Comparison between one and two-dimensional liquid chromatographic approaches for the determination of plasmatic stroke biomarkers by isotope dilution and tandem mass spectrometry.

This work presents the evaluation of one- and two-dimensional liquid chromatography for the quantification of three stroke outcome predictors in plasma. Isotopically labelled analogues of L-arginine (L-Arg), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are used to quantify the three analytes by isotope dilution and tandem mass spectrometry. Chromatographic isotope effects were not observed between natural L-Arg and its 15N-labelled analogue but they were observed between natural ADMA and SDMA and their multiple deuterated analogues. Under these conditions, bidimensional chromatography through the use of an automated multiple heart cutting mode provided unsatisfactory results for ADMA and SDMA due to the different amounts of natural and labelled compounds transferred from the first to the second chromatographic dimension. In contrast, using one dimensional liquid chromatography after a derivatization step to esterify carboxylic groups, chromatographic isotope effects did not alter the initial mass balance as full coelution of natural and labelled analogues or baseline resolution between the analytes was not required. This method was successfully validated following the Clinical & Laboratory Standards Institute guidelines and applied to the analysis of plasma samples from patients who had suffered from an intraparenchymal haemorrhagic stroke.

Challenges for Worldwide Harmonization of Newborn Screening Programs.

BACKGROUND: Inherited metabolic disorders (IMDs) are caused by a defect in a metabolic pathway, leading to malfunctioning metabolism and/or the accumulation of toxic intermediate metabolites. To date, hundreds of IMDs have been identified. Many of these diseases are potentially fatal conditions that are not apparent at birth. Newborn screening (NBS) programs involve the clinical and laboratory examination of neonates who exhibit no health problems, with the aim of discovering those infants who are, in fact, suffering from a treatable condition. CONTENT: In recent years, the introduction of tandem mass spectrometry has allowed the expansion of screening programs. However, this expansion has brought a high degree of heterogeneity in the IMDs tested among different NBS programs. An attempt to harmonize the metabolic conditions recommended to be screened has been carried out. Two uniform screening panels have been proposed in the US and European Union, by knowledgeable organizations. Here, we review current evidence-based processes to assess and expand NBS programs. We also discuss the IMDs that have recently been introduced in some screening programs, such as severe combined immunodeficiencies, lysosomal storage disorders, and adrenoleukodystrophy. SUMMARY: NBS programs have been an established public health function for more than 50 years to efficiently and cost-effectively identify neonates with severe conditions. However, NBS is not yet optimal. This review is intended to elucidate the current degree of harmonization of NBS programs worldwide as well as to describe the major controversial points and discuss the multiple challenges that must be confronted in expanded NBS strategies.

Identification of novel biomarkers of brain damage in patients with hemorrhagic stroke by integrating bioinformatics and mass spectrometry-based proteomics.

Hemorrhagic stroke (HS) is a significant cause of mortality that requires rapid diagnosis and prompt medical attention. A time-efficient diagnostic test to assist in the early classification of patients with stroke would be of great value. The aims here were to (a) select "brain-specific" proteins using a bioinformatics approach, (b) develop selected reaction monitoring (SRM) assays for candidate proteins, and (c) quantify these proteins in cerebrospinal fluid (CSF). "The Human Protein Atlas" and the "Peptide Atlas" were used to select proteins specifically and abundantly expressed in brain tissue, excluding high-abundance plasma proteins. Protein extracts from brain tissue were used for SRM assay development of proteins of interest. The levels of 68 "brain-specific" proteins were measured by SRM in 36 age-matched patients, including individuals with HS (n = 15), ischemic stroke (n = 11), and controls (n = 10). Additionally, S100B was measured using an electrochemoluminometric immunoassay. CSF levels of S100B and eight of the "brain-specific" proteins (NSE, GFAP, α-Inx, MBP, MT3, NFM, ß-Syn, and γ-Syn) were increased in a subset of samples from HS patients, especially in those individuals with intraventricular hemorrhage and poor outcome. Seven of these proteins (S100B, NSE, GFAP, α-Inx, MBP, NFM, and ß-Syn) showed significant differences between patients with and without brain hemorrhage. Novel biomarkers of brain injury (α-Inx, NFM, and ß-Syn) were identified in the CSF of patients with HS. Investigating the role of these proteins in blood with more sensitive methods is warranted.

