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BACKGROUNDInherited 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.CONTENTIn 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.SUMMARYNBS 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.

A species-specific Isotope Dilution (ID) method is described for the determination of free methionine in human blood plasma by High Performance Liquid Chromatography - Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) using 34S-labelled methionine as species-specific spike. The 34S-labelled methionine was obtained from yeasts grown in a medium enriched with 34S (>91%) in the form of sulphate. Methionine was extracted from yeasts using enzymatic digestion with protease XIV followed by isolation using preparative reverse phase HPLC. Two separate batches of the labelled methionine standard were obtained. The 34S-labelled methionine standard solutions obtained were characterised both in terms of 34S isotope enrichment (82.7 ± 0.6%) and total sulfur concentration (396 ± 6 μg g−1) by reverse Isotope Dilution HPLC-ICP-MS using a natural abundance methionine standard solution and a multicollector instrument working in the pseudo-high resolution mode to avoid spectral interference. Additionally, the identity of the 34S-labelled methionine isolated by preparative HPLC and its isotope enrichment was confirmed by Gas Chromatography-Mass Spectrometry (GC-MS). Human blood plasma samples were spiked with the 34S-labelled methionine spike. Then, plasma proteins were precipitated with trifluoroacetic acid and separated by centrifugation. Methionine was separated from the rest of sulfur containing compounds by reversed phase chromatography in isocratic mode using a mobile phase of 75 mM ammonium acetate (pH 7.4) containing 2% of methanol. The retention time of methionine (8.3 minutes) was confirmed by fortifying the samples with natural abundance methionine and also by collecting the methionine peak and further analysis by GC-MS. Finally, methionine in human blood plasma samples was determined by measuring the signals for 32S and 34S in a double focusing ICP-MS instrument working at medium resolution (R = 4000). Concentrations were calculated by integrating the methionine peak for both masses and applying the isotope dilution equation after mass bias correction. The recoveries for samples fortified at different concentration levels ranged between 98.4 and 100.5%. Additionally, good agreement was obtained between the results found with this method and those reported by the clinical laboratory using a validated routine method.

Trace elements are essential substances for the proper physiological and biochemical functioning of the organism. Hemodialysis patients are potentially at risk of deficiency or excess of these elements. The application of inductively coupled plasma mass spectrometry (ICP-MS) allows the simultaneous quantification of very small amounts of multiple trace elements. The aim was to measure the serum concentration of copper (Cu), zinc (Zn), selenium (Se), and nickel (Ni), and the whole blood concentration of arsenic (As), lead (Pb), and manganese (Mn), in patients undergoing hemodialysis as well as in controls.The study was carried out in 57 hemodialysis patients compared with 57 controls with normal renal function. Serum and whole blood samples from the dialysis group were collected before and after hemodialysis sessions and Cu, Zn, Se, Ni, As, Pb and Mn levels were determined using ICP-MS.Hemodialysis patients showed significantly lower blood levels of Cu, Zn and Se than controls (p 0.001) and higher concentrations of Ni, As and Pb (p 0.0001). The levels of Mn were similar in both groups. After performing hemodialysis, Cu, Zn, Se and Ni concentrations were significantly higher than the pre-hemodialysis levels (p 0.0001). However, the concentration of As decreased (p 0.0001) and Pb and Mn levels were not significantly altered after the dialysis session.Hemodialysis patients are at increased risk of trace elements deficiency (especially for Zn and Se) or excess (Ni) in respect to healthy subjects. Monitoring of blood levels and supplementation of some trace elements may be indicated in patients undergoing hemodialysis.

