Kolesterol transporteras i blodet med hjälp av lipoproteinpartiklar. Höga nivåer av low-density lipoprotein (LDL)-kolesterol i blodet är en riskfaktor för kardiovaskulär sjukdom. Koncentrationen av LDL-kolesterol kan beräknas med hjälp av Friedewalds formel men det finns även metoder där LDL-kolesterol kan analyseras direkt. Syftet med arbetet var att verifiera metoden direkt LDL-kolesterol på analysinstrumentet Beckman Coulter AU680. Metodens inomserie- och totalimprecision analyserades. Två korrelationsstudier utfördes mellan direkt LDL-kolesterol och beräknat LDL-kolesterol, en med 43 patientprover med triglycerider < 4,5 mmol/L och en med 11 patientprover med triglycerider > 4,5 mmol/L. Friedewalds formel ska egentligen inte användas vid triglycerider > 4,5 mmol/L, men i detta fall användes formeln ändå för att utvärdera eventuella skillnader mellan metodernas resultat vid höga triglyceridkoncentrationer. Vid analys av metodens inomserieimprecision blev variationskoefficienten (CV) omkring 0,5 % vid analys av både den låga kontrollen (A1) och den höga kontrollen (A2). CV för totalimprecisionen blev 1,21 % vid analys av A1 och 1,11 % vid analys av A2. Korrelationsstudierna visade ett linjärt samband mellan metoderna men den direkta metoden gav något högre resultat vid lägre koncentrationer och något lägre resultat vid högre koncentrationer jämfört med beräknat LDL-kolesterol. Vid triglycerider > 4,5 mmol/L gav den direkta metoden betydligt högre resultat än beräknat LDL-kolesterol. Slutsatsen blev att metoden hade god precision. Överensstämmelsen mellan metodernas resultat var relativt bra för proverna med triglycerider < 4,5 mmol/L. Vid triglycerider > 4,5 mmol/L var differensen mellan metoderna stor, troligtvis på grund av falskt för låga resultat från beräknat LDL-kolesterol. / Cholesterol is transported in the blood by lipoproteins. High levels of low-density lipoprotein (LDL)-cholesterol in the blood is a risk factor for cardiovascular disease. The concentration of LDL-cholesterol can be calculated using the Friedewald formula but there are also methods that measure LDL-cholesterol directly. The aim of this study was to verify the method P-LDL-cholesterol on a Beckman Coulter AU680 analyzer. Within-run imprecision and total imprecision were analyzed. The correlation between direct LDL-cholesterol and calculated LDL-cholesterol was examined using 43 patient samples with triglyceride levels < 4,5 mmol/L and 11 patient samples with triglyceride levels > 4,5 mmol/L. The Friedewald formula is not supposed to be used on triglyceride levels > 4,5 mmol/L, but in this case the formula was used anyway to evaluate differences between the methods at high triglyceride concentrations. The coefficient of variation (CV) for the within-run imprecision was about 0,5 %, both for the low control (A1) and the high control (A2). Total imprecision had a CV of 1,21 % for A1 and 1,11 % for A2. There was a linear relationship between the methods, but the direct method gave slightly higher results at low concentrations and slightly lower results at high concentrations compared to calculated LDL-cholesterol. At triglyceride levels > 4,5 mmol/L the results from the direct method was considerably higher than calculated LDL-cholesterol. The conclusion is that the precision of the method was good. The correlation between the results from direct LDL-cholesterol and calculated LDL-cholesterol was relatively high for samples with triglyceride levels < 4,5 mmol/L. At triglyceride levels > 4,5 mmol/L there was a big difference between the methods, probably because of falsely low results from calculated LDL-cholesterol.

