Clin Chem Lab Med 2015; 53(5): 761–770
Josef van Helden* and Ralf Weiskirchen
Experience with the first fully automated chemiluminescence immunoassay for the quantification of 1α, 25-dihydroxy-vitamin D DOI 10.1515/cclm-2014-0698 Received July 7, 2014; accepted August 27, 2014; previously published online September 30, 2014
Abstract Background: Previous studies have shown that the measurement of vitamin D and its derivatives, especially its active metabolite 1α, 25-dihydroxy-vitamin D [1,25(OH)2D], is highly complex and prone to analytical error. We have evaluated a new immunological method for detecting and quantifying of 1,25(OH)2D. This assay is fully automated, sensitive and uses a specific recombinant fusion protein for capturing of 1,25(OH)2D. The assay was originally developed by DiaSorin for the immunoassay analyzer LIAISON XL. Methods: Performance data of this assay were determined including intra- and inter-assay precision, recovery, linearity, and limit of detection of the DiaSorin 1,25(OH)2D immunoassay on the LIAISON XL analyzer. Respective data were compared from two different liquid chromatography tandem mass spectrometry (LC-MS/MS) assays and a common radioimmunoassay (RIA) using clinical samples taken from patients suffering from vitamin D deficiency, chronic renal failure, biliary atresia, hyperpara thyroidism, vitamin D-dependent rickets or sarcoidosis, as well as from pregnant women and high-level athletes. Results: The performance evaluation of 1,25(OH)2D resulted in an intra-assay and total imprecision correlation variant between 1.4% and 5.2% and 3.8%–7.1% with the new immunoassay and 3.5%–5.8% or 3.8%–7.5% with the LC-MS/MS method, respectively. Limits of detection and quantification of the immunoassay were 0.7 ng/L and 5.0 ng/L for the LIAISON XL immunoassay and 1.8 ng/L and 5.4 ng/L for the LC-MS/MS assay, respectively. *Corresponding author: Josef van Helden, MVZ Dr. Stein und Kollegen, Wallstrasse 10, 41061 Moenchengladbach, Germany, E-mail: [email protected] Ralf Weiskirchen: Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, University Hospital RWTH Aachen, Germany
Pearson’s coefficients of correlation were 0.998 and 0.952 for method comparison to different established LC-MS/ MS methods. Linear regression according to Passing and Bablok showed larger deviations to the RIA (slopes 0.64– 0.97, coefficients of correlation 0.822–0.823). Conclusions: The DiaSorin LIAISON XL 1,25(OH)2D immunoassay appears to have improved comparability to LC-MS/MS with low imprecision and limits of detection. The assay time of 65 min, the small sample volume required (75 μL) and the throughput of 90 tests/hour without manually handling time for extraction and purification procedures is superior to the LC-MS/MS method. Keywords: immunoassay; LC-MS/MS; linearity repro ducibility; vitamin D.
