Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nitric oxide (NO) is a reactive free radical, that acts as a messenger molecule, mediating several functions, including vasodilation, platelet aggregation inhibition, neurotransmission, antimicrobial and antitumor activities. In several pathological conditions, NO is associated with increased circulatory concentrations of cytokines and endotoxins in inflammatory processes in especially. Because this radical to have a short half-life, its determination is difficult, therefore, measurement of metabolites nitrite/nitrate (NOx) is most often used to evaluate NO production. The objective of this study the effect of pre-analytical interferences been investigating on the NOx and determine the limits of urinary and serum reference for a healthy population. In the simulation of bilirubin, lipemia and hemolysis on serum samples, we evaluated the pre-analytical interference in the measurement of NOx All bilirubin concentrations used (9, 19, 38, 75, 150 and 300 mg /L), Intralipid® (0.67, 1.25, 2.5, 5 and 10 g/L) and hemoglobin (0.5, 1.0, 2.0, 4.0 and 5.0 g/L) resulted in a difference between the original amount of NOx being checked greater than 10 percent %, thus, considered an analytical interference. The reference limits were evaluated following the recommendations of the International Federation of Clinical Chemistry (IFCC), and urinary values of 46.1 mmol / L to 1533.0 mmol/L, and serum values 56.8 mmol/L at 340, 3 mmol/L for a presumably healthy population. Thus, we conclude that bilirubin, lipemia and hemolysis interfere with the measurement of serum activity of NOx. The reference limits were evaluated following the recommendations of the International Federation of Clinical Chemistry (IFCC), and urinary values of 46.1 mmol/L to 1533.0 mmol/L, and serum values 56.8 mmol/L at 340, 3 mmol/L for a healthy population. Thus, we conclude that bilirubin, lipemia and hemolysis interfere with the measurement of serum activity of NOx. / O óxido nítrico (NO) é um radical livre reativo, que age como uma molécula mensageira, mediando diversas funções, incluindo vasodilatação, inibição da agregação plaquetária, neurotransmissão, atividades antimicrobianas e antitumorais. Em várias condições patológicas, o NO está associado com o aumento da concentração circulatória de citocinas e endotoxinas especialmente em processos inflamatórios. Pelo fato deste radical possuir uma meia-vida curta, a sua determinação torna-se difícil e, consequentemente, a mensuração de seus metabólitos nitrito/nitrato (NOx) é mais frequentemente utilizado para avaliar a produção de NO. Assim, o objetivo deste estudo foi investigar o efeito de interferentes pré-analíticos sobre os níveis de NOx, bem como determinar os limites de referência urinária e sérica para uma população saudável. Na simulação da icterícia, lipemia e hemólise em amostras séricas, avaliou-se a interferência pré-analítica na mensuração do NOx. Todas as concentrações utilizadas de bilirrubina (9, 19, 38, 75, 150 and 300 mg/L), de Intralipid® (0,67; 1,25; 2,5; 5 e 10 g/L) e de hemoglobina (0,5; 1,0; 2,0; 4,0 e 5,0 g/L) resultaram em uma diferença do valor original de NOx, sendo verificada uma porcentagem maior que 10%, sendo assim, considerada uma interferência analítica. Os limites de referência foram avaliados seguindo recomendações da International Federation of Clinical Chemistry (IFCC), sendo os valores urinários de 46,1 μmol/L a 1533,0 μmol/L, e os valores séricos 56,8 μmol/L a 340,3 μmol/L para uma população saudável. Também foi possível concluir que a bilirrubina, lipemia e hemólise causam interferência na mensuração da concentração sérica do NOx.

