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Short-term stability of free metanephrines in plasma and whole blood
Clinical Chemistry and Laboratory Medicine
Abstract
Background. Analysis of plasma metanephrine (MN) and normetanephrine (NMN) with liquid chromatography tandem mass spectrometry (LC-MS/MS) is the gold standard for the screening of pheochromocytomas and paragangliomas (PPGLs). As scarce information is available on the stability of MNs in diagnostic samples, this study was aimed at analyzing the short-term stability of plasma free MNs in whole blood and plasma, using LC-MS/MS.
Methods. The stability of plasma MNs was evaluated after sample collection at 1, 2 and 3 h in whole blood, and at 2, 4 and 6 h in centrifuged samples. Both studies were performed while maintaining the samples at room temperature (RT) and at 4 °C. The ClinMass Complete Kit (Recipe, Munchen, Germany) was used for measuring MNs with LC-MS/MS (Nexera X2 UHPLC-4500MD Sciex). Differences from the baseline (T0) were assessed using repeated measures one-way ANOVA, Students’ paired t-test and a comparison of the mean percentage changes with the total change limit (TCL).
Results. Statistically significant differences from T0 were found for both MNs (p < 0.001) in whole blood stored at RT, and for NMN (p = 0.028) but not MN (p = 0.220) at 4 °C. The mean difference exceeded the TCL after 1 h and 3 h at RT for MN, and after 1 h at RT for NMN. Statistically significant differences from T0 were only observed in the plasma samples for NMN at RT (p = 0.012), but the variation was within the TCL.
Conclusions. MN and NMN displayed different patterns of stability before and after centrifugation. Even short-time storage at RT in whole blood should hence be avoided.
... In addition, several reports showed that plasma catecholamines and metanephrines varied in their concentrations, depending on the seasons [8][9][10][11]. Other studies showed that the stability of catecholamines and the metabolites in whole blood and plasma is a function of storage temperature and time, as well as the conditions for centrifuging the blood [12][13][14][15]. Among all the preanalytical factors, blood collection tubes are considered to constitute a critical parameter that affects the matrix properties of the specimens, which may in turn affect the performance of assays and the reliability of their results [16][17][18]. ...
Background
Catecholamines (epinephrine; norepinephrine; and dopamine) and their O‐methylated metabolites (metanephrine; normetanephrine; and 3‐methoxytyramine) are biomarkers for pheochromocytoma and paraganglioma. Liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) was recommended by Endocrine Society for detecting these compounds. The influence of blood collection tubes on the analysis of the six analytes by LC‐MS/MS was not thoroughly investigated, which we want to clarify in our study.
Methods
Blood samples of healthy individuals were collected into serum, lithium heparin, and K2EDTA plasma tubes separately. Samples were subjected to solid phase extraction and then analyzed by LC‐MS/MS. The retention behavior and assay performance of the six analytes were assessed for samples from different collection containers. The impacts of potassium and sodium as the counter ions of EDTA on the retention time and matrix effect were also studied.
Results
Compared with O‐methylated metabolites, the results for catecholamines were more affected by the collection tubes, especially for norepinephrine, which displayed severely suppressed signal and very low extraction efficiency in K2EDTA plasma. Changing the counter ion of EDTA from potassium to sodium dramatically changed the retention behavior and matrix effect of norepinephrine.
Conclusions
It is necessary to evaluate blood collection tubes for catecholamines and their O‐methylated metabolites analyzed by LC‐MS/MS. In addition, attention should also be paid when the anticoagulant counter ion was changed.
... Plasma metadrenaline (also called metanephrine) analysis has slightly improved sensitivity and specificity than 24-hour urine metadrenaline excretion analysis for aiding the diagnosis of phaeochromocytoma and paraganglioma (60). However, 24-hour urine analysis is easier to complete in outpatient and primary care settings (62), so is usually the first test of choice in this context. ...
Background and Objective: The following article is part of a special series to aid the reader in diagnosing the cause of various electrolyte imbalances. By the end of the article, the reader should be able to order and interpret appropriate laboratory investigations when faced with a patient with hypercalcaemia or hypocalcaemia.
Methods: A narrative, focused literature review was performed using Medline, PubMed, Google Scholar and OMIM during August 2023 to November 2023 to identify references published from database inception. Reference lists from these articles, as well as expert opinion from the authors, were also used. Language was restricted to English.
Key Content and Findings: Calcium is an essential electrolyte and its blood concentration is tightly controlled by multiple homeostatic hormones, including parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D. However, calcium imbalance can occur in many disease states and can lead to significant morbidity and mortality. PTH analysis is of primary importance in identifying the cause of a calcium disturbance. It should be noted that calcium results are particularly susceptible to being falsely low or high depending on whether ionised or total calcium is analysed, therefore the validity of calcium results should always be questioned if the results are not consistent with the clinical presentation. Hypercalcaemia is most commonly associated with primary hyperparathyroidism (PHPT) or malignancy. Hypocalcaemia is much rarer than hypercalcaemia, and if no obvious cause is found—e.g., post total thyroidectomy—consider hypomagnesaemia.
