Biochemical Diagnosis of Pheochromocytoma (2024)

ContextDiagnosis of pheochromocytoma depends on biochemical evidence of catecholamineproduction by the tumor. However, the best test to establish the diagnosishas not been determined.

ObjectiveTo determine the biochemical test or combination of tests that providesthe best method for diagnosis of pheochromocytoma.

Design, Setting, and ParticipantsMulticenter cohort study of patients tested for pheochromocytoma at4 referral centers between 1994 and 2001. The analysis included 214 patientsin whom the diagnosis of pheochromocytoma was confirmed and 644 patients whowere determined to not have the tumor.

Main Outcome MeasuresTest sensitivity and specificity, receiver operating characteristiccurves, and positive and negative predictive values at different pretest prevalencesusing plasma free metanephrines, plasma catecholamines, urinary catecholamines,urinary total and fractionated metanephrines, and urinary vanillylmandelicacid.

ResultsSensitivities of plasma free metanephrines (99% [95% confidence interval{CI}, 96%-100%]) and urinary fractionated metanephrines (97% [95% CI, 92%-99%])were higher than those for plasma catecholamines (84% [95% CI, 78%-89%]),urinary catecholamines (86% [95% CI, 80%-91%]), urinary total metanephrines(77% [95% CI, 68%-85%]), and urinary vanillylmandelic acid (64% [95% CI, 55%-71%]).Specificity was highest for urinary vanillylmandelic acid (95% [95% CI, 93%-97%])and urinary total metanephrines (93% [95% CI, 89%-97%]); intermediate forplasma free metanephrines (89% [95% CI, 87%-92%]), urinary catecholamines(88% [95% CI, 85%-91%]), and plasma catecholamines (81% [95% CI, 78%-84%]);and lowest for urinary fractionated metanephrines (69% [95% CI, 64%-72%]).Sensitivity and specificity values at different upper reference limits werehighest for plasma free metanephrines using receiver operating characteristiccurves. Combining different tests did not improve the diagnostic yield beyondthat of a single test of plasma free metanephrines.

ConclusionPlasma free metanephrines provide the best test for excluding or confirmingpheochromocytoma and should be the test of first choice for diagnosis of thetumor.

Pheochromocytomas are chromaffin cell tumors typically arising in theadrenal glands and characterized by excessive production of catecholamines,often leading to increased blood pressure and symptoms of catecholamine excess.If not diagnosed or if left untreated, the excessive secretion of catecholaminesby these tumors can have devastating consequences. Thus, although pheochromocytomasare rare tumors, they must be considered in many patients with hypertension,the latter representing up to a quarter of the adult population in Westerncountries.

The diagnosis of pheochromocytoma depends crucially on demonstrationof excessive production of catecholamines.^1,2This step, however, is fraught with difficulties, in particular false-negativetest results. Moreover, due to the low prevalence of pheochromocytoma in thetested population and inadequate specificity of biochemical tests, false-positiveresults are a common and troublesome occurrence.³

The above difficulties in biochemical diagnosis indicate the need fora test that is maximally sensitive and specific to reliably exclude or confirmpheochromocytoma. Previous studies examining the performance of diagnostictests had small numbers of patients, inappropriate comparison groups usedto establish specificity, or limited comparisons of available biochemicaltests.^4-16Thus, the test or combination of tests that provides the best method for diagnosisof pheochromocytoma remains unsettled.

This study examined the diagnostic utility of several biochemical testsin large populations of patients tested for pheochromocytoma because of suggestivesigns and symptoms or a predisposition to develop the tumor. Biochemical testsincluded measurements of plasma and urinary catecholamines, urinary fractionatedmetanephrines, urinary total metanephrines, and urinary vanillylmandelic acid(VMA). These commonly used tests were compared with measurements of plasmaconcentrations of free metanephrines, normetanephrine, and metanephrine, whichis a promising new test for diagnosis of pheochromocytoma.^9,11,14

