https://orcid.org/0000-0002-2472-675X
Abstract
Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results.
125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined.
Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975–1.078), and 1.019 (range 0.963–1.081), and intercepts of −0.584 (−53.736 to 54.790) and −3.194 (−17.152 to 5.933), respectively. The mean bias between methods was 1.2% (−11.6% to 16.0%) for metanephrine and 0.1% (−18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from −32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine.
Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.
Introduction
For initial biochemical screening for pheochromocytomas and paragangliomas (PPGLs), the Endocrine Society guideline recommends measurements of plasma-free or urinary fractionated normetanephrine and metanephrine, together referred to as metanephrines (1). Addition of 3-methoxytyramine, the O-methylated metabolite of dopamine, to the test panel provides additional utility for detection of rare dopamine-producing PPGLs (2). With liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with appropriately established reference intervals and preanalytical precautions, measurements of plasma metabolites offer superior diagnostic accuracy over urine metabolites (3).
Although use of plasma-free metanephrines for diagnosis of PPGLs was established in 1995 (4), effective utilization of this test has been hampered by incorrectly applied preanalytical requirements, inappropriately established reference intervals, and inaccurate analytical methods (5–9). Poorly calibrated immunoassays that underestimate metabolite concentrations by more than 50%, in conjunction with reference intervals set too high, provide examples of how a potentially fatal tumor can be missed by incorrectly established methods (5, 10). The need for safe and effective diagnosis and management of patients with PPGLs calls for standardization, or at least harmonization, of the different laboratory-developed tests and commercial kit methods available for measurements of these metabolites.
External quality assurance (EQA) programs offer one approach for assessing interlaboratory comparability of test results to thereby inform the process of harmonization of laboratory tests and reference intervals (11–16). For immunoassays, this is made inherently difficult by the plethora of different commercial test kits, calibrators, and interferences (17, 18). In one report, harmonization of test results was termed a “mission impossible” (19). Although mass spectrometry-based methods are not immune to assay interferences (20–24), the high analytical specificity of these methods is an enabler of assay harmonization and standardization (25–27). The concept of harmonization should consider the total testing process, including preanalytical (matrix composition, sample collection, and processing), analytical (traceable calibration material, certified reference materials, a higher order reference measurement methods), and postanalytical procedures (units of reporting and reference intervals) (28).
For plasma-free metanephrines and 3-methoxytyramine, neither reference materials nor reference methods exist; therefore, standardization is not yet possible. The first step in such situations, according to a framework for test harmonization (29), is to assess the degree of measurement equivalence. As outlined earlier, participation in EQA programs (11) provides one means for comparing the trueness and comparability of results. However, such programs typically use artificially prepared, noncommutable materials that may not contain potentially interfering analytes inherently present in native patient samples. For example, interference by 3-O-methydopa in measurements of methoxytyramine results in bias in native plasma but not in samples used in EQA programs (23). The present study therefore aimed to establish interlaboratory comparability of LC-MS/MS measurements of plasma-free metanephrines and 3-methoxytyramine in patient-derived samples. The study also compared test interpretations based on the locally employed reference limits used by participating laboratories.
Materials and Methods
Participants and Analytical Methods
Twelve laboratories, including 2 in Germany (Dresden, Wuerzburg), three in Australia (Brisbane, Perth, Sydney), 2 in Switzerland (Lausanne, Zurich), 2 in the United Kingdom (Manchester, Newcastle), 2 in The Netherlands (Groningen, Nijmegen), and another in New Zealand (Auckland), participated in the study. All laboratories employed independently developed analytical settings (Supplemental Table 1). Laboratory-developed tests were used by 11 laboratories and another used a commercially available kit method (Chromsystems, Germany). A registration form was provided to all participants requesting relevant information on methods, including details of sample preparation, respective LC-MS/MS methods, and calibration materials (Supplemental Table 1).
Specimens
The 125 plasma samples used for this study were derived from larger numbers of lithium heparin anticoagulated plasma specimens left over after routine analyses at 4 different participating centers. Specimens were collected between January 2018 to August 2018 after an overnight fast and at least 20 min of supine rest. All samples were from patients undergoing biochemical testing for PPGLs and in whom measurements of plasma metanephrines were available. Due to the limited volume of samples left after these measurements, there was a need to pool specimens from different patients for subsequent aliquoting and distribution to the 12 participating laboratories. For pooling, samples were first grouped according to similar concentrations and relative differences of concentrations for each of the 3 metabolites. Specimens with similar patterns of metabolite concentrations were then thawed and pooled together to achieve specimen volumes of at least 6.5 mL; in that process samples were de-identified to satisfy ethical committee requirements. A set of 125 plasma samples was thereby prepared that covered a range of concentrations from low normal to pathologic, and for the latter, variable increases commonly observed in patients with PPGLs. Each of the 125 samples was then divided into aliquots of a little over 0.5 mL and frozen at -80 °C until distribution to laboratories. The specimen aliquots were distributed (on dry ice) in October 2018 to each participating laboratory and thereafter stored at −80 °C until analysis within 4 months.
Proficiency Testing
Participation in the study required enrollment of laboratories in the international Quality Assurance Program (QAP) of the Royal College of Pathologists of Australasia (RCPA) for plasma-free metanephrines (11). Method agreement within the RCPAQAP was examined according to results from lyophilized human serum samples assayed over the course of 2 6-month cycles of the program during 2018 (12 samples/cycle).
Data Analysis
Results for measurements of plasma metabolites were reported back to the principal investigators, compiled and shared with study participants for subsequent data review. Analysis of the data was carried out at Dresden and Sydney, with additional independent review at Singapore. Statistical analyses utilized the JMP® software package.
Lack of reference methods/reference materials for defining true target values for each measurand necessitated use of median concentrations of all laboratories for each patient sample as consensus target values (all-method medians). The RCPAQAP similarly uses all-participant medians as consensus target values. Analytical agreement between each participating laboratory and the all-laboratory medians for patient samples and all-participant medians for RCPAQAP materials was assessed using Passing-Bablok regression and Bland-Altman analyses.
Agreement in test interpretation was assessed by comparing the number of measurements falling within (nonpathological) and above (pathological) the upper reference limits (URLs) used by each participating laboratory (Supplemental Table 1). Plasma concentrations of normetanephrine increase with age (12). For this analyte, random patient ages were assigned to the study samples by the principal investigators. A result was defined “pathological” as any increase above the respective laboratory-derived URL. Interpretations were reported in conjunction with the analytical data and respective URLs used by the participants for clinical decision-making. Agreement between laboratories in the interpretation of nonpathological versus pathological results was assessed using the Fleiss-kappa inter-observer statistical test.
To evaluate the impact of method harmonization through calibration adjustment (i.e., bottom-up harmonization approach), the measurements of individual laboratories were adjusted to the regression equation derived from the all-method medians. The adjusted measurements were then compared against the original URLs of corresponding laboratories and reinterpreted. To evaluate the additive clinical impact of harmonized reference intervals, the adjusted measurements were further assessed and reinterpreted against URLs established by laboratory A according to clinical validation (3, 12).
The analytical performance specifications (APS) were adopted from the RCPAQAP (30). For normetanephrine, metanephrine, and 3-methoxytyramine, those APS depend on metabolite concentrations, with ranges of ±200 pmol/L, ±75 pmol/L, and ±36 pmol/L for observed concentrations up to 1000 pmol/L, 500 pmol/L, and 120 pmol/L, respectively, and ±20%, ±15%, and ±30% above these concentrations.
Results
Inter-Laboratory Comparisons of Patient Data
Target concentrations for metabolites, defined by medians of the 12 laboratories, ranged from 171 to 37 281 pmol/L, 33 to 9116 pmol/L and 13 to 3612 pmol/L, for normetanephrine, metanephrine, and 3-methoxytyramine, respectively (Fig. 1, Supplemental Table 2). Results for normetanephrine were reported by all 12 laboratories for all 125 samples, whereas the full set of results for metanephrine were reported by 8 laboratories. The shortfall in reported results for metanephrine reflected concentrations below lower limits of quantification (LLOQ) for 19 and 36 samples in respective laboratories D and E, and 3 and 9 samples in laboratories F and M due to other analytical problems Supplemental Table 1). Complete data for 3-methoxytyramine were reported by 5 laboratories (A, I, J, K, and L). For laboratories B, C, D, E F, H, and M, results were reported as below the LLOQ in 83, 90, 90, 91, 5, 6, and 18 respective samples.
Ranges of median concentrations and variations in data reported from measurements of normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) of native samples and external quality assurance (EQA) samples. Variations are shown as relative ratios of interquartile (IQ) ranges to medians.
Variations in reported data (interquartile range (IQR)/median) ranged from 3.4% to 66.1% (median 12.6%, IQR 8.9%–17.8%) for normetanephrine, from 3.5% to 73.5% (median 13.6%, IQR 9.4%–21.3%) for metanephrine, and from 4.9% to 327.8% (median 47.5%, IQR 28.3%–79.9%) for 3-methoxytyramine (Fig. 1). Larger variations in reported data were observed in samples with lower concentrations (Supplemental Fig. 1).
Passing-Bablok regression analyses using the all-method medians as consensus target values revealed strong correlations for normetanephrine (mean R = 0.994; 95% CI 0.986–1.000), metanephrine (mean R = 0.993; 95% CI 0.985–1.000), and 3-methoxytyramine (mean R = 0.975; 95% CI 0.949–1.000) (Table 1, Fig. 2). Estimated slopes of regression lines were 1.013 (95% CI 0.996–1.035) for normetanephrine and 1.019 (95% CI 0.988–1.053) for metanephrine, indicating no significant proportional bias. However, for 3-methoxytyramine, three laboratories (A, B, M) presented with weaker correlations to the all-method medians (R < 0.95). One laboratory (M) showed a particularly high proportional bias indicated by a slope of the regression line of 3.542; this was subsequently clarified to reflect an unknown interference that was corrected after the present data became available.
Comparison of analytical test results of normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) in 125 native patient samples measured by LC-MS/MS in 12 laboratories. A, B, C show regression lines of respective analytes derived from Passing Bablok (PB) regression analyses by applying minimum and maximum “median concentrations of all labs” for curve drawing; D, E, F show regression lines of respective analytes derived from Bland-Altman (BA) analysis of mean concentrations (all-method medians, respective laboratory derived data) and %differences of each laboratory relative to the all-method medians.
Differences in analytical test results for normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) in native samples.