Introduction Vitamin D is metabolized to several compounds and, ultimately, a small portion is converted to the most biologically active metabolite, i.e., 1α,25-dihydroxy-vitamin D (1,25(OH)2D) [1]. This steroid hormone, primarily of renal origin, is affected by factors that perturb mineral and skeletal metabolism. Additionally, circulating 1,25(OH)2D is altered in several pathophysiological states, such as parathyroid gland disorders, renal failure, vitamin D-dependent rickets types I and II, and sarcoidosis. As alterations in circulating disorders of calcium metabolism, considerable efforts have been directed towards the development of less cumbersome, more expedient assays for the quantifying of circulating 1,25(OH)2D [2, 3]. The metabolic activation of vitamin D is an intricately controlled process, subject to extensive alteration by variables including dietary calcium and phosphorus, degree of vitamin D deficiency, genetic deficiencies, parathyroid hormone (PTH) concentration, exposure to ultraviolet light and degree of renal function. 1,25(OH)2D is due to its essential role in the efficient active absorption of calcium and phosphorus, as well as their normal metabolism. In secondary hyperparathyroidism the parathyroid glands become enlarged and hyperactive. Kidney failure
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/20/15 6:39 PM
762 van Helden and Weiskirchen: First fully automated 1α, 25-dihydroxy-vitamin D immunoassay is a common case of secondary hyperparathyroidism and usually occurs as a complication of renal disease where the kidney is unable to remove the phosphorus produced by the body and is also unable to produce enough of the active form of vitamin D. The buildup of phosphorus leads to low levels of calcium in the blood, which in turn stimulates the parathyroid glands to increase PTH production, causing the parathyroid glands to enlarge. As the disease progresses the parathyroid glands no longer respond normally to calcium and vitamin D [4]. The clinical practice guidelines compiled from the Kidney Disease Outcomes Quality Initiative (K/DOQI) [5] and the Kidney Disease: Improving Global Outcomes (KDIGO) [6] recommended activated vitamin D therapeutic regimens for chronic kidney disease (CKD) patients. Consequently, the measurement of 1,25(OH)2D is rapidly becoming an efficient tool in the research of diseases and conditions that effect the normal metabolism of phosphorus and calcium. In this study a newly developed, fully automated chemiluminescence immunoassay without sample pretreatment and preparation, requiring only a short assay time of 65 min was compared to two different liquid chromatography tandem mass spectrometry (LC-MS/MS) methods and to a well-established 125J-labeled radioimmunoassay for the measurement of 1,25(OH)2D. A clinical evaluation of the new method was performed by comparing it to the routinely used AB SCIEX LCMS/MS method [7].
Materials and methods Clinical samples Unselected routine clinical left over samples of fresh serum were obtained from 60 patients undergoing medical examination. For sensitivity studies and testing of analytical specificity, frozen serum specimens were used in which multiple freezing/thawing was avoided. Defined samples for 1,25(OH)2D determination were collected in a large clinical laboratory including: 20 samples from patients with chronic renal failure, five with bilary atresia, 15 with primary hyperparathyroidism, 15 with sarcoidosis, 20 from pregnant women, and 15 from high level athletes. Unused aliquots were stored frozen at –30 °C until analysis. Samples tested with a LC-MS/MS method according to the protocols developed by Strathmann and colleagues [8] were provided by DiaSorin and shipped on dry ice for further analysis of 1,25(OH)2D with the LAISON XL immunoassay and the AB SCIEX LC-MS/MS method.
capture of the 1,25(OH)2D molecule from a 75 μL serum, EDTA- or Li-heparinate-plasma sample and a murine monoclonal antibody which specifically recognizes the complex formed by the recombinant fusion protein with the 1,25(OH)2D molecule. During the incubation period, calibrators, controls or patient samples are incubated with the recombinant protein (binding agent) in an assay buffer. Following this incubation the solid phase containing the specific monoclonal antibody is added and allowed to bind with the complex from the first incubation. After the second incubation, a wash cycle is performed to remove any unbound material. In the third step the conjugate is added and unbound material is removed by a second wash step. The starter reagents are then added and a flash chemiluminescent reaction is initiated. The light signal is measured by a photomultiplier as relative light