During the industrialization of the 19th century the negative health effects of vitamin D was discovered as children in the cities developed osteomalacia or more commonly known as rickets caused by vitamin D deficiency. Vitamin D is produced in the skin from 7-dehydrocholesterol during sun-exposure and enhances intestinal phosphor and calcium absorption thus enhancing the bone remodeling process. Now, in the 21st century, Vitamin D is still relevant as positive health effects have been recognized and with it an increased number of samples and a demand for accurate analyzing. Vitamin D is commonly believed to be sensitive to ultraviolet radiation in serum and blood samples and therefore have traditionally been kept protected from light exposure from the time of sampling until the finished analyze. However recent studies have proven 25- hydroxyvitamin D (25[OH]D) to be stable in both whole blood and serum. As previous studies have been primarily conducted in research laboratories with the aim to study vitamin D under specific research-laboratory conditions the aim of this study was to study the stability of 25[OH]D in serum and whole blood within both primary care- and hospital laboratories under normal and exaggerated conditions with the purpose to evaluate possible pre-analytical issues with everyday handling processes. The assay used was high pressure liquid chromatography-tandem mass spectrometry, HPLCMS/MS, and the sought analytes 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, 25[OH]D2 and 25[OH]D3. The results showed that 25-hydroxyvitamin D is stable in serum for 24 hours at room temperature whilst exposed to light both ultraviolet and fluorescent. The analyte is also stable for up to four freeze-thaw cycles rendering the process of light-protection and samples frozen immediately after centrifugation superfluous. The results also ensure reliable results even if samples are accidently left on benchtops or saved refrozen to be reanalyzed at a later date. / Under den industriella revolutionen på 1800 talet upptäcktes de negativa hälsoeffekterna av vitamin D-brist då barnen i städerna utvecklade rakit (osteomalaci) eller engelska sjukan som sjukdomen också kallas på grund av brist på sol och D-vitamin. Vitamin D produceras i huden från 7-dehydrokolesterol vid solexponering och ökar upptaget av fosfor och kalcium i tarmen som i sin tur förbättrar återuppbyggnaden av skelettet. Vitamin D är fortfarande aktuell även nu i vår tid men då för dess nyupptäckta hälsofrämjande egenskaper som till exempel förebyggandet av coloncancer. Detta medför även en ökning av antalet analyser och kräver därmed en adekvat analysmetod. Traditionellt har det antagits att vitamin D är ljuskänsligt i alla former därför har blod och serum ljusskyddats, från provtagningstillfället fram tills dess att analysen är utförd. Dock har nya studier visat att 25-hydroxyvitamin D (25[OH]D) är mycket stabilt bundet till vitamindbindande protein i både serum och helblod. Syftet med studien var att utvärdera om 25[OH]D i serum och helblod behöver ljusskddas genom att studera stabiliteten hos 25[OH]D i både serum och helblod under normala primärvårdslaboratorie- och sjukhuslaboratorieförhållanden samt under extrema förhållanden för att utvärdera eventuella preanalytiska problem eller fel relaterade till den vardagliga hanteringen av vitamin D prover. Proverna analyserades med högupplösande vätskekromatografi-tandem masspektrometri, HPLC-MS/MS, och de sökta analyterna var 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, 25[OH]D2 och 25[OH]D3. Resultat från studien visade att 25-hydroxyvitamin D är stabilt i serum i 24 timmar i rumstemperatur med ljusexponering från både ultraviolett och fluorescerande ljus. 25-hydroxyvitamin D är även stabil i serum upp till fyra frys- och tiningscykler. Detta gör att provhanteringen kan förenklas genom att dessa prover inte behöver ljusskyddas samt att serumet ej behöver frysas in direkt efter centrifugering. Resultatet säkerställer även tillförlitliga resultat om prover lämnas framme på bänken av misstag eller om prover behöver sparas och frysas om för att analyseras vid senare tillfälle.