Conclusions: Diagnostic flow charts are presented, and the limitations of the laboratory tests discussed. These algorithms, by focusing on the approach to the investigation of hypercalcaemia and hypocalcaemia, should support healthcare professionals to efficiently and rapidly diagnose the majority of causes of abnormal calcium states.
... It is usually used as a method to define the CRC stage. In patients with early CRC stages between 0-I, the range could be from 57% to 64% [34]. The mSEPT9 positivity rate increased in advanced CRC cases [26]. ...
Worldwide, colon cancer is the third most frequent malignancy and the second most common cause of death. Although it can strike anybody at any age, colon cancer mostly affects the elderly. Small, non-cancerous cell clusters inside the colon, commonly known as polyps, are typically where colon cancer growth starts. But over time, if left untreated, these benign polyps may develop into malignant tissues and develop into colon cancer. For the diagnosis of colon cancer, with routine inspection of the colon region for polyps, several techniques, including colonoscopy and cancer scanning, are used. In the case identifying the polyps in the colon area, efforts are being taken to surgically remove the polyps as quickly as possible before they become malignant. If the polyps become malignant, then colon cancer treatment strategies, such as surgery, chemotherapy, targeted therapy, and immunotherapy, are applied to the patients. Despite the recent improvements in diagnosis and prognosis, the treatment of colorectal cancer (CRC) remains a challenging task. The objective of this review was to discuss how CRC is initiated, and its various developmental stages, pathophysiology, and risk factors, and also to explore the current state of colorectal cancer diagnosis and treatment, as well as recent advancements in the field, such as new screening methods and targeted therapies. We examined the limitations of current methods and discussed the ongoing need for research and development in this area. While this topic may be serious and complex, we hope to engage and inform our audience on this important issue.
... Metanephrines are not stable in whole blood at room temperature due to the ongoing action of COMT in blood cells (converting the catecholamines to their methylated metabolites) but also due to degradation. It is possible to observe either falsely elevated or falsely low results due to this mixture of effects on stability [76,77]. Blood samples for plasma metanephrines should therefore be separated as soon as possible after collection and ideally kept on ice before and during centrifugation. ...
Phaeochromocytomas (PC) and sympathetic paragangliomas (PGL) are potentially malignant tumours arising from the adrenal medulla (PC) or elsewhere in the sympathetic nervous system (PGL). These tumours usually secrete catecholamines and are associated with significant morbidity and mortality, so accurate and timely diagnosis is essential. The initial diagnosis of phaeochromocytoma/paraganglioma (PPGL) is often dependent on biochemical testing. There is a range of pre-analytical, analytical and post-analytical factors influencing the analytical and diagnostic performance of biochemical tests for PPGL. Pre-analytical factors include patient preparation, sample handling and choice of test. Analytical factors include choice of methodology and the potential for analytical interference from medications and other compounds. Important factors in the post-analytical phase include provision of appropriate reference ranges, an understanding of the potential effects of various medications on metanephrine concentrations in urine and plasma and a consideration of PPGL prevalence in the patient population being tested. This article reviews these pre-analytical, analytical and post-analytical factors that must be understood in order to provide effective laboratory services for biochemical testing in the diagnosis of PPGL.
... The current clinical guidelines for the sampling of venous blood for the quantification of plasma MNs recommend rest in a supine position for approximately 30 min [2], which is time consuming and hard to realize under typical hospital test centre conditions. Another important consideration in favour of DBS sample collection for the analysis of metanephrines is that DBS samples may actually be more stable for short term storage than whole blood which is often stored at room temperature for extended periods of time prior to centrifugation (for plasma or serum) and either analysis or storage e which is known to lead to bias in the measurement of these analytes [3,4]. ...
The quantitation of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3MT) – referred to as metanephrines -- by LC-MS/MS is the gold-standard for screening for pheochromocytoma and paragangliomas (PPGLs), tumors of the adrenal gland and the peripheral nervous system. An assay for metanephrines from dried blood spots (DBSs) would be of high clinical utility as it simplifies sample collection, enables remote sampling, and could increase compliance with the clinical recommendation for supine sampling. Moreover, DBS sampling facilitates the measurement of blood-derived metanephrines in pediatric patients – where DBSs are well-established – in order to diagnose neuroblastomas.
Here, we adapted an established derivatization-based LC-MRM-MS assay for plasma catecholamines, and optimized the sample extraction, LC, and MS parameters to produce a fast, sensitive, and robust method for the measurement of metanephrines from DBSs, including 3-methoxytyramine. The DBS samples were excised, derivatized with phenyl isothiocyanate (PITC) on-spot, extracted, and measured by LC-MRM-MS. To validate assay suitability and performance, we assessed the linearity, precision, accuracy, recovery, and matrix effects of the method, and determined the stability of metanephrines in DBSs under different storage conditions. Assay performance for NMN, MN, and 3MT was sufficient for quantitation from a single DBS within a linear range from 40 – 2000 pg/mL MN and NMN were stable in DBSs for 2 weeks, whereas 3MT was stable for one week regardless of storage temperature. Altogether, this work represents the first quantitative LC-MS/MS method for metanephrines from DBSs and provides a novel opportunity for the diagnosis of PPGLs and neuroblastomas in the future.