| Passing-Bablok-Regression (Y = m*(all-method median) + b) | Bland-Altman-Analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96 SD | mean −1.96 SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 125 | 1.078 | 1.066 | 1.093 | 44.28 | 33.92 | 58.60 | 0.9982 | 16.0 | 18.5 | 52.3 | −20.3 | −0.004 | 0.0497 | 15.949 | <0.0001 | −0.1769 | <0.0001 |
| Lab B | 125 | 0.988 | 0.974 | 1.002 | −15.67 | −26.74 | −5.68 | 0.9988 | −4.0 | 20.2 | 35.6 | −43.6 | 0.0002 | −4.490 | 0.0247 | 0.0681 | ||
| Lab C | 125 | 0.994 | 0.985 | 1.003 | −5.21 | −10.45 | −0.95 | 0.9997 | −3.1 | 11.0 | 18.4 | −24.5 | 0.0001 | −3.351 | 0.0021 | 0.0642 | 0.0458 | |
| Lab D | 125 | 1.035 | 1.006 | 1.057 | −18.26 | −32.47 | −2.42 | 0.9953 | −0.2 | 9.7 | 18.9 | −19.3 | 0.0003 | −0.830 | 0.1590 | 0.0221 | ||
| Lab E | 121 | 1.074 | 1.053 | 1.086 | −12.57 | −24.24 | −0.62 | 0.9994 | 5.9 | 10.5 | 26.4 | −14.6 | −0.0001 | 6.079 | <0.0001 | −0.0387 | 0.0023 | |
| Lab F | 120 | 1.028 | 1.000 | 1.071 | −20.00 | −43.12 | −4.33 | 0.9504 | −4.8 | 19.4 | 33.4 | −42.9 | −0.0003 | −4.024 | 0.0413 | −0.0836 | 0.0091 | |
| Lab H | 125 | 0.996 | 0.980 | 1.025 | 54.79 | 34.05 | 80.24 | 0.9978 | 13.5 | 19.5 | 51.8 | −24.8 | −0.0010 | 0.0049 | 15.665 | <0.0001 | −0.2530 | 0.0009 |
| Lab I | 125 | 1.021 | 1.007 | 1.063 | 14.18 | −19.19 | 27.87 | 0.9972 | 9.3 | 21.9 | 52.2 | −33.6 | −0.0004 | 10.137 | <0.0001 | −0.0912 | ||
| Lab J | 125 | 0.978 | 0.967 | 0.986 | −3.05 | −9.93 | 3.37 | 0.9995 | −4.5 | 8.3 | 11.6 | −20.7 | 0.0001 | −4.728 | <0.0001 | 0.0512 | ||
| Lab K | 125 | 0.979 | 0.960 | 0.996 | 15.65 | 2.93 | 26.86 | 0.9981 | 0.7 | 12.3 | 24.8 | −23.3 | −0.0002 | 1.180 | −0.0800 | |||
| Lab L | 125 | 0.975 | 0.965 | 0.994 | −7.42 | −21.16 | −1.97 | 0.9980 | −2.8 | 11.3 | 19.3 | −25.0 | 0.0002 | −3.326 | 0.0031 | 0.1038 | 0.0102 | |
| Lab M | 123 | 1.010 | 0.986 | 1.039 | −53.74 | −67.42 | −27.08 | 0.9958 | −11.6 | 22.7 | 33.0 | −56.1 | 0.0006 | −12.891 | <0.0001 | 0.1404 | <0.0001 | |
| MN | ||||||||||||||||||
| Lab A | 125 | 1.081 | 1.054 | 1.131 | 1.18 | −5.13 | 5.57 | 0.9976 | 9.5 | 12.7 | 34.4 | −15.5 | 0.0000 | 9.472 | <0.0001 | 0.0033 | ||
| Lab B | 125 | 0.981 | 0.941 | 1.040 | −6.28 | −15.65 | 2.98 | 0.9450 | −5.0 | 28.0 | 49.8 | −59.9 | 0.0024 | −6.055 | 0.0247 | 0.0995 | 0.0181 | |
| Lab C | 125 | 0.963 | 0.946 | 0.978 | −0.75 | −3.27 | 2.01 | 0.9996 | −2.4 | 13.7 | 24.5 | −29.2 | −0.0002 | −2.303 | −0.0154 | |||
| Lab D | 106 | 0.997 | 0.963 | 1.039 | 0.43 | −5,97 | 6.89 | 0.9977 | 1.6 | 8.8 | 18.9 | −15.6 | −0.0002 | 1.688 | −0.0233 | |||
| Lab E | 89 | 1.022 | 0.986 | 1.067 | 5.93 | −4.46 | 13.83 | 0.9993 | 7.0 | 8.4 | 23.4 | −9.5 | −0.0008 | 7.421 | <0.0001 | −0.1350 | ||
| Lab F | 122 | 0.974 | 0.936 | 1.018 | −17.15 | −25.66 | −12.37 | 0.9987 | −18.0 | 18.7 | 18.7 | −54.7 | 0.0027 | −19.154 | <0.0001 | 0.1710 | <0.0001 | |
| Lab H | 125 | 1.047 | 1.026 | 1.062 | −2.83 | −4.93 | 1.79 | 0.9979 | 5.7 | 13.8 | 32.8 | −21.3 | 0.0001 | 5.674 | <0.0001 | 0.0129 | ||
| Lab I | 125 | 1.008 | 0.970 | 1.046 | 1.98 | −3.94 | 7.63 | 0.9947 | 3.7 | 18.1 | 39.3 | −31.8 | −0.0003 | 3.890 | 0.0264 | −0.0222 | ||
| Lab J | 125 | 0.985 | 0.969 | 0.992 | −2.76 | −4.24 | −0.15 | 0.9998 | −5.7 | 10.9 | 15.6 | −27.0 | 0.0007 | −5.969 | <0.0001 | 0.0759 | ||
| Lab K | 125 | 1.071 | 1.044 | 1.099 | 0.57 | −4.88 | 4.96 | 0.9996 | 7.4 | 13.2 | 33.3 | −18.5 | −0.0004 | 7.559 | <0.0001 | −0.0349 | ||
| Lab L | 125 | 1.045 | 1.009 | 1.069 | −10.35 | −15.79 | −5.03 | 0.9917 | −2.8 | 14.1 | 24.8 | −30.4 | 0.0017 | −3.559 | 0.0080 | 0.1563 | <0.0001 | |
| Lab M | 123 | 1.055 | 1.013 | 1.094 | −8.29 | −14.83 | −1.01 | 0.9981 | −0.3 | 20.7 | 40.4 | −41.0 | 0.0004 | −0.467 | 0.0239 | |||
| MTY | ||||||||||||||||||
| Lab A | 125 | 1.273 | 1.186 | 1.388 | 3.78 | −2.25 | 7.51 | 0.9430 | 32.4 | 31.3 | 93.8 | −29.0 | 0.0037 | 31.660 | <0.0001 | 0.0664 | ||
| Lab B | 42 | 0.842 | 0.711 | 0.956 | −1.34 | −14.69 | 11.80 | 0.9326 | −12.9 | 54.9 | 94.7 | −120.5 | 0.0091 | −16.135 | 0.1156 | |||
| Lab C | 35 | 0.976 | 0.878 | 1.042 | 18.25 | 3.63 | 37.49 | 0.9891 | 27.5 | 45.0 | 115.8 | −60.8 | −0.0115 | 32.957 | 0.0006 | −0.2078 | 0.0064 | |
| Lab D | 35 | 1.013 | 0.991 | 1.054 | −2.74 | −12.68 | 0.81 | 0.9986 | −1.2 | 9.4 | 17.3 | −19.7 | 0.0026 | −2.468 | 0.2227 | |||
| Lab E | 34 | 0.926 | 0.871 | 0.968 | 12.31 | 2.79 | 25.08 | 0.9986 | −1.2 | 18.0 | 34.2 | −36.5 | −0.0042 | 1.190 | −0.1925 | 0.0019 | ||
| Lab F | 120 | 0.977 | 0.958 | 1.007 | −9.79 | −11.44 | −7.63 | 0.9994 | −32.2 | 34.4 | 35.2 | −99.5 | 0.0168 | 0.0135 | −34.876 | <0.0001 | 0.2250 | <0.0001 |
| Lab H | 119 | 1.111 | 1.060 | 1.158 | −10.11 | −14.33 | −7.67 | 0.9981 | −17.2 | 42.3 | 65.8 | −100.1 | 0.0145 | −19.684 | <0.0001 | 0.1661 | <0.0001 | |
| Lab I | 125 | 1.020 | 0.991 | 1.067 | −6.55 | −8.60 | −4.78 | 0.9983 | −13.5 | 40.8 | 66.2 | −93.5 | 0.0091 | −14.930 | 0.0002 | 0.1034 | <0.0001 | |
| Lab J | 125 | 0.988 | 0.968 | 1.018 | −2.13 | −3.93 | −0.20 | 0.9995 | −7.9 | 26.2 | 43.5 | −59.4 | 0.0028 | −8.397 | 0.0010 | 0.0487 | 0.0019 | |
| Lab K | 125 | 1.051 | 1.004 | 1.148 | 13.74 | 9.87 | 17.91 | 0.9967 | 33.0 | 31.6 | 95.0 | −29.0 | −0.0235 | 0.0003 | 36.971 | <0.0001 | −0.3219 | 0.0025 |
| Lab L | 125 | 1.107 | 1.074 | 1.140 | −7.52 | −9.31 | −5.90 | 0.9973 | −11.8 | 24.9 | 36.9 | −60.6 | 0.0148 | 0.0007 | −14.392 | <0.0001 | 0.2997 | <0.0001 |
| Lab M | 109 | 3.542 | 2.126 | 5.724 | −67.71 | −165.5 | −12.64 | 0.8491 | 64.0 | 72.4 | 206.0 | −78.0 | 0.0096 | 61.069 | <0.0001 | 0.0976 | 0.0004 | |
| Passing-Bablok-Regression (Y = m*(all-method median) + b) | Bland-Altman-Analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96 SD | mean −1.96 SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 125 | 1.078 | 1.066 | 1.093 | 44.28 | 33.92 | 58.60 | 0.9982 | 16.0 | 18.5 | 52.3 | −20.3 | −0.004 | 0.0497 | 15.949 | <0.0001 | −0.1769 | <0.0001 |
| Lab B | 125 | 0.988 | 0.974 | 1.002 | −15.67 | −26.74 | −5.68 | 0.9988 | −4.0 | 20.2 | 35.6 | −43.6 | 0.0002 | −4.490 | 0.0247 | 0.0681 | ||
| Lab C | 125 | 0.994 | 0.985 | 1.003 | −5.21 | −10.45 | −0.95 | 0.9997 | −3.1 | 11.0 | 18.4 | −24.5 | 0.0001 | −3.351 | 0.0021 | 0.0642 | 0.0458 | |
| Lab D | 125 | 1.035 | 1.006 | 1.057 | −18.26 | −32.47 | −2.42 | 0.9953 | −0.2 | 9.7 | 18.9 | −19.3 | 0.0003 | −0.830 | 0.1590 | 0.0221 | ||
| Lab E | 121 | 1.074 | 1.053 | 1.086 | −12.57 | −24.24 | −0.62 | 0.9994 | 5.9 | 10.5 | 26.4 | −14.6 | −0.0001 | 6.079 | <0.0001 | −0.0387 | 0.0023 | |
| Lab F | 120 | 1.028 | 1.000 | 1.071 | −20.00 | −43.12 | −4.33 | 0.9504 | −4.8 | 19.4 | 33.4 | −42.9 | −0.0003 | −4.024 | 0.0413 | −0.0836 | 0.0091 | |
| Lab H | 125 | 0.996 | 0.980 | 1.025 | 54.79 | 34.05 | 80.24 | 0.9978 | 13.5 | 19.5 | 51.8 | −24.8 | −0.0010 | 0.0049 | 15.665 | <0.0001 | −0.2530 | 0.0009 |
| Lab I | 125 | 1.021 | 1.007 | 1.063 | 14.18 | −19.19 | 27.87 | 0.9972 | 9.3 | 21.9 | 52.2 | −33.6 | −0.0004 | 10.137 | <0.0001 | −0.0912 | ||
| Lab J | 125 | 0.978 | 0.967 | 0.986 | −3.05 | −9.93 | 3.37 | 0.9995 | −4.5 | 8.3 | 11.6 | −20.7 | 0.0001 | −4.728 | <0.0001 | 0.0512 | ||
| Lab K | 125 | 0.979 | 0.960 | 0.996 | 15.65 | 2.93 | 26.86 | 0.9981 | 0.7 | 12.3 | 24.8 | −23.3 | −0.0002 | 1.180 | −0.0800 | |||
| Lab L | 125 | 0.975 | 0.965 | 0.994 | −7.42 | −21.16 | −1.97 | 0.9980 | −2.8 | 11.3 | 19.3 | −25.0 | 0.0002 | −3.326 | 0.0031 | 0.1038 | 0.0102 | |
| Lab M | 123 | 1.010 | 0.986 | 1.039 | −53.74 | −67.42 | −27.08 | 0.9958 | −11.6 | 22.7 | 33.0 | −56.1 | 0.0006 | −12.891 | <0.0001 | 0.1404 | <0.0001 | |
| MN | ||||||||||||||||||
| Lab A | 125 | 1.081 | 1.054 | 1.131 | 1.18 | −5.13 | 5.57 | 0.9976 | 9.5 | 12.7 | 34.4 | −15.5 | 0.0000 | 9.472 | <0.0001 | 0.0033 | ||