Introdução: O Estudo Longitudinal de Saúde do Adulto é um estudo de coorte multicêntrico com o objetivo de identificar os fatores de risco associados ao diabetes tipo 2 e à doença cardiovascular na população brasileira. Nosso principal objetivo é explicar a concepção e implementação das rotinas do laboratório central e biobanco do Estudo Longitudinal de Saúde do Adulto (ELSA-Brasil); destacando as forças e limitações do protocolo. O segundo objetivo é descrever os pré-testes usados para validar o protocolo de antes do início do estudo. Métodos: O estudo optou pela centralização dos exames em um laboratório central no Hospital Universitário da Universidade de São Paulo (USP), Com base em dados recentes que confirma a estabilidade da glicose em amostras congeladas, até os testes de glicose no sangue foram centralizadas. Porém o processamento das amostras foi realizado nos laboratórios locais o que reduziu os custos do transporte das amostras para o laboratório central. A estratégia implicou na necessidade de implementação de equipes nos laboratórios em cada Centro de Investigação para coletar e processar as amostras antes do transporte. O estudo incluiu exames para a avaliação do metabolismo da glicose, resistência à insulina, perfil lipídico, eletrólitos, ácido úrico, hormônios da tireóide, função hepática e contagem total de células do sangue. Além disso, as amostras de DNA, urina plasma, e de soro foram colhidas e Além desses exames, o estudo também extraiu DNA de leucócitos, colheu e estocou amostras de urina, plasma e soro. Para garantir a homogeneidade do protocolo todos os membros das equipes locais passaram por treinamento e certificação centralizado, com visitas cruzadas entre os centros durante o campo para controle de qualidade. Resultados: A opção por um laboratório central garantiu a uniformidade da metodologia utilizada para a realização dos exames, evitando as variações inevitáveis entre laboratórios. Durante 26 meses, cerca de 375.000 testes foram realizados no laboratório central. Não houve perda de amostras biológicas durante o estudo. A implementação do biobanco usando palhetas armazenados em repositórios de nitrogênio foi realizada sem problemas importantes desde 2008 até agora. Conclusão: O ELSA-Brasil mostrou a exequibilidade de exames multicêntricos no Brasil com todos os exames realizados em um laboratório central, de uma maneira custo-efetiva. A logística de armazenamento de amostras biológicas foi feito com custos aceitáveis e de qualidade, sendo um modelo para estudos futuros / Background: The Longitudinal Study of Adult Health is a multicenter cohort study aimed to identify risk factors associated with type 2 diabetes and cardiovascular disease in Brazilian population. Our main objective is to explain the conception and implementation of the routines of the central laboratory and biobank of the Longitudinal Study of Adult Health (ELSA-Brasil) highlighting the strength and the limitations of the protocol The second objective is to describe the pre-tests used to validate the protocol before the start of the study. Methods: The study made an option to centralize the exams in one central laboratory at the University Hospital, São Paulo University (\"USP\"). Based on recent data that confirms the stability of glucose in freeze samples, even blood glucose tests were centralized. However, biological samples were processed in the local laboratories, reducing the weight of the material to be transported, and diminishing costs of transportation to the central lab. Especially trained local teams collected and processed biological samples before transportation. The study included tests for glucose metabolism, insulin resistance, lipid profile, electrolytes, uric acid, thyroid hormones, hepatic function, and total blood cell count. In addition, DNA, urine, plasma and serum samples were collected and stored. In order to guarantee protocol homogeneity, all team members underwent centralized training and certification, and cross-visits in each research center were done. Results: The choice of a central laboratory assured uniformity of the methodology used for the exams, avoiding the variations between laboratories During 26 months, approximately 375,000 tests were done in the central- laboratory. There was no loss of biological samples during the study. The implementation of the biobank using straws stored in nitrogen repositories was performed without important problems since 2008 until now. Conclusion: The ELSA-Brazil showed the feasibility of a multicenter study in Brazil with all the analyses performed in a central laboratory, in a cost-effective way. The logistic of storage of biological samples was done with acceptable costs and quality being a model for future studies