Evaluation of the first 5 years of a glutaric aciduria type I neonatal screening programme in Asturias.

INTRODUCTION: . Neonatal screening of glutaric aciduria type 1 (GA-1) has brought radical changes in the course and outcomes of this disease. This study analyses the outcomes of the first 5 years (2015-2019) of the AGA1 neonatal screening programme in our autonomous community. MATERIAL: . We conducted an observational, descriptive and retrospective study. All neonates born between January 1, 2015 and December 31, 2019 that participated in the neonatal screening programme were included in the study. The glutarylcarnitine (C5DC) concentration in dry blood spot samples was measured by means of tandem mass spectrometry applying a cut-off point of 0.25 µmol/L. RESULTS: . A total of 30 120 newborns underwent screening. We found differences in the C5DC concentration based on gestational age, type of feeding and hours of life at sample collection. These differences were not relevant for screening purposes. There were no differences between neonates with weights smaller and greater than 1500 g. Screening identified 2 affected patients and there were 3 false positives. There were no false negatives. The diagnosis was confirmed by genetic testing. Patients have been in treatment since diagnosis and have not developed encephalopathic crises in the first 4 years of life. CONCLUSIONS: . Screening allowed early diagnosis of two cases of GA-1 in the first 5 years since its introduction in our autonomous community. Although there were differences in C5DC levels based on gestational age, type of feeding and hours of life at blood extraction, they were not relevant for screening.

Value of the soluble urokinase-type plasminogen activator receptor as a predictor of prognosis in patients attended in hospital emergency departments. / Capacidad del biomarcador suPAR para estratificar el pronóstico en pacientes atendidos en servicios de urgencias hospitalarios.

OBJECTIVES: To determine the value of the soluble urokinase-type plasminogen activator receptor (suPAR) for predicting outcomes in emergency department (ED) patients. Secondary objectives were 1) to measure the predictive value of the usual decision points, 2) to identify patients at low risk for mortality who could be safely discharged from the ED, and 3) to measure the correlation between suPAR and other biomarkers. MATERIAL AND METHODS: Prospective observational cohort study of patients attended in the EDs of participating hospitals. We recorded sociodemographic variables, comorbidity, variables related to the acute episode, prognostic markers commonly used in EDs, and suPAR concentration. Outcome variables were the need for hospital admission during the index episode, ED revisits within 90 days, and 90-day mortality. RESULTS: A total of 990 patients with a median (interquartile range) age of 68 (53-81 years) were studied; 50.8% were men. The median suPAR concentration was 3.8 (2.8-6.0) ng/mL, and 112 patients (11.31%) required admission. At 90 days there were 276 revisits (27.9% of the cohort), and 47 patients (4.74%) had died. Mortality was lower (1%) in patients with suPAR concentrations less than 4 ng/mL (52.5%), and fewer of these patients revisited (24.4%) or required hospitalization (20.6%) than patients with suPAR concentrations higher than 6 ng/mL (mortality, 13.5%; revisits, 39.6%; admissions, 56.3%). A suPAR concentration over 6 ng/mL was associated with 90-day mortality and revisits (adjusted hazard ratios and 95% CIs of 4.61 [1.68-12.67] and 1.59 [1.13-2.10]), respectively. The high suPAR concentration was also associated with hospital admission (odds ratio, 1.62 [0.99-2.62]). CONCLUSION: A suPAR concentration of less than 4 ng/mL identifies patients at low risk of 90-day mortality and revisits or need for hospitalization, whereas a suPAR concentration higher than 6 ng/mL is associated with higher risk for these outcomes.