| Lab B | 125 | 0.981 | 0.941 | 1.040 | −6.28 | −15.65 | 2.98 | 0.9450 | −5.0 | 28.0 | 49.8 | −59.9 | 0.0024 | −6.055 | 0.0247 | 0.0995 | 0.0181 | |
| Lab C | 125 | 0.963 | 0.946 | 0.978 | −0.75 | −3.27 | 2.01 | 0.9996 | −2.4 | 13.7 | 24.5 | −29.2 | −0.0002 | −2.303 | −0.0154 | |||
| Lab D | 106 | 0.997 | 0.963 | 1.039 | 0.43 | −5,97 | 6.89 | 0.9977 | 1.6 | 8.8 | 18.9 | −15.6 | −0.0002 | 1.688 | −0.0233 | |||
| Lab E | 89 | 1.022 | 0.986 | 1.067 | 5.93 | −4.46 | 13.83 | 0.9993 | 7.0 | 8.4 | 23.4 | −9.5 | −0.0008 | 7.421 | <0.0001 | −0.1350 | ||
| Lab F | 122 | 0.974 | 0.936 | 1.018 | −17.15 | −25.66 | −12.37 | 0.9987 | −18.0 | 18.7 | 18.7 | −54.7 | 0.0027 | −19.154 | <0.0001 | 0.1710 | <0.0001 | |
| Lab H | 125 | 1.047 | 1.026 | 1.062 | −2.83 | −4.93 | 1.79 | 0.9979 | 5.7 | 13.8 | 32.8 | −21.3 | 0.0001 | 5.674 | <0.0001 | 0.0129 | ||
| Lab I | 125 | 1.008 | 0.970 | 1.046 | 1.98 | −3.94 | 7.63 | 0.9947 | 3.7 | 18.1 | 39.3 | −31.8 | −0.0003 | 3.890 | 0.0264 | −0.0222 | ||
| Lab J | 125 | 0.985 | 0.969 | 0.992 | −2.76 | −4.24 | −0.15 | 0.9998 | −5.7 | 10.9 | 15.6 | −27.0 | 0.0007 | −5.969 | <0.0001 | 0.0759 | ||
| Lab K | 125 | 1.071 | 1.044 | 1.099 | 0.57 | −4.88 | 4.96 | 0.9996 | 7.4 | 13.2 | 33.3 | −18.5 | −0.0004 | 7.559 | <0.0001 | −0.0349 | ||
| Lab L | 125 | 1.045 | 1.009 | 1.069 | −10.35 | −15.79 | −5.03 | 0.9917 | −2.8 | 14.1 | 24.8 | −30.4 | 0.0017 | −3.559 | 0.0080 | 0.1563 | <0.0001 | |
| Lab M | 123 | 1.055 | 1.013 | 1.094 | −8.29 | −14.83 | −1.01 | 0.9981 | −0.3 | 20.7 | 40.4 | −41.0 | 0.0004 | −0.467 | 0.0239 | |||
| MTY | ||||||||||||||||||
| Lab A | 125 | 1.273 | 1.186 | 1.388 | 3.78 | −2.25 | 7.51 | 0.9430 | 32.4 | 31.3 | 93.8 | −29.0 | 0.0037 | 31.660 | <0.0001 | 0.0664 | ||
| Lab B | 42 | 0.842 | 0.711 | 0.956 | −1.34 | −14.69 | 11.80 | 0.9326 | −12.9 | 54.9 | 94.7 | −120.5 | 0.0091 | −16.135 | 0.1156 | |||
| Lab C | 35 | 0.976 | 0.878 | 1.042 | 18.25 | 3.63 | 37.49 | 0.9891 | 27.5 | 45.0 | 115.8 | −60.8 | −0.0115 | 32.957 | 0.0006 | −0.2078 | 0.0064 | |
| Lab D | 35 | 1.013 | 0.991 | 1.054 | −2.74 | −12.68 | 0.81 | 0.9986 | −1.2 | 9.4 | 17.3 | −19.7 | 0.0026 | −2.468 | 0.2227 | |||
| Lab E | 34 | 0.926 | 0.871 | 0.968 | 12.31 | 2.79 | 25.08 | 0.9986 | −1.2 | 18.0 | 34.2 | −36.5 | −0.0042 | 1.190 | −0.1925 | 0.0019 | ||
| Lab F | 120 | 0.977 | 0.958 | 1.007 | −9.79 | −11.44 | −7.63 | 0.9994 | −32.2 | 34.4 | 35.2 | −99.5 | 0.0168 | 0.0135 | −34.876 | <0.0001 | 0.2250 | <0.0001 |
| Lab H | 119 | 1.111 | 1.060 | 1.158 | −10.11 | −14.33 | −7.67 | 0.9981 | −17.2 | 42.3 | 65.8 | −100.1 | 0.0145 | −19.684 | <0.0001 | 0.1661 | <0.0001 | |
| Lab I | 125 | 1.020 | 0.991 | 1.067 | −6.55 | −8.60 | −4.78 | 0.9983 | −13.5 | 40.8 | 66.2 | −93.5 | 0.0091 | −14.930 | 0.0002 | 0.1034 | <0.0001 | |
| Lab J | 125 | 0.988 | 0.968 | 1.018 | −2.13 | −3.93 | −0.20 | 0.9995 | −7.9 | 26.2 | 43.5 | −59.4 | 0.0028 | −8.397 | 0.0010 | 0.0487 | 0.0019 | |
| Lab K | 125 | 1.051 | 1.004 | 1.148 | 13.74 | 9.87 | 17.91 | 0.9967 | 33.0 | 31.6 | 95.0 | −29.0 | −0.0235 | 0.0003 | 36.971 | <0.0001 | −0.3219 | 0.0025 |
| Lab L | 125 | 1.107 | 1.074 | 1.140 | −7.52 | −9.31 | −5.90 | 0.9973 | −11.8 | 24.9 | 36.9 | −60.6 | 0.0148 | 0.0007 | −14.392 | <0.0001 | 0.2997 | <0.0001 |
| Lab M | 109 | 3.542 | 2.126 | 5.724 | −67.71 | −165.5 | −12.64 | 0.8491 | 64.0 | 72.4 | 206.0 | −78.0 | 0.0096 | 61.069 | <0.0001 | 0.0976 | 0.0004 | |
Missing reported results observed in respective samples are majorly caused by measurements <lower limits of quantification, or failures during the sample extraction (CL, confidence limit; SD, standard deviation).
Differences in analytical test results for normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) in native samples.
| Passing-Bablok-Regression (Y = m*(all-method median) + b) | Bland-Altman-Analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96 SD | mean −1.96 SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 125 | 1.078 | 1.066 | 1.093 | 44.28 | 33.92 | 58.60 | 0.9982 | 16.0 | 18.5 | 52.3 | −20.3 | −0.004 | 0.0497 | 15.949 | <0.0001 | −0.1769 | <0.0001 |
| Lab B | 125 | 0.988 | 0.974 | 1.002 | −15.67 | −26.74 | −5.68 | 0.9988 | −4.0 | 20.2 | 35.6 | −43.6 | 0.0002 | −4.490 | 0.0247 | 0.0681 | ||
| Lab C | 125 | 0.994 | 0.985 | 1.003 | −5.21 | −10.45 | −0.95 | 0.9997 | −3.1 | 11.0 | 18.4 | −24.5 | 0.0001 | −3.351 | 0.0021 | 0.0642 | 0.0458 | |
| Lab D | 125 | 1.035 | 1.006 | 1.057 | −18.26 | −32.47 | −2.42 | 0.9953 | −0.2 | 9.7 | 18.9 | −19.3 | 0.0003 | −0.830 | 0.1590 | 0.0221 | ||
| Lab E | 121 | 1.074 | 1.053 | 1.086 | −12.57 | −24.24 | −0.62 | 0.9994 | 5.9 | 10.5 | 26.4 | −14.6 | −0.0001 | 6.079 | <0.0001 | −0.0387 | 0.0023 | |
| Lab F | 120 | 1.028 | 1.000 | 1.071 | −20.00 | −43.12 | −4.33 | 0.9504 | −4.8 | 19.4 | 33.4 | −42.9 | −0.0003 | −4.024 | 0.0413 | −0.0836 | 0.0091 | |
| Lab H | 125 | 0.996 | 0.980 | 1.025 | 54.79 | 34.05 | 80.24 | 0.9978 | 13.5 | 19.5 | 51.8 | −24.8 | −0.0010 | 0.0049 | 15.665 | <0.0001 | −0.2530 | 0.0009 |
| Lab I | 125 | 1.021 | 1.007 | 1.063 | 14.18 | −19.19 | 27.87 | 0.9972 | 9.3 | 21.9 | 52.2 | −33.6 | −0.0004 | 10.137 | <0.0001 | −0.0912 | ||
| Lab J | 125 | 0.978 | 0.967 | 0.986 | −3.05 | −9.93 | 3.37 | 0.9995 | −4.5 | 8.3 | 11.6 | −20.7 | 0.0001 | −4.728 | <0.0001 | 0.0512 | ||
| Lab K | 125 | 0.979 | 0.960 | 0.996 | 15.65 | 2.93 | 26.86 | 0.9981 | 0.7 | 12.3 | 24.8 | −23.3 | −0.0002 | 1.180 | −0.0800 | |||
| Lab L | 125 | 0.975 | 0.965 | 0.994 | −7.42 | −21.16 | −1.97 | 0.9980 | −2.8 | 11.3 | 19.3 | −25.0 | 0.0002 | −3.326 | 0.0031 | 0.1038 | 0.0102 | |
| Lab M | 123 | 1.010 | 0.986 | 1.039 | −53.74 | −67.42 | −27.08 | 0.9958 | −11.6 | 22.7 | 33.0 | −56.1 | 0.0006 | −12.891 | <0.0001 | 0.1404 | <0.0001 | |
| MN | ||||||||||||||||||
| Lab A | 125 | 1.081 | 1.054 | 1.131 | 1.18 | −5.13 | 5.57 | 0.9976 | 9.5 | 12.7 | 34.4 | −15.5 | 0.0000 | 9.472 | <0.0001 | 0.0033 | ||
| Lab B | 125 | 0.981 | 0.941 | 1.040 | −6.28 | −15.65 | 2.98 | 0.9450 | −5.0 | 28.0 | 49.8 | −59.9 | 0.0024 | −6.055 | 0.0247 | 0.0995 | 0.0181 | |
| Lab C | 125 | 0.963 | 0.946 | 0.978 | −0.75 | −3.27 | 2.01 | 0.9996 | −2.4 | 13.7 | 24.5 | −29.2 | −0.0002 | −2.303 | −0.0154 | |||
| Lab D | 106 | 0.997 | 0.963 | 1.039 | 0.43 | −5,97 | 6.89 | 0.9977 | 1.6 | 8.8 | 18.9 | −15.6 | −0.0002 | 1.688 | −0.0233 | |||
| Lab E | 89 | 1.022 | 0.986 | 1.067 | 5.93 | −4.46 | 13.83 | 0.9993 | 7.0 | 8.4 | 23.4 | −9.5 | −0.0008 | 7.421 | <0.0001 | −0.1350 | ||
| Lab F | 122 | 0.974 | 0.936 | 1.018 | −17.15 | −25.66 | −12.37 | 0.9987 | −18.0 | 18.7 | 18.7 | −54.7 | 0.0027 | −19.154 | <0.0001 | 0.1710 | <0.0001 | |
| Lab H | 125 | 1.047 | 1.026 | 1.062 | −2.83 | −4.93 | 1.79 | 0.9979 | 5.7 | 13.8 | 32.8 | −21.3 | 0.0001 | 5.674 | <0.0001 | 0.0129 | ||
| Lab I | 125 | 1.008 | 0.970 | 1.046 | 1.98 | −3.94 | 7.63 | 0.9947 | 3.7 | 18.1 | 39.3 | −31.8 | −0.0003 | 3.890 | 0.0264 | −0.0222 | ||
| Lab J | 125 | 0.985 | 0.969 | 0.992 | −2.76 | −4.24 | −0.15 | 0.9998 | −5.7 | 10.9 | 15.6 | −27.0 | 0.0007 | −5.969 | <0.0001 | 0.0759 | ||
| Lab K | 125 | 1.071 | 1.044 | 1.099 | 0.57 | −4.88 | 4.96 | 0.9996 | 7.4 | 13.2 | 33.3 | −18.5 | −0.0004 | 7.559 | <0.0001 | −0.0349 | ||
| Lab L | 125 | 1.045 | 1.009 | 1.069 | −10.35 | −15.79 | −5.03 | 0.9917 | −2.8 | 14.1 | 24.8 | −30.4 | 0.0017 | −3.559 | 0.0080 | 0.1563 | <0.0001 | |
| Lab M | 123 | 1.055 | 1.013 | 1.094 | −8.29 | −14.83 | −1.01 | 0.9981 | −0.3 | 20.7 | 40.4 | −41.0 | 0.0004 | −0.467 | 0.0239 | |||
| MTY | ||||||||||||||||||
| Lab A | 125 | 1.273 | 1.186 | 1.388 | 3.78 | −2.25 | 7.51 | 0.9430 | 32.4 | 31.3 | 93.8 | −29.0 | 0.0037 | 31.660 | <0.0001 | 0.0664 | ||
| Lab B | 42 | 0.842 | 0.711 | 0.956 | −1.34 | −14.69 | 11.80 | 0.9326 | −12.9 | 54.9 | 94.7 | −120.5 | 0.0091 | −16.135 | 0.1156 | |||