Introdução: O Estudo Longitudinal de Saúde do Adulto é um estudo de coorte multicêntrico com o objetivo de identificar os fatores de risco associados ao diabetes tipo 2 e à doença cardiovascular na população brasileira. Nosso principal objetivo é explicar a concepção e implementação das rotinas do laboratório central e biobanco do Estudo Longitudinal de Saúde do Adulto (ELSA-Brasil); destacando as forças e limitações do protocolo. O segundo objetivo é descrever os pré-testes usados para validar o protocolo de antes do início do estudo. Métodos: O estudo optou pela centralização dos exames em um laboratório central no Hospital Universitário da Universidade de São Paulo (USP), Com base em dados recentes que confirma a estabilidade da glicose em amostras congeladas, até os testes de glicose no sangue foram centralizadas. Porém o processamento das amostras foi realizado nos laboratórios locais o que reduziu os custos do transporte das amostras para o laboratório central. A estratégia implicou na necessidade de implementação de equipes nos laboratórios em cada Centro de Investigação para coletar e processar as amostras antes do transporte. O estudo incluiu exames para a avaliação do metabolismo da glicose, resistência à insulina, perfil lipídico, eletrólitos, ácido úrico, hormônios da tireóide, função hepática e contagem total de células do sangue. Além disso, as amostras de DNA, urina plasma, e de soro foram colhidas e Além desses exames, o estudo também extraiu DNA de leucócitos, colheu e estocou amostras de urina, plasma e soro. Para garantir a homogeneidade do protocolo todos os membros das equipes locais passaram por treinamento e certificação centralizado, com visitas cruzadas entre os centros durante o campo para controle de qualidade. Resultados: A opção por um laboratório central garantiu a uniformidade da metodologia utilizada para a realização dos exames, evitando as variações inevitáveis entre laboratórios. Durante 26 meses, cerca de 375.000 testes foram realizados no laboratório central. Não houve perda de amostras biológicas durante o estudo. A implementação do biobanco usando palhetas armazenados em repositórios de nitrogênio foi realizada sem problemas importantes desde 2008 até agora. Conclusão: O ELSA-Brasil mostrou a exequibilidade de exames multicêntricos no Brasil com todos os exames realizados em um laboratório central, de uma maneira custo-efetiva. A logística de armazenamento de amostras biológicas foi feito com custos aceitáveis e de qualidade, sendo um modelo para estudos futuros / Background: The Longitudinal Study of Adult Health is a multicenter cohort study aimed to identify risk factors associated with type 2 diabetes and cardiovascular disease in Brazilian population. Our main objective is to explain the conception and implementation of the routines of the central laboratory and biobank of the Longitudinal Study of Adult Health (ELSA-Brasil) highlighting the strength and the limitations of the protocol The second objective is to describe the pre-tests used to validate the protocol before the start of the study. Methods: The study made an option to centralize the exams in one central laboratory at the University Hospital, São Paulo University (\"USP\"). Based on recent data that confirms the stability of glucose in freeze samples, even blood glucose tests were centralized. However, biological samples were processed in the local laboratories, reducing the weight of the material to be transported, and diminishing costs of transportation to the central lab. Especially trained local teams collected and processed biological samples before transportation. The study included tests for glucose metabolism, insulin resistance, lipid profile, electrolytes, uric acid, thyroid hormones, hepatic function, and total blood cell count. In addition, DNA, urine, plasma and serum samples were collected and stored. In order to guarantee protocol homogeneity, all team members underwent centralized training and certification, and cross-visits in each research center were done. Results: The choice of a central laboratory assured uniformity of the methodology used for the exams, avoiding the variations between laboratories During 26 months, approximately 375,000 tests were done in the central- laboratory. There was no loss of biological samples during the study. The implementation of the biobank using straws stored in nitrogen repositories was performed without important problems since 2008 until now. Conclusion: The ELSA-Brazil showed the feasibility of a multicenter study in Brazil with all the analyses performed in a central laboratory, in a cost-effective way. The logistic of storage of biological samples was done with acceptable costs and quality being a model for future studies

Snabb och korrekt artidentifiering är avgörande för effektiv behandling av svampinfektioner, särskilt bland immunsupprimerade patienter. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) används rutinmässigt på kliniska laboratorier för identifiering av karaktäristiska proteinmönster hos bakterier och jästsvampar genom tolkning av proteinspektra i en masspektradatabas för korrekt artidentifiering. Mögelsvamparnas hårda cellvägg och heterogena växtsätt med varierande proteinuttryck beroende på mognadsstadie, försvårar identifiering med MALDI-TOF MS. Metodens tänkbara fördelar mot traditionella metoden mikroskopering är förkortade svarstider, säkrare artidentifiering av fler arter och mindre beroende av subjektiv morfologisk bedömning. Studiens syfte var att undersöka om MALDI-TOF MS kunde anpassas och användas för identifieringen av mögelsvamp i klinisk rutindiagnostik. Fyra referensstammar (Aspergillus niger, A. fumigatus, A.terreus, A.flavus) och ett kliniskt isolat (A.terreus) undersöktes. Preparationsmetoderna (I) fullständig myrsyraextraktion, (II) direktapplicering och (III) suspension i destillerat vatten användes för analys av sporer och frontmycel hos yngre och äldre mögelkulturer. Två olika masspektradatabaser för artidentifiering jämfördes; rutindatabasen BDAL och den specialiserade mögeldatabasen Filamentous Fungi Library. Även plocktekniken av mögelmaterial inför analys med MALDI-TOF MS utvärderades. Vid vissa tillfällen förbättrades artidentifieringen efter extraktion av mögelkulturerna, medan i andra fall var direktapplicering fullt tillräcklig. Mögelmaterial med mycket sporer tenderade ge något fler artidentifieringar i BDAL oavsett kulturernas ålder.  