Harmonization study of results between biochemical analyzers Labmax 240® and Labmax 240 Premium® / Estudo de harmonização de resultados entre analisadores bioquímicos Labmax 240® e Labmax 240 Premium®

ABSTRACT Introduction: The harmonization of equipment is recommended in clinical laboratory practice aiming for the homogeneity of results when similar or equivalent analyzers are used to perform routine testing. Objectives: To conduct a study of equivalence between the biochemical analyzers Labmax 240® (E1) and Labmax 240 Premium® (E2) through the matching results and the statistical value analysis of dosages. Materials and methods: We evaluated tests with glucose, total cholesterol, triglycerides, uric acid, aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH), all with 40 repeated measurements, performed in both equipments. The Clinical and Laboratory Standards Institute (CLSI) EP09-3A protocol was used to conduct the comparison test between E1 and E2 equipment, with subsequent evaluation of the results for statistical analysis determining the Pearson correlation coefficient (r) and indexes comparison error with EP Evaluator® software. Results: Regarding the values of the Pearson correlation coefficient, all tests showed a strong correlation between equipment with r > 0.989, except for the dosage of LDH (r = 0.982). This dosage failed not because the value of r, but due to the values obtained in the error index being larger than the total errors index allowed. Discussion: Compared to clinical criteria, the results of the analyzers are approximately equal, but this control process must be done continuously in order to prevent and track random errors within the laboratory routine. Conclusion: The process of harmonization of multiple devices that perform the same laboratory parameters is essential for ensuring quality and reliability of laboratory results and should be standardized and included in routine clinical analysis laboratories.

RESUMO Introdução: No laboratório clínico, é recomendável a harmonização de equipamentos que visem à homogeneidade dos resultados, quando analisadores similares ou equivalentes são utilizados para desempenho da rotina de realização dos testes. Objetivos: Realizar um estudo de equivalência entre os analisadores bioquímicos Labmax 240® (E1) e Labmax 240 Premium® (E2). Materiais e métodos: Foram avaliados os testes glicose (GLI), colesterol total (COL), triglicerídeos (TRI), ácido úrico , aspartato aminotransferase (AST), alanina aminotransferase (ALT) e lactato desidrogenase (LDH), todos com 40 dosagens repetidas, realizadas em ambos os equipamentos. O protocolo do Clinical and Laboratory Standards Institute (CLSI) EP09-3A foi utilizado para conduzir o teste de comparação, com posterior avaliação dos resultados pela análise estatística no software EP Evaluator®, com determinação do coeficiente de correlação de Pearson (r) e comparação de índices de erro. Resultados: Em relação aos valores do coeficiente de correlação de Pearson, todos os testes apresentaram forte correlação entre os equipamentos, com r > 0,989, exceto para a dosagem de LDH (r = 0,982), que foi reprovada, não em função do valor de r, mas devido aos valores obtidos em relação ao índice de erro, o qual é maior do que os índices de erro total permitido. Discussão: Diante dos critérios clínicos, os resultados dos analisadores são aproximadamente iguais, porém esse controle do processo deve ser feito continuamente a fim de impedir e rastrear erros aleatórios dentro da rotina laboratorial. Conclusão: O processo de harmonização de múltiplos equipamentos que realizam os mesmos parâmetros laboratoriais é fundamental para a garantia da qualidade e da confiabilidade dos resultados laboratoriais, devendo ser padronizado e incluído na rotina dos laboratórios de análises clínicas.

Methionine and methionine sulfoxide treatment induces M1/classical macrophage polarization and modulates oxidative stress and purinergic signaling parameters.