... Thereafter, the focus shifts on to a single metabolite, trimethylamine N-oxide (or TMAO), to better understand its characteristics and to establish reference values across a generalized, disease-free population [16]. From here, we venture into steroid [17] and hormone [18] analyses, specifically the stability of parent and metabolite molecules within biofluid samples. The studies presented highlight important information regarding the need to correctly process and/or store samples in the hours following collection. ...
Pheochromocytoma and paraganglioma (PPGL) require prompt consideration and efficient diagnosis and treatment to minimize associated morbidity and mortality. Once considered, appropriate biochemical testing is key to diagnosis. Advances in understanding catecholamine metabolism clarified why measurements of the O-methylated catecholamine metabolites rather than the catecholamines themselves are important for effective diagnosis. These metabolites, normetanephrine and metanephrine, produced respectively from norepinephrine and epinephrine, can be measured in plasma or urine, with choice according to available methods or presentation of patients. For patients with signs and symptoms of catecholamine excess, either test will invariably establish the diagnosis, whereas the plasma test provides higher sensitivity than urinary metanephrines for patients screened due to an incidentaloma or genetic predisposition, particularly for small tumors or in patients with an asymptomatic presentation. Additional measurements of plasma methoxytyramine can be important for some tumors, such as paragangliomas and for surveillance of patients at risk of metastatic disease. Avoidance of false-positive test results is best achieved by plasma measurements with appropriate reference intervals and preanalytical precautions, including sampling blood in the fully supine position. Follow-up of positive results, including optimization of preanalytics for repeat tests or whether to proceed directly to anatomic imaging or confirmatory clonidine tests, depends on the nature of test results, which can also suggest likely size, adrenal versus extra-adrenal location, underlying biology or even metastatic involvement of a suspected tumor. Modern biochemical testing now makes diagnosis of PPGL relatively simple. Integration of artificial intelligence into the process should make it possible to fine-tune these advances.
Background
Phaeochromocytomas and paragangliomas (PPGL) are catecholamine secreting tumours associated with significant morbidity and mortality. Timely diagnosis and management are essential. A range of laboratory tests can be utilised in the investigation of PPGL. There is scope for significant variation in practice between centres. We aimed to investigate how the laboratory investigation of PPGL is performed in laboratories across the United Kingdom.
Methods
A questionnaire consisting of 21 questions was circulated to Clinical Biochemistry laboratories in the United Kingdom via the Association for Clinical Biochemistry and Laboratory Medicine office. The survey was designed to allow audit against Endocrine Society Guidelines on the Investigation and Management of PPGL and to obtain information on other important aspects not included in these guidelines.
Results
Responses were received from 58 laboratories and the data were compiled. The majority of laboratories use either urine or plasma metanephrines in first-line testing for PPGL, although a number of different combinations of biochemistry tests are utilised in different centres. All laboratories measuring metanephrines or catecholamines in-house use LC or LC-MS/MS methods. There are some marked differences between laboratories in urine metanephrines reference ranges used and sample requirements.
Conclusions
There is evidence of good practice in UK laboratories (as assessed against Endocrine Society Guidelines) such as widespread use of urine/plasma metanephrines and appropriate analytical methodologies used. However, there is also evidence of variations in practice in some areas that should be addressed.
Background
Accurate diagnosis of pheochromocytoma and paraganglioma (PPGLs) is highly dependent on the detection of metanephrines and catecholamines. However, the systematic investigation on influencing factors including specimen (plasma or whole blood), anticoagulant, storage conditions, and interference factors need further confirmation.
Methods
Blood with heparin-lithium or EDTA-K2 were collected, stability of epinephrine (EPI), norepinephrine (NE), dopamine (DA), metanephrine (MN), normetanephrine (NMN), 3-methoxytyramine (3-MT) in whole blood and plasma at room temperature and 4 °C for different storage times, stability of plasma MN, NMN and 3-MT at −20 °C and −80 °C were investigated. Plasma with hemoglobin (1 g/L, 2 g/L, 3 g/L, 4 g/L, 6 g/L), TG (<5 mmol/L, 5–8 mmol/L, >8 mmol/L) were prepared.
Results
EPI, NE, DA were prone to degrade at room temperature, samples should be centrifuged at 4 °C. EPI and NE were stable in whole blood at 4 °C for 4 h and in plasma for 2 h. For MN, NMN, 3-MT, plasma can be stable at room temperature and 4 °C for at least 6 h, which is better than whole blood; there was no significant difference when stored at −20 °C and −80 °C for 7 days. Heparin-lithium had a slight advantage over EDTA-K2. EPI, NE, DA should not be performed when Hb > 1 g/L or TG > 5 mmol/L. MN, NMN, 3-MT should not be performed when Hb > 2 g/L, whereas TG had no interference.
