Background: A blood analysis with the particular focus of the blood gas status is often performed on critically ill patients to investigate whether there are any potential metabolic or respiratory causes underlying their condition. Utilizing a point-of-care analysis system for blood analysis close to the patient can enable faster analysis and thus accelerate medical decision-making. The aim of this project was to validate two point-of-care blood analysis instruments, EPOC and iSTAT. The blood analysis comprises 13 parameters (e.g. blood gases, hemoglobin, electrolytes), and some of which are calculated based on the original parameters.   Method and materials: The study was performed on blood samples taken from 20 volunteers after informed consent. The volunteers were regular patients who were referred to the hospital to leave a blood test: at the phlebotomy department the patients were informed about the purpose of the study, and for those who agreed, additional sample designated for this study were taken. The samples were initially analyzed using the two point-of-care instruments (Epoc and iSTAT) and thereafter with two verification instruments (ABL90-flex PLUS and Cobas Pure), which were located in the laboratory of Bollnäs hospital. Precision measurements were conducted by doing five replicates for four of the parameters (sodium, base excess, pCO2, potassium) on five different occasions.  Result: Both the Epoc and iSTAT-blood analysis system displayed satisfactory correlation with the reference instruments for each analyzed parameter. Further, the precision measurements showed that both Epoc and iSTAT displayed equivalent variation coefficient (CV%) as the reference methods.  Conclusion: The result of this study showed that the point-of-care instruments performed well and displayed a high correlation with the reference instruments.

The liver is one of the largest organs within the human body and it handles many vital tasks such as nutrient processing, toxin removal, and synthesis of important proteins. The number of people suffering from chronic liver disease is on the rise, likely due to the present ‘western’ lifestyle. As disease develops in the liver there are pathophysiological manifestations within the liver parenchyma that are both common and important to monitor. These manifestations include inflammation, fatty infiltration (steatosis), excessive scar tissue formation (fibrosis and cirrhosis), and iron loading. Importantly, as the disease progresses there is concurrent loss of liver function. Furthermore, postoperative liver function insufficiency is an important concern when planning surgical treatment of the liver, because it is associated with both morbidity and mortality. Liver function can also be hampered due to drug-induced injuries, an important aspect to consider in drug-development. Currently, an invasive liver needle biopsy is required to determine the aetiology and to stage or grade the pathophysiological manifestations. There are important limitations with the biopsy, which include, risk of serious complications, mortality, morbidity, inter- and intra-observer variability, sampling error, and sampling variability. Cleary, it would be beneficial to be able investigate the pathophysiological manifestations accurately, non-invasively, and on regional level. Current available laboratory liver function blood panels are typically insufficient and often only indicate damage at a late stage. Thus, it would be beneficial to have access to biomarkers that are both sensitive and responds to early changes in liver function in both clinical settings and for the pharmaceutical industry and regulatory agencies. The main aim of this thesis was to develop and evaluate methods that can be used for a ‘non-invasive liver biopsy’ using magnetic resonance (MR). We also aimed to develop sensitive methods for measure liver function based on gadoxetate-enhanced MR imaging (MRI). The presented work is primarily based on a prospective study on c. 100 patients suffering from chronic liver disease of varying aetiologies recruited due to elevated liver enzyme levels, without clear signs of decompensated cirrhosis. Our results show that the commonly used liver fat cut-off for diagnosing steatosis should be lowered from 5% to 3% when using MR proton-density fat fraction (PDFF). We also show that MR elastography (MRE) is superior in staging fibrosis. Finally we presented a framework for quantifying liver function based on gadoxetate-enhanced MRI. The method is based on clinical images and a clinical approved contrast agent (gadoxetate). The framework consists of; state-of the-art image reconstruction and correction methods, a mathematical model, and a precise model parametrization method. The model was developed and validated on healthy subjects. Thereafter the model was found applicable on the chronic liver disease cohort as well as validated using gadoxetate levels in biopsy samples and blood samples. The liver function parameters correlated with clinical markers for liver function and liver fibrosis (used as a surrogate marker for liver function). In summary, it should be possible to perform a non-invasive liver biopsy using: MRI-PDFF for liver fat and iron loading, MRE for liver fibrosis and possibly also inflammation, and measure liver function using the presented framework for analysing gadoxetate-enhanced MRI. With the exception of an MREtransducer no additional hardware is required on the MR scanner. The liver function method is likely to be useful both in a clinical setting and in pharmaceutical trials.

Urinvägsinfektion är en av de mest förekommande infektionerna hos människan. För att visualisera urinvägspatogener kan det kromogena mediet UriSelectTM4 användas för diagnostik. Det primära syftet med studien var att utvärdera om det är möjligt att inkubera det kromogena mediet UriSelectTM4 i 5 % koldioxid istället för aerob miljö utan att totalväxt och morfologi påverkas. Vidare utvärderades totalväxt och antal fria kolonier vid odling på en halv UriSelectTM4-agarplatta med två odlingstekniker för att undersöka om fortsatt diagnostik är möjligt. Urinprover inkuberades i aerob miljö och i 5 % koldioxid och jämfördes visuellt utifrån totalväxt, antal fria kolonier, morfologi samt färgförändring på kolonier och agarn. Resultatet visade att totalväxt och antal fria kolonier endast skiljer i liten grad mellan inkubationsmiljöerna. Däremot förekom skillnader i morfologi och färg. Vidare kunde en halv agarplatta användas vid odling och fortsatt diagnostik. Studien visar därmed att UriSelectTM4 kan inkuberas i 5 % koldioxid utan att totalväxt, fria kolonier och agarn påverkas. / Urinary tract infection is one of the most common infections among humans. For diagnostics, the chromogenic media UriSelectTM4 can be used to visualize the urinary tract pathogens. The primary purpose of the study was to evaluate if the chromogenic media UriSelectTM4 could be incubated in 5% carbon dioxide instead of aerobic environment without impacting total growth and morphology. Furthermore, total growth and number of free colonies was evaluated when cultivating on a half UriSelectTM4 agar media with two streak patterns to examine if further diagnostics is possible. Urine samples were incubated in aerobic environment and in 5% carbon dioxide and visually compared for total growth, number of free colonies, morphology and color change of bacterial colonies and the agar media. The results showed that total growth and free colonies only had slight differences between the incubation environments. On the other hand, morphology and color of the colonies may vary. Further a half agar media could be used for cultivation and further diagnostics. Consequently, the study shows that UriSelectTM4 can be incubated in 5% carbon dioxide without any impact on total growth, free colonies or of the chromogenic media.