| Lab C | 35 | 0.976 | 0.878 | 1.042 | 18.25 | 3.63 | 37.49 | 0.9891 | 27.5 | 45.0 | 115.8 | −60.8 | −0.0115 | 32.957 | 0.0006 | −0.2078 | 0.0064 | |
| Lab D | 35 | 1.013 | 0.991 | 1.054 | −2.74 | −12.68 | 0.81 | 0.9986 | −1.2 | 9.4 | 17.3 | −19.7 | 0.0026 | −2.468 | 0.2227 | |||
| Lab E | 34 | 0.926 | 0.871 | 0.968 | 12.31 | 2.79 | 25.08 | 0.9986 | −1.2 | 18.0 | 34.2 | −36.5 | −0.0042 | 1.190 | −0.1925 | 0.0019 | ||
| Lab F | 120 | 0.977 | 0.958 | 1.007 | −9.79 | −11.44 | −7.63 | 0.9994 | −32.2 | 34.4 | 35.2 | −99.5 | 0.0168 | 0.0135 | −34.876 | <0.0001 | 0.2250 | <0.0001 |
| Lab H | 119 | 1.111 | 1.060 | 1.158 | −10.11 | −14.33 | −7.67 | 0.9981 | −17.2 | 42.3 | 65.8 | −100.1 | 0.0145 | −19.684 | <0.0001 | 0.1661 | <0.0001 | |
| Lab I | 125 | 1.020 | 0.991 | 1.067 | −6.55 | −8.60 | −4.78 | 0.9983 | −13.5 | 40.8 | 66.2 | −93.5 | 0.0091 | −14.930 | 0.0002 | 0.1034 | <0.0001 | |
| Lab J | 125 | 0.988 | 0.968 | 1.018 | −2.13 | −3.93 | −0.20 | 0.9995 | −7.9 | 26.2 | 43.5 | −59.4 | 0.0028 | −8.397 | 0.0010 | 0.0487 | 0.0019 | |
| Lab K | 125 | 1.051 | 1.004 | 1.148 | 13.74 | 9.87 | 17.91 | 0.9967 | 33.0 | 31.6 | 95.0 | −29.0 | −0.0235 | 0.0003 | 36.971 | <0.0001 | −0.3219 | 0.0025 |
| Lab L | 125 | 1.107 | 1.074 | 1.140 | −7.52 | −9.31 | −5.90 | 0.9973 | −11.8 | 24.9 | 36.9 | −60.6 | 0.0148 | 0.0007 | −14.392 | <0.0001 | 0.2997 | <0.0001 |
| Lab M | 109 | 3.542 | 2.126 | 5.724 | −67.71 | −165.5 | −12.64 | 0.8491 | 64.0 | 72.4 | 206.0 | −78.0 | 0.0096 | 61.069 | <0.0001 | 0.0976 | 0.0004 | |
| Passing-Bablok-Regression (Y = m*(all-method median) + b) | Bland-Altman-Analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96 SD | mean −1.96 SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 125 | 1.078 | 1.066 | 1.093 | 44.28 | 33.92 | 58.60 | 0.9982 | 16.0 | 18.5 | 52.3 | −20.3 | −0.004 | 0.0497 | 15.949 | <0.0001 | −0.1769 | <0.0001 |
| Lab B | 125 | 0.988 | 0.974 | 1.002 | −15.67 | −26.74 | −5.68 | 0.9988 | −4.0 | 20.2 | 35.6 | −43.6 | 0.0002 | −4.490 | 0.0247 | 0.0681 | ||
| Lab C | 125 | 0.994 | 0.985 | 1.003 | −5.21 | −10.45 | −0.95 | 0.9997 | −3.1 | 11.0 | 18.4 | −24.5 | 0.0001 | −3.351 | 0.0021 | 0.0642 | 0.0458 | |
| Lab D | 125 | 1.035 | 1.006 | 1.057 | −18.26 | −32.47 | −2.42 | 0.9953 | −0.2 | 9.7 | 18.9 | −19.3 | 0.0003 | −0.830 | 0.1590 | 0.0221 | ||
| Lab E | 121 | 1.074 | 1.053 | 1.086 | −12.57 | −24.24 | −0.62 | 0.9994 | 5.9 | 10.5 | 26.4 | −14.6 | −0.0001 | 6.079 | <0.0001 | −0.0387 | 0.0023 | |
| Lab F | 120 | 1.028 | 1.000 | 1.071 | −20.00 | −43.12 | −4.33 | 0.9504 | −4.8 | 19.4 | 33.4 | −42.9 | −0.0003 | −4.024 | 0.0413 | −0.0836 | 0.0091 | |
| Lab H | 125 | 0.996 | 0.980 | 1.025 | 54.79 | 34.05 | 80.24 | 0.9978 | 13.5 | 19.5 | 51.8 | −24.8 | −0.0010 | 0.0049 | 15.665 | <0.0001 | −0.2530 | 0.0009 |
| Lab I | 125 | 1.021 | 1.007 | 1.063 | 14.18 | −19.19 | 27.87 | 0.9972 | 9.3 | 21.9 | 52.2 | −33.6 | −0.0004 | 10.137 | <0.0001 | −0.0912 | ||
| Lab J | 125 | 0.978 | 0.967 | 0.986 | −3.05 | −9.93 | 3.37 | 0.9995 | −4.5 | 8.3 | 11.6 | −20.7 | 0.0001 | −4.728 | <0.0001 | 0.0512 | ||
| Lab K | 125 | 0.979 | 0.960 | 0.996 | 15.65 | 2.93 | 26.86 | 0.9981 | 0.7 | 12.3 | 24.8 | −23.3 | −0.0002 | 1.180 | −0.0800 | |||
| Lab L | 125 | 0.975 | 0.965 | 0.994 | −7.42 | −21.16 | −1.97 | 0.9980 | −2.8 | 11.3 | 19.3 | −25.0 | 0.0002 | −3.326 | 0.0031 | 0.1038 | 0.0102 | |
| Lab M | 123 | 1.010 | 0.986 | 1.039 | −53.74 | −67.42 | −27.08 | 0.9958 | −11.6 | 22.7 | 33.0 | −56.1 | 0.0006 | −12.891 | <0.0001 | 0.1404 | <0.0001 | |
| MN | ||||||||||||||||||
| Lab A | 125 | 1.081 | 1.054 | 1.131 | 1.18 | −5.13 | 5.57 | 0.9976 | 9.5 | 12.7 | 34.4 | −15.5 | 0.0000 | 9.472 | <0.0001 | 0.0033 | ||
| Lab B | 125 | 0.981 | 0.941 | 1.040 | −6.28 | −15.65 | 2.98 | 0.9450 | −5.0 | 28.0 | 49.8 | −59.9 | 0.0024 | −6.055 | 0.0247 | 0.0995 | 0.0181 | |
| Lab C | 125 | 0.963 | 0.946 | 0.978 | −0.75 | −3.27 | 2.01 | 0.9996 | −2.4 | 13.7 | 24.5 | −29.2 | −0.0002 | −2.303 | −0.0154 | |||
| Lab D | 106 | 0.997 | 0.963 | 1.039 | 0.43 | −5,97 | 6.89 | 0.9977 | 1.6 | 8.8 | 18.9 | −15.6 | −0.0002 | 1.688 | −0.0233 | |||
| Lab E | 89 | 1.022 | 0.986 | 1.067 | 5.93 | −4.46 | 13.83 | 0.9993 | 7.0 | 8.4 | 23.4 | −9.5 | −0.0008 | 7.421 | <0.0001 | −0.1350 | ||
| Lab F | 122 | 0.974 | 0.936 | 1.018 | −17.15 | −25.66 | −12.37 | 0.9987 | −18.0 | 18.7 | 18.7 | −54.7 | 0.0027 | −19.154 | <0.0001 | 0.1710 | <0.0001 | |
| Lab H | 125 | 1.047 | 1.026 | 1.062 | −2.83 | −4.93 | 1.79 | 0.9979 | 5.7 | 13.8 | 32.8 | −21.3 | 0.0001 | 5.674 | <0.0001 | 0.0129 | ||
| Lab I | 125 | 1.008 | 0.970 | 1.046 | 1.98 | −3.94 | 7.63 | 0.9947 | 3.7 | 18.1 | 39.3 | −31.8 | −0.0003 | 3.890 | 0.0264 | −0.0222 | ||
| Lab J | 125 | 0.985 | 0.969 | 0.992 | −2.76 | −4.24 | −0.15 | 0.9998 | −5.7 | 10.9 | 15.6 | −27.0 | 0.0007 | −5.969 | <0.0001 | 0.0759 | ||
| Lab K | 125 | 1.071 | 1.044 | 1.099 | 0.57 | −4.88 | 4.96 | 0.9996 | 7.4 | 13.2 | 33.3 | −18.5 | −0.0004 | 7.559 | <0.0001 | −0.0349 | ||
| Lab L | 125 | 1.045 | 1.009 | 1.069 | −10.35 | −15.79 | −5.03 | 0.9917 | −2.8 | 14.1 | 24.8 | −30.4 | 0.0017 | −3.559 | 0.0080 | 0.1563 | <0.0001 | |
| Lab M | 123 | 1.055 | 1.013 | 1.094 | −8.29 | −14.83 | −1.01 | 0.9981 | −0.3 | 20.7 | 40.4 | −41.0 | 0.0004 | −0.467 | 0.0239 | |||
| MTY | ||||||||||||||||||
| Lab A | 125 | 1.273 | 1.186 | 1.388 | 3.78 | −2.25 | 7.51 | 0.9430 | 32.4 | 31.3 | 93.8 | −29.0 | 0.0037 | 31.660 | <0.0001 | 0.0664 | ||
| Lab B | 42 | 0.842 | 0.711 | 0.956 | −1.34 | −14.69 | 11.80 | 0.9326 | −12.9 | 54.9 | 94.7 | −120.5 | 0.0091 | −16.135 | 0.1156 | |||
| Lab C | 35 | 0.976 | 0.878 | 1.042 | 18.25 | 3.63 | 37.49 | 0.9891 | 27.5 | 45.0 | 115.8 | −60.8 | −0.0115 | 32.957 | 0.0006 | −0.2078 | 0.0064 | |
| Lab D | 35 | 1.013 | 0.991 | 1.054 | −2.74 | −12.68 | 0.81 | 0.9986 | −1.2 | 9.4 | 17.3 | −19.7 | 0.0026 | −2.468 | 0.2227 | |||
| Lab E | 34 | 0.926 | 0.871 | 0.968 | 12.31 | 2.79 | 25.08 | 0.9986 | −1.2 | 18.0 | 34.2 | −36.5 | −0.0042 | 1.190 | −0.1925 | 0.0019 | ||
| Lab F | 120 | 0.977 | 0.958 | 1.007 | −9.79 | −11.44 | −7.63 | 0.9994 | −32.2 | 34.4 | 35.2 | −99.5 | 0.0168 | 0.0135 | −34.876 | <0.0001 | 0.2250 | <0.0001 |
| Lab H | 119 | 1.111 | 1.060 | 1.158 | −10.11 | −14.33 | −7.67 | 0.9981 | −17.2 | 42.3 | 65.8 | −100.1 | 0.0145 | −19.684 | <0.0001 | 0.1661 | <0.0001 | |
| Lab I | 125 | 1.020 | 0.991 | 1.067 | −6.55 | −8.60 | −4.78 | 0.9983 | −13.5 | 40.8 | 66.2 | −93.5 | 0.0091 | −14.930 | 0.0002 | 0.1034 | <0.0001 | |
| Lab J | 125 | 0.988 | 0.968 | 1.018 | −2.13 | −3.93 | −0.20 | 0.9995 | −7.9 | 26.2 | 43.5 | −59.4 | 0.0028 | −8.397 | 0.0010 | 0.0487 | 0.0019 | |
| Lab K | 125 | 1.051 | 1.004 | 1.148 | 13.74 | 9.87 | 17.91 | 0.9967 | 33.0 | 31.6 | 95.0 | −29.0 | −0.0235 | 0.0003 | 36.971 | <0.0001 | −0.3219 | 0.0025 |
| Lab L | 125 | 1.107 | 1.074 | 1.140 | −7.52 | −9.31 | −5.90 | 0.9973 | −11.8 | 24.9 | 36.9 | −60.6 | 0.0148 | 0.0007 | −14.392 | <0.0001 | 0.2997 | <0.0001 |
| Lab M | 109 | 3.542 | 2.126 | 5.724 | −67.71 | −165.5 | −12.64 | 0.8491 | 64.0 | 72.4 | 206.0 | −78.0 | 0.0096 | 61.069 | <0.0001 | 0.0976 | 0.0004 | |
Missing reported results observed in respective samples are majorly caused by measurements <lower limits of quantification, or failures during the sample extraction (CL, confidence limit; SD, standard deviation).
Bland-Altman analyses of results from individual laboratories for normetanephrine compared to the all-methods median (Table 1) showed a mean difference less than that for the RCPAQAP APS of ±20% for all laboratories. For metanephrine, all laboratories met the RCPAQAP APS of ±15% except for Laboratory F (mean difference = −18%). For 3-methoxytyramine, most laboratories met the RCPAQAP APS requirements of ±30% except for laboratories A (32%), F (−32%), K (33%), and M (64%) (Table 1, Fig. 2).