Conclusions
According to the actual clinical application scenario, this study provided a reliable basis for the accurate diagnosis of PPGLs.
Background
Measurement of metanephrines (MNs: metanephrine [MN] and normetanephrine [NMN]) is recommended for the initial biochemical diagnosis of pheochromocytoma and paraganglioma. Despite some drawbacks, plasma is commonly used for sampling. Here, we determined the feasibility of using serum, as an alternative to plasma, by comparing MNs in plasma and serum and evaluating the stability of MNs in serum. MNs obtained from serum, EDTA plasma, and heparin plasma were measured using LC-MS/MS immediately or after storage at 4 °C for 24 h, 72 h, and 7 days, and at −80 °C for 7 days, after sample collection. The differences between sample stability at given time points were compared using one-way ANOVA and Students’ paired t-test, and the mean percent deviation was compared with total change limit (TCL). No significant difference was observed in MN and NMN between serum and EDTA plasma, and the mean percent deviation of the results obtained from serum compared to that from EDTA plasma was within the TCL. However, the difference of MN between EDTA plasma and heparin plasma exceeded the TCL. Both MNs in EDTA plasma and heparin plasma showed a significant decreasing trend at 4 °C with time (p < .01), while those in serum were relatively stable, with the mean percent deviation not exceeding the TCL at any time point or temperature. In conclusion, MNs measurement did not significantly differ between EDTA plasma and serum when measured immediately after collection, and MNs in serum were more stable than that in plasma.
Background: Plasma or urinary metanephrines are recommended for screening of pheochromocytomas and paragangliomas (PPGLs). Measurements of urinary free rather than deconjugated metanephrines and additional measurements of methoxytyramine represent other developments. For all measurements there is need for reference intervals. Methods: Plasma free, urinary free and urinary deconjugated O-methylated catecholamine metabolites were measured by LC-MS/MS in specimens from 590 hypertensives and normotensives. Reference intervals were optimized using data from 2,056 patients tested for PPGLs. Results: Multivariate analyses, correcting for age and body surface area, indicated higher plasma and urinary metanephrine in males than females and sex differences in urinary normetanephrine and free methoxytyramine that largely reflected body size variation. There were positive associations of age with plasma metabolites, but negative relationships with urinary free metanephrine and methoxytyramine. Plasma and urinary normetanephrine were higher in hypertensives than normotensives, but differences were small. Optimization of reference intervals using the data from patients tested for PPGLs indicated that age was the most important consideration for plasma normetanephrine and sex most practical for urinary metabolites. Conclusion: This study clarifies impacts of demographic and anthropometric variables on catecholamine metabolites, verifies use of age-specific reference intervals for plasma normetanephrine and establishes sex-specific reference intervals for urinary metabolites.
Background
The sympatho-adrenergic activation during exercise is implicated in many cardiovascular respiratory and metabolic adaptations which have been thought to partially explain the different levels of performance observed between trained and untrained subjects. To date, no evidence exists about the association between competition performance and markers of “acute stress response”. We designed this study to investigate; (i) the acute sympatho-adrenergic activation during endurance exercise in recreational runners by measuring plasma levels of free metanephrine (MN) and normethanephrine (NMN) before and after a half-marathon run; (ii) the association between the metanephrines levels and the running time.
Methods
26 amateur runners (15 males, 11 females) aged 30 to 63 years were enrolled. The quantification of MN and NMN was performed by LC-MS/MS. Anthropometric ergonomic and routine laboratory data were recorded. Statistical analyses included paired T-test, univariate and multivariate regressions.
Results
The post-run values of MN and NMN displayed a nearly 3.5 and 7 fold increase respectively compared to the baseline values (p < 0.0001 for both). NMN pre-run values and pre/post run delta values showed a significant direct and inverse association (p = 0.021 and p = 0.033, respectively) with running performance. No correlations were found for MN values.
Conclusion
NMN is a reliable marker of sympatho-adrenergic activation by exercise and can predict endurance performance in the individual athlete. Adaptation phenomenon occurring not only in the adrenal medulla might represent the biological mechanism underlying this association. Further studies on sympatho-adrenergic activation, competition performance and training status should contemplate the measurement of these metabolites instead of their unstable precursors.
Background
Blood specimens are transported from clinical departments to the biochemistry laboratory by hospital courier service, sometimes over long distances. The aim of this study was to assess the stability of common biochemical analytes in venous blood under our routine transport conditions and to evaluate analyte stability after prompt or delayed centrifugation.
Methods
We investigated pre- and postanalytical contributions of 32 biochemical analytes in plasma and serum samples from 10 patients (healthy adults and patients from intensive care units). Differences in analyte concentrations between baseline (T0) and different time intervals (2, 4, 6, 8, 12 and 24 h) following storage after prompt and delayed centrifugation were reported. Evaluation was against the total change limit as described by Oddoze et al. (Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma. Clin Biochem 2012;45:464–9).