The study population was selected from a total of 1003 patients testedfor pheochromocytoma using plasma free metanephrines. Patients were testedbetween 1994 and 2001 at 4 referral centers (National Institutes of Health,Bethesda, Md; St Radboud University Medical Center, Nijmegen, the Netherlands;Sahlgren's University Hospital, Göteborg, Sweden; and University of Florence,Florence, Italy). Patients were either tested as part of routine screeningfor hereditary pheochromocytoma or after referral to 1 of the 4 centers becauseof a suspicion of pheochromocytoma based on a previous history of the tumor,the finding of an adrenal mass, or more often because of suggestive signs(eg, therapy-resistant or paroxysmal hypertension) and symptoms (eg, sweating,headache, palpitations). Procedures were approved by the intramural researchboard or hospital ethics committee of the centers in which patients were studiedand all patients provided informed consent.

For the purposes of patient selection into the study, the results ofbiochemical tests could not be used to exclude or confirm pheochromocytoma,since by definition this would bias the analyses of test performance. Therefore,selection of patients for inclusion in the final analyses was based on whetherpheochromocytoma could be excluded or confirmed by standard criteria thatwere necessarily independent of the diagnostic biochemical tests being evaluated.

Confirmation of pheochromocytoma required pathological examination ofsurgically resected or biopsied tumor tissue or a diagnosis of inoperablemalignant pheochromocytoma based on findings of metastatic disease by imagingstudies. Exclusion of pheochromocytoma required lack of radiological evidenceof a tumor by computed tomography or magnetic resonance imaging, pathologicalexamination of a surgically resected or biopsied adrenal mass, or lack ofpheochromocytoma on patient follow-up 2 or more years after initial testing.Using the above criteria, pheochromocytoma was confirmed in 214 patients andexcluded in 644 patients, all of whom were included in the final analyses(Table 1).

Among the 145 patients who did not fulfill the criteria for exclusionor confirmation of pheochromocytoma, and who were not included in the finalanalyses, 25 patients had a high likelihood of pheochromocytoma but had notbeen operated on at the time of analysis. All 25 patients had evidence ofa small adrenal mass by imaging studies. All had some biochemical evidenceof pheochromocytoma and most had a hereditary predisposition to the tumorbut were asymptomatic and normotensive. Pheochromocytomas in the remaininggroup of 120 patients were unlikely based on findings that did not fulfillthe criteria of the study for selection of patients into the final analyses(eg, patients in whom pheochromocytoma was excluded solely on the basis ofnegative biochemical test results).

The patients with (n = 214) and without (n = 644) pheochromocytoma whowere selected into the final analyses were further divided into 2 subgroupsbased on whether testing was performed because of a hereditary predispositionfor pheochromocytoma or because of clinical suspicion and no hereditary predisposition(Table 1). Among patients withhereditary pheochromocytoma, 48 had the tumor due to von Hippel-Lindau syndrome,23 due to multiple endocrine neoplasia type 2, and 3 due to neurofibromatosistype 1. The mutation remained undetermined in 2 patients.

To validate the use of negative imaging studies as a criterion for exclusionof pheochromocytoma, patients were followed up if they had been tested morethan 1 year previously. Follow-up information confirming lack of pheochromocytomawas obtained in 330 of the 546 patients in whom imaging studies were usedto exclude pheochromocytoma (mean follow-up, 2.5 years; range, 1-7.8 years).

Only 1 case of pheochromocytoma was confirmed by follow-up in a patientwith a previously negative computed tomographic scan result. This patientwas diagnosed with metastatic pheochromocytoma 3½ years after the negativescan and 16½ years after removal of the primary tumor.

Blood samples were collected from all patients using a forearm venouscannula with patients supine for at least 20 minutes before sampling. Patientswere instructed to fast and abstain from caffeinated and decaffeinated beveragesovernight and to avoid taking acetaminophen for 5 days before blood sampling.Collection of blood for measurements of plasma free metanephrines was performedprospectively in 819 of the 858 patients included in the final analyses. Inthe remaining patients, all with pheochromocytoma, measurements of plasmafree metanephrines were performed after removal of tumors. Twenty-four hoururine collections were obtained from 790 of the 858 patients included in thefinal analyses. Urine samples were usually subjected to assays of 2 to 3 differentanalytes.