Inter-Laboratory Comparison of Proficiency Testing Derived Data
Median concentrations of metanephrines measured in RCPAQAP samples by the 12 laboratories ranged from 399 to 2562 pmol/L for normetanephrine and 228 to 1374 pmol/L for metanephrine (Fig. 1, Supplemental Table 2). For 3-methoxytyramine, median concentrations derived from the 11 laboratories that reported these measurements ranged from 122 to 1205 pmol/L. Variations in reported RCPAQAP data ranged from 2.8% to 16.6% (median 7.6%, IQR 6.6%–10.4%) for normetanephrine, from 2.0% to 13.2% (median 6.4%, IQR 5.5%–8.5%) for metanephrine, and from 6.2% to 15.6% (median 11.0%, IQR 8.3%–12.7%) for 3-methoxytyramine (Fig. 1). In contrast to results for patient samples, there were no differences in variation according to the target concentration ranges (online Supplemental Fig. 1).
Passing-Bablok regression analyses in RCPAQAP samples revealed excellent correlations (R = 0.993; R = 0.994; R = 0.990) for all 3 catecholamine metabolites with observed slopes of 1.018 (95% CI 0.958–1.093), 1.015 (95% CI 0.969–1.070), and 1.001 (95% CI 0.963–1.087) for normetanephrine, metanephrine, and 3-methoxytyramine, respectively (Fig. 3, Table 2). Bland-Altman analyses revealed mean observed biases for the respective metabolites of 1.2% (range −7.9% to 12.1%), −0.8% (range −7.2% to 6.6%) and 1.7% (range −7.4% to 13.6%) in comparison to target values (Fig. 3, Table 2). Therefore, all laboratories met RCPAQAP APS requirements for measurements of all 3 metabolites (Supplemental Fig. 2).
Differences in analytical test results for normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) in samples derived from an external quality assurance scheme
| Passing-Bablok-regression (Y = m*(all-method median) + b) | Bland-Altman-analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96SD | mean -1.96SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 24 | 0.990 | 0.958 | 1.013 | 13.57 | −16.93 | 58.73 | 0.9985 | 0.1 | 2.9 | 5.8 | −5.6 | −0.0008 | 1.364 | −0.2111 | |||
| Lab B | 22 | 1.022 | 0.983 | 1.070 | −74.35 | −151.81 | −29.63 | 0.9940 | −5.7 | 6.4 | 6.8 | −18.2 | 0.0050 | 0.0087 | −12.889 | 0.0001 | 0.5452 | 0.0039 |
| Lab C | 22 | 1.038 | 0.999 | 1.100 | −21.02 | −83.38 | 11.62 | 0.9963 | 1.6 | 4.3 | 10.0 | −6.8 | 0.0019 | −1.274 | 0.3247 | |||
| Lab D | 24 | 1.028 | 0.991 | 1.074 | −20.00 | −77.91 | 19.15 | 0.9957 | 1.7 | 4.1 | 9.6 | −6.3 | 0.0009 | 0.344 | 0.1678 | |||
| Lab E | 12 | 1.153 | 1.074 | 1.227 | −24.62 | −129.86 | 51.09 | 0.9958 | 12.1 | 4.1 | 20.2 | 4.1 | 0.0011 | 10.412 | 0.0049 | 0.2192 | ||
| Lab F | 24 | 1.096 | 0.951 | 1.335 | −44.30 | −337.00 | 90.36 | 0.9929 | −2.1 | 16.8 | 30.9 | −35.0 | 0.0000 | 2.472 | 0.0040 | |||
| Lab H | 24 | 0.936 | 0.885 | 0.994 | −12.30 | −75.55 | 48.90 | 0.9931 | −7.9 | 6.2 | 4.2 | −20.0 | −0.0007 | −6.866 | 0.0272 | −0.0843 | ||
| Lab I | 24 | 1.048 | 0.973 | 1.171 | −85.04 | −265.58 | 18.75 | 0.9834 | −2.3 | 10.6 | 18.6 | −23.1 | 0.0026 | −6.044 | 0.1855 | |||
| Lab J | 24 | 0.967 | 0.886 | 1.020 | −24.73 | −69.56 | 42.17 | 0.9861 | −6.2 | 7.0 | 7.6 | −20.0 | −0.0004 | −5.581 | −0.0419 | |||
| Lab K | 24 | 0.984 | 0.942 | 1.045 | 11.52 | −44.69 | 74.03 | 0.9944 | 0.3 | 4.8 | 9.6 | −9.1 | −0.0015 | 2.400 | −0.2216 | |||
| Lab L | 24 | 0.970 | 0.948 | 0.991 | −12.62 | −33.72 | 11.79 | 0.9990 | −4.1 | 2.6 | 1.0 | −9.2 | 0.0009 | −5.391 | 0.0002 | 0.2498 | ||
| Lab M | 24 | 0.985 | 0.902 | 1.072 | −6.80 | −98.93 | 6.94 | 0.9832 | −1.4 | 9.9 | 18.0 | −20.8 | −0.0024 | 2.069 | −0.1741 | |||
| MN | ||||||||||||||||||
| Lab A | 24 | 0.932 | 0.893 | 0.963 | 10.69 | −11.53 | 49.47 | 0.9955 | −5.3 | 4.4 | 3.3 | −13.8 | −0.0046 | −1.765 | −0.3859 | |||
| Lab B | 22 | 1.009 | 0.981 | 1.078 | −12.36 | −43.88 | 6.02 | 0.9956 | −0.7 | 4.2 | 7.6 | −9.0 | 0.0035 | −3.465 | 0.3053 | |||
| Lab C | 22 | 1.055 | 0.966 | 1.129 | −9.87 | −72.28 | 37.80 | 0.9862 | 2.2 | 8.3 | 18.4 | −14.1 | −0.0011 | 3.029 | −0.0480 | |||
| Lab D | 24 | 1.011 | 0.966 | 1.079 | −17.95 | −60.38 | 7.89 | 0.9925 | −1.1 | 5.5 | 9.7 | −11.9 | 0.0029 | −3.381 | 0.2048 | |||
| Lab E | 12 | 1.106 | 1.059 | 1.167 | −12.26 | −70.08 | 8.03 | 0.9985 | 6.6 | 3.3 | 13.1 | 0.1 | 0.0033 | 3.930 | 0.4134 | |||
| Lab F | 24 | 1.033 | 1.003 | 1.067 | −36.95 | −48.97 | −8.18 | 0.9977 | −2.0 | 5.0 | 7.8 | −11.7 | 0.0071 | 0.0047 | −7.526 | 0.0009 | 0.5565 | 0.0053 |
| Lab H | 24 | 1.012 | 0.978 | 1.043 | −10.79 | −33.97 | 12.44 | 0.9963 | −0.6 | 5.1 | 9.4 | −10.6 | −0.0002 | −0.473 | −0.0130 | |||
| Lab I | 24 | 1.022 | 0.965 | 1.078 | −10.61 | −46.66 | 19.12 | 0.9937 | −0.3 | 6.1 | 11.8 | −12.3 | 0.0027 | −2.423 | 0.1691 | |||
| Lab J | 24 | 1.000 | 0.947 | 1.050 | −35.80 | −69.42 | −4.74 | 0.9928 | −7.2 | 6.7 | 6.0 | −20.4 | 0.0073 | 0.0453 | −12.715 | 0.0002 | 0.4122 | 0.0285 |
| Lab K | 24 | 1.041 | 1.001 | 1.094 | −6.75 | −51.09 | 14.99 | 0.9955 | 2.7 | 5.1 | 12.8 | −7.4 | 0.0009 | 1.969 | 0.0697 | |||
| Lab L | 24 | 0.981 | 0.951 | 1.019 | −3.74 | −36.71 | 20.16 | 0.9968 | −2.5 | 5.2 | 7.8 | −12.7 | −0.0002 | −2.332 | −0.0145 | |||
| Lab M | 24 | 0.975 | 0.921 | 1.066 | 6.75 | −53.10 | 44.99 | 0.9871 | −1.8 | 7.7 | 13.4 | −16.9 | −0.0030 | 0.563 | −0.1465 | |||
| MTY | ||||||||||||||||||
| Lab A | 24 | 0.999 | 0.974 | 1.022 | 6.61 | −6.03 | 21.25 | 0.9977 | 2.0 | 4.7 | 11.2 | −7.1 | −0.0049 | 5.304 | 0.0098 | −0.3880 | ||
| Lab B | 22 | 0.955 | 0.909 | 1.011 | −8.23 | −39.34 | 20.41 | 0.9960 | −4.5 | 5.8 | 6.9 | −16.0 | −0.0024 | −3.043 | −0.1391 | |||
| Lab C | 22 | 1.090 | 1.024 | 1.153 | −14.66 | −54.58 | 28.26 | 0.9924 | 4.9 | 5.9 | 16.5 | −6.6 | 0.0034 | 2.637 | 0.2098 | |||
| Lab D | 24 | 1.054 | 0.993 | 1.107 | −7.27 | −35.83 | 11.20 | 0.9950 | 3.1 | 4.7 | 12.3 | −6.1 | 0.0021 | 1.733 | 0.1648 | |||
| Lab E | 12 | 1.098 | 1.048 | 1.183 | −5.49 | −68.32 | 21.98 | 0.9972 | 6.9 | 6.6 | 19.7 | −6.0 | 0.0033 | 4.571 | 0.2009 | |||
| Lab F | 24 | 1.076 | 1.046 | 1.110 | −5.18 | −23.23 | 10.99 | 0.9978 | 5.1 | 5.2 | 15.3 | −5.0 | 0.0052 | 1.634 | 0.3814 | |||
| Lab H | 24 | 0.961 | 0.889 | 1.029 | −8.05 | −34.32 | 24.56 | 0.9874 | −7.4 | 8.4 | 9.1 | −23.8 | 0.0046 | −10.278 | 0.0085 | 0.1953 | ||
| Lab I | 24 | 0.941 | 0.867 | 1.043 | −2.45 | −55.58 | 27.26 | 0.9738 | −6.2 | 10.2 | 13.8 | −26.2 | 0.0048 | −9.230 | 0.0429 | 0.1721 | ||
| Lab J | ||||||||||||||||||
| Lab K | 23 | 1.054 | 0.983 | 1.129 | 10.83 | −37.82 | 60.64 | 0.9915 | 7.8 | 7.7 | 22.8 | −7.3 | −0.0041 | 10.650 | 0.0066 | 0.1961 | ||
| Lab L | 24 | 0.944 | 0.924 | 0.969 | −1.16 | −14.54 | 7.21 | 0.9975 | −6.4 | 4.4 | 2.3 | −15.0 | 0.0038 | −8.797 | <0.0001 | 0.3129 | ||
| Lab M | 24 | 1.070 | 0.933 | 1.206 | 34.10 | −19.85 | 99.48 | 0.9678 | 13.6 | 15.7 | 44.3 | −17.1 | −0.0174 | 0.0457 | 25.603 | 0.0006 | −0.4116 | |
| Passing-Bablok-regression (Y = m*(all-method median) + b) | Bland-Altman-analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96SD | mean -1.96SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 24 | 0.990 | 0.958 | 1.013 | 13.57 | −16.93 | 58.73 | 0.9985 | 0.1 | 2.9 | 5.8 | −5.6 | −0.0008 | 1.364 | −0.2111 | |||
| Lab B | 22 | 1.022 | 0.983 | 1.070 | −74.35 | −151.81 | −29.63 | 0.9940 | −5.7 | 6.4 | 6.8 | −18.2 | 0.0050 | 0.0087 | −12.889 | 0.0001 | 0.5452 | 0.0039 |
| Lab C | 22 | 1.038 | 0.999 | 1.100 | −21.02 | −83.38 | 11.62 | 0.9963 | 1.6 | 4.3 | 10.0 | −6.8 | 0.0019 | −1.274 | 0.3247 | |||
| Lab D | 24 | 1.028 | 0.991 | 1.074 | −20.00 | −77.91 | 19.15 | 0.9957 | 1.7 | 4.1 | 9.6 | −6.3 | 0.0009 | 0.344 | 0.1678 | |||