Results
The majority of analytes were stable with delayed separation up to 12 h, except for potassium, C-peptide, osteocalcin, parathyroid hormone (PTH), bicarbonate and LDH. After prompt centrifugation and storage at 4°C, stability was greatly increased up to 48 h for most analytes. LDH and bicarbonate had the lowest stability after centrifugation; therefore, no reanalysis of these analytes in a centrifuged tube can be allowed.
Conclusions
Knowledge of analyte stability is crucial to interpret biological analysis with confidence. However, centrifugation prior to transport is time consuming, and the transfer of plasma or serum from a primary tube to a secondary tube increases the risk of preanalytical errors. For analytes that are stable in whole blood for 24 h or more, it seems that there is no benefit to centrifuge before transport.
Despite all technical progress in modern diagnostic methods and treatment modalities of pheochromocytoma/paraganglioma, early consideration of the presence of these tumors remains the pivotal link towards the best possible outcome for patients. A timely diagnosis and proper treatment can prevent the wide variety of potentially catastrophic cardiovascular complications. Modern biochemical testing should include tests that offer the best available diagnostic performance, measurements of metanephrines and 3-methoxytyramine in plasma or urine. To minimize false-positive test results particular attention should be paid to pre-analytical sampling conditions. In addition to anatomical imaging by computed tomography (CT) or magnetic resonance imaging, new promising functional imaging modalities of photon emission tomography/CT using with somatostatin analogues such as ⁶⁸Ga-DOTATATE (⁶⁸Ga-labeled DOTA(0)-Tyr(3)-octreotide) will probably replace ¹²³I-MIBG (iodine-123-metaiodobenzylguanidine) in the near future. As nearly half of all pheochromocytoma patients harbor a mutation in one of the 14 tumor susceptibility genes, genetic testing and counseling should at least be considered in all patients with a proven tumor. Post-surgical annual follow-up of patients by measurements of plasma or urinary metanephrines should last for at least 10 years for timely detection of recurrent or metastatic disease. Patients with a high risk for recurrence or metastatic disease (paraganglioma, young age, multiple or large tumors, genetic background) should be followed up lifelong.
PurposeTo determine the accuracy of biochemical tests for the diagnosis of pheochromocytoma and paraganglioma. MethodsA search of the PubMed database was conducted for English-language articles published between October 1958 and December 2016 on the biochemical diagnosis of pheochromocytoma and paraganglioma using immunoassay methods or high-performance liquid chromatography with coulometric/electrochemical or tandem mass spectrometric detection for measurement of fractionated metanephrines in 24-h urine collections or plasma-free metanephrines obtained under seated or supine blood sampling conditions. ResultsApplication of the Standards for Reporting of Diagnostic Studies Accuracy Group criteria yielded 23 suitable articles. Summary receiver operating characteristic analysis revealed sensitivities/specificities of 94/93% and 91/93% for measurement of plasma-free metanephrines and urinary fractionated metanephrines using high-performance liquid chromatography or immunoassay methods, respectively. Partial areas under the curve were 0.947 vs. 0.911. Irrespective of the analytical method, sensitivity was significantly higher for supine compared with seated sampling, 95 vs. 89% (p < 0.02), while specificity was significantly higher for supine sampling compared with 24-h urine, 95 vs. 90% (p < 0.03). Partial areas under the curve were 0.942, 0.913, and 0.932 for supine sampling, seated sampling, and urine. Test accuracy increased linearly from 90 to 93% for 24-h urine at prevalence rates of 0.0–1.0, decreased linearly from 94 to 89% for seated sampling and was constant at 95% for supine conditions. Conclusions
Current tests for the biochemical diagnosis of pheochromocytoma and paraganglioma show excellent diagnostic accuracy. Supine sampling conditions and measurement of plasma-free metanephrines using high-performance liquid chromatography with coulometric/electrochemical or tandem mass spectrometric detection provides the highest accuracy at all prevalence rates.
There is substantial evidence that plasma concentrations of the free (unconjugated) metanephrines metanephrine (MN) and normetanephrine (NMN) are better than other indices of catecholamine excess for detecting pheochromocytomas (1)(2)(3). However, it currently is unknown how stable these compounds are after blood collection and after separation of plasma as well as during storage. To investigate this, we modified the original method by Lenders et al. (4), which consists of HPLC with electrochemical detection, preceded by a prepurification step on cation-exchange columns, to increase the procedural recovery and thereby sensitivity. The principal adjustments were as follows:
Before plasma was passed through the cation-exchange column, 1 mL of Aqua Dest and 135 μL of a solution of 0.2 mol/L ammonium acetate (pH 6.0) were added to 1 mL of plasma. The 135 μL of ammonium acetate solution included 100 μL of internal standard solution [108.7 nmol/L 3-ethoxy-4-hydroxyphenylethanolamine oxalate (EHPEA)] and 35 μL of ammonium acetate containing, only for addition experiments, MN and NMN. The calibrator mixture consisted of 19.5 nmol/L MN, 18.1 nmol/L NMN, and 108.7 nmol/L EHPEA in the aforementioned ammonium acetate solution, of which 140 μL was injected directly. After column elution, dried residues were dissolved in 150 μL of the ammonium acetate solution, of which 140 μL was injected. In all plasma samples assayed, MN and NMN peaks were completely separated from surrounding peaks by virtue of a slight increase in polarity of the mobile phase.