Plasma was analyzed, using high-performance liquid chromatography (HPLC),for concentrations of catecholamines, norepinephrine and epinephrine, andfree metanephrines, normetanephrine, and metanephrine.^17-19Metanephrines in urine were analyzed as fractionated normetanephrine and metanephrineby HPLC or as total metanephrines by spectrophotometry.^10,20Urinary VMA was measured spectrophotometrically and urinary catecholaminesby HPLC.^21,22 Upper referencelimits for biochemical tests are provided as established by the principallaboratories responsible for each test (Table 2).¹¹

All 858 assays of plasma free metanephrines and 91% of the 855 assaysof plasma catecholamines were performed at the National Institutes of Health.Seventy-five percent of the 557 assays of urinary fractionated metanephrineswere performed by Quest Diagnostics (Collegeville, Pa). Seventy-two percentof the 710 assays of urinary catecholamines, 74% of the 297 assays of urinarytotal metanephrines, and 79% of the 616 assays of urinary VMA were performedby Mayo Medical Laboratories (Rochester, Minn). The remaining assays wereperformed at different laboratories depending on the center where patientswere evaluated.

For biochemical tests involving pairs of measurements (eg, normetanephrineand metanephrine or norepinephrine and epinephrine in plasma or urine), afalse-negative result in a patient with pheochromocytoma or a true-negativeresult in a patient without pheochromocytoma was defined as a value for eachmeasurement lower than the upper reference limit. A true-positive result forpairs of measurements in a patient with pheochromocytoma or a false-positiveresult in a patient without pheochromocytoma was defined as a value for eitheror both measurements equal to or higher than the appropriate upper referencelimit.

Sensitivity was calculated from the percentage of true-positive overthe total of true-positive plus false-negative test results in patients withpheochromocytoma. Specificity was calculated from the percentage of true-negativeover the total of true-negative plus false-positive test results in patientswithout pheochromocytoma. Differences in sensitivity and specificity wereexamined using the McNemar test and are illustrated using 95% confidence intervals(CIs).

For each analyte, a receiver operating characteristic (ROC) curve wasconstructed from the relationship between true-positive and false-positiveresults (ie, sensitivity vs 1 − specificity) at different upper referencelimits for each analyte.²³ As summary measuresof the diagnostic utility of each test, independent of upper reference limits,areas under ROC curves were calculated and differences among tests examinedaccording to the method by Hanley and McNeil.²⁴

Negative predictive values were calculated from the percentage of true-negativeover the total of true-negative plus false-negative test results. Positivepredictive values were calculated from the percentage of true-positive overthe total of true-positive plus false-positive results. Positive and negativepredictive values of each test were calculated at different prevalences ofpheochromocytoma to establish posttest probabilities of pheochromocytoma atdifferent pretest probabilities of the tumor.

Relative to patients in whom pheochromocytoma was excluded, median plasmaconcentrations of free metanephrines were increased by 7-fold in patientswith hereditary pheochromocytoma and 21-fold in patients with sporadic pheochromocytoma(Table 2). These increases wereconsistently larger than those of plasma norepinephrine (3-fold and 5-foldincreases), urinary norepinephrine (5-fold and 6-fold increases), urinaryfractionated normetanephrine (4-fold and 8-fold increases), urinary totalmetanephrines (3-fold and 7-fold increases), and urinary VMA (2-fold and 4-foldincreases). Increases in all analytes were larger in patients with sporadicrather than hereditary pheochromocytoma.

The sensitivities of diagnostic tests for detection of hereditary orsporadic pheochromocytoma ranged from a low of 46% (95% CI, 34%-59%) for useof urinary VMA in the detection of hereditary pheochromocytoma to a high of99% (95% CI, 96%-100%) for use of plasma free metanephrines in the detectionof sporadic pheochromocytoma (Table 3).

