison's disease. In practice, immunoassays are the easiest and most commonly used methods for cortisol measurements in serum and urine and are widely available in most clinical laboratories on different analyzers [1]. Over the years, random-access automated immunoassays have provided many benefits. Most cortisol immunoassays performed in clinical laboratories are direct assays and require no initial extraction of steroids from the specimen [1]. Such extraction techniques are not always suitable for large numbers of samples and require significant experience to yield useful results [2]. Although highly specific antibodies are available for immunoassays, lack of specificity and cross-reactivity with both endogenous and exogenous compounds are common concerns. Limit of detection, linearity, imprecision, and reference interval were determined for the cortisol immunoassay on the Architect i2000SR (Abbott Diagnostics, Abbott Park, IL). Method comparison testing was performed using the Advia® Centaur (Siemens Medical Solutions Diagnostics, Tarrytown, NY), AxSYM® (Abbott Diagnostics, Abbott Park, IL), Immulite® 2000 (Siemens Medical Solutions Diagnostics, Los Angeles, CA), Modular® E170 (Roche Diagnostics, Indianapolis, IN), and UniCel® DxI 800 (Beckman Coulter, Fullterton, CA). All testing was performed according to manufacturers' instructions. Urine samples were tested without organic solvent extraction. A urine cortisol method was not available on the Immulite 2000 analyzer. Limit of detection was determined by averaging 3 separate runs with each run consisting of 20 replicates of the 0 calibrator (Calibrator A, 0 nmol/l) and 15 replicates of non-zero material (3 serum pools ranging between 6.9 to 20.7 nmol/l). 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.11.005
Specimens used for linearity testing were collected following completion of clinical testing. A high patient serum pool with an analyte concentration near the upper reportable limit was diluted with a low pool of cortisol-free stripped serum to yield concentrations of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0% of the original high pool. Five replicates of each dilution were tested. Imprecision was assessed using 3 levels of manufacturer's quality control material. Controls were run twice a day, for 5 days, in replicates of 2, using a fresh aliquot for each run. A minimum of 2 h separated each run for a total of 20 replicates for each control level. Interference studies were performed as previously described [4]. Briefly, a serum pool with a cortisol concentration of 822 nmol/l was supplemented with the following: (1) RBC hemolysate to a final hemoglobin concentration of 37.0 g/l, (2) bilirubin to a final concentration of 1157 μmol/l (677 mg/l), and (3) Intralipid to a final triglycerides concentration of 46.7 mmol/l (41.3 g/l). A deviation of N15% from the target cortisol concentration was considered a clinically significant degree of interference. Serum specimens for a reference interval study were collected from apparently healthy subjects, not taking any prescription medications, drawn between 8:00 AM and 10:00 AM from 75 males and 75 females. All studies using samples obtained from human subjects were approved by the Institutional Review Board of the University of Utah. Samples were retrieved from − 70 °C storage, thawed, mixed thoroughly, and analyzed on the Architect. Method comparison testing was performed with 80 residual serum and 104 residual urine specimens that had been submitted for clinical testing. Some of the serum samples were obtained from hospitalized subjects. The albumin and total protein concentrations of each serum sample were determined using a Modular P analyzer (Roche Diagnostics) with Roche reagents. All serum and urine specimens were tested for cortisol by all analyzers except urine specimens were not tested by the Immulite analyzer because this sample type is not listed as an acceptable sample type in the package insert for this method. A previously described LC-MS/MS was used as the comparison method for urine samples [5]. Prior to testing, specimens were thawed, mixed thoroughly, subjected to centrifugation at 3000 ×g for 10 min, and serum samples were checked for clots. EP Evaluator Release 5 software (David G. Rhoads Associates, Kennett Square, PA) was used to calculate limit of detection, linearity, imprecision, and reference intervals. Passing-Bablok and linear regression analysis was performed using Analyse-It, ver. 1.71 (Analyse-It Software, Leeds, England).
220
Letter to the Editor
Fig. 1. Method comparison using serum samples. The Architect method was compared to all methods using Passing-Bablok analysis. For graphs B and D, arrow points to repeated outliers included in regression analysis.
The limit of detection was calculated and compared with the manufacturer's claimed value. The average limit of detection was 3.6 nmol/l with a manufacturer's claim of ≤22.1 nmol/l. The target value for each linearity sample was calculated based
on the samples with the lowest and highest concentrations within the analytic measurement range. The Architect had a maximum deviation from the target recovery of 14.9% from a high patient serum pool diluted with cortisol-free stripped
Fig. 2. Method comparison using urine samples. Urinary free cortisol by LC-MS/MS was compared to all methods using Passing-Bablok analysis.