| Lab E | 12 | 1.153 | 1.074 | 1.227 | −24.62 | −129.86 | 51.09 | 0.9958 | 12.1 | 4.1 | 20.2 | 4.1 | 0.0011 | 10.412 | 0.0049 | 0.2192 | ||
| Lab F | 24 | 1.096 | 0.951 | 1.335 | −44.30 | −337.00 | 90.36 | 0.9929 | −2.1 | 16.8 | 30.9 | −35.0 | 0.0000 | 2.472 | 0.0040 | |||
| Lab H | 24 | 0.936 | 0.885 | 0.994 | −12.30 | −75.55 | 48.90 | 0.9931 | −7.9 | 6.2 | 4.2 | −20.0 | −0.0007 | −6.866 | 0.0272 | −0.0843 | ||
| Lab I | 24 | 1.048 | 0.973 | 1.171 | −85.04 | −265.58 | 18.75 | 0.9834 | −2.3 | 10.6 | 18.6 | −23.1 | 0.0026 | −6.044 | 0.1855 | |||
| Lab J | 24 | 0.967 | 0.886 | 1.020 | −24.73 | −69.56 | 42.17 | 0.9861 | −6.2 | 7.0 | 7.6 | −20.0 | −0.0004 | −5.581 | −0.0419 | |||
| Lab K | 24 | 0.984 | 0.942 | 1.045 | 11.52 | −44.69 | 74.03 | 0.9944 | 0.3 | 4.8 | 9.6 | −9.1 | −0.0015 | 2.400 | −0.2216 | |||
| Lab L | 24 | 0.970 | 0.948 | 0.991 | −12.62 | −33.72 | 11.79 | 0.9990 | −4.1 | 2.6 | 1.0 | −9.2 | 0.0009 | −5.391 | 0.0002 | 0.2498 | ||
| Lab M | 24 | 0.985 | 0.902 | 1.072 | −6.80 | −98.93 | 6.94 | 0.9832 | −1.4 | 9.9 | 18.0 | −20.8 | −0.0024 | 2.069 | −0.1741 | |||
| MN | ||||||||||||||||||
| Lab A | 24 | 0.932 | 0.893 | 0.963 | 10.69 | −11.53 | 49.47 | 0.9955 | −5.3 | 4.4 | 3.3 | −13.8 | −0.0046 | −1.765 | −0.3859 | |||
| Lab B | 22 | 1.009 | 0.981 | 1.078 | −12.36 | −43.88 | 6.02 | 0.9956 | −0.7 | 4.2 | 7.6 | −9.0 | 0.0035 | −3.465 | 0.3053 | |||
| Lab C | 22 | 1.055 | 0.966 | 1.129 | −9.87 | −72.28 | 37.80 | 0.9862 | 2.2 | 8.3 | 18.4 | −14.1 | −0.0011 | 3.029 | −0.0480 | |||
| Lab D | 24 | 1.011 | 0.966 | 1.079 | −17.95 | −60.38 | 7.89 | 0.9925 | −1.1 | 5.5 | 9.7 | −11.9 | 0.0029 | −3.381 | 0.2048 | |||
| Lab E | 12 | 1.106 | 1.059 | 1.167 | −12.26 | −70.08 | 8.03 | 0.9985 | 6.6 | 3.3 | 13.1 | 0.1 | 0.0033 | 3.930 | 0.4134 | |||
| Lab F | 24 | 1.033 | 1.003 | 1.067 | −36.95 | −48.97 | −8.18 | 0.9977 | −2.0 | 5.0 | 7.8 | −11.7 | 0.0071 | 0.0047 | −7.526 | 0.0009 | 0.5565 | 0.0053 |
| Lab H | 24 | 1.012 | 0.978 | 1.043 | −10.79 | −33.97 | 12.44 | 0.9963 | −0.6 | 5.1 | 9.4 | −10.6 | −0.0002 | −0.473 | −0.0130 | |||
| Lab I | 24 | 1.022 | 0.965 | 1.078 | −10.61 | −46.66 | 19.12 | 0.9937 | −0.3 | 6.1 | 11.8 | −12.3 | 0.0027 | −2.423 | 0.1691 | |||
| Lab J | 24 | 1.000 | 0.947 | 1.050 | −35.80 | −69.42 | −4.74 | 0.9928 | −7.2 | 6.7 | 6.0 | −20.4 | 0.0073 | 0.0453 | −12.715 | 0.0002 | 0.4122 | 0.0285 |
| Lab K | 24 | 1.041 | 1.001 | 1.094 | −6.75 | −51.09 | 14.99 | 0.9955 | 2.7 | 5.1 | 12.8 | −7.4 | 0.0009 | 1.969 | 0.0697 | |||
| Lab L | 24 | 0.981 | 0.951 | 1.019 | −3.74 | −36.71 | 20.16 | 0.9968 | −2.5 | 5.2 | 7.8 | −12.7 | −0.0002 | −2.332 | −0.0145 | |||
| Lab M | 24 | 0.975 | 0.921 | 1.066 | 6.75 | −53.10 | 44.99 | 0.9871 | −1.8 | 7.7 | 13.4 | −16.9 | −0.0030 | 0.563 | −0.1465 | |||
| MTY | ||||||||||||||||||
| Lab A | 24 | 0.999 | 0.974 | 1.022 | 6.61 | −6.03 | 21.25 | 0.9977 | 2.0 | 4.7 | 11.2 | −7.1 | −0.0049 | 5.304 | 0.0098 | −0.3880 | ||
| Lab B | 22 | 0.955 | 0.909 | 1.011 | −8.23 | −39.34 | 20.41 | 0.9960 | −4.5 | 5.8 | 6.9 | −16.0 | −0.0024 | −3.043 | −0.1391 | |||
| Lab C | 22 | 1.090 | 1.024 | 1.153 | −14.66 | −54.58 | 28.26 | 0.9924 | 4.9 | 5.9 | 16.5 | −6.6 | 0.0034 | 2.637 | 0.2098 | |||
| Lab D | 24 | 1.054 | 0.993 | 1.107 | −7.27 | −35.83 | 11.20 | 0.9950 | 3.1 | 4.7 | 12.3 | −6.1 | 0.0021 | 1.733 | 0.1648 | |||
| Lab E | 12 | 1.098 | 1.048 | 1.183 | −5.49 | −68.32 | 21.98 | 0.9972 | 6.9 | 6.6 | 19.7 | −6.0 | 0.0033 | 4.571 | 0.2009 | |||
| Lab F | 24 | 1.076 | 1.046 | 1.110 | −5.18 | −23.23 | 10.99 | 0.9978 | 5.1 | 5.2 | 15.3 | −5.0 | 0.0052 | 1.634 | 0.3814 | |||
| Lab H | 24 | 0.961 | 0.889 | 1.029 | −8.05 | −34.32 | 24.56 | 0.9874 | −7.4 | 8.4 | 9.1 | −23.8 | 0.0046 | −10.278 | 0.0085 | 0.1953 | ||
| Lab I | 24 | 0.941 | 0.867 | 1.043 | −2.45 | −55.58 | 27.26 | 0.9738 | −6.2 | 10.2 | 13.8 | −26.2 | 0.0048 | −9.230 | 0.0429 | 0.1721 | ||
| Lab J | ||||||||||||||||||
| Lab K | 23 | 1.054 | 0.983 | 1.129 | 10.83 | −37.82 | 60.64 | 0.9915 | 7.8 | 7.7 | 22.8 | −7.3 | −0.0041 | 10.650 | 0.0066 | 0.1961 | ||
| Lab L | 24 | 0.944 | 0.924 | 0.969 | −1.16 | −14.54 | 7.21 | 0.9975 | −6.4 | 4.4 | 2.3 | −15.0 | 0.0038 | −8.797 | <0.0001 | 0.3129 | ||
| Lab M | 24 | 1.070 | 0.933 | 1.206 | 34.10 | −19.85 | 99.48 | 0.9678 | 13.6 | 15.7 | 44.3 | −17.1 | −0.0174 | 0.0457 | 25.603 | 0.0006 | −0.4116 | |
Numbers lower than 24 caused by laboratory-specific participation in the external quality assurance program (CL, confidence limits; SD, standard deviation).
Differences in analytical test results for normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) in samples derived from an external quality assurance scheme
| Passing-Bablok-regression (Y = m*(all-method median) + b) | Bland-Altman-analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96SD | mean -1.96SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 24 | 0.990 | 0.958 | 1.013 | 13.57 | −16.93 | 58.73 | 0.9985 | 0.1 | 2.9 | 5.8 | −5.6 | −0.0008 | 1.364 | −0.2111 | |||
| Lab B | 22 | 1.022 | 0.983 | 1.070 | −74.35 | −151.81 | −29.63 | 0.9940 | −5.7 | 6.4 | 6.8 | −18.2 | 0.0050 | 0.0087 | −12.889 | 0.0001 | 0.5452 | 0.0039 |
| Lab C | 22 | 1.038 | 0.999 | 1.100 | −21.02 | −83.38 | 11.62 | 0.9963 | 1.6 | 4.3 | 10.0 | −6.8 | 0.0019 | −1.274 | 0.3247 | |||
| Lab D | 24 | 1.028 | 0.991 | 1.074 | −20.00 | −77.91 | 19.15 | 0.9957 | 1.7 | 4.1 | 9.6 | −6.3 | 0.0009 | 0.344 | 0.1678 | |||
| Lab E | 12 | 1.153 | 1.074 | 1.227 | −24.62 | −129.86 | 51.09 | 0.9958 | 12.1 | 4.1 | 20.2 | 4.1 | 0.0011 | 10.412 | 0.0049 | 0.2192 | ||
| Lab F | 24 | 1.096 | 0.951 | 1.335 | −44.30 | −337.00 | 90.36 | 0.9929 | −2.1 | 16.8 | 30.9 | −35.0 | 0.0000 | 2.472 | 0.0040 | |||
| Lab H | 24 | 0.936 | 0.885 | 0.994 | −12.30 | −75.55 | 48.90 | 0.9931 | −7.9 | 6.2 | 4.2 | −20.0 | −0.0007 | −6.866 | 0.0272 | −0.0843 | ||
| Lab I | 24 | 1.048 | 0.973 | 1.171 | −85.04 | −265.58 | 18.75 | 0.9834 | −2.3 | 10.6 | 18.6 | −23.1 | 0.0026 | −6.044 | 0.1855 | |||
| Lab J | 24 | 0.967 | 0.886 | 1.020 | −24.73 | −69.56 | 42.17 | 0.9861 | −6.2 | 7.0 | 7.6 | −20.0 | −0.0004 | −5.581 | −0.0419 | |||
| Lab K | 24 | 0.984 | 0.942 | 1.045 | 11.52 | −44.69 | 74.03 | 0.9944 | 0.3 | 4.8 | 9.6 | −9.1 | −0.0015 | 2.400 | −0.2216 | |||
| Lab L | 24 | 0.970 | 0.948 | 0.991 | −12.62 | −33.72 | 11.79 | 0.9990 | −4.1 | 2.6 | 1.0 | −9.2 | 0.0009 | −5.391 | 0.0002 | 0.2498 | ||
| Lab M | 24 | 0.985 | 0.902 | 1.072 | −6.80 | −98.93 | 6.94 | 0.9832 | −1.4 | 9.9 | 18.0 | −20.8 | −0.0024 | 2.069 | −0.1741 | |||
| MN | ||||||||||||||||||
| Lab A | 24 | 0.932 | 0.893 | 0.963 | 10.69 | −11.53 | 49.47 | 0.9955 | −5.3 | 4.4 | 3.3 | −13.8 | −0.0046 | −1.765 | −0.3859 | |||
| Lab B | 22 | 1.009 | 0.981 | 1.078 | −12.36 | −43.88 | 6.02 | 0.9956 | −0.7 | 4.2 | 7.6 | −9.0 | 0.0035 | −3.465 | 0.3053 | |||
| Lab C | 22 | 1.055 | 0.966 | 1.129 | −9.87 | −72.28 | 37.80 | 0.9862 | 2.2 | 8.3 | 18.4 | −14.1 | −0.0011 | 3.029 | −0.0480 | |||
| Lab D | 24 | 1.011 | 0.966 | 1.079 | −17.95 | −60.38 | 7.89 | 0.9925 | −1.1 | 5.5 | 9.7 | −11.9 | 0.0029 | −3.381 | 0.2048 | |||
| Lab E | 12 | 1.106 | 1.059 | 1.167 | −12.26 | −70.08 | 8.03 | 0.9985 | 6.6 | 3.3 | 13.1 | 0.1 | 0.0033 | 3.930 | 0.4134 | |||
| Lab F | 24 | 1.033 | 1.003 | 1.067 | −36.95 | −48.97 | −8.18 | 0.9977 | −2.0 | 5.0 | 7.8 | −11.7 | 0.0071 | 0.0047 | −7.526 | 0.0009 | 0.5565 | 0.0053 |
| Lab H | 24 | 1.012 | 0.978 | 1.043 | −10.79 | −33.97 | 12.44 | 0.9963 | −0.6 | 5.1 | 9.4 | −10.6 | −0.0002 | −0.473 | −0.0130 | |||
| Lab I | 24 | 1.022 | 0.965 | 1.078 | −10.61 | −46.66 | 19.12 | 0.9937 | −0.3 | 6.1 | 11.8 | −12.3 | 0.0027 | −2.423 | 0.1691 | |||