Methionine is an essential amino acid involved in critical metabolic process, and regulation of methionine flux through metabolism is important to supply this amino acid for cell needs. Elevation in plasma methionine commonly occurs due to mutations in methionine-metabolizing enzymes, such as methionine adenosyltransferase. Hypermethioninemic patients exhibit clinical manifestations, including neuronal and liver disorders involving inflammation and tissue injury, which pathophysiology is not completely established. Here, we hypothesize that alterations in macrophage inflammatory response may contribute to deleterious effects of hypermethioninemia. To this end, macrophage primary cultures were exposed to methionine (1 mM) and/or its metabolite methionine sulfoxide (0.5 mM), and M1/proinflammatory or M2/anti-inflammatory macrophage polarization was evaluated. In addition, inflammation-related pathways including oxidative stress parameters, as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities; reactive oxygen species (ROS) production, and purinergic signaling, as ATP/ADP/AMPase activities, were investigated. Methionine and/or methionine sulfoxide induced M1/classical macrophage activation, which is related to proinflammatory responses characterized by increased iNOS activity and TNF-α release. Further experiments showed that treatments promoted alterations on redox state of macrophages by differentially modulated SOD and CAT activities and ROS levels. Finally, methionine and/or methionine sulfoxide treatment also altered the extracellular nucleotide metabolism, promoting an increase of ATPase/ADPase activities in macrophages. In conclusion, these findings contribute to better understand the participation of proinflammatory responses in cell injury observed in hypermethioninemic patients.

Performance analysis of software for identification of intestinal parasites / Análise de desempenho de software para identificação de parasitas intestinais

ABSTRACTIntroduction:Intestinal parasites are among the most frequent diagnoses worldwide. An accurate clinical diagnosis of human parasitic infections depends on laboratory confirmation for specific differentiation of the infectious agent.Objectives:To create technological solutions to help parasitological diagnosis, through construction and use of specific software.Material and method:From the images obtained from the sediment, the software compares the morphometry, area, perimeter and circularity, and uses the information on specific morphological and staining characteristics of parasites and allows the potential identification of parasites.RESULTS:Our results demonstrate satisfactory performance, from a total of 204 images analyzed, 81.86% had the parasite correctly identified by the computer system, and 18.13% could not be identified, due to the large amount of fecal debris in the sample evaluated.Discussion:Currently the techniques used in Parasitology area are predominantly manual, probably being affected by variables, such as attention and experience of the professional. Therefore, the use of computerization in this sector can improve the performance of parasitological analysis.Conclusions:This work contributes to the computerization of healthcare area, and benefits both health professionals and their patients, in addition to provide a more efficient, accurate and secure diagnosis.

RESUMOIntrodução:As parasitoses intestinais figuram entre os diagnósticos mais frequentes no mundo. Um diagnóstico clínico acurado das infecções parasitárias humanas depende da confirmação laboratorial para diferenciação específica do agente infeccioso.Objetivos:Criar formas tecnológicas para auxiliar no diagnóstico parasitológico por meio da construção e da utilização de um software específico.Material e método:A partir das imagens obtidas do sedimento, o software compara a morfometria, a área, o perímetro e a circularidade, além de utilizar informações sobre características específicas de morfologia e coloração dos parasitos e permitir a identificação dos possíveis parasitas.Resultados:Nossos resultados apontam desempenho satisfatório, sendo que do total de 204 imagens analisadas, 81,86% tiveram o parasita identificado corretamente pelo sistema computacional e 18,13% não puderam ser identificados, em função da grande quantidade de detritos fecais na amostra avaliada.Discussão:Atualmente, as técnicas realizadas no setor de Parasitologia são predominantemente manuais, sendo afetadas possivelmente por variáveis como atenção e experiência do profissional. Portanto, a utilização da informatização deste setor pode melhorar a performance das análises parasitológicas.Conclusão:O presente trabalho contribui para a informatização da área da saúde e beneficia tanto os profissionais da saúde como também seus clientes, além de proporcionar um diagnóstico mais eficiente, preciso e seguro.

Discrepancies among three laboratory methods for Clostridium difficile detection and a proposal for their optimal use.

Clostridium difficile is the major cause of nosocomial diarrhoea. Several detection methods are available for the laboratory diagnosis of C. difficile, but these vary in terms of sensitivity and specificity. In this study, we compared the performance of three following laboratory tests to detect C. difficile: in-house real-time PCR aiming for toxin B gene (tcdB), EIA for detection of toxins A and B (Premier Toxins A & B) and C. difficile culture in selective medium (bioMerieux). Our results were grouped into three categories as follows: (1) C. difficile-associated diarrhoea (CDAD); (2) asymptomatic carriers; and (3) negative results. Among the 113 patients included in the study, 9 (8.0%) were classified as CDAD, 19 (16.8%) were asymptomatic carriers, 76 (67.2%) had negative results and 9 (8.0%) could not be categorized (positive test for C. difficile toxins only). PCR was found to be the most sensitive diagnostic test in our study, with the potential to be used as a screening method for C. difficile colonization/CDAD. Diagnosis of CDAD would be better performed by a combination of PCR and EIA tests.