Filamentous Fungi Library tenderade i vissa fall ge bättre resultat jämfört med BDAL för yngre kulturer. Fler studier krävs för att utvärdera och optimera MALDI-TOF MS som metod för artidentifiering av mögelsvamp. / Rapid and accurate species identification is crucial for successful treatment of fungal infections, especially among immunosuppressed patients. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used routinely at clinical laboratories to identify characteristic protein patterns of bacteria and yeast by the interpretation of protein spectra in a database for accurate species identification. The hard cell wall of the mold and the heterogeneous growth with varying protein expression due to maturation, complicates identification with MALDI-TOF MS. The potential benefits of this method compared to microscopy as traditional method are shortened turn-around times, safer species identification of more species that is independent on subjective morphological assessment. The purpose of the study was to investigate whether MALDI-TOF MS could be adapted and used for the identification of molds in clinical routine diagnostics. Four reference strains (Aspergillus niger, A.fumigatus, A.terreus, A.flavus) and a clinical isolate (A.terreus) were examined. The preparation methods (I) complete formic acid extraction, (II) direct application and (III) suspension in distilled water were used for analysis of spores and frontmycelium from younger and older mold cultures. Two different masspektradatabases for species identification were compared; routine database BDAL and the specialized mold database, Filamentous Fungi Library. Also the collecting technique of mold prior to analysis with MALDI-TOF MS was evaluated. Sometimes, the species identification improved after extraction of mold cultures, while in other cases direct application was sufficient. Cultures with a lot of spores tended to give slightly more species identifications in BDAL regardless of the age of cultures. Filamentous Fungi Library, in some cases, tended to improve the performance compared to BDAL for younger cultures. More studies are required to evaluate and optimize MALDI-TOF MS as a method of mold identification.

Trigger finger is one of the most common hand conditions, with a prevalence of almost 3%. The aetiology remains unclear even though many causes have been suggested. The prevailing paradigm is that the pathogenesis of trigger finger is ascribed to primary changes in the first fibrous condensation of the tendon sheath (A1-pulley). Several studies have investigated pathology in the pulley, but few have investigated the tendon. The general aim of this thesis was to find out if there is pathology in the trigger finger tendon and to define it. We first looked at trigger finger tendon biopsies in a light microscope, and found that they were histologically different from healthy tendons. They showed signs of micro-ruptures, collagen degradation, increased amounts of ground substance, both hyper- and hypo-cellular areas, round active cell nuclei and absence of inflammatory cells, all similar to tendinosis. The histological picture was further assessed by using a scoring system for Achilles tendinosis. The trigger finger tendons scored high, suggesting a similar histopathology. Next, we performed a quantitative real-time polymerase chain reaction (qPCR) on trigger finger tendons. We assessed the mRNA expression of 10 genes, which have been described to be differently expressed in Achilles tendinosis (collagen 1 and 3, versican, decorin, biglycan, aggrecan, MMP-2, MMP-3, ADAMTS-5, and TIMP-3). The overall expression pattern agreed with previous studies on Achilles tendinosis, suggesting that the cellular function in trigger finger tendons is disturbed in a similar way as in Achilles tendinosis. Recent experimental and observational research has suggested potential side effects of statin treatment on tendons, but firm evidence was lacking. We performed an epidemiological study on two large population-based cohorts. Statin use was found to increase the risk of both trigger finger and tendinosis in the shoulder and Achilles tendons, especially among men. This suggests a similar pathology in trigger finger and tendinosis. We have also studied the time to treatment effect after a single injection of glucocorticoid in trigger finger. Our results suggest that 60-80% of patients can expect resolution of the triggering within 14 days, and half of them within seven days. This result allows correct information to be given to the patient and proper planning of follow-ups. In conclusion, the pathology in trigger finger tendons is similar to tendinosis in other tendons.

