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Assessing the Best Sampling Procedures for PPGL

Study Finds Supine, Fasting Conditions With Corresponding Cutoffs Yields Best Results

By Genna Rollins

Measurement of plasma-free metanephrines to detect pheochromocytoma and paraganglioma (PPGL) has been shown to have higher sensitivity than other tests for PPGL. However, many pre-analytical factors, including blood sampling conditions, affect the results. Researchers recently compared the diagnostic performance of plasma-free metanephrine testing under supine and fasting versus seated and nonfasting sampling conditions, and their findings are the subject of this issue of Strategies.

PPGL tumors arise from the adrenomedullary or extra-adrenal chromaffin cells and release excess amounts of the catecholamines epinephrine, norepinephrine, and dopamine. PPGL causes hypertension and related symptoms such as headaches, sweatiness, and palpitations. Without treatment, PPGL can lead to cardiovascular-related morbidity and mortality; a minority of PPGL become malignant.

Standard diagnostic testing for PPGL involves either plasma-free or urinary fractionated metanephrines, the O-methylated metabolites of norepinephrine and epinephrine. Evidence has shown plasma-free metanephrine testing to have higher sensitivity than other methods. However, the catecholamines and their metabolites are formed from cells of the autonomic nervous system, which is activated by posture changes and even certain foods, so without adherence to exacting sampling protocols, spurious results can occur.

“A very small proportion of patients actually are found with the tumors compared to the number of patients who are tested, and you really want to be clear that when you have a patient with the tumor, the test is positive,” explained Graeme Eisenhofer, PhD, professor and chief of clinical neurochemistry at University Hospital in Dresden, Germany. “Correctly excluding tumors based on negative results is important because then you don’t have to do any other expensive testing. And you want to catch PPGLs because if they’re missed the patient often goes away for several years, and lo-and-behold has an episode and rolls over dead because of hypertensive crisis.”

Given the complicated sampling conditions for plasma-free metanephrine testing, some labs offer urinary-fractioned testing which is less-sensitive but requires less rigorous sampling procedures. Others offer plasma-free metanephrine testing, but do so without obtaining supine or fasting samples, and by using cutoffs based on seated samples, according to Eisenhofer.

Many labs have been slow to implement plasma-free metanephrine testing under supine and fasting sample conditions, along with appropriate cutoffs, added Eisenhofer. “Almost all laboratories in the United States and Europe have set up their methods using seated upper cutoffs, and it takes time, energy, and effort to change one’s reference intervals,” he explained. “It’s also difficult at many testing centers to do these samplings in the supine position, because of the way the phlebotomy centers are set up. Basically, [it boils down to] the setup of the infrastructure, which dictates sampling in the seated position as the most practical method.”

Lingering questions about the impact of fasting and supine versus non-fasting and seated sampling for PPGL testing led Eisenhofer and his colleagues to study and document differences between the methodologies (Clin Endocrinol 2014;80:478–86). Their study involved 762 patients undergoing evaluation for PPGL at six European centers. This investigation took advantage of a multicenter trial already in progress, in which two of four participating centers initially did not follow supine and fasting blood sampling procedures. Standard operating procedures for the study required patients to fast overnight and refrain from both caffeinated and decaffeinated beverages. Labs were asked to obtain samples after patients had been supine for 30 minutes, drawing the samples from a previously inserted intravenous cannula. In the centers that did not adhere to these procedures, patients were either seated or in a semi-recumbent position during blood draws via needlestick and might not have fasted.

Overall, 129 patients were diagnosed with PPGL, including 62 under compliant conditions and 67 under noncompliant conditions. The researchers found that in patients with PPGL, plasma concentrations of normetanephrine and methoxytyramine did not differ between compliant and noncompliant sampling conditions, but they were approximately 50% higher in patients without PPGL when they were sampled under noncompliant conditions. Upper cutoffs established from noncompliant sampling lowered diagnostic sensitivity from 98% to 85%, whereas upper cutoffs established from compliant sampling conditions but used with noncompliant conditions decreased specificity from 95% to 71.3%.

The researchers concluded that “high diagnostic sensitivity of plasma normetanephrine, metanephrine, and methoxytyramine for the detection of PPGL can only be guaranteed using upper cutoffs of reference intervals established with blood sampling under supine fasting conditions.”

Eisenhofer suggested that the common practices of seated sampling along with inappropriate cutoffs not only increases the number of false-positive results, but also leads to a kind of false-positive fatigue. “There have already been some studies showing that what typically happens is that when there are too many false positive results, clinicians ignore those false positive results and don’t even follow up. They become complacent,” he said.

Stefan Grebe, MD, PhD, a professor of laboratory medicine and pathology at the Mayo Clinic in Rochester, Minnesota, agreed that because of the rarity of PPGL, physicians who specialize in the diagnosis and treatment of this disorder are likely to see many false-positives for every true positive. He also emphasized the importance of following up on any positive results, and suggested that specialty practices are likely to send patients with positive plasma results directly for follow-up imaging. However, he argued that other clinical settings might take a different approach based on Eisenhofer’s findings.

“If you’re in a lab serving a primary care constituency, then you’re probably best off recommending to your referring physicians fractionated urine metanephrine testing if you can’t be compliant with supine and fasting sampling conditions,” said Grebe.

Eisenhofer emphasized that labs should take a close look at their metanephrine cutoffs. “If a lab is measuring plasma-free metanephrines, they should rethink their reference interval cutoffs, and establish them in the supine position,” he advised. Eisenhofer’s own lab has taken things a step further by developing age-adjusted cutoffs. “We’re now doing personalized upper cutoffs. This really is terrific for keeping the diagnostic specificity high, thereby reducing false-positives,” he observed.

A newly released guideline from the Endocrine Society developed in cooperation with AACC recommends initial biochemical testing for PPGL with either plasma-free or urinary fractionated metanephrines. The guideline emphasizes that plasma metanephrine samples should be drawn from patients in the supine position and use reference intervals established from the supine position. Both Eisenhofer and Grebe served on this guideline committee, with Grebe the official AACC representative.

Eisenhofer’s study along with the Endocrine Society guideline already is prompting some institutions to reconsider their procedures. One example is Hamilton Health Services in Hamilton, Ontario, Canada. “Up until now, we’ve not been convinced that the blood testing is better than the urine testing, but I’m certainly going to discuss this with colleagues here,” said Andrew Don-Wauchope, MB BCh, MD, a consultant medical biochemist and associate professor of pathology and molecular medicine. “This is something that I would pay attention to, and we might start looking at changing our practice.”

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