Performance of plasma loose metanephrines in 168pg.net prognosis of pheochromocytomas and paragangliomas inside the populace of Asturias
Rendimiento de las metanefrinas libres plasmáticas en el diagnóstico de los feocromocitomas y paragangliomas en l. A. Población asturiana
, Nuria Valdés Gallegob, Edwin Eguia Ángelesa, Juan Carlos Fernández Fernándeza, Belén Prieto Garcíaa,c, Francisco V. Álvareza,c
a Laboratorio de Medicina, Servicio de Bioquímica Clínica, Hospital Universitario Central de Asturias, Oviedo, Spain
b Servicio de Endocrinología, Hospital Universitario Central de Asturias, Oviedo, Spain
c Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Oviedo, Spain
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Pheochromocytoma and paraganglioma are unusual tumours whose exceptional regarded symptoms consist of excessive blood stress, palpitations, headache, and sweating. Clinical identification is not easy, but, and calls for biochemical assessments that allow for early analysis, such as measurement of metanephrines degrees. The goal of this examine was to assess the diagnostic overall performance of plasma unfastened metanephrines (PMETs) and to verify the transferability of the reference values used.
PMETs ranges have been measured with the aid of liquid chromatography coupled to tandem mass spectrometry. Other biochemical tests evaluated (plasma catecholamine, urine metanephrine, catecholamine and vanilmandelic acid ranges) had been executed by means of liquid chromatography with electrochemical detection. Requests of these assessments from 01/09/2015 to 31/10/2017 were reviewed, and each the reference values (file EP28-A3c) and the parameters of biological version (Fraser technique) for PMETs were expected.
The look at pattern consisted of 1279 patients (61.3% females) elderly 0–ninety years, consisting of 19 with pheochromocytoma/paraganglioma. Tests requested covered: PMETs (n=662), catecholamines (n=589), metanephrines (n=586), and vanilmandelic acid (n=513) in urine, and plasma catecholamines (n=228). Tests with better sensitivity were urinary fractionated metanephrines (ninety one.7%) and PMETs (82.4%). When overall performance was in comparison in sufferers with each assessments (n=243), they detected the equal range of tumours (ninety.Nine%), however PMETs confirmed more specificity (93.Five% vs. 88.8%). Plasma normetanephrine levels showed a sizeable affiliation with age (rho=0.19, p<zero.0001).
PMETs and urinary fractionated metanephrines are the biochemical checks with better overall performance in diagnosis of pheochromocytomas/paragangliomas.
Los feocromocitomas y paragangliomas son tumores percent frecuentes cuyos síntomas más conocidos son hipertensión arterial, palpitaciones, cefalea y diaforesis. Sin embargo, su identificación clínica no es fácil. Por ello, se utilizan pruebas bioquímicas que permitan un diagnóstico precoz, destacando las metanefrinas. El objetivo de este estudio fue evaluar el rendimiento diagnóstico de las metanefrinas libres plasmáticas (MLP) y verificar l. A. Transferibilidad de los valores de referencia utilizados.
Las MLP fueron cuantificadas mediante cromatografía líquida de alta resolución acoplada a espectrometría de masas. Otras pruebas bioquímicas evaluadas (catecolaminas en plasma, metanefrinas, catecolaminas y ácido vanilmandélico en orina) fueron analizadas por cromatografía líquida de alta resolución con detección electroquímica. Se revisaron las solicitudes de dichas pruebas del 01/09/2015 al 31/10/2017 y se estimaron los valores de referencia (documento EP28-A3c) y los parámetros de variabilidad biológica (método de Fraser) de las MLP.
Se estudiaron 1.279 pacientes (sixty one,3% mujeres), con edades entre 0-ninety años, incluyendo 19 casos de feocromocitoma/paraganglioma. Las solicitudes bioquímicas fueron: MLP (n=662), catecolaminas urinarias (n=589), metanefrinas urinarias (n=586), ácido vanilmandélico urinario (n=513) y catecolaminas plasmáticas (n=228). Las pruebas con mayor sensibilidad fueron las metanefrinas fraccionadas urinarias (ninety one,7%) y las MLP (82,4%). Cuando se comparó el rendimiento en pacientes con ambas pruebas (n=243), estas detectaron los mismos casos (90,nine%), pero las MLP fueron más específicas (ninety three,5 vs. 88,eight%). Para los angeles normetanefrina plasmática se observó una asociación significativa con l. A. Edad (rho=zero,19; p<0,0001).
Las MLP y las metanefrinas fraccionadas urinarias son las pruebas bioquímicas que ofrecen un mayor rendimiento en el diagnóstico de los feocromocitomas/paragangliomas.