Among all patients with pheochromocytoma, sensitivities were the highestfor measurements of plasma free metanephrines at 99% (95% CI, 96%-100%), followedclosely by urinary fractionated metanephrines at 97% (95% CI, 92%-99%). Sensitivitiesof both the above tests considerably (P<.001)exceeded those for urinary catecholamines at 86% (95% CI, 80%-91%), plasmacatecholamines at 84% (95% CI, 78%-89%), urinary total metanephrines at 77%(95% CI, 68%-85%), and urinary VMA at 64% (95% CI, 55%-71%).

The above variations in sensitivities of diagnostic tests showed similarpatterns in patients with hereditary and sporadic pheochromocytoma (Table 3). Plasma free metanephrines andurinary fractionated metanephrines offered the highest sensitivities. Plasmaand urinary catecholamines had intermediate sensitivities. Urinary total metanephrinesand VMA consistently showed the lowest sensitivities in both groups of patients.The sensitivities of all tests were higher for detection of sporadic pheochromocytomathan for detection of hereditary pheochromocytoma.

Specificities of biochemical tests ranged widely from 45% (95% CI, 36%-51%)for urinary fractionated metanephrines in patients tested for sporadic pheochromocytomato 99% (95% CI, 98%-100%) for urinary VMA in patients tested for hereditarypheochromocytoma (Table 3).

Among all patients tested for pheochromocytoma, the highest specificitieswere 95% (95% CI, 93%-97%) for tests of urinary VMA and 93% (95% CI, 89%-97%)for tests of urinary total metanephrines. Specificities were intermediatefor tests of plasma free metanephrines at 89% (95% CI, 87%-92%), urinary catecholaminesat 88% (95% CI, 85%-91%), and plasma catecholamines at 81% (95% CI, 78%-84%),and lower than those of all other tests (P<.001)for urinary fractionated metanephrines at 69% (95% CI, 64%-72%).

In contrast to sensitivities, specificities of all tests were higherin patients tested for hereditary pheochromocytoma than for sporadic pheochromocytoma(Table 3). In both groups, urinaryVMA and total metanephrines offered the highest specificities and urinaryfractionated metanephrines the lowest specificities.

Only 2 of the 76 patients with hereditary pheochromocytoma and 1 ofthe 138 patients with sporadic pheochromocytoma had normal levels of plasmafree metanephrines. Both hereditary cases were in patients who were normotensiveand asymptomatic and had no other biochemical evidence of the tumor. Bothwere patients with von Hippel-Lindau syndrome and had single adrenal tumorsof less than 1 cm in diameter, which were identified and removed coincidentallyduring surgery for renal carcinoma.

The single false-negative result for plasma free metanephrines in patientswith sporadic pheochromocytoma involved a patient tested for possible tumorrecurrence 13 years after the removal of a large extra-adrenal pheochromocytoma.Computed tomography and all biochemical tests yielded negative results. Thepatient was subsequently diagnosed with metastatic pheochromocytoma 3½years later. Since there was no evidence for a hereditary basis for the patient'sdisease, it was presumed that the malignancy developed secondary to remainingdisease that went undetected for more than 16 years after the original tumorwas removed. Thus, despite the considerable time between biochemical testingand final diagnosis, all tests performed were designated as providing false-negativeresults.

Integrated comparison of sensitivity and specificity using ROC curvesshowed that the diagnostic power of plasma free metanephrines was superiorto that of all other tests (Figure 1).The areas under the ROC curves for plasma catecholamines (0.927), urinarycatecholamines (0.931), urinary total metanephrines (0.919), and urinary VMA(0.896) were all significantly lower (P<.001)than the area for plasma free metanephrines (0.985). Although closer, thearea under the ROC curve for urinary fractionated metanephrines (0.960) wasalso lower (P = .01) than that for plasma free metanephrines(0.985).

Areas under the ROC curves were only marginally improved when testsof urinary fractionated metanephrines were combined with those for urinarycatecholamines (0.965) or plasma catecholamines (0.969) or when tests of urinarytotal metanephrines and catecholamines were combined (0.949) (Figure 1). Thus, combining tests for different analytes did notimprove diagnostic efficacy beyond that of a single test of plasma free metanephrines.