units (RLU) and is proportional to the concentration of 1,25(OH)2D present in the calibrators, controls and patient samples. The test principle of the LIAISON XL 1,25 Dihydroxyvitamin D assay is displayed in Figure 1. The first LC-MS/MS method (AB SCIEX) used was performed according to a modified method of Casetta and colleagues [7]. Briefly, 100 μL sample were added to 10 μL of the internal standard solution of d6-1α, 25(OH)2-vitamin D3. After vortex mixing, 200 μL of acetonitrile was added for the protein precipitation. After further vortex mixing and centrifugation at 12,000 × g for 5 min, 200 μL of the supernatant was collected and placed into an autosampler for a subsequent injection of 100 μL for a two-dimensional liquid chromatographic (2DLC) process. The API4000TM tandem mass spectrometer (AB SCIEX, Framingham, MA) was operated using the Turbo VTM source in positive ion electrospray mode. Multiple reaction monitoring (MRM) of the transition m/z 423/369 for the 1α,25(OH)2-vitamin D3 and the transmission m/z 429/393 for the internal standard d6-1α,25(OH)2vitamin D3 was used throughout. The second LC-MS/MS method used was performed according to a method published by Strathmann et al. [8]. Briefly, preparation of 400 μL serum or plasma samples consisted of protein precipitation, immunoextraction with solid phase anti-1,25(OH)2D antibody and derivatization with 4-pheny-1,2,4-triazoline-3,5-dione. Analytes were resolved using reverse-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. A commercial RIA assay IDS 1,25-Dihydroxy Vitamin D RIA kit (Immunodiagnostic Systems, Frankfurt, Germany) that includes a complete assay system intended for the purification of
H3C H3C
The LIAISON XL 1,25 Dihydroxyvitamin D assay is a modified threestep sandwich assay that uses a recombinant fusion protein for
OH CH3
1,25-(OH)2D3
3rd step Chemiluminescent reaction
H CH2 1
RLU
OH
HO
Complex detection with a MAB Recombinant 1,25-(OH)2D3 binding protein
Test principles
CH3
25
H
1st
step
Conformational change
2nd step
[1,25-(OH)2D]
Figure 1 Test principle of the LIAISON XL 1,25 Dihydroxyvitamin D immunoassay.
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van Helden and Weiskirchen: First fully automated 1α, 25-dihydroxy-vitamin D immunoassay 763 1,25-dihydroxyvitamin D in a 400 μL human serum or plasma sample by immunoextraction with solid phase anti-1,25(OH)2D antibody followed by quantitation by 125I radioimmunoassay, was essentially performed according to the manufacturer’s instructions.
Precision The precision study was carried out using the kit control of two levels provided by the manufacturer, control level 1 range 27–33 ng/L, level 2 range 104–140 ng/L, and five self-prepared pool sera at different concentrations (range 22.1–184.7 ng/L) distributed over the whole measuring range of the assay for the performance evaluation of LIAISON XL. The precision study was additionally carried out with the ABI SCIEX LC-MS/MS method to get an impression to the corresponding performance data of one clinically evaluated LC-MS/MS method which is often considered as reference method for many analytes. Within-run precision was determined by 21 replicate measurements in a single run. Mean, standard deviation (SD), and coefficients of variation (CV) were calculated. Repeatability and intermediate precision was calculated according to the CLSI/NCCLS guideline EP5-A2 [9] using aliquots of control material and the five self-prepared pool sera at different concentrations which were analyzed in three determinations per run and two runs per day on 10 days (n = 60 measurements). Repeatability and intermediate precision was estimated using frozen (–20 °C) human pool sera as well as aliquots of the controls thawed immediately before the analysis as recommended by the manufacturer.
Limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) LoB was determined as the 95th percentile value from n = 60 determinations of five 1,25(OH)2D free serum samples in single determination over six independent series on the LIAISON XL immunoassay analyzer and the API4000TM tandem mass spectrometer, respectively. LoD was determined based on the LoB and the individual standard deviations of five low level 1,25(OH)2D sera. LoD corresponds to the lowest 1,25(OH)2D concentration which can be detected above the LoB with a probability of 95%. The LoQ defined as the lowest amount of 1,25(OH)2D that can be accurately quantitated with an allowable error (square root model) of ≤ 20% was determined with three low level 1,25(OH)2D samples diluted each in four dilution steps with analytefree dilution serum pool to approximately 4–5 ng/L 1,25(OH)2D. One assay run per day over 6 days on both instruments was performed.