OBJETIVO: Determinar la capacidad del receptor soluble del activador del plasminógeno tipo uroquinasa (suPAR) para la estratificación pronóstica en pacientes atendidos en servicios de urgencias hospitalarios (SUH). Los objetivos secundarios son: 1) medir la capacidad de los `puntos de decisión habituales, 2) identificar una población de bajo riesgo de mortalidad que puede darse de alta de forma segura desde el SUH, y 3) medir la correlación entre suPAR y otros biomarcadores. METODO: Estudio observacional de cohortes prospectivo de pacientes atendidos en SUH. Se registraron variables sociodemográficas, de comorbilidad, datos del episodio agudo, biomarcadores de uso común en urgencias y suPAR. Las variables de resultado fueron la necesidad de ingreso en el episodio índice, reconsulta al SUH y mortalidad a los 90 días. RESULTADOS: Se incluyeron 990 pacientes, la edad fue de 68 (53-81) años, 50,8% eran hombres, la mediana de suPAR fue de 3,8 (2,8-6,0) ng/ml, 112 pacientes (11,31%) requirieron ingreso. En el seguimiento a 90 días hubo 276 reconsultas (27,9%) y 47 pacientes (4,74%) fallecieron. Los pacientes con suPAR 4 ng/ml (52,5%) tenían menor mortalidad (1%), menor reconsulta (24,4%) y menor necesidad de ingreso hospitalario (20,6%), que pacientes con suPAR 6 ng/ml (mortalidad 13,5%, reconsulta 39,6% e ingreso 56,3%). Un suPAR 6 ng/ml mostró una hazard ratio (IC 95%) ajustada de 4,61 (1,68-12,67) para predecir mortalidad a 90 días y de 1,59 (1,13-2,10) para la reconsulta, y una odds ratio de 1,62 (0,99-2,62) para la necesidad de ingreso hospitalario. CONCLUSIONES: Un valor de suPAR 4 ng/ml identifica pacientes con riesgo bajo de mortalidad a 90 días, de reconsulta y de necesidad de ingreso, mientras que los pacientes con suPAR 6 ng/ml tienen mayor mortalidad, reconsulta y necesidad de ingreso.

Purple urine in Berdon syndrome: a rare finding in the clinic laboratory.

INTRODUCTION: Purple Urine Bag Syndrome (PUBS) is a rare disorder seen in elderly persons, wherein the urinary bag and the tubing turn in to purple colour. It is usually seen in patients who are on urinary catheters for a long time. It consists of a change in the colour of the urine that turns purple in a very specific context. CASE REPORT: We report the case of a paediatric female patient with Berdon Syndrome with symptoms consistent with urinary tract infection and purple urine discolouration. Urine test revealed leukocyturia and bacteriuria. DISCUSSION: Several risk factors have been proposed regarding this syndrome. Among them the commonest are female gender, advanced age, kind of diet (increased dietary tryptophan), alkaline urine and diverse situations that leads to urinary retentions which allows bacteria to work on their substrate for a longer time. Although it is a process that is not associated with gravity, recognizing it is important as treatment is simple and can minimize patient and family distress.

Assessment of coagulation assays on Roche Cobas t711 analyzer: performance and clinical implications.