| Lab J | 24 | 1.000 | 0.947 | 1.050 | −35.80 | −69.42 | −4.74 | 0.9928 | −7.2 | 6.7 | 6.0 | −20.4 | 0.0073 | 0.0453 | −12.715 | 0.0002 | 0.4122 | 0.0285 |
| Lab K | 24 | 1.041 | 1.001 | 1.094 | −6.75 | −51.09 | 14.99 | 0.9955 | 2.7 | 5.1 | 12.8 | −7.4 | 0.0009 | 1.969 | 0.0697 | |||
| Lab L | 24 | 0.981 | 0.951 | 1.019 | −3.74 | −36.71 | 20.16 | 0.9968 | −2.5 | 5.2 | 7.8 | −12.7 | −0.0002 | −2.332 | −0.0145 | |||
| Lab M | 24 | 0.975 | 0.921 | 1.066 | 6.75 | −53.10 | 44.99 | 0.9871 | −1.8 | 7.7 | 13.4 | −16.9 | −0.0030 | 0.563 | −0.1465 | |||
| MTY | ||||||||||||||||||
| Lab A | 24 | 0.999 | 0.974 | 1.022 | 6.61 | −6.03 | 21.25 | 0.9977 | 2.0 | 4.7 | 11.2 | −7.1 | −0.0049 | 5.304 | 0.0098 | −0.3880 | ||
| Lab B | 22 | 0.955 | 0.909 | 1.011 | −8.23 | −39.34 | 20.41 | 0.9960 | −4.5 | 5.8 | 6.9 | −16.0 | −0.0024 | −3.043 | −0.1391 | |||
| Lab C | 22 | 1.090 | 1.024 | 1.153 | −14.66 | −54.58 | 28.26 | 0.9924 | 4.9 | 5.9 | 16.5 | −6.6 | 0.0034 | 2.637 | 0.2098 | |||
| Lab D | 24 | 1.054 | 0.993 | 1.107 | −7.27 | −35.83 | 11.20 | 0.9950 | 3.1 | 4.7 | 12.3 | −6.1 | 0.0021 | 1.733 | 0.1648 | |||
| Lab E | 12 | 1.098 | 1.048 | 1.183 | −5.49 | −68.32 | 21.98 | 0.9972 | 6.9 | 6.6 | 19.7 | −6.0 | 0.0033 | 4.571 | 0.2009 | |||
| Lab F | 24 | 1.076 | 1.046 | 1.110 | −5.18 | −23.23 | 10.99 | 0.9978 | 5.1 | 5.2 | 15.3 | −5.0 | 0.0052 | 1.634 | 0.3814 | |||
| Lab H | 24 | 0.961 | 0.889 | 1.029 | −8.05 | −34.32 | 24.56 | 0.9874 | −7.4 | 8.4 | 9.1 | −23.8 | 0.0046 | −10.278 | 0.0085 | 0.1953 | ||
| Lab I | 24 | 0.941 | 0.867 | 1.043 | −2.45 | −55.58 | 27.26 | 0.9738 | −6.2 | 10.2 | 13.8 | −26.2 | 0.0048 | −9.230 | 0.0429 | 0.1721 | ||
| Lab J | ||||||||||||||||||
| Lab K | 23 | 1.054 | 0.983 | 1.129 | 10.83 | −37.82 | 60.64 | 0.9915 | 7.8 | 7.7 | 22.8 | −7.3 | −0.0041 | 10.650 | 0.0066 | 0.1961 | ||
| Lab L | 24 | 0.944 | 0.924 | 0.969 | −1.16 | −14.54 | 7.21 | 0.9975 | −6.4 | 4.4 | 2.3 | −15.0 | 0.0038 | −8.797 | <0.0001 | 0.3129 | ||
| Lab M | 24 | 1.070 | 0.933 | 1.206 | 34.10 | −19.85 | 99.48 | 0.9678 | 13.6 | 15.7 | 44.3 | −17.1 | −0.0174 | 0.0457 | 25.603 | 0.0006 | −0.4116 | |
| Passing-Bablok-regression (Y = m*(all-method median) + b) | Bland-Altman-analysis | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| na | slope | Lower CL (slope) | Upper CL (slope) | inter- cept | Lower CL (inter- cept) | Upper CL (inter- cept) | R | mean difference [%] | SD | mean + 1.96SD | mean -1.96SD | slope | P | intercept | P | R | P | |
| NMN | ||||||||||||||||||
| Lab A | 24 | 0.990 | 0.958 | 1.013 | 13.57 | −16.93 | 58.73 | 0.9985 | 0.1 | 2.9 | 5.8 | −5.6 | −0.0008 | 1.364 | −0.2111 | |||
| Lab B | 22 | 1.022 | 0.983 | 1.070 | −74.35 | −151.81 | −29.63 | 0.9940 | −5.7 | 6.4 | 6.8 | −18.2 | 0.0050 | 0.0087 | −12.889 | 0.0001 | 0.5452 | 0.0039 |
| Lab C | 22 | 1.038 | 0.999 | 1.100 | −21.02 | −83.38 | 11.62 | 0.9963 | 1.6 | 4.3 | 10.0 | −6.8 | 0.0019 | −1.274 | 0.3247 | |||
| Lab D | 24 | 1.028 | 0.991 | 1.074 | −20.00 | −77.91 | 19.15 | 0.9957 | 1.7 | 4.1 | 9.6 | −6.3 | 0.0009 | 0.344 | 0.1678 | |||
| Lab E | 12 | 1.153 | 1.074 | 1.227 | −24.62 | −129.86 | 51.09 | 0.9958 | 12.1 | 4.1 | 20.2 | 4.1 | 0.0011 | 10.412 | 0.0049 | 0.2192 | ||
| Lab F | 24 | 1.096 | 0.951 | 1.335 | −44.30 | −337.00 | 90.36 | 0.9929 | −2.1 | 16.8 | 30.9 | −35.0 | 0.0000 | 2.472 | 0.0040 | |||
| Lab H | 24 | 0.936 | 0.885 | 0.994 | −12.30 | −75.55 | 48.90 | 0.9931 | −7.9 | 6.2 | 4.2 | −20.0 | −0.0007 | −6.866 | 0.0272 | −0.0843 | ||
| Lab I | 24 | 1.048 | 0.973 | 1.171 | −85.04 | −265.58 | 18.75 | 0.9834 | −2.3 | 10.6 | 18.6 | −23.1 | 0.0026 | −6.044 | 0.1855 | |||
| Lab J | 24 | 0.967 | 0.886 | 1.020 | −24.73 | −69.56 | 42.17 | 0.9861 | −6.2 | 7.0 | 7.6 | −20.0 | −0.0004 | −5.581 | −0.0419 | |||
| Lab K | 24 | 0.984 | 0.942 | 1.045 | 11.52 | −44.69 | 74.03 | 0.9944 | 0.3 | 4.8 | 9.6 | −9.1 | −0.0015 | 2.400 | −0.2216 | |||
| Lab L | 24 | 0.970 | 0.948 | 0.991 | −12.62 | −33.72 | 11.79 | 0.9990 | −4.1 | 2.6 | 1.0 | −9.2 | 0.0009 | −5.391 | 0.0002 | 0.2498 | ||
| Lab M | 24 | 0.985 | 0.902 | 1.072 | −6.80 | −98.93 | 6.94 | 0.9832 | −1.4 | 9.9 | 18.0 | −20.8 | −0.0024 | 2.069 | −0.1741 | |||
| MN | ||||||||||||||||||
| Lab A | 24 | 0.932 | 0.893 | 0.963 | 10.69 | −11.53 | 49.47 | 0.9955 | −5.3 | 4.4 | 3.3 | −13.8 | −0.0046 | −1.765 | −0.3859 | |||
| Lab B | 22 | 1.009 | 0.981 | 1.078 | −12.36 | −43.88 | 6.02 | 0.9956 | −0.7 | 4.2 | 7.6 | −9.0 | 0.0035 | −3.465 | 0.3053 | |||
| Lab C | 22 | 1.055 | 0.966 | 1.129 | −9.87 | −72.28 | 37.80 | 0.9862 | 2.2 | 8.3 | 18.4 | −14.1 | −0.0011 | 3.029 | −0.0480 | |||
| Lab D | 24 | 1.011 | 0.966 | 1.079 | −17.95 | −60.38 | 7.89 | 0.9925 | −1.1 | 5.5 | 9.7 | −11.9 | 0.0029 | −3.381 | 0.2048 | |||
| Lab E | 12 | 1.106 | 1.059 | 1.167 | −12.26 | −70.08 | 8.03 | 0.9985 | 6.6 | 3.3 | 13.1 | 0.1 | 0.0033 | 3.930 | 0.4134 | |||
| Lab F | 24 | 1.033 | 1.003 | 1.067 | −36.95 | −48.97 | −8.18 | 0.9977 | −2.0 | 5.0 | 7.8 | −11.7 | 0.0071 | 0.0047 | −7.526 | 0.0009 | 0.5565 | 0.0053 |
| Lab H | 24 | 1.012 | 0.978 | 1.043 | −10.79 | −33.97 | 12.44 | 0.9963 | −0.6 | 5.1 | 9.4 | −10.6 | −0.0002 | −0.473 | −0.0130 | |||
| Lab I | 24 | 1.022 | 0.965 | 1.078 | −10.61 | −46.66 | 19.12 | 0.9937 | −0.3 | 6.1 | 11.8 | −12.3 | 0.0027 | −2.423 | 0.1691 | |||
| Lab J | 24 | 1.000 | 0.947 | 1.050 | −35.80 | −69.42 | −4.74 | 0.9928 | −7.2 | 6.7 | 6.0 | −20.4 | 0.0073 | 0.0453 | −12.715 | 0.0002 | 0.4122 | 0.0285 |
| Lab K | 24 | 1.041 | 1.001 | 1.094 | −6.75 | −51.09 | 14.99 | 0.9955 | 2.7 | 5.1 | 12.8 | −7.4 | 0.0009 | 1.969 | 0.0697 | |||
| Lab L | 24 | 0.981 | 0.951 | 1.019 | −3.74 | −36.71 | 20.16 | 0.9968 | −2.5 | 5.2 | 7.8 | −12.7 | −0.0002 | −2.332 | −0.0145 | |||
| Lab M | 24 | 0.975 | 0.921 | 1.066 | 6.75 | −53.10 | 44.99 | 0.9871 | −1.8 | 7.7 | 13.4 | −16.9 | −0.0030 | 0.563 | −0.1465 | |||
| MTY | ||||||||||||||||||
| Lab A | 24 | 0.999 | 0.974 | 1.022 | 6.61 | −6.03 | 21.25 | 0.9977 | 2.0 | 4.7 | 11.2 | −7.1 | −0.0049 | 5.304 | 0.0098 | −0.3880 | ||
| Lab B | 22 | 0.955 | 0.909 | 1.011 | −8.23 | −39.34 | 20.41 | 0.9960 | −4.5 | 5.8 | 6.9 | −16.0 | −0.0024 | −3.043 | −0.1391 | |||
| Lab C | 22 | 1.090 | 1.024 | 1.153 | −14.66 | −54.58 | 28.26 | 0.9924 | 4.9 | 5.9 | 16.5 | −6.6 | 0.0034 | 2.637 | 0.2098 | |||
| Lab D | 24 | 1.054 | 0.993 | 1.107 | −7.27 | −35.83 | 11.20 | 0.9950 | 3.1 | 4.7 | 12.3 | −6.1 | 0.0021 | 1.733 | 0.1648 | |||
| Lab E | 12 | 1.098 | 1.048 | 1.183 | −5.49 | −68.32 | 21.98 | 0.9972 | 6.9 | 6.6 | 19.7 | −6.0 | 0.0033 | 4.571 | 0.2009 | |||
| Lab F | 24 | 1.076 | 1.046 | 1.110 | −5.18 | −23.23 | 10.99 | 0.9978 | 5.1 | 5.2 | 15.3 | −5.0 | 0.0052 | 1.634 | 0.3814 | |||
| Lab H | 24 | 0.961 | 0.889 | 1.029 | −8.05 | −34.32 | 24.56 | 0.9874 | −7.4 | 8.4 | 9.1 | −23.8 | 0.0046 | −10.278 | 0.0085 | 0.1953 | ||
| Lab I | 24 | 0.941 | 0.867 | 1.043 | −2.45 | −55.58 | 27.26 | 0.9738 | −6.2 | 10.2 | 13.8 | −26.2 | 0.0048 | −9.230 | 0.0429 | 0.1721 | ||
| Lab J | ||||||||||||||||||
| Lab K | 23 | 1.054 | 0.983 | 1.129 | 10.83 | −37.82 | 60.64 | 0.9915 | 7.8 | 7.7 | 22.8 | −7.3 | −0.0041 | 10.650 | 0.0066 | 0.1961 | ||
| Lab L | 24 | 0.944 | 0.924 | 0.969 | −1.16 | −14.54 | 7.21 | 0.9975 | −6.4 | 4.4 | 2.3 | −15.0 | 0.0038 | −8.797 | <0.0001 | 0.3129 | ||
| Lab M | 24 | 1.070 | 0.933 | 1.206 | 34.10 | −19.85 | 99.48 | 0.9678 | 13.6 | 15.7 | 44.3 | −17.1 | −0.0174 | 0.0457 | 25.603 | 0.0006 | −0.4116 | |
Numbers lower than 24 caused by laboratory-specific participation in the external quality assurance program (CL, confidence limits; SD, standard deviation).