Linearity in dilution The dilution linearity of the different assay was evaluated with two different serum samples containing 1,25(OH)2D levels up to 180 ng/L. The samples were diluted using an analyte-free sample diluent. Each dilution series contained nine linear dilution steps (9+1, 8+2, 7+3, 6+4, etc). In each run, measurements of the dilutions were performed in duplicate, the undiluted serum and the analyte-free sample were analyzed in six replicate measurements. The method was considered to be linear, if the measured 1,25(OH)2D values differed by
Josef van Helden* and Ralf Weiskirchen
Experience with the first fully automated chemiluminescence immunoassay for the quantification of 1α, 25-dihydroxy-vitamin D DOI 10.1515/cclm-2014-0698 Received July 7, 2014; accepted August 27, 2014; previously published online September 30, 2014
Abstract Background: Previous studies have shown that the measurement of vitamin D and its derivatives, especially its active metabolite 1α, 25-dihydroxy-vitamin D [1,25(OH)2D], is highly complex and prone to analytical error. We have evaluated a new immunological method for detecting and quantifying of 1,25(OH)2D. This assay is fully automated, sensitive and uses a specific recombinant fusion protein for capturing of 1,25(OH)2D. The assay was originally developed by DiaSorin for the immunoassay analyzer LIAISON XL. Methods: Performance data of this assay were determined including intra- and inter-assay precision, recovery, linearity, and limit of detection of the DiaSorin 1,25(OH)2D immunoassay on the LIAISON XL analyzer. Respective data were compared from two different liquid chromatography tandem mass spectrometry (LC-MS/MS) assays and a common radioimmunoassay (RIA) using clinical samples taken from patients suffering from vitamin D deficiency, chronic renal failure, biliary atresia, hyperpara thyroidism, vitamin D-dependent rickets or sarcoidosis, as well as from pregnant women and high-level athletes. Results: The performance evaluation of 1,25(OH)2D resulted in an intra-assay and total imprecision correlation variant between 1.4% and 5.2% and 3.8%–7.1% with the new immunoassay and 3.5%–5.8% or 3.8%–7.5% with the LC-MS/MS method, respectively. Limits of detection and quantification of the immunoassay were 0.7 ng/L and 5.0 ng/L for the LIAISON XL immunoassay and 1.8 ng/L and 5.4 ng/L for the LC-MS/MS assay, respectively. *Corresponding author: Josef van Helden, MVZ Dr. Stein und Kollegen, Wallstrasse 10, 41061 Moenchengladbach, Germany, E-mail: [email protected] Ralf Weiskirchen: Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, University Hospital RWTH Aachen, Germany
Pearson’s coefficients of correlation were 0.998 and 0.952 for method comparison to different established LC-MS/ MS methods. Linear regression according to Passing and Bablok showed larger deviations to the RIA (slopes 0.64– 0.97, coefficients of correlation 0.822–0.823). Conclusions: The DiaSorin LIAISON XL 1,25(OH)2D immunoassay appears to have improved comparability to LC-MS/MS with low imprecision and limits of detection. The assay time of 65 min, the small sample volume required (75 μL) and the throughput of 90 tests/hour without manually handling time for extraction and purification procedures is superior to the LC-MS/MS method. Keywords: immunoassay; LC-MS/MS; linearity repro ducibility; vitamin D.
Introduction Vitamin D is metabolized to several compounds and, ultimately, a small portion is converted to the most biologically active metabolite, i.e., 1α,25-dihydroxy-vitamin D (1,25(OH)2D) [1]. This steroid hormone, primarily of renal origin, is affected by factors that perturb mineral and skeletal metabolism. Additionally, circulating 1,25(OH)2D is altered in several pathophysiological states, such as parathyroid gland disorders, renal failure, vitamin D-dependent rickets types I and II, and sarcoidosis. As alterations in circulating disorders of calcium metabolism, considerable efforts have been directed towards the development of less cumbersome, more expedient assays for the quantifying of circulating 1,25(OH)2D [2, 3]. The metabolic activation of vitamin D is an intricately controlled process, subject to extensive alteration by variables including dietary calcium and phosphorus, degree of vitamin D deficiency, genetic deficiencies, parathyroid hormone (PTH) concentration, exposure to ultraviolet light and degree of renal function. 1,25(OH)2D is due to its essential role in the efficient active absorption of calcium and phosphorus, as well as their normal metabolism. In secondary hyperparathyroidism the parathyroid glands become enlarged and hyperactive. Kidney failure
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/20/15 6:39 PM