Letter to the Editor
serum. To answer the question of a possible matrix effect, linearity studies were also performed with a low patient serum pool rather than cortisol-free stripped serum and the maximum deviation from the target recovery did not improve. All levels of quality control material demonstrated total imprecision of ≤ 10%. Interestingly, the highest level of control gave the highest CV of 10.4% at a mean cortisol concentration of 649 nmol/l. The lowest level of control had a CV of 6.0% and the medium control level had the lowest CV at 3.4%. Total imprecision for all control levels on the Architect fall within the range of previously observed CVs for other automated immunoassays [3]. Interference studies for hemolysis, icterus, and lipemia demonstrated b15% interference detected by the maximum concentration of each substance tested. Trending shows a likely negative effect on cortisol concentrations above the maximum concentrations tested for all interferents. The nonparametric reference interval for serum samples drawn from healthy volunteers between 8:00 AM and 10:00 AM was 110 to 613 nmol/l. This confirms the manufacturer's serum AM reference interval of 101 to 536 nmol/l. Many specimen types can be used for cortisol testing such as serum, plasma, urine, and saliva; however, not all specimen types are approved for every method. The Architect was compared against other methods for serum and urine specimens. For serum samples used for the method comparison study, the median albumin concentration was 45 g/l with a range of albumin concentrations of 31–52 g/l. The median total protein concentration for these samples was 70 g/l with a range of total protein concentrations of 48–86 g/l. Furthermore, the range of serum cortisol concentrations was 75–1570 nmol/l, indicating varying degrees of activation of the adrenal glands. These data indicate that in addition to relatively healthy patients, some of the serum samples were obtained from patients with significant illness. Serum specimens demonstrated favorable agreement with correlation coefficients of 0.98 or better and slopes ranging from 0.92 to 1.11 (Fig. 1). Method comparison results for serum are comparable to what has been observed previously for other automated immunoassays [3]. It is noteworthy that for the method comparison with serum specimens there was one sample with an Architect result of 676 nmol/l and AxSYM and Modular E170 results of 1212 and 1256 nmol/l, respectively (Fig. 1). Repeat testing of this sample confirmed the initial results of each of the three methods. Interestingly, this sample did not appear to be an outlier with any of the other cortisol methods. No clinical history was available on the subject from whom this sample was collected. The possibility of an interfering substance, either endogenous or exogenous, cannot be excluded. Urine specimens demonstrated varying degrees of agreement with correlation coefficients of 0.57 to 0.81 and slopes ranging from 1.77 to 18.5 using LC-MS/MS as the comparison method (Fig. 2). One explanation may be the limited range of urine cortisol concentrations tested. Also, due to the lack of clinical data associated with collected specimens, we are unable to identify the possibility of biologic factors causing interference. Observed method comparison results for urine specimens may reflect matrix effects and/or differing specificities towards endogenous
221
steroids, which have been concerns for other automated immunoassays [6,7]. In conclusion, the Architect cortisol immunoassay shows excellent sensitivity, acceptable imprecision, and compares favorably against all methods, particularly for serum specimens. Differences in correlation of urine specimens are unlikely to be improved by calibration standardization but are likely due to variable cross-reactivities of endogenous substances. Variation between lots of reagents and calibrators reagent lots and variability in individual calibrations presents obstacles to assay harmonization in the field. Although most methods claim standardization to the Institute for Reference Material and Measurements/International Federation for Clinical Chemistry 451 serum reference panel, continuing standardization efforts have proven beneficial since an earlier study was conducted [3]. Acknowledgements Support for this study was provided by Abbott Diagnostics and the ARUP Institute for Clinical and Experimental Pathology. We gratefully acknowledge Abbott Diagnostics and Roche Diagnostics for providing instrumentation to perform testing using their methods. We thank Mamak Vedadi for her assistance with sample collection and de-identification. References [1] Demers LM. The adrenal cortex. 4th ed. St Louis, MO: Elsevier Saunders; 2005 [2034–7pp.]. [2] Holder G. Measurement of glucocorticoids in biological fluids. Methods Mol Biol 2006;324:141–57. [3] Roberts RF, Roberts WL. Performance characteristics of five automated serum cortisol immunoassays. Clin Biochem 2004;37:489–93. [4] Owen WE, Roberts WL. Performance characteristics of the IMMUNLITE 2000 erythropoietin assay. Clin Chim Acta 2004;340:213–7. [5] Kushnir MM, Rockwood AL, Nelson GJ, Terry AH, Meikle AW. Liquid chromatography-tandem mass spectrometry analysis of urinary free cortisol. Clin Chem 2003;49:965–7. [6] Gray G, Shakerdi L, Wallace AM. Poor specificity and recovery of urinary free cortisol as determined by the Bayer Advia Centaur extraction method. Ann Clin Biochem 2003;40:563–5. [7] Horie H, Kidowaki T, Koyama Y, et al. Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations. Clin Chim Acta 2007;378:66–70.
Sonia L. La'ulu ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States William L. Roberts Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT, United States E-mail address: [email protected]. Corresponding author. c/o 500 Chipeta Way, Salt Lake City, UT 84108, United States. Tel.: +1 800 242 2787x2086; fax: +1 801 584 5207. 24 August 2007