True-positive rates (ie, test sensitivity) at higher upper referencelimits when false-positive rates were zero (ie, when test specificity equaled100%) were higher for plasma free metanephrines than for other tests (Figure 1). None of the 644 patients withoutpheochromocytoma had a plasma concentration of normetanephrine above 2.19nmol/L or of metanephrine above 1.20 nmol/L, whereas 79% of patients withpheochromocytoma had plasma concentrations of normetanephrine or metanephrineabove these levels (Table 4).

Negative predictive values of tests of plasma and urinary metanephrinesat different prevalences of pheochromocytoma showed that negative test resultsfor plasma free and urinary fractionated metanephrines provided the highestprobabilities for excluding pheochromocytoma at all pretest prevalences ofthe tumor (Figure 2). However, theposttest probability of a pheochromocytoma from a positive test result forplasma free metanephrines, although similar to that for urinary total metanephrines,was consistently higher than that from a positive test result for urinaryfractionated metanephrines at all pretest prevalences of the tumor.

The present examination of biochemical tests used in the diagnosis ofpheochromocytoma provides several advances over previous studies. First, thisstudy comprehensively compared measurements of plasma free metanephrines withall other commonly available biochemical tests used to diagnose excess catecholamineproduction. Second, these comparisons were made in large populations of patientswith and without pheochromocytoma, who were tested for the tumor because ofclinically appropriate predisposing conditions or suspicious symptoms andsigns. Finally, standard criteria that were independent of the biochemicaltests being compared were used to assign patients into groups with and withoutthe tumor.

The present study confirms the findings of several other reports thatmeasurements of plasma free metanephrines or urinary fractionated metanephrinesoffer higher sensitivity for diagnosis of pheochromocytoma than measurementsof plasma or urinary catecholamines or of urinary total metanephrines or VMA.^8,9,11,14,16Our comparisons further establish that among all tests, including urinaryfractionated metanephrines, measurements of plasma free metanephrines providethe best test for excluding or confirming pheochromocytoma.

Since measurements of urinary fractionated metanephrines and plasmafree metanephrines offer similarly high sensitivity, a negative result foreither test is equally effective for excluding pheochromocytoma. However,because urinary fractionated metanephrines have low specificity, tests ofplasma free metanephrines exclude pheochromocytoma in many more patients withoutthe tumor than do tests of urinary fractionated metanephrines.

The above considerations illustrate the importance of ROC curves forcomparing different tests. At equivalent levels of sensitivity, the specificityof plasma free metanephrines is higher than that of other tests. At equivalentlevels of specificity, the sensitivity of plasma free metanephrines is alsohigher than that of other tests, including urinary fractionated metanephrines.

To minimize the risk of missing a patient with pheochromocytoma, cliniciansoften use multiple biochemical tests during the initial diagnostic workupof patients with suspected tumors. Although this may increase sensitivity,it is at the cost of decreased specificity. Thus, tests involving pairs ofmeasurements, such as fractionated catecholamines or metanephrines, have lowerspecificity and higher sensitivity than tests involving single measurements,such as urinary VMA or total metanephrines (Table 3).⁸ As shown by ROC curves,the diagnostic utility of tests of plasma free metanephrines remains superiorto that of other tests even when the latter are used in combination.

The above considerations lead us to recommend against use of multiplebiochemical tests to exclude pheochromocytoma in favor of a single test ofplasma free metanephrines. In patients with negative test results for plasmafree metanephrines, indiscriminate use of extra tests is unlikely to improvediagnostic efficacy. If multiple biochemical tests have been run, the decisionto exclude pheochromocytoma should be based on whether plasma free metanephrinesshow a negative test result, regardless of whether other test results arepositive or negative.