Since laboratory tests influence as much as 70% of the medical decisions, errors throughout the blood sampling process may jeopardize the safety of the patient. Research has shown that the pre-analytical phase of the blood sampling process is where the most errors occur. Moreover, this phase is said to be the most influential part of the total testing process, due to maximal involvement of humans. In this study, the pre-analytical errors in the blood sample handling process and their causes were investigated. Therefore, a literature study was performed, and first-hand data was collected through interviews. A total of 8 interviews was conducted in Sweden, Belgium, and the Netherlands. From these interviews, qualitative data was collected and analysed with a thematic approach. The 5 themes that were obtained are: nurse vs. lab technician, sample identification, logistics, location dependency of sample quality, and hospital management. Each theme included multiple observations and conclusions. One specific observation was a lack of communication and knowledge management amongst nurses and lab technicians. To minimize this gap between nurse and lab technician from a knowledge transfer perspective, a conceptual framework was constructed. This framework can be used both from an academic and practical point of view. This research has both academic and practical implications. Within the academic perspective, empirical data is gathered among hospitals in three different countries, which is compared to data found in academic literature. Moreover, with the analysis of the empirics gathered in this study and the framework that is proposed, a contribution is made to knowledge management within hospitals. From a practical perspective, a contribution is made in helping to minimise the pre-analytical errors in their blood sampling process, while giving the hospital a better understanding of the lack of communication and knowledge management amongst their key employees. By strengthening the communication and knowledge transfer, errors can be avoided. This would not only benefit the speed and accuracy of the diagnose process, but also benefit the nurses and lab technicians in workload. / Eftersom laboratorietester påverkar så mycket som 70 % av de medicinska besluten kan fel i blodprovstagningen äventyra patientsäkerheten. Forskning visar att det är i den föranalytiska fasen av blodprovstagningsprocessen som de flesta felen uppstår. Dessutom sägs denna fas vara den mest inflytelserika delen av den totala provtagningsprocessen, på grund av den höga graden av människors inblandning. Denna studie undersökte de föranalytiska felen i blodprovshanteringen och deras orsaker. En litteraturstudien genomfördes, och förstahandsuppgifter samlades in genom intervjuer. Totalt åtta intervjuer genomfördes i Sverige, Belgien och Nederländerna. Från dessa intervjuer samlades kvalitativa data in och analyserades med en tematisk ansats. De fem teman som erhölls var: sjuksköterska jämte laboratorietekniker, providentifiering, logistik, platsberoende provkvalitet och sjukhusledning. Varje tema innehöll flera observationer och slutsatser. En specifik observation var bristen på kommunikation och kunskapshantering mellan sjuksköterskor och laboratorietekniker. För att minimera denna klyfta mellan sjuksköterska och laboratorietekniker ur ett kunskapsöverföringsperspektiv konstruerades en konceptuell ram. Denna ram kan användas både ur akademisk och praktisk synvinkel. Denna forskning har både akademiska och praktiska konsekvenser. Utifrån det akademiska perspektivet samlades empiriska data in bland sjukhus i tre olika länder, som jämförs med data i den existerande litteraturen. Med analysen av den empiri som samlats in i den här studien och det ramverk som föreslås ges dessutom ett bidrag till kunskapshantering inom sjukhus. Ur ett praktiskt perspektiv bidrar detta till att minimera de föranalytiska felen i blodprovstagningsprocessen, samtidigt som sjukhuset får en bättre förståelse för bristen på kommunikation och kunskapshantering bland sina nyckelpersoner. Genom att stärka kommunikationen och kunskapsöverföringen kan fel undvikas. Detta skulle inte bara gynna diagnostikprocessens snabbhet och noggrannhet, utan även avlasta sjuksköterskorna och laboratorieteknikernas arbetsbörda.