OBJECTIVES: We performed an analytical assessment of five coagulation tests [i.e. prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, thrombin time (TT) and D-dimer] on the Roche Cobas t711 analyzer and a comparison study with the methodology in use at our laboratory (i.e. Werfen ACL Top 750 analyzer), expanding the analysis to the clinical implications of Cobas t711 implementation. METHODS: Imprecision studies were performed following the Clinical and Laboratory Standards Institute (CLSI) H57 A:2008 guideline. Linearity of D-dimer and fibrinogen tests was analysed according to the CLSI EP06-A: 2003 recommendations. For method comparison, the results were analyzed using the Bland-Altman plot and Passing-Bablok regression. RESULTS: Imprecision met manufacturer claims for PT, aPTT and TT. D-dimer and fibrinogen tests showed a coefficient of variation (CV)% over manufacturer claims at certain concentration levels. Linearity ranges could not be verified. Comparison study revealed that results are not interchangeable for any test, a lower correlation for aPTT test and lower D-dimer results from Roche Cobas t711. CONCLUSION: The strength of this study relies on the analysis of the clinical implications of reporting Cobas t711 results compared to those obtained with the methodology in use at our laboratory. Different sensibility to factor deficiency, anticoagulant therapy and interferences might explain lower correlation rates obtained for the aPTT test. Different monoclonal antibodies used for D-dimer determination might explain the lower results obtained with the Cobas t711 analyzer. This aspect needs further studies given the relevance of D-dimer test to exclude thrombotic events and reinforces the need of harmonization in the haemostasis laboratory.

Neonatal TSH as a marker of iodine nutrition status. Effect of maternal ioduria and thyroid function on neonatal TSH.

INTRODUCTION: Neonatal thyroid stimulating hormone (nTSH) is a marker of iodine nutrition status in the population. The WHO considers a prevalence of less than 3% of nTSH levels greater than 5 mIU/L in samples obtained within 72h from birth indicative of iodine sufficiency. The aim of this study was to determine the prevalence of nTSH levels greater than 5 mIU/L in an iodine-sufficient population and its association with maternal, neonatal and obstetric factors. MATERIALS AND METHODS: A total of 243 pregnant women were recruited between May and June 2017 in our health area. A questionnaire of iodine intake was administered, in addition to determination of ioduria, thyroid function and autoimmunity in the first trimester of gestation. We analysed nTSH levels in samples collected between 48 and 72h post birth and other obstetric and neonatal factors. RESULTS: The mean nTSH level (standard deviation) was 2.43 (1.68 mIU/L), with 7.8% of neonates having levels greater than 5 mIU/L. The highest nTSH levels corresponded to neonates of mothers with insufficient ioduria (P = 0.021) or TSH levels greater than 2.5 mIU/L, in both the case of negative (P = 0.049) and positive (P = 0.006) thyroid autoimmunity results. Maternal ioduria less than 150 µg/L was a risk factor for nTSH levels greater than 5 mIU/L (3.70 [1.06-14.60]; P = 0.046), while a neonatal weight of 2500 g or greater was a protective factor (0.14 [0.02-1.00]; P = 0.038). CONCLUSIONS: The prevalence of nTSH levels greater than 5 mIU/L in our health area was high based on the WHO recommendations. Maternal iodine deficiency was associated with a higher risk of nTSH levels greater than 5 mIU/L. Given that nTSH is currently measured before 72h post birth, we need new cut-off points to keep on using nTSH as a marker of iodine nutritional status.

A Dynamic Approach for Early Risk Prediction of Gram-Negative Bloodstream Infection and Systemic Inflammatory Response Syndrome in Febrile Pediatric Hemato-Oncology Patients.

Background: The aim of this study was to evaluate the usefulness of C-Reactive Protein (CRP), Procalcitonin (PCT), and Interleukine 6 (IL6) biomarkers in predicting the existence of high-risk episodes (HRE) during the first 24 h of fever in pediatric cancer patients. HRE were defined as the presence of Gram-negative bloodstream infections or Systemic Inflammatory Response Syndrome. Methods: The study included 103 consecutive fever episodes in 44 hemato-oncological pediatric patients, from whom samples for biomarkers were taken upon initial evaluation (CRP-1, PCT-1 and IL6-1) and then between 12 and 24 h afterward (CRP-2, PCT-2 and IL6-2). Results: An IL6-1 value higher than 164 pg/mL showed an area under the curve (AUC) of 0.890 (0.791−0.989) and OR of 48.68 (7.92−951.42, p < 0.001) to detect HRE in multivariate analysis. A PCT-1 higher than 0.32 ng/mL showed an AUC of 0.805 (0.700−0.910) and OR of 4.55 (0.90−27.84, p = 0.076). A PCT-2 higher than 0.94 ng/mL showed an AUC of 0.836 (0.725−0.947) and OR of 13.01 (1.82−149.13, p = 0.018), and an increase in CRP between the first and second sample (CRP-2vs1) higher than 291% also showed an AUC of 0.785 (0.655−0.915) and OR of 31.09 (4.87−355.33, p = 0.001). Conclusions: IL6-1, PCT-2, and CRP-2vs1 showed a strong and independent correlation with HREs in pediatric cancer patients. CRP variations over the first 24 h provide an improvement in predictive models that are especially useful if IL-6 and PCT are not available.