Pheochromocytomas (PCTs) and paragangliomas (PGLs) are infrequent tumours, with 1–2 cases per 100,000 inhabitants and year, and are mainly derived from the chromaffin cells of the sympathetic nervous system.1 These neoplasms are located in the adrenal gland medulla (80–85% of all PCTs) or in the sympathetic nervous system ganglia of the chest, abdomen and pelvis (15–20% of all PGLs), and usually produce abnormally high levels of catecholamines and their metabolites. Paragangliomas located in the head and skull base are of parasympathetic origin.2
The typical signs and symptoms of PCTs/PGLs are well known, but are not very specific. The episodic secretion of catecholamines is responsible for most of the symptoms, including major fluctuations in blood pressure3,4 and the classic triad of palpitations (58%), headache (52%) and perspiration (49%).5 However, only 30–40% of all patients experience these three symptoms.3,6 The clinical identification of PCT/PGL is not easy, because approximately half of all patients experience paroxysmal arterial hypertension (AHT) or have normal blood pressure,7,8 and necropsy studies have shown that approximately 0.05% of all deceased individuals have undiagnosed PCT/PGL.9 At present, up to 40% of all PCTs/PGLs are considered to be hereditary, while the remaining 60% correspond to spontaneous cases.10
Pheochromocytomas/paragangliomas occur more frequently in individuals between 40 and 50 years of age, with a slight female predominance (55%).11 The prevalence differs according to the population studied. Thus, in the general population, the prevalence of PCT/PGL is less than 0.1%,8 while in patients with AHT the figure increases to 0.2–0.6%, and in patients with adrenal incidentalomas the prevalence is usually 3–7%2 but can reach 20%.12
The most common reasons for the biochemical study of PCT/PGL are the presence of AHT and episodic symptoms attributable to excess catecholamine output, as well as treatment-resistant AHT. Other indications are the determination of functionality of an adrenal incidentaloma, the assessment of genetic predisposition, the exclusion of tumour recurrence,13 young patients with AHT, and individuals with a history of hypertensive crises during anaesthesia. The traditionally used biochemical techniques have been the analysis of vanillylmandelic acid (VMA) and total catecholamines and metanephrines (MNs) in urine, and plasma catecholamines.
However, in recent years several publications have shown fractionated MNs in 24-h urine and plasma free metanephrines (PFMs) to afford a better diagnostic performance.14–18
In fact, according to the current recommendations of the American Society of Endocrinology, the initial biochemical study of PCT/PGL should include one of these two tests.19
Since PCTs/PGLs are potentially fatal tumours, especially if the patient suffers an adrenergic crisis, the establishment of an early diagnosis is very important.20 The availability of biochemical tests with a good diagnostic performance, in which sample extraction is fast and simple, is very useful for obtaining such an early diagnosis. To the best of our knowledge, the Medicine laboratory of the Department of Clinical Biochemistry of Hospital Universitario Central de Asturias (HUCA) was the first laboratory in Spain to incorporate the mass spectrometry measurement of PFMs within its range of services. The objective of the present study was to clinically validate the PFM test, introduced in 2015, based on a review of its diagnostic performance during its first two years of use, and to compare it with other classical biochemical tests used for the same purpose. In addition, the transferability of the reference values used for plasma MNs and normetanephrine (NMN) was verified and their biological variability parameters were assessed.
Material and methods
A sample collection for the determination of plasma MNs and catecholamines was made in a purple stopper tube containing K3-EDTA. The patients were instructed to avoid chocolate, bananas, vanilla, pineapple, nuts, coffee and tea before sampling. They were also instructed to avoid alcohol consumption. In addition, the suppression of drugs such as levodopa, hydrazine and its derivatives, monoamine oxidase inhibitors (MAOIs) and prochlorperazine was requested. Sampling was performed with the patient in the supine position after at least 15min of rest.
Urinary MNs, catecholamines and VMA were determined from 24-h urine collected in specific 3-l amber containers with a vacuum port, holding 10ml of HCl 6N as a stabilizing preservative.
Plasma free metanephrines were quantified with a triple quadrupole mass spectrometer (QTRAP 5500, AB Sciex) coupled to a liquid chromatograph (Ekspert ultra LC 100, Eksigent). The method was developed and validated in our laboratory. Briefly, the technique includes the initial separation of MNs (500μl of plasma EDTA) using solid phase extraction cartridges (SampliQ WCX, 30mg, 1ml [Agilent]). After extraction, the MNs are subjected to gradient chromatographic separation using two mobile phases: ammonium formate with 0.2% formic acid, pH=3.2 (solution A) and methanol with 0.2% formic acid (solution B). These mobile phases are prepared with high purity reagents: LiChrosolv® methanol (Merck-Millipore), ultrapure Milli-Q® water, Suprapur® formic acid (Merck-Millipore) and ammonium formate, purity ≥99% (Fluka). The chromatographic precolumn and column used are: Pursuit 3 PFP MetaGuard 10mm×2mm and Pursuit 3 PFP, 2mm×150mm (Agilent), respectively. The calibrators are prepared from certified reference material: Catecholamine Mix 2 (Metanephrines) solution (Sigma–Aldrich). In addition, isotopic (deuterium) labelled internal standards are used for the standardization of all the steps of the analytical process: (±)-Metanephrine-D3 hydrochloride solution and (±)-Normetanephrine-D3 hydrochloride solution, 100μg/ml in methanol (Sigma–Aldrich). Software is used to operate the mass spectrometer and analyze the results: Analyst® (version 1.6.2) and MultiQuant™ (version 2.1.1296.0).
The linearity of the method is 20–2000pg/ml for both MN and NMN, with correlation coefficients (r2)>0.995. The inter-assay coefficients of variation (CV) are ≤4.1% and six.2% for MN and NMN, respectively. The limit of quantitation (LoQ) turned into mounted at 20pg/ml.
The final biochemical tests evaluated (catecholamines in plasma and MN, catecholamines and VMA in urine) were analyzed the usage of commercial analytical methods formerly implemented in our laboratory. Specifically, these tests are quantified the usage of high-performance liquid chromatography (HPLC) with electrochemical detection. The system used is a liquid chromatograph (version 1200 collection, Agilent) with an electro