Warfarin är ett läkemedel som används för att förhindra att högriskpatienter såsom de med förmaksflimmer får tromboembolism. Denna verkan uppnås genom att hämma de K-vitaminberoende faktorerna VII, X och protrombin och på så sätt minska blodets förmåga att koagulera. Att hitta rätt dosering av läkemedlet för warfarinbehandlade patienter har visat sig vara svårt eftersom det kräver regelbunden provtagning och påverkas av mat- och levnadsvanor. Det vanligaste sättet att mäta protrombinkomplexhalten är med venös plasma men det är även möjligt att använda sig av kapillär plasma. Helblod kan användas för mekaniska metoder som inte använder sig av optisk detektion. Fördelen är att helblod inte kräver centrifugering. Studiens syfte var att undersöka om det fanns en signifikant skillnad (p≤0,05) mellan helblod och plasma som används i den nuvarande metoden för kapillära prover och om det finns en skillnad i stabiliteten av dessa prov. Dubbla prover togs från 30 warfarinbehandlade patienter och 5 icke warfarinbehandlade individer. Ett av proven centrifugerades och analyserades på plasma, det andra analyserades på helblod. Resultaten visade att det fanns en signifikant skillnad (p≤0,05) mellan metoderna. Bland-Altman diagrammet visade att 95 % av helblodsproverna inte var högre än 0,25 INR och lägre än 0,14 INR. Detta har en låg klinisk inverkan. 4 Proverna förvarades i rumstemperatur i upp till 24 timmar och analyserades sedan om. Ingen förändring över 10 % kunde observeras i hållbarheten. Studien visade att trots att det finns en signifikant skillnad är det möjligt att ersätta den nuvarande metoden med plasma och använda helblod istället. / Warfarin is a drug used to prevent high-risk patients such as those with atrial fibrillation from thromboembolisms. This effect is achieved by suppressing vitamin-K dependent factors VII, X and prothrombin and therefore decreasing the bloods ability to clot. Finding the right dosage of the drug for warfarin treated patients has proven difficult, as it demands regular blood draws to monitor their prothrombin complex level, which is affected by dietary and living habits. The most common way to measure prothrombin complex levels is by using venous plasma but it is also possible to use capillary plasma. Whole blood can be used for mechanical methods, which don’t use optical detection. The benefit is that whole blood doesn’t require centrifugation. The aim of this study was to investigate if there was a significant difference (p≤0,05) between using whole blood and plasma which is the existing method for capillary sample and also if there is any differences between the stability of these samples. Double samples from 30 warfarin treated patients and 5 non-treated persons were taken. One of the samples were centrifuged and analyzed on plasma and the other analyzed on whole blood. The results showed that there was a significant difference (p≤0,05) between the methods. Bland-Altman plot comparison showed that 95 % of the whole blood samples would not be higher than 0,25 INR and lower than 0,14 INR. This has low clinical impact. The samples were stored at room temperature for up to 24 hours and reanalyzed. No changes over 10 % in INR values were observed. This study showed that even though there is a significant difference, it is possible to replace the existing method which using plasma with the whole blood instead.