Biomarkers and Fever in Children with Cancer: Kinetics and Levels According to Final Diagnosis.

We investigated the kinetics of CRP, PCT, IL-6 and MR-proADM in a cohort of consecutive febrile patients with cancer in order to test the hypothesis that higher plasma concentrations and the absence of a rapid decrease in peak values would be associated with disease severity. (1) Method: A prospective descriptive and analytical study of patients with cancer and fever (≤18 years of age) at a University Hospital was carried out between January 2018 and December 2019. Information collected: sex, age, diagnosis, date and symptoms at diagnosis and medical history. The episodes were classified into three groups: bacterial infection, non-bacterial infection and systemic inflammatory response syndrome (SIRS). (2) Results: One hundred and thirty-four episodes were included. Bacterial infection criteria were met in 38 episodes. Biomarkers were measured at four different points: baseline, at 12-24 h, at 25-48 h and at 49-72 h. All the biomarkers evaluated decreased after the peak level was reached. IL-6 and MR-proADM showed a trend towards higher levels in the SIRS group although this rise was statistically significant only for IL-6 (p < 0.005). Bacterial infections more frequently presented values of PCT above the cut-off point (>0.5 ng/mL) at 12-24 h. (3) Conclusion: In our experience, IL-6 kinetics is faster than PCT kinetics and both are faster than CRP in patients with fever and cancer who present a good outcome. Patients with a good evolution show a rapid increase and decrease of PCT and particularly of IL-6 levels.

Midregional proadrenomedullin safely reduces hospitalization in a low severity cohort with infections in the ED: a randomized controlled multi-centre interventional pilot study.

INTRODUCTION: The midregional fragment of proadrenomedullin (MR-proADM) is known to provide accurate short-, mid- and long term prognostic information in the triage and multi-dimensional risk assessment of patients in the emergency department (ED). In two independent observational cohorts MR-proADM values identified low disease severity patients without risk of disease progression in the ED with no 28 days mortality that wouldn´t require hospitalization. In this interventional study we want to show that the combination of an MR-proADM algorithm with clinical assessment is able to identify low risk patients not requiring hospitalization to safely reduce the number of hospital admissions. METHODS: A randomized-controlled interventional multicenter study in 4 EDs in Spain. The study protocol was approved by Ethics Committees. Control arm patients received Standard Care. MR-proADM guided arm patients with low MR-proADM value (≤0.87 nmol/L) were treated as out-patients, with high MR-proADM value (>0.87 nmol/L) were hospitalized. The hospitalization rate was compared between the study arms. RESULTS: Two hundred patients with suspicion of infection were enrolled. In the MR-proADM guided arm the hospital admission rate in the intention-to-treat (ITT) population was 17% lower than in the control arm (40.6% vs. 57.6%, p=0.024) and 20% lower in the per protocol (PP) population (37.2% vs. 57.6%, p=0.009). No deaths of out-patients and no significant difference for the safety endpoints readmission and representation rates were observed. The readmission rate was only slightly higher in the MR-proADM guided arm compared to the control arm (PP population: at 14 days 9.3% vs. 7.1%, difference 2.1% (95% CI: -11.0% to 15.2%); and at 28 days 11.1% vs. 9.5%, difference 1.6% (95% CI: -12.2% to 15.4%)). The rate of 28 days representation was slightly lower in the MR-proADM guided arm compared to the control arm (20.4% vs. 26.2%, difference -5.8% (95% CI: -25.0% to 13.4%); PP population). CONCLUSIONS: Implementing a MR-proADM algorithm optimizes ED workflows efficiently and sustainably. Hospitals can highly benefit from a reduced rate of hospitalizations by 20% using MR-proADM. The safety in the MR-proADM guided study arm was similar to the Standard Care arm. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03770533.