Within-assay SDs, estimated from duplicate measurements of various samples (n = 11) containing 98–351 pmol/L MN and 129–350 pmol/L NMN, were 12.3 pmol/L for MN and 11.6 pmol/L for NMN (mean CVs, 7.0% and 4.5%, respectively). Single measurements of a control sample in 15 separate runs gave mean (SD) values of 207 (22.9) pmol/L (CV, 11%) for MN and 277 …
Objective:
The aim was to formulate clinical practice guidelines for pheochromocytoma and paraganglioma (PPGL).
Participants:
The Task Force included a chair selected by the Endocrine Society Clinical Guidelines Subcommittee (CGS), seven experts in the field, and a methodologist. The authors received no corporate funding or remuneration.
Evidence:
This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe both the strength of recommendations and the quality of evidence. The Task Force reviewed primary evidence and commissioned two additional systematic reviews.
Consensus process:
One group meeting, several conference calls, and e-mail communications enabled consensus. Committees and members of the Endocrine Society, European Society of Endocrinology, and Americal Association for Clinical Chemistry reviewed drafts of the guidelines.
Conclusions:
The Task Force recommends that initial biochemical testing for PPGLs should include measurements of plasma free or urinary fractionated metanephrines. Consideration should be given to preanalytical factors leading to false-positive or false-negative results. All positive results require follow-up. Computed tomography is suggested for initial imaging, but magnetic resonance is a better option in patients with metastatic disease or when radiation exposure must be limited. (123)I-metaiodobenzylguanidine scintigraphy is a useful imaging modality for metastatic PPGLs. We recommend consideration of genetic testing in all patients, with testing by accredited laboratories. Patients with paraganglioma should be tested for SDHx mutations, and those with metastatic disease for SDHB mutations. All patients with functional PPGLs should undergo preoperative blockade to prevent perioperative complications. Preparation should include a high-sodium diet and fluid intake to prevent postoperative hypotension. We recommend minimally invasive adrenalectomy for most pheochromocytomas with open resection for most paragangliomas. Partial adrenalectomy is an option for selected patients. Lifelong follow-up is suggested to detect recurrent or metastatic disease. We suggest personalized management with evaluation and treatment by multidisciplinary teams with appropriate expertise to ensure favorable outcomes.
Different methodological aspects on the assay of human erythrocyte catechol-O-methyltransferase (COMT) activity were studied. No temporal variations were found either over a 24 hour period or over one month. Erythrocytes from whole blood collected with any of the anticoagulants heparin, EDTA or citrate could be used as the enzyme source provided the cells were washed in saline. The COMT activity in lysed erythrocytes was rapidly lost when the lysate was stored at +4 degrees C and -20 degrees C. Intact erythrocytes could be stored up to one week in +4 degrees C without considerable loss of activity. The COMT activity was stable for at least two years when storing the cells at -85 degrees C. Freeze-thawing and hypotonic disruption of the erythrocytes resulted in the same activity and neither freeze-thawing nor sonication altered the apparent Km for the substrate. Noradrenaline and 3,4-dihydroxybenozic acid (DBA) could both be used as substrates although DBA gave higher activity values and had a higher affinity to the enzyme. The COMT activity increased with increasing concentration of the methyl-donor S-adenosyl-1-methionine up to approximately 0.1 mM. Preincubation at 47 degrees C decreased the COMT activity whereas the apparent Km values remained unchanged. The present COMT assay was convenient and reproducible and could be used with small amounts of blood with different kinds of anticoagulants. Interactions with plasma factors were avoided by washing the erythrocytes with isotonic sodium chloride.
The impact of blood sampling in sitting vs supine positions on measurements of plasma metanephrines for diagnosis of pheochromocytoma is unknown.
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.
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%.
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.
Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range.
We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode.
Total run-time including sample cleanup was 8 min. Intra- and interassay analytical variation (CV) varied from 2.0% to 4.7% and 1.6% to 13.5%, respectively, whereas biological intra- and interday variation ranged from 9.4% to 45.0% and 8.4% to 23.2%. Linearity in the 0 to 20 nmol/L calibration range was excellent (R(2) > 0.99). For all compounds, recoveries ranged from 74.5% to 99.6%, and detection limits were <0.10 nmol/L. Reference intervals for 120 healthy adults were 0.07 to 0.33 nmol/L (MN), 0.23 to 1.07 nmol/L (NMN), and <0.17 nmol/L (3-methoxytyramine).
This automated high-throughput XLC-MS/MS method for the measurement of plasma free MNs is precise and linear, with short analysis time and low variable costs. The method is attractive for routine diagnosis of pheochromocytoma because of its high analytical sensitivity, the analytical power of MS/MS, and the high diagnostic accuracy of free MNs.
