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Technical Briefs |
Departments of1 Internal Medicine, 2 Chemical Endocrinology, and 3 Endocrinology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; 4 Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke and 5 Reproductive Biology and Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD;
aaddress correspondence to this author at: Department of Internal Medicine, Radboud University Nijmegen Medical Center, Geert Grooteplein, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands; fax 31-24-3541734, e-mail j.lenders{at}aig.umcn.nl
Abstract
Background: The impact of blood sampling in sitting vs supine positions on measurements of plasma metanephrines for diagnosis of pheochromocytoma is unknown.
Methods: We compared plasma concentrations of free metanephrines in samples from patients with primary hypertension obtained after supine rest with those obtained in the sitting position without preceding rest. We also assessed the effects on diagnostic test performance retrospectively in patients with and without pheochromocytoma, and we calculated cost-effectiveness for pheochromocytoma testing.
Results: Upper reference limits of plasma free metanephrines were higher in samples obtained from seated patients without preceding rest than from supine patients with preceding rest. Application of these higher upper reference limits to samples from supine patients with pheochromocytoma decreased the diagnostic sensitivity from 99% to 96%. In patients without pheochromocytoma, adjusting the plasma concentration for the effects of sitting while preserving the 99% sensitivity by use of the supine upper reference limits increased the number of false-positive test results from 9% to 25%.
Conclusions: To preserve high diagnostic sensitivity we recommend the use of upper reference limits determined from blood samples collected in the supine position. Under these conditions, negative test results for blood samples obtained with patients sitting are as effective for ruling out pheochromocytoma as negative results from samples obtained after supine rest. Repeat testing with samples obtained in the supine position offers a cost-effective approach for dealing with the increased numbers of false-positive results expected after initial sampling in the sitting position.
Measurements of plasma free metanephrines (normetanephrine and metanephrine) provide a particularly sensitive test for the diagnosis of pheochromocytoma (1)(2)(3)(4). Suboptimal specificity in some studies may be attributable to blood sampling conditions(5). We therefore examined diagnostic test performance for samples obtained from patients who were either supine or sitting.
Study participants were 60 patients [38 females, mean (SD) age 45 (13.3) years, range 22–78 years; blood pressure 154 (15)/96 (8) mmHg] with primary hypertension (off medication for 2–3 weeks) and normal renal function. A first blood sample was drawn through an antecubital cannula immediately after patients sat down and a 2nd blood sample after 30 min of supine rest. Plasma samples were assayed for concentrations of metanephrines (6).
Gaussian distributions of metanephrines were obtained after logarithmic transformation of the data. The antilogarithm of the mean + 2 SD of the transformed data of the 60 patients was used as an estimate of the 97.5th percentile, which was adopted as the upper reference limit.
The data obtained from sitting and supine conditions were used in a retrospective analysis of a dataset of samples drawn after at least 20 min of supine rest from patients with (n = 228) and without pheochromocytoma (n = 644) (1). We used the upper reference limits of samples obtained from the 60 hypertensives in the sitting and supine positions to recalculate all diagnostic indices in the retrospective dataset. To further assess the potential influences of posture on diagnostic specificity, we adjusted the test results in the 644 patients without pheochromocytoma for the influence of sitting. We did not do so for the pheochromocytoma patients because of the likely negligible influence of posture on the already considerably increased plasma concentrations of metanephrines in these patients.
The distributions of the 4 variables from sampling in supine and sitting positions were tested for normality using the Shapiro–Wilk test (SPSS v12). The effect of the different positions was tested by conventional paired t-test.
Samples obtained from seated hypertensive patients had 30% higher (P <0.001) values of plasma normetanephrine and 27% higher (P <0.001) values for plasma metanephrine than samples obtained from patients after 30 min of supine rest (data not shown). These results indicated higher upper reference limits in the sitting than the supine position for both plasma free normetanephrine (0.73 vs 0.62 nmol/L) and metanephrine (0.50 vs 0.35 nmol/L). In the present study the upper reference limits for supine plasma metanephrines for the 60 hypertensive patients were similar to those we previously estimated in a mixed population of 178 normotensive and hypertensive patients, 0.62 nmol/L for normetanephrine and 0.31 nmol/L for metanephrine.
Applying the supine upper reference limits of the present study to the supine data of our previous study in patients with and without pheochromocytoma resulted in no or little change in diagnostic sensitivity (99% vs 99%) or specificity (87% vs 91%) for plasma metanephrines (1). In contrast, applying the upper reference limits derived from the samples obtained from sitting patients resulted in a 3-fold increase in false-negative test results (3 to 9 patients), leading to a change in diagnostic sensitivity for plasma free metanephrines from 99% to 96% (Table 1
). Adjusting the supine data in the 644 patients without pheochromocytoma for the projected 30% and 27% sitting-associated increases in plasma normetanephrine and metanephrine resulted in a diagnostic specificity of 89%. Applying the supine upper reference limits to the same posture-adjusted values in the patients without pheochromocytoma led to a 2.8-fold increase in false-positive test results and a drop in diagnostic specificity to 75%, with a preserved sensitivity of 99% (Table 1
).