[Beginnings, evolution and current situation of the Newborn Screening Programs in Spain.] / Inicio, evolución y situación actual de los Programas de Cribado Neonatal en España.

Newborn Screening Programs (NSP) in Spain were born in the city of Granada in 1968. Till the 1980s, they were developed around the so-called "National Plan for Preventing Subnormality", covering up to 30% of the Spanish newborns. From 1982, when the health system management was transferred to the different autonomous regions, the NSP began to expand, and the bases to transform them into an organized and multidisciplinary activity, integrated and coordinated from the National Health System were settled. Despite this expansion, it is not until the 1990s when their coverage reaches almost 100% newborns in Spain. NSP grew up asymmetrically across the different autonomous regions. In 2005 and 2006 the scientific societies SEQC (Spanish Society of Clinical Chemistry) and AECNE (Spanish Society of Newborn Screening), coordinated by the Health Promotion Area of the General Directorate of Public Health, gathered together the necessary information to elaborate a report on the NSP in Spain addressed to the Interterritorial Council of the National Health System. In July 2013, that Council approved the seven diseases that should be part of each region newborn screening panel, being the first step towards the NSP harmonization in Spain. Currently, the NSP include between 8 and 29 diseases in their panels, thus more still more efforts are needed in order to achieve a higher uniformity.

Los Programas de Cribado Neonatal (PCN) nacen en España en Granada en el año 1968. Posteriormente, y hasta los años 80, se fueron desarrollando en torno al llamado "Plan Nacional de Prevención de la Subnormalidad" con una cobertura cercana al 30% de los recién nacidos españoles. A partir de 1982, con el inicio de la gestión de la sanidad a las comunidades autónomas (CCAA), los PCN se expandieron y se comenzaron a sentar las bases para que éstos se convirtieran en una actividad organizada y multidisciplinar, integrados y coordinados desde el Sistema de Salud. A pesar de dicha expansión no es hasta el inicio de la década de los 90 cuando se consigue una cobertura próxima al 100% de los RN en España. Los PCN fueron creciendo de forma muy asimétrica en las diferentes CCAA y en los años 2005 y 2006 las Sociedades Científicas SEQC (Sociedad Española de Química Clínica) y AECNE (Asociación Española de Cribado Neonatal), con la coordinación del Área de Promoción de la Salud de la Dirección General de Salud Pública, recopilaron la información y elaboraron un informe, sobre los PCN en España para el Consejo Interterritorial del sistema Nacional de Salud (CISNS). En julio de 2013 este Consejo aprobó las siete enfermedades que debían formar parte del panel de detección de los PCN territoriales, primer paso hacia la armonización de estos programas. Actualmente, los PCN incluyen entre 8 y 29 enfermedades por lo que es necesario seguir trabajando para conseguir una mayor uniformidad.

Cell-free DNA as a noninvasive acute rejection marker in renal transplantation.