Background:
Pheochromocytomas and paragangliomas (PPGLs) are potentially lethal yet usually surgically curable causes of endocrine hypertension; therefore, once clinical suspicion is aroused it is imperative that clinicians choose the most appropriate laboratory tests to identify the tumors.
Content:
Compelling evidence now indicates that initial screening for PPGLs should include measurements of plasma free metanephrines or urine fractionated metanephrines. LC-MS/MS offers numerous advantages over other analytical methods and is the method of choice when measurements include methoxytyramine, the O-methylated metabolite of dopamine. The plasma test offers advantages over the urine test, although it is rarely implemented correctly, rendering the urine test preferable for mainstream use. To ensure optimum diagnostic sensitivity for the plasma test, reference intervals must be established for blood samples collected after 30 min of supine rest and after an overnight fast when measurements include methoxytyramine. Similarly collected blood samples during screening, together with use of age-adjusted reference intervals, further minimize false-positive results. Extents and patterns of increases in plasma normetanephrine, metanephrine, and methoxytyramine can additionally help predict size and adrenal vs extraadrenal locations of tumors, as well as presence of metastases and underlying germline mutations of tumor susceptibility genes.
Summary:
Carried out correctly at specialist endocrine centers, collection of blood for measurements of plasma normetanephrine, metanephrine, and methoxytyramine not only provides high accuracy for diagnosis of PPGLs, but can also guide clinical decision-making about follow-up imaging strategies, genetic testing, and therapeutic options. At other centers, measurements of urine fractionated metanephrines will identify most PPGLs.
Pheochromocytomas are dangerous tumors that, although a rare cause of hypertension, require consideration among large numbers of patients. The resulting low prevalence of the tumor among tested populations and the inadequacies of commonly used biochemical tests make excluding or confirming the tumor an often difficult and time-consuming task. Recognition that catecholamines are metabolized to free metanephrines within pheochromocytoma tumor cells, and that this process is independent of catecholamine release, provides a rationale for use of these metabolites in the biochemical diagnosis of pheochromocytoma. Here we briefly review the history of biochemical diagnosis of pheochromocytoma in relation to recent data about the diagnostic utility of plasma free metanephrines for detection of these tumors. Measurements of urinary or plasma catecholamines have reasonable sensitivity for detection of most pheochromocytomas, particularly those in patients with sustained hypertension. False-negative test results can, however, occur in asymptomatic patients tested because of an adrenal incidentaloma or a familial predisposition for pheochromocytoma, or when sampling is carried out between episodes of paroxysmal hypertension. Measurements of urinary total metanephrines or vanillylmandelic acid are less reliable and are of little value as initial screening tests. In contrast, measurements of plasma concentrations or free metanephrines or 24-hour urinary outputs of fractionated normetanephrine and metanephrine almost always reveal the tumor. Although, both tests have similarly high sensitivity, the relatively low specificity of urinary fractionated metanephrines means that pheochromocytomas can be more efficiently excluded or confirmed using measurements of plasma free metanephrines.
We studied the pre-analytical stability of 81 analytes based on the variables of delay before processing, storage as whole blood or serum/plasma, the storage temperature and the type of tube the sample was stored in.
The mean difference between assays for samples from 10 subjects was calculated with the samples being kept under different storage conditions and for different times between sampling time and analysis: up to 24h for biochemistry, coagulation and hematology, and up to 72 h for hormonology. This difference was compared to the acceptable limits derived from the analytical and the intra individual biological variation.
Most of the analytes investigated remained stable up to 24h under all storage conditions prior to centrifugation. However, some analytes were significantly affected either by delay, tube type or temperature, such as potassium, inorganic phosphorus, magnesium, LD, glucose, lactate, mean corpuscular volume, mean corpuscular hemoglobin, activated partial thromboplastin time, insulin, C-peptide, PTH, osteocalcin, C-telopeptide and ACTH.
This study may be useful to help define acceptable delay times and storage conditions when a short time between sample collection and processing is not possible.
The aim of the study was to define the analytical and diagnostic performance of the Labor Diagnostica Nord (LDN) 2-Met plasma ELISA assay for fractionated plasma metanephrines in the biochemical diagnosis of phaeochromocytoma.
The stated manufacturer's performance characteristics were assessed. Clinical utility was evaluated against liquid chromatography tandem mass spectrometry (LC-MS/MS) using bias, sensitivity and specificity outcomes. Samples (n=73) were collected from patients in whom phaeochromocytoma had been excluded (n=60) based on low probability of disease, repeat negative testing for urinary fractionated catecholamines and metanephrines, lack of radiological and histological evidence of a tumour and from a group (n=13) in whom the tumour had been histologically confirmed. Blood collected into k(2)EDTA tubes was processed within 30 min. Separated plasma was aliquoted (×2) and frozen at -40°C prior to analyses. One aliquot was analysed for plasma metanephrines using the LDN 2-Met ELISA and the other by LC-MS/MS.
The mean bias of -32% for normetanephrine (ELISA) when compared to the reference method (LC-MS/MS) makes under-diagnosis of phaeochromocytoma likely. The sensitivity of the assay (100%) was equal to the reference method, but specificity (88.3%) lower than the reference method (95%), making it less than optimum for the biochemical diagnosis of phaeochromocytoma.