Este estudo de caráter qualitativo e quantitativo, tipo exploratório-descritivo, trata da caracterização do trabalho de enfermagem em laboratórios de análises clínicas. O objetivo geral visa identificar os aspectos da atuação da enfermagem nos laboratórios de análises clínicas, que permitam caracterizar o processo de trabalho da enfermagem. Os objetivos específicos buscaram identificar os trabalhadores da saúde que atuam na coleta de exames; reconhecer as atividades desempenhadas pelos diferentes agentes da enfermagem e conhecer sua inserção na estrutura organizacional. O referencial teórico adotado pautou-se nos estudos do processo de trabalho e de recursos humanos em saúde e em enfermagem. Para a coleta de dados utilizou-se um questionário e a amostra foi composta por 45 instituições. A análise dos resultados revelou que, quanto à caracterização dos laboratórios, 15,6% não realizam treinamento em serviço e 60% fazem-no de modo isolado, não continuado; o enfermeiro é o profissional que assume majoritariamente a responsabilidade por essa ação. Quanto aos recursos humanos, 77,8% são auxiliares de enfermagem, 13% enfermeiros e 9,1% técnicos de enfermagem. Evidencia-se a divisão social e técnica do trabalho, no qual os auxiliares executam o cuidado direto, o enfermeiro gerencia o processo e os técnicos desempenham ambas ações, sem diferenças relevantes entre as atividades dos auxiliares e técnicos de enfermagem. Além disso, há outros profissionais que compartilham das mesmas atividades realizadas pela enfermagem e esta se encontra, em grande parte, subordinada a outras áreas de atuação, com escassa autonomia na estrutura organizacional. / This qualitative and quantitative study, an exploratory-descriptive study, examines the characteristics regarding the work performed by nursing professionals in clinical laboratories. The general goal aims to identify roles played by nursing professionals in clinical laboratories that allow us to characterize the nursing work process. The specific goals seek to identify health workers that are responsible for collecting samples, to distinguish the activities played by different nursing professionals and to learn how they are inserted in the organizational structure. The theoretical reference adopted is based on studies regarding work procedures and human resources in health and nursing. A questionnaire was used to collect data and the sample comprised 45 institutions. Regarding the clinical laboratories, result analysis revealed that 15.6% of them do not offer in-service training and 60% do not do it on a continuous manner; nurses basically take on the responsibility for training other nursing professionals. Regarding human resources, 77.8% are nursing assistants, 13% are nurses, and 9.1% are practical nurses. There is evidence of a social and technical division of the workload: nursing assistants provide direct care, nurses manage the processes, and practical nurses perform both activities. No relevant differences were observed between the activities played by nursing assistants from those played by practical nurses. Furthermore, there are other professionals that share the same activities played by those nursing professionals. In most cases, nursing professionals are subordinated to other areas and have little autonomy in the organizational structure.

Abstract Laboratory medicine is under constant pressure from changes in the operating environment. Organisational changes and tendering processes have led to a trend towards shorter turn-around times and more cost-effective choices. Analysis tools that were previously only available at research laboratories, such as the mass spectrometer and polymerace chain reaction (PCR), have now made their way to university hospital laboratories and even mid-sized laboratories. Organisational changes have increased the need to monitor the pre-analytical steps. The specimen can be drawn from the patient in a satellite laboratory, which may be located several hours from the central laboratory. The increased transportation times may change the analytical properties of the specimens, which is why the stability of different analytes should be investigated thoroughly in different temperatures. It should be born in mind that doctors are treating the patients based on the results they receive from the laboratory. To avoid possible malpractice, the analytical properties should remain reliable. Traditionally, some analyses have been carried out manually, which is known to be time-consuming and carries the possibility of wide intra-observatory mistakes. For that reason, it would be reasonable to perform some manual analyses, such as body fluid analysis, in an automated manner. Automating the manual steps taken in the laboratory would release labour for other tasks and may increase the cost-effectiveness of the work. Organisational changes have redirected the needs of a clinical laboratory towards automated options instead of manual ones and finding more economically-based alternatives to replace or complement traditional methods. / Tiivistelmä Laboratoriolääketiede on jatkuvan muutospaineen alla. Organisaatiomuutokset ja kilpailutus ovat saaneet aikaan sen, että laboratorioiden analytiikkatarjonnan tulee olla kilpailukykyistä niin hinnan kuin tulosten vastausnopeuden suhteen. Aikaisemmin pelkästään tutkimuskäytössä olleet menetelmät, kuten PCR ja massaspektrometri, ovat jalkautuneet jo keskussairaalatasoiseen tutkimusvalikoimaan. Organisaatiomuutokset ovat saaneet aikaan myös sen, että näytteet voidaan ottaa potilaasta alueellisissa toimipisteissä ja kuljettaa päivän aikana keskuslaboratorioon analysoitavaksi. Kuljetusmatkat ja -ajat saattavat olla hyvinkin pitkiä. Tämän johdosta on erittäin tärkeää selvittää näytteiden säilyvyys niin, että tulokset pysyvät luotettavina eikä potilaan hoito kärsi. Perinteisesti osa tutkimuksista, kuten punktionesteen solut, on tehty käsin mikroskopoimalla, jonka tiedetään olevan aikaa vievää ja näin ollen myös kallista analysointia. Kyseisen tutkimuksen siirtäminen analysaattoreille tehtäväksi voi tuoda laboratoriolle taloudellisen säästön lisäksi työvoiman vapautumista manuaalisesti suoritettavalta mikroskopoinnilta. Muutospaineet laboratoriotoiminnoissa ovat saaneet aikaan tarpeen automatisaation lisääntymiselle ja taloudellisempien vaihtoehtojen löytämiselle perinteisten menetelmien rinnalle tai niiden sijaan.