762 van Helden and Weiskirchen: First fully automated 1α, 25-dihydroxy-vitamin D immunoassay is a common case of secondary hyperparathyroidism and usually occurs as a complication of renal disease where the kidney is unable to remove the phosphorus produced by the body and is also unable to produce enough of the active form of vitamin D. The buildup of phosphorus leads to low levels of calcium in the blood, which in turn stimulates the parathyroid glands to increase PTH production, causing the parathyroid glands to enlarge. As the disease progresses the parathyroid glands no longer respond normally to calcium and vitamin D [4]. The clinical practice guidelines compiled from the Kidney Disease Outcomes Quality Initiative (K/DOQI) [5] and the Kidney Disease: Improving Global Outcomes (KDIGO) [6] recommended activated vitamin D therapeutic regimens for chronic kidney disease (CKD) patients. Consequently, the measurement of 1,25(OH)2D is rapidly becoming an efficient tool in the research of diseases and conditions that effect the normal metabolism of phosphorus and calcium. In this study a newly developed, fully automated chemiluminescence immunoassay without sample pretreatment and preparation, requiring only a short assay time of 65 min was compared to two different liquid chromatography tandem mass spectrometry (LC-MS/MS) methods and to a well-established 125J-labeled radioimmunoassay for the measurement of 1,25(OH)2D. A clinical evaluation of the new method was performed by comparing it to the routinely used AB SCIEX LCMS/MS method [7].
Materials and methods Clinical samples Unselected routine clinical left over samples of fresh serum were obtained from 60 patients undergoing medical examination. For sensitivity studies and testing of analytical specificity, frozen serum specimens were used in which multiple freezing/thawing was avoided. Defined samples for 1,25(OH)2D determination were collected in a large clinical laboratory including: 20 samples from patients with chronic renal failure, five with bilary atresia, 15 with primary hyperparathyroidism, 15 with sarcoidosis, 20 from pregnant women, and 15 from high level athletes. Unused aliquots were stored frozen at –30 °C until analysis. Samples tested with a LC-MS/MS method according to the protocols developed by Strathmann and colleagues [8] were provided by DiaSorin and shipped on dry ice for further analysis of 1,25(OH)2D with the LAISON XL immunoassay and the AB SCIEX LC-MS/MS method.
capture of the 1,25(OH)2D molecule from a 75 μL serum, EDTA- or Li-heparinate-plasma sample and a murine monoclonal antibody which specifically recognizes the complex formed by the recombinant fusion protein with the 1,25(OH)2D molecule. During the incubation period, calibrators, controls or patient samples are incubated with the recombinant protein (binding agent) in an assay buffer. Following this incubation the solid phase containing the specific monoclonal antibody is added and allowed to bind with the complex from the first incubation. After the second incubation, a wash cycle is performed to remove any unbound material. In the third step the conjugate is added and unbound material is removed by a second wash step. The starter reagents are then added and a flash chemiluminescent reaction is initiated. The light signal is measured by a photomultiplier as relative light units (RLU) and is proportional to the concentration of 1,25(OH)2D present in the calibrators, controls and patient samples. The test principle of the LIAISON XL 1,25 Dihydroxyvitamin D assay is displayed in Figure 1. The first LC-MS/MS method (AB SCIEX) used was performed according to a modified method of Casetta and colleagues [7]. Briefly, 100 μL sample were added to 10 μL of the internal standard solution of d6-1α, 25(OH)2-vitamin D3. After vortex mixing, 200 μL of acetonitrile was added for the protein precipitation. After further vortex mixing and centrifugation at 12,000 × g for 5 min, 200 μL of the supernatant was collected and placed into an autosampler for a subsequent injection of 100 μL for a two-dimensional liquid chromatographic (2DLC) process. The API4000TM tandem mass spectrometer (AB SCIEX, Framingham, MA) was operated using the Turbo VTM source in positive ion electrospray mode. Multiple reaction monitoring (MRM) of the transition m/z 423/369 for the 1α,25(OH)2-vitamin D3 and the transmission m/z 429/393 for the internal standard d6-1α,25(OH)2vitamin D3 was used throughout. The second LC-MS/MS method used was performed according to a method published by Strathmann et al. [8]. Briefly, preparation of 400 μL serum or plasma samples consisted of protein precipitation, immunoextraction with solid phase anti-1,25(OH)2D antibody and derivatization with 4-pheny-1,2,4-triazoline-3,5-dione. Analytes were resolved using reverse-phase UPLC and quantified using positive ion electrospray ionization-tandem mass spectrometry. A commercial RIA assay IDS 1,25-Dihydroxy Vitamin D RIA kit (Immunodiagnostic Systems, Frankfurt, Germany) that includes a complete assay system intended for the purification of
H3C H3C
The LIAISON XL 1,25 Dihydroxyvitamin D assay is a modified threestep sandwich assay that uses a recombinant fusion protein for
OH CH3
1,25-(OH)2D3
3rd step Chemiluminescent reaction
H CH2 1
RLU
OH
HO
Complex detection with a MAB Recombinant 1,25-(OH)2D3 binding protein
Test principles
CH3
25
H
1st
step
Conformational change
2nd step
[1,25-(OH)2D]
Figure 1 Test principle of the LIAISON XL 1,25 Dihydroxyvitamin D immunoassay.