Plasma-free metanephrines as measured by Labor Diagnostica Nord (LDN) 2-Met ELISA do not display test characteristics that would support their introduction or continuation as part of a screening protocol for the biochemical detection of phaeochromocytoma unless the calibration problem identified is corrected and other more accurate and analytically specific methods remain unavailable.
Previous stability studies of plasma free metanephrines do not extend beyond one month. For retrospective evaluation and documentation purposes, knowledge of stability for more prolonged storage periods is required.
A panel of seven plasma samples was aliquoted and stored at -20 and -80°C. Aliquots were thawed and assayed by high-performance liquid chromatography with electrochemical detection at regular intervals during three years. A final set was assayed after five years at -80°C. Results were evaluated by repeated-measures analysis of variance.
After a stable period of over one year, an upward trend for plasma free metanephrine and a downward trend for normetanephrine was observed. For both analytes, measurement results were parallel between samples during the study. Concentrations in samples stored at -20°C did not differ from those stored at -80°C.
Storage at either -20 or -80°C must be considered as safe for at least one year. The residual variation with respect to time of the concentrations of metanephrines was identical in all of the samples assayed and almost did not exceed previously determined within-run variation. This suggests that between-assay variability is the cause of the overall trends and not sample deterioration. Moreover, between-assay variability manifesting itself as drift or trend remained within the range of earlier observed between-assay variation. Despite this, the assay for plasma metanephrines remains capable of detecting catecholamine overproduction in plasma samples that have been stored for prolonged periods of time.
Measurements of plasma free metanephrines have been advocated as first-line tests for phaeochromocytoma. The aim of the study was to assess the impact of potential confounding variables.
Comparative study between 2008 and 2009.
Hundred and eighty healthy subjects.
The effects of age, BMI, gender, menstrual cycle (sampling every 2 days), time of day (sampling every 2 h), venepunture (0, 15, 30, 60, 90 and 120 min), physical exercise (0, 15 and 30 min), coffee (0 and 60 min), breakfast (0 and 60 min) and various body positions (standing and supine rest, each 0 and 120 min) were evaluated. In addition, whole blood and plasma samples were stored at 4 degrees C or at 22 degrees C for 0, 1, 3, 24 and 72 h. Plasma free metanephrines were measured using radioimmunoassay (LDN).
While metanephrine was significantly influenced by sex and age, BMI and sex were significant predictors of normetanephrine. Coffee (+20%) and food (+8%) elevated normetanephrine significantly (P < 0.05), while metanephrine remained stable. Physical exercise increased metanephrine (+82%) as well as normetanephrine (+84%) significantly (P < 0.005). Supine rest significantly decreased both metanephrine (-34%) and normetanephrine (-19%) when compared to standing rest (P < 0.01). Metanephrine and normetanephrine were not significantly influenced by time of day, menstrual cycle or venepuncture. When plasma samples were stored at 4 degrees C, metanephrine and normetanephrine were stable for 72 h.
Physical exercise may lead to relevant changes in metanephrine and normetanephrine and should therefore be avoided prior to sampling. Although effects of age, sex and BMI were small, these variables should be considered when interpreting biochemical results. Blood should be taken in the supine position, and samples should be immediately centrifuged and stored at 4 degrees C to improve stability.
Pheochromocytomas are dangerous tumors that, although a rare cause of hypertension, require consideration among large numbers of patients. The resulting low prevalence of the tumor among tested populations and the inadequacies of commonly used biochemical tests make excluding or confirming the tumor an often difficult and time-consuming task. Recognition that catecholamines are metabolized to free metanephrines within pheochromocytoma tumor cells, and that this process is independent of catecholamine release, provides a rationale for use of these metabolites in the biochemical diagnosis of pheochromocytoma. Here we briefly review the history of biochemical diagnosis of pheochromocytoma in relation to recent data about the diagnostic utility of plasma free metanephrines for detection of these tumors. Measurements of urinary or plasma catecholamines have reasonable sensitivity for detection of most pheochromocytomas, particularly those in patients with sustained hypertension. False-negative test results can, however, occur in asymptomatic patients tested because of an adrenal incidentaloma or a familial predisposition for pheochromocytoma, or when sampling is carried out between episodes of paroxysmal hypertension. Measurements of urinary total metanephrines or vanillylmandelic acid are less reliable and are of little value as initial screening tests. In contrast, measurements of plasma concentrations or free metanephrines or 24-hour urinary outputs of fractionated normetanephrine and metanephrine almost always reveal the tumor. Although, both tests have similarly high sensitivity, the relatively low specificity of urinary fractionated metanephrines means that pheochromocytomas can be more efficiently excluded or confirmed using measurements of plasma free metanephrines.
Enzyme-linked immunoassay for plasma-free metanephrines in the biochemical diagnosis of phaeochromocytoma in adults is not ideal
- F Mullins
- P Shea
- R Fitzgerald
- W Tormey