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We calculated the financial implications of sampling under both conditions (sitting and supine) to test for pheochromocytoma in 100 000 hypothetical patients. From a 3rd-payer perspective, the charges in US dollars were assumed to be $20 for a venipuncture, $100 for an assay of plasma free metanephrines, and $200 dollars for the supine rest test. The calculation was carried out with the assumption of a prevalence rate of pheochromocytoma of 0.1% (7), a diagnostic sensitivity of 99%, and a specificity of 75% for plasma free metanephrines (Table 1
). The total cost of initial supine rest tests in all patients was calculated to be $30 000 000. If all 100 000 patients had initial samples drawn in the sitting position, however, 25 074 patients (99 true positives and 24 975 false-positives) would have positive test results for plasma metanephrines and should have additional supine tests. The total costs of this latter approach would be $19 522 200, reducing the cost by nearly $10 000 000.
The effect of posture on plasma concentrations of free metanephrines must be considered in establishing reference intervals and interpreting test results for diagnosis of pheochromocytoma. The higher plasma concentrations of metanephrines associated with blood sampling from patients in seated rather than in the supine positions are not unexpected given the known effects of posture on plasma catecholamines. The findings are also consistent with previous observations that during sympathoadrenal activation plasma free metanephrines show similar, albeit smaller, directional changes compared to plasma catecholamines (8)(9)(10). Despite such influences it has become a relatively common practice to measure plasma metanephrines in blood samples obtained from seated patients(2).
Obtaining blood samples from seated rather than supine patients may be more practical for phlebotomists, but the sympathoadrenal activating effects of upright posture may compromise the diagnostic accuracy of measurements of plasma metanephrines. As we show here, sitting-associated increases in plasma metanephrines in patients without pheochromocytoma decreases the signal-to-noise ratio for a true-positive compared to a false-positive test result, thereby increasing the difficulty of distinguishing increases in metanephrines due to a tumor from those due to seated posture. More importantly, as we also show, use of reference intervals established from samples obtained from seated patients can be expected to result in increased false-negative test results and loss of diagnostic sensitivity.
Although pheochromocytomas are rare, they can have deadly consequences if undiagnosed. Initial testing should therefore reliably provide a positive test result in that rare patient with the tumor. This conversely also means that a negative result reliably excludes the tumor, thereby avoiding the need for multiple or repeat biochemical testing and costly and unnecessary imaging studies. The projected 3-fold increase in false-negative test results associated with reference intervals established from blood samples drawn in the seated position erodes confidence that a negative test result for plasma free metanephrines reliably excludes pheochromocytoma.
The disadvantage of upper reference intervals established for samples obtained from supine patients is an expected increase in false-positive test results when samples for diagnosis of pheochromocytoma are drawn from seated patients, the condition most convenient for phlebotomists. The diagnostic sensitivity, however, is preserved at 99%. Retrospective analysis of the present study indicated a likely 2.8-fold increase in false-positive results, from 9% to 25%. In the additional 16% of patients with posture-dependent false-positive results, the tumor may be ruled out by true-negative test results after additional testing with blood samples taken after 30 min of supine rest.
What are the practical implications of initial blood sampling from patients in the diagnostically ideal supine position vs the more convenient and less costly seated position? Provided that upper reference limits obtained after supine rest are used, negative test results for blood samples from seated patients are as effective in ruling out pheochromocytoma as negative results obtained from patients after supine rest. Repeat testing with samples obtained in the supine position offers a cost-effective approach for dealing with the increased numbers of false-positive results expected after initial sampling in the sitting position.
References
The following articles in journals at HighWire Press have cited this article:
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  W. H. A. de Jong, G. Eisenhofer, W. J. Post, F. A. J. Muskiet, E. G. E. de Vries, and I. P. Kema Dietary Influences on Plasma and Urinary Metanephrines: Implications for Diagnosis of Catecholamine-Producing Tumors J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 2841 - 2849. [Abstract] [Full Text] [PDF]
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 R. T Peaston and S. Ball Biochemical detection of phaeochromocytoma: why are we continuing to ignore the evidence? Ann Clin Biochem, January 1, 2008; 45(1): 6 - 10. [Abstract] [Full Text] [PDF]
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A. Algeciras-Schimnich, C. M. Preissner, W. F. Young Jr., R. J. Singh, and S. K. G. Grebe Plasma Chromogranin A or Urine Fractionated Metanephrines Follow-Up Testing Improves the Diagnostic Accuracy of Plasma Fractionated Metanephrines for Pheochromocytoma J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 91 - 95. [Abstract] [Full Text] [PDF]
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 A. Karagiannis, D. P Mikhailidis, V. G Athyros, and F. Harsoulis Pheochromocytoma: an update on genetics and management Endocr. Relat. Cancer, December 1, 2007; 14(4): 935 - 956. [Abstract] [Full Text] [PDF]
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 R. J. Singh and G. Eisenhofer High-Throughput, Automated, and Accurate Biochemical Screening for Pheochromocytoma: Are We There Yet? Clin. Chem., September 1, 2007; 53(9): 1565 - 1567. [Full Text] [PDF]
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W. H.A. de Jong, K. S. Graham, J. C. van der Molen, T. P. Links, M. R. Morris, H. A. Ross, E. G.E. de Vries, and I. P. Kema Plasma Free Metanephrine Measurement Using Automated Online Solid-Phase Extraction HPLC Tandem Mass Spectrometry Clin. Chem., September 1, 2007; 53(9): 1684 - 1693. [Abstract] [Full Text] [PDF]
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