BACKGROUND: Acute rejection (AR) is a key conditioning factor for long-term graft function and survival in renal transplantation patients. The standard care with creatinine measurements and biopsy upon allograft dysfunction implies that AR is usually detected at advanced stages. Rapid noninvasive biomarkers of rejection are needed to improve the management of these patients. We assessed whether total cell-free DNA (tCF-DNA) and donor-derived cell-free DNA (ddCF-DNA) were useful markers for this purpose, both in plasma and in urine. METHODS: Plasma and urine samples from 100 renal transplant recipients were obtained during the first 3 months after transplantation. tCF-DNA and ddCF-DNA were analyzed by quantitative PCR for the HBB (hemoglobin, beta) and the TSPY1 (testis specific protein, Y-linked 1) genes, respectively. We observed 19 episodes of AR, as well as other complications, such as acute tubular necrosis, nephrotoxicity, and infections. RESULTS: Plasma tCF-DNA concentrations increased markedly during AR episodes, often before clinical diagnosis, and returned to reference values after antirejection treatment. A cutoff plasma tCF-DNA concentration of 12 000 genome equivalents/mL correctly classified AR and non-AR episodes in 86% of posttransplantation complications (diagnostic sensitivity, 89%; specificity, 85%). Although similar increases were observed during severe posttransplantation infections, use of the combination of plasma tCF-DNA and procalcitonin (PCT), a specific marker of sepsis, significantly improved the diagnostic specificity (to 98%; 95% CI, 92%-100%), with 97% of the episodes being correctly classified. Use of transrenal DNA and ddCF-DNA concentrations did not add relevant information. CONCLUSIONS: Given that renal biopsy is the gold standard for detecting AR, analysis of both plasma tCF-DNA and PCT could permit a more selective use of this invasive procedure.

Performance of plasma free metanephrines in diagnosis of pheochromocytomas and paragangliomas in the population of Asturias. / Rendimiento de las metanefrinas libres plasmáticas en el diagnóstico de los feocromocitomas y paragangliomas en la población asturiana.

INTRODUCTION: Pheochromocytoma and paraganglioma are uncommon tumors whose best known symptoms include high blood pressure, palpitations, headache, and sweating. Clinical identification is not easy, however, and requires biochemical tests that allow for early diagnosis, including measurement of metanephrines levels. The aim of this study was to assess the diagnostic performance of plasma free metanephrines (PMETs) and to verify the transferability of the reference values used. METHODS: PMETs levels were measured by liquid chromatography coupled to tandem mass spectrometry. Other biochemical tests evaluated (plasma catecholamine, urine metanephrine, catecholamine and vanilmandelic acid levels) were performed by liquid chromatography with electrochemical detection. Requests of these tests from 01/09/2015 to 31/10/2017 were reviewed, and both the reference values (document EP28-A3c) and the parameters of biological variation (Fraser method) for PMETs were estimated. RESULTS: The study sample consisted of 1,279 patients (61.3% females) aged 0-90 years, including 19 with pheochromocytoma/paraganglioma. Tests requested included: PMETs (n=662), catecholamines (n=589), metanephrines (n=586), and vanilmandelic acid (n=513) in urine, and plasma catecholamines (n=228). Tests with higher sensitivity were urinary fractionated metanephrines (91.7%) and PMETs (82.4%). When performance was compared in patients with both tests (n=243), they detected the same number of tumors (90.9%), but PMETs showed greater specificity (93.5% vs 88.8%). Plasma normetanephrine levels showed a significant association with age (rho=0.19, P<.0001). CONCLUSION: PMETs and urinary fractionated metanephrines are the biochemical tests with better performance in diagnosis of pheochromocytomas/paragangliomas.

Predictive capacity of a multimarker strategy to determine short-term mortality in patients attending a hospital emergency Department for acute heart failure. BIO-EAHFE study.