Quinolinic acid-induced seizures stimulate glutamate uptake into synaptic vesicles from rat brain: effects prevented by guanine-based purines.

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5'-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80-100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.

Diferenciação entre Entamoeba histolytica e Entamoeba dispar por meio de ensaio imunoenzimático para pesquisa de antígenos em amostras fecais / Differentiation between Entamoeba histolytica and Entamoeba dispar by means of immunoenzymatic assay for antigens detection in children fecal samples

A diferenciação do agente patogênico causador de amebíase obteve grande importância desde que Entamoeba histolytica (patogênica) foi considerada como espécie distinta de Entamoeba dispar (não patogênica). No presente estudo, foi realizada a pesquisa de antígenos de E. histolytica em amostras fecais de crianças residentes na cidade de São Leopoldo, Rio Grande do Sul, Brasil, utilizando-se ensaio imunoenzimático, ELISA (E. histolytica Test, TechLab Inc., Blacksburg, EUA) disponível no comércio. Foram analisadas 262 amostras de fezes pela técnica de Hoffman, Pons e Janer (HPJ), em que três amostras apresentaram positividade para o complexo E. histolytica/E. dispar. Do total de amostras, 91 (incluindo aquelas positivas pela técnica de HPJ) foram analisadas por meio de ELISA. Houve discordância entre os resultados obtidos no exame coproparasitológico e no ELISA, pois todas amostras foram não reagentes no ELISA. Estes dados indicam a presença de E. dispar e ausência de E. histolytica nas três amostras positivas pela técnica de HPJ. Os dados do presente estudo mostram a importância de utilização de técnica mais específica para efetuar identificação e diferenciação da amebíase intestinal.

In vivo quinolinic acid increases synaptosomal glutamate release in rats: reversal by guanosine.

Glutamate, the main excitatory neurotransmitter in the mammalian central nervous system (CNS), plays important role in brain physiological and pathological events. Quinolinic acid (QA) is a glutamatergic agent that induces seizures and is involved in the etiology of epilepsy. Guanine-based purines (GBPs) (guanosine and GMP) have been shown to exert neuroprotective effects against glutamatergic excitotoxic events. In this study, the influence of QA and GBPs on synaptosomal glutamate release and uptake in rats was investigated. We had previously demonstrated that QA "in vitro" stimulates synaptosomal L-[3H]glutamate release. In this work, we show that i.c.v. QA administration induced seizures in rats and was able to stimulate synaptosomal L-[3H]glutamate release. This in vivo neurochemical effect was prevented by i.p. guanosine only when this nucleoside prevented QA-induced seizures. I.c.v. QA did not affect synaptosomal L-[3H]glutamate uptake. These data provided new evidence on the role of QA and GBPs on glutamatergic system in rat brain.

The effects of ebselen on [3H]glutamate uptake by synaptic vesicles from rat brain.

Ebselen is a selenium organic compound, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effects of ebselen on the functionality of the glutamatergic system are still poorly investigated. In this study, by using synaptic vesicle preparation, we investigated the effects of ebselen (0.3 to 10 microM) on (i) vesicular glutamate uptake, (ii) bafilomycin-sensitive H+ -ATPase activity, and (iii) proton gradient formation (DeltapH). Ebselen presented a dual effect on glutamate uptake: the 1 microM concentration resulted in a 60% increase of the uptake, while the 10 microM concentration resulted in 60% inhibition. We also observed that ebselen (10 microM) inhibited the H+ -ATPase activity and dissipated the DeltapH. The inhibitory effects of ebselen were prevented by dithiothreitol (DTT). These findings suggest that high concentrations of ebselen may oxidize the essential thiol groups of the H+ -ATPase, which in turn affect its activity and compromise the vesicular glutamate uptake, and consequently lead to an impairment of the neural homeostasis.

