Calibration of fractionated metanephrines in urine: still an issue?

Measurement of fractionated total metanephrines in urine provides important information for the diagnosis of pheochromocytoma (1). Reference intervals that distinguish patients who harbor a pheochromocytoma from those with similar symptoms not actually caused by this disease are highly variable (2– 4 ). Such differences may be caused by variations in analytical methods, the selection of negative controls for establishing reference values, age, and sex (4 ). A group of 3 laboratories have reported in this journal significantly higher results than expected for urinary fractionated total metanephrines during 3 successive proficiency surveys in 2003 and 2004. These differences were due to assigned values for the Bio-Rad Laboratories calibrator that were 24%–33% lower than the actual concentration (5 ). To determine whether the concentration of the Bio-Rad calibrator has been corrected, we examined proficiency-test data obtained from ProBioQual, a French qualityassurance center, to assess the accuracy of urine metanephrine results collected from 48 laboratories during the 2004–2006 period. We included only 8 surveys with target metanephrine concentrations between 1000 and 1800 nmol/L in this retrospective analysis to exclude the uncertainty produced by measurements of very low or very high concentrations. Forty-eight laboratories were requested to specify the calibrator used, and 34 laboratories actually returned this information. Ten laboratories used an in-house calibrator, and calibrators manufactured by Chromsystems Instruments & Chemicals (Munich, Germany), Bio-Rad, and Recipe Chemicals Instruments (Munich, Germany) were used by 15, 7, and 2 laboratories, respectively. An ANOVA of the results for the 8 surveys showed no statistical differences, with the exceptions of survey no. 6 in 2005, which revealed higher normetanephrine concentrations in laboratories that used Chromsystems reagents than in those that used an in-house calibrator (difference, 15.6%; P 0.048), and survey no. 3 in 2006, which found higher metanephrine concentrations in laboratories that used Chromsystems and Bio-Rad calibrators than in those that used an in-house calibrator (differences of 9% and 15.4%, respectively; P 0.005). The number of users of Recipe calibrators was too low (0–2) to evaluate. Despite the differences in these 2 surveys, we concluded that the metanephrine concentrations obtained by the laboratories were roughly similar. A runs test on variation coefficients revealed an improvement in precision for metanephrine (P 0.02) beginning in June 2005, but not for normetanephrine (P 0.45). Because of the limited number of runs, these results must certainly be interpreted with caution, but they are indicative of decreasing dispersion for metanephrine among the participating laboratories (Fig. 1). We then determined whether the concentrations of fractionated total metanephrines measured by HPLC with amperometric detection were similar for the urine calibrator from Bio-Rad, 2 commercially available sources (Recipe and Chromsystems), and our inhouse calibrator, which is prepared by weighing normetanephrine and metanephrine obtained from Sigma-Aldrich. In addition, we used the Bio-Rad calibrator to measure metanephrine concentration across all the samples to determine whether 2 concentrations of the internal quality controls (typical and abnormal) from Chromsystems and Recipe were in agreement with their nominal values. We repeated the experiments 10 times within 1 month with freshly prepared calibrators and controls. Our results showed mean systematic departures between found and expected concentrations ranging from 91% to 100% among calibrators and internal quality controls for both metanephrines and normetanephrines, whereas the CVs observed within the 10 determinations for metanephrines ranged between 2% and 9%. An ANOVA revealed significant differences between the groups for both normetanephrine (P 0.0026) and metanephrine (P 0.0001). Post hoc Scheffé tests indicated that our in-house calibrator exhibits a 9% higher normetanephrine concentration than the Chromsystems calibrator (P 0.008). The Chromsystems metanephrine calibrator exhibited 5% higher values than the Recipe calibrator (P 0.0001). The metanephrine concentrations observed for the Chromsystems abnormalconcentration internal quality control were also 10% higher than expected compared with those from Recipe (P 0.008). Despite the statistical significance of this difference (P 0.0026), it is not expected to affect the medical decision to a clinically relevant degree. In conclusion, we did not notice any crucial differences between 0 2 4 6 8 10 12 14 16