Selenium, an essential trace element, is involved in the complex system of defense against oxidative stress through selenium-dependent glutathione peroxidases (GPx) and other selenoproteins. Because of its antioxidant properties, selenium or its selenospecies at appropriate levels could hinder oxidative stress and so development of diabetes. In this vein, quantitative speciation of selenium in human plasma samples from healthy and diabetic patients (controlled and non-controlled) was carried out by affinity chromatography (AF) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) and isotope dilution analysis (IDA). Similarly, it is well known that patients with diabetes who exhibit poor control of blood glucose show a decreased total antioxidant activity. Thus, we evaluated the enzymatic activity of GPx in diabetic and healthy individuals, using the Paglia and Valentine enzymatic method, observing a significant difference (p

Resumen Mujer que presento un importante incremento de la hormona estimulante del tiroides (TSH) (62,2 mU/L) con hormonas tiroideas dentro de los intervalos de referencia. La paciente se encontraba eutiroidea y no presentaba bocio. Se realizo un estudio inicial para determinar la posible causa del incremento en la concentracion de TSH. La recuperacion de TSH tras precipitacion con polietilenglicol fue del 1%, sugiriendo la presencia de alguna molecula de elevado peso molecular que podria interferir en la determinacion. Mediante cromatografia de exclusion, se confirmo la presencia de macro-TSH, un complejo autoinmune formado por TSH unido a una Inmunoglobulina G que es inmunorreactivo pero biologicamente inactivo, por lo que, si no se detecta, induce a una interpretacion erronea de la concentracion de TSH.

Resumen Introduccion El trasplante cardiaco es una opcion terapeutica disponible para algunos pacientes con insuficiencia cardiaca avanzada. Es importante establecer el pronostico que presentan estos pacientes para poder diferenciar aquellos con peor expectativa de vida y adoptar medidas adicionales en estos casos. Los peptidos natriureticos cardiacos han demostrado su utilidad diagnostica y pronostica en diferentes enfermedades. El objetivo de este estudio es valorar la utilidad del extremo aminoterminal del propeptido natriuretico tipo B (NT-proBNP) postoperatorio en el pronostico de los pacientes trasplantados cardiacos a corto plazo. Materiales y metodos Se determino la concentracion de NT-proBNP a los 15 dias postoperatorios en 50 pacientes que recibieron trasplante cardiaco para valorar la utilidad pronostica de mortalidad a 6 meses de seguimiento. Resultados Los pacientes que fallecieron mostraron concentraciones de NT-proBNP significativamente superiores que los que sobrevivieron, con una mediana de la concentracion de NT-proBNP a los 15 dias postrasplante de 20.632 ng/l (intervalo interculartilico: 6.183 a 37.925 ng/l) frente a 3.923 ng/l (intervalo interculartilico: 1.752 a 6.890 ng/l) respectivamente. Se observo que el hazard-ratio de mortalidad se multiplica por 8,5 veces (IC del 95%: 1,7 – 44,2) en el grupo de pacientes con concentraciones de NT-proBNP, cuantificadas a los 15 dias postrasplante, superiores al valor discriminatorio de 7.500 ng/l.

Resumen En los ultimos anos, la procalcitonina (PCT) ha demostrado ser la prueba mas sensible para el manejo del proceso infeccioso, el diagnostico y la estratificacion de la sepsis. Esta aporta valor pronostico de complicaciones en pacientes postoperados y criticos, e incluso es util como guia para la retirada del tratamiento antibiotico. Recientemente se esta comercializando un nuevo inmunoensayo de electroquimioluminiscencia para la determinacion de PCT (Elecsys BRAHMS PCT) con caracteristicas similares a la prueba mas eficaz descrita hasta ahora (PCT KRYPTOR). En el presente trabajo se procesaron 140 especimenes de pacientes con concentraciones de PCT de entre 0,02 y 47,13 ng/ml, en un Cobas e411 y en un KRYPTOR; se observo que los resultados no son transferibles entre ambos metodos. Si bien la clasificacion diagnostica con los puntos de corte actuales no varia sensiblemente, es necesario un estudio mas amplio para redefinir estos puntos de corte con el nuevo metodo. Por otra parte, se ha verificado que, aunque no hay efecto de arrastre significativo para una concentracion de PCT cercana al limite superior del intervalo dinamico de la prueba, si se observa arrastre para concentraciones mas altas. Sin embargo, la modificacion producida para una concentracion cercana al limite de decision clinica de 0,5 ng/ml es clinicamente irrelevante, por lo que no parece necesario incorporar ninguna accion correctiva para evitar este arrastre.