Bakgrund: Provmaterial för joniserat kalcium är känsligt för pH-förändringar och med tanke på svårstuckna patienter är det betydelsefullt att undersöka lägre fyllnadsnivåers påverkan på analysresultatet. På grund av olika pre-analytiska faktorer kan tidsgränsen (4 timmar) för analys av standardbikarbonat överskridas. Förvaring av post-analytiska serumprover medför att kompletteringsanalyser kan beställas. Begränsad dokumentation finns om avkorkade serumprovers stabilitet i rumstemperatur. Syfte: Syftet var att undersöka hur lägre fyllnadsnivåer av serum påverkar analysresultatet för joniserat kalcium, om standardbikarbonatsprover på helblod kan analyseras senare än 4 timmar och hur länge serumprover kan stå i rumstemperatur utan kork för eventuella kompletteringsanalyser. Metod: Koncentrationen av analyten joniserat kalcium i serumprover med fyllnadsnivåerna 1 mL och 2 mL jämfördes med maximalt fyllda provrör. Kylskåpsförvarade helblodsprover analyserades för standardbikarbonat efter 4-7 timmar. Avkorkade serumprover analyserades för 10 biokemiska analyter efter att ha stått i rumstemperatur 2-8 timmar. Genomsnittlig procentuell avvikelse jämfördes med en analytisk och biologisk imprecisionsgräns för att bedöma analyters stabilitet. Resultat och slutsatser: Analysresultat av joniserat kalcium i lägre fyllnadsnivåer var tillförlitliga. Stabiliteten av standardbikarbonatsproverna kunde inte bedömas och därmed kunde inte en eventuell tidsgränsändring rekommenderas. De biokemiska analyterna var stabila upp till 8 timmar i rumstemperatur. / Background: Ionized calcium concentrations decrease when samples are exposed to air. Due to pre-analytical factors, the 4 hour time limit for analysis of standard bicarbonate, can sometimes be exceeded. There is limited documentation about additional analyses on post-analytic decapped serum samples stored at room temperature. Aim: The aim was to examine how lower sample volumes affect the concentration of ionized calcium, if the time limit for analysis of standard bicarbonate on whole blood can be prolonged and how long decapped serum samples can be stored at room temperature for eventual additional analyses. Methods: The concentration of ionized calcium was analyzed on serum samples filled with 1 mL and 2 mL and were compared to maximally filled samples. Refrigerated whole blood samples were analyzed for standard bicarbonate after 4-7 hours. Ten biochemical analytes were measured in decapped serum samples after 2-8 hours of storage at room temperature. The mean percentage deviation was compared to an analytical and biological imprecision limit to determine analyte stability. Results and conclusions: Ionized calcium concentrations in lower sample volumes were reliable. The stability of standard bicarbonate could not be determined, therefore a longer possible time limit could not be recommended. The biochemical analytes were stable for 8 hours.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the major non-protein thiol body and is involved in cellular antioxidant defense. The free glutathione is present mainly in its reduced form (GSH) and can be converted to the oxidized form (GSSG) in the presence of reactive oxygen species (ROS). The GSH/GSSG ratio high is very important for the cellular redox state and a reduction of this ratio is often used as an indicator of oxidative stress. The enzyme glutathione reductase (GR) catalyzes the reduction of GSSG to GSH using NADPH. Because of the great importance of the antioxidant glutathione and considering that it is present in almost all organisms, numerous studies involving several attempts to detect GSH, GSSG and GR in biological systems has been performed. The objective of this study was to validate an automated analytical method, based on a spectrophotometric method proposed by Mannervick and Carlberg in 1985 to measurement the GR activity which is the oxidation of NADPH, which is monitored spectrophotometrically at a wavelength of 340 nm using the automated analyzer Cobas Mira®, determine its limits of reference for a healthy population and further evaluate the pre-analytical interference that influence the analytical phase of hemoglobin, bilirubin and lipemia. The automated method for measuring