Why do plasma free metanephrines provide the best test to diagnose pheochromocytoma?First, plasma free metanephrines are produced continuously by metabolism ofcatecholamines within pheochromocytoma tumor cells.^25-27This contrasts with episodic secretion of catecholamines. Second, sympathoadrenalexcitation causes large increases in catecholamine release, whereas plasmafree metanephrines remain relatively unaffected.^14,25,27,28Third, VMA and the total and fractionated metanephrines measured in urineare different metabolites from the free metanephrines measured in plasma,and are produced in different parts of the body by metabolic processes notdirectly related to the tumor itself.^28-30Urinary total and fractionated metanephrines are measured after a deconjugationstep and largely reflect levels of conjugated metanephrines that are producedoutside of tumor tissue. Similarly, VMA is produced mainly in the liver.

The lower sensitivity and higher specificity of biochemical tests forhereditary compared with sporadic pheochromocytoma reflect different reasonsfor testing in the 2 groups.³¹ Routine screeningfor pheochromocytoma in patients with a hereditary predisposition to the tumoroften leads to detection of small tumors that release catecholamines in amountsthat are insufficient to produce signs or symptoms of the tumor. In contrast,sporadic pheochromocytoma is typically suspected because of signs and symptomsof catecholamine excess, produced by larger more easily detected tumors thanfound by routine screening in hereditary pheochromocytoma. Moreover, patientstested for sporadic pheochromocytoma who do not have the tumor are often symptomaticof some condition associated with sympathoadrenal activation, leading to relativelyhigh numbers of false-positive results.

The consistently lower specificities of biochemical tests in patientstested for sporadic rather than for hereditary pheochromocytoma may also reflectreferral of patients in the former group with previously determined positivebiochemical tests. Thus, specificities of biochemical tests for detectionof sporadic pheochromocytoma in the present study are likely to be lower thanin unselected populations tested by commercial laboratories, but should reflectthose expected in populations tested at referral centers.

Apart from patients at risk for hereditary pheochromocytoma, patientswith previously resected tumors are another at-risk group who should be testedperiodically for the tumor regardless of signs and symptoms. The importanceof this group is illustrated by the single patient who tested negative forpheochromocytoma by all tests 3½ years before metastatic disease wasfinally diagnosed and 16 years after removal of the primary tumor. The sensitivityof plasma free metanephrines is not always sufficient for detection of microscopicrecurrent or metastatic disease or small tumors (<1 cm) in patients withhereditary pheochromocytoma.

Typically fewer than 1% of hypertensive patients tested for pheochromocytomahave the tumor. In some patient groups, such as those with hypertension andan adrenal mass, the pretest probability of a pheochromocytoma may be higher.The probability that a negative test result excludes pheochromocytoma or thata positive test result confirms the tumor depends in part on the pretest probabilityof the disease. These posttest probabilities therefore require calculationof positive and negative predictive values at different pretest probabilities(prevalences) of the tumor.

As shown in Figure 2, a negativetest result for plasma free metanephrines or urinary fractionated metanephrinesprovides a high probability of excluding pheochromocytoma at all clinicallyrelevant pretest probabilities of the tumor. In contrast, at the typicallylow prevalences of pheochromocytoma, the likelihood of the tumor after a singlepositive test remains low even for tests with up to 95% specificity. A routinepractice to further increase or decrease the likelihood of pheochromocytomainvolves use of additional biochemical tests.³²

In patients with positive results for an initial test of plasma freemetanephrines, extra tests can be useful, but judging the likelihood of apheochromocytoma should first take into account results of ROC curves. Inparticular, at higher upper reference limits, in which test specificity is100%, plasma concentrations of free normetanephrine higher than 2.19 nmol/Lor of free metanephrine higher than 1.20 nmol/L unequivocally confirm a pheochromocytomain 79% of patients with the tumor (Table4), a higher proportion than for other tests (Figure 1). The probability of pheochromocytoma in these patientsis so high that further biochemical tests to confirm the tumor may be unnecessary.In the remaining patients, in whom increased levels are insufficient to unequivocallyconfirm a tumor, additional judiciously selected follow-up tests are appropriate,with additional attention focused on possible causes of false-positive testresults.^32-35

Because of the low prevalence of pheochromocytoma in the patient groupsusually tested for the tumor, false-positive results can be expected to outnumbertrue-positive results for all biochemical tests, including plasma free metanephrines.There are 3 potential sources of false-positive test results: diet, drugs,and stressors.