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats.

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.

Anticonvulsant effect of GMP depends on its conversion to guanosine.

Studies on the purinergic system normally deal with adenine-based purines, namely, adenine nucleotides and adenosine. However, a guanine-based purinergic system may also have important neuromodulatory roles. Guanine-based purines exert trophic effects on neural cells, protect brain slices in a model of hypoxia and stimulate glutamate uptake. In vivo, both guanosine 5'-monophosphate (GMP) and guanosine (GUO) protected against seizures. In this study, we investigated if the anticonvulsant effect of GMP is mediated by guanosine and if guanosine or GMP treatments were able to increase adenosine levels. Intraperitoneal (i.p.) treatments with 7.5 mg/kg GMP or guanosine prevented 50% of seizures by quinolinic acid (QA) and increased guanosine cerebrospinal fluid (CSF) levels around twofold and threefold, respectively; GMP and adenosine levels remained unchanged. Intracerebroventricular treatment with 960 nmol GMP prevented 80% of seizures and the 5'-nucleotidase inhibitor alpha-beta-methyleneadenosine 5'-diphosphate (AOPCP), when injected 3 min before, reduced this anticonvulsant effect to 30% protection as well as significantly decreased the conversion of GMP into guanosine measured in the CSF. This study shows that the previously reported effect of GMP as an anticonvulsant seems to be related to its ability to generate guanosine through the action of ecto-5'-nucleotidase.

Guanine derivatives modulate L-glutamate uptake into rat brain synaptic vesicles.

Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.

Methylmercury inhibits glutamate uptake by synaptic vesicles from rat brain.

Methylmercury (MeHg) is an environmental contaminant that continues to cause risk to human health. The toxic effects of MeHg on the CNS implicate the involvement of glutamatergic system. In this study, we evaluated the effects of MeHg on [3H]glutamate uptake by synaptic vesicles. MeHg inhibited [3H]glutamate uptake in a concentration dependent manner. Since glutamate uptake by synaptic vesicles is driven by an electrochemical gradient, formed across the vesicle membrane by a bafilomycin A(1)-sensitive H+-ATPase, we further investigated the effect of MeHg on activity of this enzyme. MeHg inhibited the H+-ATPase activity and also dissipated the proton gradient (DeltapH), indicating that MeHg decreased [3H]glutamate uptake involving the H+-ATPase activity. Until now, the toxic effects of MeHg on CNS were attributed mainly to an impairment of glial glutamate transporters. These findings contribute for the understanding of the neurotoxicity by MeHg, pointing to the involvement of vesicular glutamate.

Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes.

Quinolinic acid (QA) is an endogenous neurotoxin involved in various neurological diseases, whose action seems to be exerted via glutamatergic receptors. However, the exact mechanism responsible for the neurotoxicity of QA is far from being understood. We have previously reported that QA inhibits vesicular glutamate uptake. In this work, investigating the effects of QA on the glutamatergic system from rat brain, we have demonstrated that QA (from 0.1 to 10mM) had no effect on synaptosomal L-[3H]glutamate uptake. The effect of QA on glutamate release in basal (physiological K+ concentration) or depolarized (40 mM KCl) conditions was evaluated. QA did not alter K+-stimulated glutamate release, but 5 and 10mM QA significantly increased basal glutamate release. The effect of dizolcipine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor on glutamate release was investigated. MK-801 (5 microM) did not alter glutamate release per se, but completely abolished the QA-induced glutamate release. NMDA (50 microM) also stimulated glutamate release, without altering QA-induced glutamate release, suggesting that QA effects were exerted via NMDA receptors. QA (5 and 10mM) decreased glutamate uptake into astrocyte cell cultures. Enhanced synaptosomal glutamate release, associated with inhibition of glutamate uptake into astrocytes induced by QA could contribute to increase extracellular glutamate concentrations which ultimately lead to overstimulation of the glutamatergic system. These data provide additional evidence that neurotoxicity of QA may be also related to disturbances on the glutamatergic transport system, which could result in the neurological manifestations observed when this organic acid accumulates in the brain.