Brought to you by | Purdue University Libraries Authenticated Download Date | 5/20/15 6:39 PM
van Helden and Weiskirchen: First fully automated 1α, 25-dihydroxy-vitamin D immunoassay 763 1,25-dihydroxyvitamin D in a 400 μL human serum or plasma sample by immunoextraction with solid phase anti-1,25(OH)2D antibody followed by quantitation by 125I radioimmunoassay, was essentially performed according to the manufacturer’s instructions.
Precision The precision study was carried out using the kit control of two levels provided by the manufacturer, control level 1 range 27–33 ng/L, level 2 range 104–140 ng/L, and five self-prepared pool sera at different concentrations (range 22.1–184.7 ng/L) distributed over the whole measuring range of the assay for the performance evaluation of LIAISON XL. The precision study was additionally carried out with the ABI SCIEX LC-MS/MS method to get an impression to the corresponding performance data of one clinically evaluated LC-MS/MS method which is often considered as reference method for many analytes. Within-run precision was determined by 21 replicate measurements in a single run. Mean, standard deviation (SD), and coefficients of variation (CV) were calculated. Repeatability and intermediate precision was calculated according to the CLSI/NCCLS guideline EP5-A2 [9] using aliquots of control material and the five self-prepared pool sera at different concentrations which were analyzed in three determinations per run and two runs per day on 10 days (n = 60 measurements). Repeatability and intermediate precision was estimated using frozen (–20 °C) human pool sera as well as aliquots of the controls thawed immediately before the analysis as recommended by the manufacturer.
Limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) LoB was determined as the 95th percentile value from n = 60 determinations of five 1,25(OH)2D free serum samples in single determination over six independent series on the LIAISON XL immunoassay analyzer and the API4000TM tandem mass spectrometer, respectively. LoD was determined based on the LoB and the individual standard deviations of five low level 1,25(OH)2D sera. LoD corresponds to the lowest 1,25(OH)2D concentration which can be detected above the LoB with a probability of 95%. The LoQ defined as the lowest amount of 1,25(OH)2D that can be accurately quantitated with an allowable error (square root model) of ≤ 20% was determined with three low level 1,25(OH)2D samples diluted each in four dilution steps with analytefree dilution serum pool to approximately 4–5 ng/L 1,25(OH)2D. One assay run per day over 6 days on both instruments was performed.
Linearity in dilution The dilution linearity of the different assay was evaluated with two different serum samples containing 1,25(OH)2D levels up to 180 ng/L. The samples were diluted using an analyte-free sample diluent. Each dilution series contained nine linear dilution steps (9+1, 8+2, 7+3, 6+4, etc). In each run, measurements of the dilutions were performed in duplicate, the undiluted serum and the analyte-free sample were analyzed in six replicate measurements. The method was considered to be linear, if the measured 1,25(OH)2D values differed by