The pre-analytical phase is known for being the most important step in the laboratory process to reach reliable test results. If handling, transport or preparation of the sample is performed incorrectly the results can deviate from the true value. Today, sampling tubes contains various additives to stabilize concentration levels. The aim of this study was to test a new sampling tube containing fluoride/citrate for glucose, lactate and homocysteine. It was also of interest to evaluate the stability of those three analytes in lithium-heparin, sodium-fluoride/potassium oxalate and fluoride/citrate tubes. To perform the sustainability study, a method verification was done for homocysteine in plasma. The study was performed in a hospital laboratory on the routine instrument Roche Cobas 6000 analyzer. Blood was drawn from 20 patients and was analyzed at the hospital laboratory in Gävle. The blood samples were transported frozen to the laboratory in Hudiksvall and were used in the method verification. For the sustainability study, blood was drawn from 10 healthy volunteers in lithium-heparin, sodium-fluoride/potassium oxalate and fluoride/citrate tubes. The method verification was approved. The results showed that glucose was stable for up to 72 hours in Vacuette Glycaemia tube with fluoride/citrate and this tube also gave more accurate results. Lactate and homocysteine were also stable in fluoride/citrate, but needs further studies. All three analytes were more stable if the sample tubes were centrifuged as soon as possible after blood collection. Fluoride/citrate tubes were stable without centrifugation directly.

Neisseria gonorrhoeae (NG) har successivt utvecklat resistens mot många antimikrobiella medel och betraktas som ett av de tre reella hoten bland antibiotikaresistenta bakterier. Ciprofloxacin är ett bredspektrum-antibiotikum tillhörande gruppen kinoloner som, förutom att behandla urinvägsinfektioner, används mot NG och infektioner i mage och tarm. Dock har det rapporterats att ca 30 % av NG-isolat som samlats in genom gonokock-isolatövervakningsprojekt (GISP) under 2017 var resistenta mot ciprofloxacin. På molekylnivå är resistens mot ciprofloxacin starkt associerad med en enda mutation i kodon 91 i gyras-genen (gyrA). Detta projekt har undersökt om det går att använda molekylärbiologiska metoder för att detektera NG-isolat med gyrA mutationen. Analysen gjordes med två olika PCR-system, ”7500 Fast Real-Time PCR System” från Applied Biosystems (ABI) och Panther Fusion från Hologic. Proberna som användes designades för påvisning av vildtyp gyrA (ciprofloxacin-känslig) och mutant gyrA (ciprofloxacin-resistent) hos NG. I projektet analyserades 50 NG-positiva prov (analyserade med screeningtest APTIMA COMBO2 från Hologic), från 43 patienter som provtagits under januari-februari 2020 i Region Skåne. Några patienter testades flera gånger vid olika tillfällen. NG-odling hade utförts parallellt från motsvarande tagna prov från patienterna. ABI-metoden påvisade genen hos 90 % (45/50) av NG-positiva prover (APTIMA COMBO2) medan endast 24 av de 49 proven (49 %) kunde odlas med traditionell metodik för att därefter resistensbestämmas. Av de 45 prov där gyras-genen kunde detekteras med ABI-metoden, uppvisade 28 (62 %) av proven en muterad gen och därmed en potentiell resistens för ciprofloxacin. Panther Fusion-metoden påvisade genen hos 80 % (40/50) av NG-positiva prover (APTIMA COMBO2), och såsom tidigare nämnts, kunde endast 24 av de 49 proven (49 %) odlas med traditionell metodik för att därefter resistensbestämmas. Av de 40 prov där gyras-genen kunde detekteras med Panther Fusion-metoden, uppvisade 26 av proven (65 %) en muterad gen och därmed en potentiell resistens för ciprofloxacin. En jämförelse mellan resultaten från PCR-metoderna och odlingarna visar att av de 24 odlingarna som kunde resistensbestämmas fick ABI-metoden resultat för 23 och Panther Fusion för 22. PCR-metodernas resultat överensstämde perfekt med resultaten från odling med samma 8 känsliga och 15 respektive 14 resistenta NG-isolat som odling. De båda PCR-metoderna och traditionell