Results of normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (MTY) measured by LC-MS/MS in 12 laboratories in comparison to Royal College of Pathologists of Australasia-Quality Assurance Program (RCPAQAP) target medians. A, B, C show regression lines of respective analytes derived from Passing Bablok (PB) regression analyses by applying minimum and maximum “RCPA medians” for curve drawing; D, E, F show regression lines of respective analytes derived from Bland-Altman (BA) analysis of mean concentrations (RCPAQAP median, respective laboratory derived data) and %differences of each laboratory relative to the RCPAQAP median.
Comparisons of Native Patient vs Proficiency Testing Sample Data
Median concentrations of normetanephrine, metanephrine, and 3-methoxytyramine were 2.7-fold (P = 0.006), 4.7-fold and 14.5-fold (P ≤ 0.001) higher in RCPAQAP samples than in samples from patients (Fig. 1). In contrast, median variations for the 3 metabolites were 1.7-fold (7.6% vs 12.6%), 2.1-fold (6.4% vs 13.6%), and 3.6-fold (11.0% vs 40.1%) (P ≤ 0.001), respectively, lower in RCPAQAP samples than in patient samples.
Agreement in Test Interpretation
In order to assess agreement among laboratories in interpretation of test results, we examined data according to interpretations achieved according to the URLs used by each laboratory. Seven of 12 laboratories used age-dependent URLs for normetanephrine, whereas 5 laboratories used fixed URLs ranging from 710 pmol/L to 1180 pmol/L (Supplemental Table 1, Supplemental Table 2). Mean inter-rater agreement in test interpretation was 90.6% (113/125) (95% CI 89.2%–92.0%) and ranged from 83.7% to 93.3% (104/125 to 116/125) (Fig. 4).
Inter-rater agreement in interpretations of 125 test results for normetanephrine (A) and metanephrine (B) using i) the analyzed raw data and participants own upper reference limits (black circles), ii) normalized data and participants own upper reference limits (black filled triangles) and iii) normalized data in conjunction with normalized upper reference limits derived from laboratory A (black rectangles). Whiskers represent the lower and upper 95% confidence intervals. 19 and 36 results for metanephrine reported below the lower limit of quantification by laboratories D and E, respectively, are assumed nonpathologic, 3 and 9 missing results due to analytical problems from respective laboratories F and M are not considered in the analysis for those laboratories.
After normalization of analytical data to the all-method medians, the agreement in test interpretation for normetanephrine increased slightly from 90.6% (113/125) to 91.2% (114/125) (95% CI 89.8%–92.6%), with a range of 84.9% to 93.6% (106/125 to 117/125). Further normalization of analytical data in conjunction with the use of the same but normalized URLs improved the agreement in test interpretations to 94.4% (118/125) (95% CI 93.5%–95.3%) ranging from 91.9% to 96.4% (115/125 to 120/125). This improvement was generally observed for all participating laboratories and particularly for laboratory C, which used the highest fixed URL of 1180 pmol/L.
For metanephrine, all laboratories used fixed URLs to distinguish nonpathological from pathological test results; these ranged widely from 299 pmol/L to 560 pmol/L (Supplemental Table 1, Supplemental Table 2). After applying each laboratories’ own URLs first to the analytical raw data and then the normalized data, the observed agreement in test interpretation increased marginally from 97.0% (121/125) (95% CI 96.3%–97.7%) for the raw data to 97.2% (122/125) (95% CI 96.5%–97.9%) for the normalized data (Fig. 4). After application of the same but a normalized URL, the agreement in test interpretation improved further to 98.3% (123/125) (95% CI 97.9%–98.7%). This increase was largest for laboratories C, H, J, and L, which used particularly high (510 and 560 pmol/L) or low (299 and 280 pmol/L) URLs.
Discussion
Using patient plasma samples, this study demonstrates good agreement between 12 laboratories for measurements of normetanephrine and metanephrine with respective mean biases from target of 1.2% and 0.1%. All laboratories performed well within the APS of the international RCPAQAP. Agreement for 3-methoxytyramine was, however, suboptimal, with differences from target ranging from −32% to 64% in patient samples. Since 3-methoxytyramine circulates at lower concentrations than the metanephrines, inaccuracy and imprecision for this biomarker can be expected, but does indicate need for improved analytical methods. Although analytical results for normetanephrine and metanephrine seem to be harmonized, differences exist in test interpretation based on laboratory-specific URLs; this variability in test interpretation may be improved by harmonized reference intervals.
Standardization of analytical methods requires reference materials, reference methods, and reference laboratories. Additionally, URLs should be transferable across methods for harmonized reporting (31). Some degree of harmonization can be achieved through participation in EQA programs that use commutable samples for proficiency testing (29). EQA programs, such as the RCPAQAP, are particularly valuable for measurements of plasma-free metanephrines and 3-methoxytyramine, for which there is lack of commutable reference materials, reference methods, and reference laboratories. Through these programs, assessments of performance are defined according to quality specifications and in relation to APS (30).
According to those APS criteria, measurements of normetanephrine in all 12 laboratories are well harmonized and should, therefore, allow use of harmonized URLs for discrimination of pathological and nonpathological test results. Similar findings for metanephrine in all except one laboratory should also allow use of harmonized URLs for this metabolite. In contrast, for 3-methoxytyramine, one-third of all laboratories had a mean bias higher than ±30%. In addition, due to differences in assay-specific LLOQs, some laboratories failed to detect 3-methoxytyramine in the clinically relevant range of patient samples (2). Nevertheless, in samples distributed by the RCPA program, all 3 metabolites met the APS criteria with minimal mean biases of less than ±2%. In relation to the clinically relevant concentration range, it is important to highlight that the metabolite concentrations in RCPA samples were considerably higher than in patient samples. Variations observed in analytical test results for patient and RCPA samples were similar at concentration ranges observed in RCPA samples, but much higher for lower concentration ranges commonly observed in samples from patients without PPGLs (3). Therefore, the true clinical performance of these tests cannot be assessed purely on the basis of such an EQA program.
The finding of a positive relationship for differences in bias for measurements of 3-methoxytyramine in native plasma compared to RCPA samples indicates systematic discordance in the analysis of this metabolite among laboratories; therefore, participation in an EQA program remains informative and beneficial for improving measurement accuracy for 3-methoxytyramine. However, lack of relationships of bias for normetanephrine and metanephrine point to the presence of factors within patient specimens that influence patient results but that cannot be captured using the artificial sample matrix employed in the RCPAQAP program; this is, however, a common weakness of EQA observed with many laboratory tests. Altogether, the findings of this study call for a commutable QAP material that covers the clinically relevant concentration ranges and matrix composition, including potentially interfering endogenous substances that may not be present in artificial sample matrix.
Besides agreement in analytical test results, it is also important for patient care that results are interpreted among different laboratories with consistent diagnostic accuracy. Inter-rater analysis demonstrated generally good agreement in clinical interpretation for both normetanephrine and metanephrine. However, diagnostic agreement was higher for metanephrine than for normetanephrine. The variation of literature-reported values for URLs of normetanephrine largely reflects higher concentrations during seated than supine sampling, with the latter recommended by the Endocrine Society guideline (1). Within the 12 laboratories of the present study, only one used URLs determined for seated blood sampling (10); accordingly, that laboratory showed the least agreement in test interpretation for normetanephrine. Differential adoption of fixed vs age-specific URLs may contribute to other disparities in interpretation of results for normetanephrine.
The laboratories with the least agreements for metanephrine had particularly low or high decision limits. The lower limits may reflect use of URLs for this metabolite determined by 97.5 percentiles for supine sampling, while higher values are associated with seated sampling or established to minimize false-positive results (32). Use of the latter has been justified by findings that most patients with PPGLs present with increases of normetanephrine, with or without increases of metanephrine (3); only occasionally do patients with PPGLs present with solitary increases of metanephrine. Thus, accurate diagnosis of PPGLs depends more on URLs used for normetanephrine than for metanephrine.
After data were normalized according to a single set of published URLs (32), agreement in test interpretation improved beyond that achieved by normalizing for bias, indicating that differences in URLs are more crucial for test interpretation than differences in analytical test performance. This strengthens the argument for common use of URLs as a next step towards harmonized reporting. Age-specific URLs for plasma-free normetanephrine have been proposed (12) and further validated in a comprehensive prospective diagnostic accuracy study involving more than 2000 patients including 236 confirmed PPGL cases (3). Age-specific URLs, with lower decision limits for younger patients and continuously increasing cut-offs with age, assist in reducing false-positive test results in older patients and false-negative test results in younger patients (12).
By demonstrating that the analytical methods for metanephrine and normetanephrine were reasonably harmonized among 12 laboratories, there can be confidence that already established age-specific reference limits (12, 32) are transferable to all laboratories of this study. In fact, the majority of laboratories within this study are already using age-specific URLs. Nevertheless, full transference of continuous age-dependent URLs into laboratory information management systems was reported to be difficult by several participants. This indicates a need to modernize existing systems to meet emerging demands for personalized interpretation of test results that will thereby achieve improved diagnostic accuracy.
For 3-methoxytyramine, the situation was quite different to that of normetanephrine and metanephrine, with highly variable URLs ranging from 36 to 200 pmol/L. Consistencies in bias according to patient and EQA materials point to systematic discordances in measurement methods. Interferences from endogenous substances (23) and cross-reaction with other metabolites, such as metanephrine when chromatographic separation is limited (33), may contribute to some of the discordance. Nevertheless, most of the discordance likely reflects imprecision of most methods for which the LLOQ was often close to the upper range of reference intervals. This points to the need for improved and more sensitive analytical methods, such as recently described by van Faassen et al. (34). Only through such methods is the full potential of plasma 3-methoxytyramine likely to be realized for applications involving patients with PPGLs or neuroblastoma (35–37).
Limitations of the study include lack of universal calibrators to exclude proportional bias due to differences in calibration, and lack of multiple aliquots of the same patient sample to establish variance within methods. Despite these limitations, the differences in agreement for normetanephrine and metanephrine were minor. Future harmonization studies should nevertheless include a universal set of calibrators and sample material replicates to assess precision. Another limitation involved the lack of true clinical diagnosis for the 125 pooled patient samples in order to better reconcile differences in test interpretation based on URLs. Relevant endogenous interfering substances, only present in some patient samples, may have also been diluted in the pooling process. Finally, some participating laboratories described LLOQs higher than required for precise measurement of low endogenous concentrations of 3-methoxytyramine and metanephrine. Therefore, such laboratories reported incomplete analytical test results that may have led to overestimated analytical accuracy. Nevertheless, LLOQs for metanephrine were always well below the applied ULRs for clinical test interpretation and therefore not expected to have clinical relevance for this metabolite.
In conclusion, analytical results by LC-MS/MS and interpretation of results seem harmonizable for metanephrine and normetanephrine. However, differences exist in test interpretation based on laboratory URLs, particularly for normetanephrine. Adoption of common reference limits that have been validated in large studies and that can be easily verified locally using widely accepted criteria (38) may therefore improve clinical decision-making for the diagnosis of PPGLs by laboratories that show minimal bias in biochemical test results. Proficiency testing organizations may also consider a focus on distributing commutable QAP materials that cover concentration ranges relevant for clinical decision-making. In this way, laboratories can continuously better assess their analytical quality in conjunction with the transferability and stability of their clinical decision limits. This should help ensure high diagnostic accuracy of laboratory services.
Supplemental Material
Supplemental material is available at Clinical Chemistry online.
Nonstandard Abbreviations
PPGL, pheochromocytoma and paraganglioma; LC-MS/MS, liquid chromatography-tandem mass spectrometry; EQA, external quality assurance; QAP, Quality Assurance Program; RCPA, Royal College of Pathologists of Australasia; URL, upper reference limit; APS, analytical performance specifications; LLOQ, lower limit of quantification.
Author Contributions
All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
M. Peitzsch, financial support, administrative support, statistical analysis, provision of study material or patients; T. Novos, statistical analysis, provision of study material or patients; A.E. van Herwaarden, provision of study material or patients; D.M. Mueller, provision of study material or patients; M. Fassnacht, financial support, provision of study material or patients; F. Sweep, provision of study material or patients; A.R. Horvath, financial support, statistical analysis, administrative support, provision of study material or patients; G. Eisenhofer, financial support, statistical analysis, provision of study material or patients.
Authors’ Disclosures or Potential Conflicts of Interest
Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership
None declared.
Consultant or Advisory Role
None declared.
Stock Ownership
None declared.
Honoraria
None declared.
Research Funding
This work was supported by the Deutsche Forschungsgemeinschaft (DFG), German Research Foundation Project number 314061271-CRC/TRR 205/1.
Expert Testimony
None declared.
Patents
None declared.
Role of Sponsor
The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.
References
CLSI.