OBJECTIVE: A multimarker strategy may help determine the prognosis of patients with acute heart failure (AHF). The aim of this study was to evaluate the capacity of mid-regional pro-adrenomedullin (MRproADM), copeptin and interleukin-6 (IL-6) combined with conventional clinical and biochemical markers to predict the 30-day mortality of patients with AHF. METHODS: We performed an observational, multicenter, prospective study of patients attended in the emergency department (ED) for AHF. We collected clinical and biochemical data as well as comorbidities and biomarker values. The endpoint variable was mortality at 7, 14, 30, 90 and 180days. The clinical model included: gender, age, blood pressure values, hemoglobin, sodium <135mmol/L and estimated glomerular filtration <60mL/min/1.73m2. We made receiver operating curves (ROC) curves, and areas under the curve (AUC) and survival analysis for each model and calculated the hazard ratio (HR) and its 95% confidence interval. RESULTS: A total of 547 individuals were included: 55.6% were women with a mean age of 79.9 (9.5) years. Copeptin alone showed greater discriminatory power for 30-mortality [AUC 0.70 (0.62-0.78)]. The AUC for 30-day mortality of the clinical model plus copeptin and NTproBNP was 0.75 (0.67-0.83), being better than the clinical model alone with 0.67 (0.58-0.76; p=0.19). The discriminatory power of the different biomarkers alone, in combination or together with the clinical model decreased over time. CONCLUSIONS: The combination of a clinical model with copeptin and NTproBNP, which are available in the ED, is able to prognose early mortality in patients with an episode of AHF.

Elevated transrenal DNA (cell-free urine DNA) in patients with urinary tract infection compared to healthy controls.

OBJECTIVE: To study whether levels of transrenal DNA (Tr-DNA) are influenced by the presence of urinary tract infections (UTI). METHODS: Tr-DNA concentrations were measured in 124 donors and 55 patients with UTI. Two methods for DNA extraction were compared. RESULTS: UTI patients showed higher concentrations than the donors (normal range: 0-147 GE/mL). QIAamp MiniElute Virus Spin Kit was more efficient than QIAamp DNA Blood Mini Kit. CONCLUSION: Increased concentrations of Tr-DNA urine DNA are shown in presence of UTI.

Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR.

BACKGROUND: Recently cell-free plasma DNA has been described as a marker of apoptosis during hemodialysis (HD), but little is known about how different dialysis membranes may contribute to this process or whether pre-HD levels are restored afterwards. Here we evaluate the influence of the dialysis membrane on cell-free plasma DNA levels and investigate the clearance of plasma circulating DNA after HD. METHODS: Cell-free plasma DNA was measured using a real-time quantitative PCR for the beta-globin gene. Reference values for plasma DNA were established in a group of 100 healthy voluntary blood donors. Pre- and post-HD levels were also measured in 30 patients with end-stage renal disease on regular HD (52 sessions; 104 samples). The sessions lasted for 2.5-5 h. Different dialysis membranes were compared: high-flux (n=37) vs. low-flux (n=15) and polysulfone (n=42) vs. modified cellulose (n=10). To determine the time at which pre-HD levels are restored, DNA was quantified in serial plasma samples obtained from 10 of these 30 patients, just before and immediately after HD, as well as at 30, 60 and 120 min after HD. RESULTS: Reference plasma DNA values for healthy blood donors ranged from 112 to 2452 gEq/mL (median 740 gEq/mL). Cell-free plasma DNA levels significantly increased during HD (Wilcoxon test for paired samples, p<0.0001), with increases of more than four-fold observed in 75% of the patients after HD. There was no significant linear association between the length of the HD session (between 2.5 and 5 h) and the increase in cell-free plasma DNA concentration (Pearson correlation). No significant differences were observed between different types of membranes (Mann-Whitney U-test). Plasma DNA returned to pre-HD levels by 30 min after HD, regardless of the starting concentration. CONCLUSIONS: Plasma DNA levels significantly increase after a conventional 2.5-5-h HD session. Therefore, HD patients require special consideration for correct interpretation of plasma DNA concentrations. This parameter can be considered a reliable diagnostic tool for certain pathologies when measured at least 30 min after a HD session without further complications. The different dialysis membranes used in this study had no influence on cell-free plasma DNA concentrations, so the level of circulating DNA is not an appropriate marker of dialysis membrane biocompatibility.