odling uppvisade jämförbara resultat. Av de 24 prov som kunde odlas och därmed resistensbestämmas, detekterades med ABI-metoden gyras-genen i 23 av dessa prov och i 22 av proven med Panther Fusion-metoden. Resistens mot ciprofloxacin uppvisades genom odling i 16 av de 24 odlingsbara prov, och av dessa 24 odlingsbara prov uppvisade ABI-metoden en muterad gen i 15 av proven och Panther Fusion-metoden en muterad gen i 14 av proven. Traditionell odling kunde bara genomföras på 24 av proven och PCR-metoderna identifierade signifikant fler prov innehållande vildtyp eller muterad gyras-gen, 45 respektive 40 prov. Projektet visade tydligt att PCR-metoderna kan identifiera fler prov än genom traditionell odling och kan därmed upptäcka fler prov med förväntad ciprofloxacin-resistens än vad som kan bestämmas genom traditionell odling. / Neisseria gonorrhoeae (NG) has been developing a resistance towards several different antibiotics and is viewed as one of the three real threats among resistant bacteria. Ciprofloxacin is a broad-spectrum-antibiotic belonging to the group quinolone antibiotics which, in addition to being used to treat urinal infections, is used to treat NG and infections in the stomach and intestines. However, it has been reported that 30 % of NG-isolates that have been gathered through the Gonococcal Isolate Surveillance Project (GISP) throughout 2017 were resistant to ciprofloxacin. On a molecular level, resistance to ciprofloxacin is strongly associated with a single mutation in kodon 91 in the gyras-gene (gyrA). This project sought to examine if it is possible to use methods from molecular biology to detect which NG that have the gyrA-mutation. The test was done using two different PCR-systems, ”7500 Fast Real-Time PCR System” from Applied Biosystems (ABI) and Panther Fusion from Hologic. The probes used were designed to show wild type gyrA (ciprofloxacin sensitive), and mutated gyrA (ciprofloxacin resistant) in NG. In this project 50 NG-positive samples (analysed with screentest APTIMA COMBO2 from Hologic), from 43 patients that had been tested during January-February 2020 in Region Skåne, were analysed. Some patients were tested several times, within the time period. NG-cultivation had been done in parallel from corresponding samples taken from the patients. The ABI-method showed the gene in 90 % (45/50) of NG-positive samples (APTIMA COMBO2) while only 24 of the 49 samples (49 %) could be cultivated by traditional methodology, and then tested for resistance. Of the 45 samples where the gyras-gene could be detected with the ABI-method, 28 samples (62 %) exhibited a mutated gene and thus a potential resistance to ciprofloxacin. The Panther fusion-method showed the gene in 80 % (40/50) of NG-positive samples (APTIMA COMBO2), and as mentioned earlier, only 24 of the 49 samples (49 %) could be cultivated by traditional methodology to then be tested for resistance. Of the 40 samples where the gyras-gene could be detected with the Panther Fusion-method, 26 samples (65 %) exhibited a mutated gene and thus a potential resistance to ciprofloxacin. The two PCR-methods and traditional cultivation exhibited comparable results. Of the 24 samples that could be cultivated and thus tested for resistance, the ABI-method detected the gyras-gene in 23 of these samples and the Panther Fusion-method detected the gene in 22 of the samples. Cultivation exhibited resistance to ciprofloxacin in 16 of the 24 samples that could be cultivated, and of these 24 cultivatable samples the ABI method exhibited a mutated gene in 15 of the samples and the Panther Fusion-method exhibited a mutated gene in 14 of the samples. Traditional cultivation could only be done on 24 of the samples and the PCR-methods could identify significantly more samples containing either wild type or mutated gyras-gene, 45 and 40 samples, respectively. The project clearly showed that more samples can be identified with the PCR-methods than through traditional cultivation, and thereby discover more samples with expected ciprofloxacin-resistance, than can be determined through traditional cultivation.