of the GR activity was validated as recommended by the EMEA and ANVISA. Since it was linear (r2 = 0.990), precise, with a coefficient of variation (CV) in precision intraassay of 5.7% (50 U/L) and 3.4% (100 U/L) and precision interassay CV of 9.5% (50 U/L) and 9.9% (100 U/L). In addition, we observed a recovery of 114.1% with this method considered accurate. The reference limits were evaluated as recommended by the International Federation of Clinical Chemistry (IFCC), and were 21.7 U / L to 60.3 U / L, for a healthy population. In the simulation of hemolysis, lipemia and jaundice in plasma samples, we evaluated the pre-analytical interference in the activity of the GR. All concentrations of Intralipid® (0.67, 1.25, 2.5, 5 and 10 mg / dL), hemoglobin standard (0.0625, 0.125, 0.25, 0.5 and 1 g / dL), and bilirubin (0.9, 1.9, 3.8, 7.5, 15 and 30 mg / dL) resulted in a difference from the original value of GR, and verified a percentage greater than 5%, and this percentage considered for enzymes, analytical interference. Thus, it was concluded that the automated method developed was linear, precise, accurate, simple and inexpensive, and can be adapted to the Cobas Mira® analyzer. The reference limits for a healthy population were established. Furthermore, it was demonstrated that hemoglobin, lipemia and bilirubin interfere in the measurement of the GR activity. / A glutationa (L-γ-glutamil-L-cysteinylglycine) é o principal tiol não proteico do organismo e está envolvida na defesa celular antioxidante. A glutationa livre está presente principalmente na sua forma reduzida (GSH) e pode ser convertida para a forma oxidada (GSSG) na presença de espécies reativas de oxigênio (EROs). A razão GSH/GSSG elevada é muito importante para o estado redox celular e uma redução desta razão é frequentemente utilizada como um indicador do estresse oxidativo. A enzima glutationa redutase (GR) catalisa a redução de GSSG a GSH utilizando NADPH. Devido a grande importância antioxidante da glutationa e considerando que a mesma está presente em quase todos os organismos, numerosas pesquisas envolvendo as mais diversas tentativas de detecção de GSH, GSSG e GR em sistemas biológicos tem sido realizadas. Assim, o objetivo deste estudo foi validar um método analítico automatizado, baseado em um método espectrofotométrico proposto por Mannervick e Carlberg em 1985 para a mensuração da atividade da GR que consiste na oxidação do NADPH, o qual é monitorado espectrofotometricamente no comprimento de onda de 340 nm utilizando o analisador automatizado Cobas Mira®, determinar seus limites de referência para uma população saudável e ainda avaliar a interferência pré-analítica que influenciam na fase analítica da hemoglobina, lipemia e bilirrubina. O método automatizado para a mensuração da atividade da enzima GR foi validado seguindo recomendações da ANVISA e EMEA. Sendo que o mesmo foi linear (r2=0,990), preciso, apresentando um coeficiente de variação (CV) na precisão intraensaio de 5,7% (50 U/L) e 3,4% (100 U/L) e na precisão interensaio um CV de 9,5% (50U/L) e 9,9% (100 U/L). Além disso, foi observada uma recuperação de 114,1%, sendo este método considerado exato. Os limites de referência foram avaliados seguindo recomendações da International Federation of Clinical Chemistry (IFCC), sendo que foram de 21,7 U/L a 60,3 U/L, para uma população saudável. Na simulação da hemólise, lipemia e icterícia em amostras de plasma, avaliou-se a interferência pré-analítica na atividade da GR. Todas as concentrações utilizadas de Intralipid® (0,67; 1,25; 2,5; 5 e 10 mg/dL), de padrão de hemoglobina (0,0625; 0,125; 0,25; 0,5 e 1 g/dL) e de bilirrubina (0,9; 1,9; 3,8; 7,5; 15 e 30 mg/dL) resultaram em uma diferença do valor original de GR, sendo verificada uma porcentagem maior que 5%, sendo essa porcentagem considerada, para enzimas, interferência analítica. Dessa forma, foi possível concluir que o método automatizado desenvolvido foi linear, preciso, exato, simples e de baixo custo, podendo ser adaptado ao analisador Cobas Mira®. Os limites de referência para uma população saudável também foram estabelecidos. Além disso, foi demonstrado que a hemoglobina, a lipemia e a bilirrubina interferem na mensuração da atividade da GR.