Caffeic acid, a catechol found in coffee (including decaffeinated coffee),and its derivative dihydrocaffeic acid are dietary substances known to interferewith assays of plasma catecholamines.³⁶ Moreover,both catechols are excellent substrates for catechol O-methyltransferase,the enzyme that converts catecholamines to metanephrines, and this easilycould affect plasma levels of metanephrines. There are many other unidentifieddietary constituents that can influence the results of HPLC assays. The simplestway to avoid these sources of false-positive results is by drawing blood samplesin patients who have fasted.

Acetaminophen is the only direct source of interference with assaysof plasma free metanephrines that we have identified to date.¹⁹However, caffeine and nicotine both increase plasma levels of catecholaminesand should also be avoided. In our series, treatment with tricyclic antidepressantsor phenoxybenzamine (dibenzyline) were major causes of false-positive testresults for norepinephrine and its metabolites, presumably due to presynapticactions on sympathetic nerves. Phenoxybenzamine, a nonselective α-adrenoceptorblocker commonly used to treat patients with pheochromocytoma, can be particularlytroublesome.

Although plasma levels of free metanephrines are less sensitive to changesin sympathoadrenal activity than are levels of the parent amines, these metabolitesare nevertheless influenced by many of the same stimuli and drugs that influenceplasma catecholamines.^25-28Upright posture and emotional stress are well-known to stimulate release ofcatecholamines from sympathetic nerves and the adrenal medulla. To minimizethe possibility of false-positive test results, we collected blood samplesfor plasma free metanephrines under the same conditions used for collectionof samples for measurements of plasma catecholamines. Blood samples were drawnwith patients in the supine position, through an in-dwelling intravenous catheter,and after an overnight fast.

The major strength of this study—all patients were examined becauseof clinical suspicion of pheochromocytoma—was associated with the limitationthat exclusion of pheochromocytoma required use of methods other than thebiochemical tests normally used in clinical practice. Although computed tomographyand magnetic resonance imaging offer high sensitivity for detecting adrenaltumors, sensitivity decreases for detecting extra-adrenal disease. We thereforefollowed up patients for an average of 2.5 years to further exclude pheochromocytomain patients with negative imaging. Only 1 patient in whom pheochromocytomawas initially excluded by imaging studies was subsequently found to have thedisease at follow-up. It remains possible, however, that other patients inwhom pheochromocytoma was excluded according to the criteria of our studymay have had undiagnosed disease. Even so and unless these numbers were large,it is unlikely that incorrect categorization of these patients would makea significant difference to the results and conclusions of the study.

A related potential limitation of the study was the need to omit fromthe analyses 145 patients who did not meet the research criteria for exclusionor confirmation of pheochromocytoma. Separate analyses of how inclusion ofthe data from these patients would affect test performance revealed littleinfluence on the results and conclusions of the study.

Another potential limitation of the study involved the multicenter natureof patient recruitment and subsequent measurements of urinary analytes bydifferent laboratories compared with the single laboratory used for plasmafree metanephrines. However, separate analysis of data derived from singlelaboratories revealed no obvious influences. Thus, rather than a limitation,the multicenter nature of the study is a strength establishing that many ofthe findings (eg, low specificity of urinary fractionated metanephrines, lowsensitivity of urinary VMA) were independent of the laboratory where testswere run.

Plasma free metanephrines constitute the best test for excluding orconfirming pheochromocytoma and should be the test of first choice for diagnosisof the tumor. A negative test result virtually excludes pheochromocytoma.In such patients, representing more than 80% of those tested, no immediatefurther tests for the tumor are necessary. Furthermore, in about 80% of patientswith pheochromocytoma, the magnitude of increase in plasma free metanephrinesis so large that the tumor can be confirmed with close to 100% probability.In these patients, the immediate task is to locate the tumor.