Prenatal diagnosis is based on genetic analysis of invasively obtained fetal tissue. Amniocentesis and chorial biopsy are the most used techniques, but carry a potential risk of miscarriage, from 0.5–1% up to 3.9% respectively. Isolation of fetal cells from maternal blood allows a non-invasive obtention of fetal genetic material, but their scarcity difficults an efficient diagnosis method. The relative abundance of fetal DNA in maternal blood, as well as the sensitivity of quantitative PCR based methods, constitute an attractive alternative method for X-linked diseases and RhD status diagnosis. In this paper, a review of the present state of non-invasive prenatal diagnosis methods is described.

This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

Background: Brain injury is a medical emergency that\ud needs to be diagnosed and treated promptly. Several proteins\ud have been studied as biomarkers of this medical condition. The aims of this study were to: 1) evaluate the selectivity and precision of a commercial ELISA kit for neurofilament medium polypeptide (NFM) protein; and 2) evaluate the concentration in cerebrospinal fluid (CSF) and serum of healthy individuals and patients with brain damage.\ud Methods: An ELISA from Elabscience was used. The selectivity was evaluated using size-exclusion chromatography and mass spectrometry. Intra- and inter-batch coefficients of variation (CV) were also studied. Fifty-one CSF samples from 36 age-matched patients with hemorrhagic stroke (HS) (n = 30), ischemic stroke (IS) (n = 11) and healthy individuals (n = 10) were assayed. In addition, serum samples from healthy volunteers (n = 47), 68 serum samples from seven patients with HS, 106 serum samples from 12 patients with traumatic brain injury (TBI) and 68 serum samples from 68 patients with mild traumatic brain injury (mTBI) were also analyzed.\ud Results: NFM was identified in the chromatographic\ud fraction with highest immunoreactivity. The intra- and\ud inter-batch CVs were ≤ 10% and ≤ 13%, respectively. The\ud CSF-NFM concentration in HS was significantly higher\ud (p < 0.0001) than in IS and controls. Serum NFM concentration ranged from 0.26 to 8.57 ng/mL in healthy\ud individuals (median = 2.29), from 0.97 to 42.4 ng/mL in\ud HS (median = 10.8) and from 3.48 to 45.4 ng/mL in TBI\ud (median = 14.7). Finally, 44% of patients with mTBI had\ud increased NFM concentration, with significantly higher\ud levels (p = 0.01) in patients with polytrauma.\ud Conclusions: To our knowledge this is the first study\ud describing increased NFM levels in CSF and serum from\ud patients with brain damage.

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.

Sepsis is the primary cause of death in the intensive care unit. The prevention of sepsis complications requires an early and accurate diagnosis as well as the appropriate monitoring. A deep knowledge of the immunologic basis of sepsis is essential to better understand the scope of incorporating a new marker into clinical practice. Besides revising this theoretical aspect, the current available tools for bacterial identification have been briefly reviewed as well as a variety of new markers showing either well-recognized or potential usefulness for diagnosis and prognosis of infections in critically ill patients. Particular conditions such as community-acquired pneumonia, pediatric sepsis, or liver transplantation, among others, have been separately treated, since the optimal approaches and markers might be different in these special cases.

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, β-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 β-Syn) showed significant differences between patients with and without brain hemorrhage. Novel biomarkers of brain injury (α-Inx, NFM, and β-Syn) were identified in the CSF of patients with HS. Investigating the role of these proteins in blood with more sensitive methods is warranted.

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 p-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