Apixaban och lågmolekylärt heparin (LMH) är antikoagulantia som förhindrar blodproppsbildning genom att hämma faktor Xa. Allt mer patienter använder apixaban och LMH, vilket gör att laboratoriemedicin på länssjukhuset Ryhov är i behov av att utvärdera analysmetoder för apixaban och LMH för att kunna implementera analyserna i klinisk rutin. Syftet med studien var att utvärdera analysmetoden för bestämning av anti-FXa aktivitet i plasma hos patienter behandlade med apixaban eller LMH med hjälp av kromogen substratmetod. Metodutvärderingen bestod av fyra steg: repeterbarhet, mellanliggande precision, överensstämmelse med validerad metod och analys av normalpopulation. Utvärderingen genomfördes med hjälp av Sysmex CS-2100 där det analyserades 20 respektive 40 patientprover för apixaban och LMH samt 10 normalprover. Aktivitet av faktor Xa bestämdes kvantitativt med användning av ljusabsorption vid 405 nm. Repeterbarhet och mellanliggande precision visade låg CV. Patientprover visade överensstämmande resultat med referensvärden från andra laboratorium där r2 för apixaban och LMH var 0,95. Avvikande resultat kan bero på mätfel eller förväxling mellan prover. Analys av normalpopulation visade att värden låg under det lägsta tillförlitliga värdet. Utvärdering av analysmetoden apixaban och LMH på Ryhovs laboratorium visade goda resultat vilket bekräftar att analysmetoden kan användas i klinisk rutin. / Introduction: Apixaban and low molecular weight heparin (LMWH), are anticoagulants that prevent clot formation by inhibiting factor Xa. Increasingly more patients use apixaban and LMWH, for this reason the laboratory medicine at the county hospital Ryhov needs to evaluate methods of analysis for apixaban and LMWH to be able to implement the analyzes in clinical routine. Aim: The purpose of the study was to evaluate the assay method for determining anti-FXa activity in plasma in patients treated with apixaban or LMWH using chromogenic substrate method. Method: The method evaluation consisted of four steps: repeatability, intermediate precision measures, compliance with validated method and analysis of normal population. The evaluation was performed using Sysmex CS-2100 where 20 respective 40 patient samples were analyzed for apixaban and LMWH as well as 10 normal population samples. Factor Xa activity was quantitatively determined using light absorption at 405 nm.Result and discussion: Repeatability and intermediate precision showed low CV. Patient samples showed consistent results with reference values from other laboratories where r2 for apixaban and LMWH were 0.95. Deviant results may be due to measurement errors or confusion between samples. Analysis of normal population showed that values were below the lowest reliable value. Conclusion: Evaluation of the analysis method apixaban and LMWH at Ryhov's laboratory showed good results, which confirms that the assay method can be used in clinical routine.

Blodgassprutor beställs för att undersöka en patients syra-bas-status, laktatkoncentration och elektrolytkoncentration. Några orsaker till balansrubbningar kan vara trauma, syrebrist, infektion, intoxikation eller svält. År 2018 installerades ett nytt rörtransportsystem på Västerviks sjukhus och syftet med studien var att undersöka om det är möjligt att transportera blodgassprutor i det nya rörsystemet utan att provresultat påverkas. Analyser som studerades var pH (power of hydrogen), syretryck, koldioxidtryck, syrgasmättnad, natriumjoner, kaliumjoner, fria kalciumjoner, standardbikarbonat, basöverskott och laktat. Studien omfattade 27 arteriella dubbelprover där det ena provet transporterades i rörtransportsystemet och det andra transporterades manuellt till laboratoriet. Proverna analyserades på instrumentet ABL 800 Flex, inom 30 minuter efter provtagning, med analysmetoderna potentiometri, amperometri och spektrofotometri. Resultaten jämfördes i korrelationsdiagram med en regressionslinje för att påvisa samband mellan proverna. Korrelationsdiagrammen visade positiv linjär korrelation hos samtliga analyser och ett samband kunde påvisas (r = 0,930-0,998). Om resultatet från proverna som transporterats manuellt ökade, ökade även resultatet från proverna som transporterats i rörpost och tvärtom. Ett stapeldiagram skapades för att visualisera skillnader i medelvärde som visade en liten skillnad på basöverskott som ökade efter transport i rörpostsystemet. Ett tvåsidigt parat t-test utfördes för att påvisa om någon signifikant skillnad förelåg mellan analysresultaten. T-testet visade en statistisk signifikant skillnad på syretrycket (p = 0,04), syrgasmättnaden (p = 0,04), basöverskott (p = 0,001) och standardbikarbonat (p = 0,006), då medelvärdet ökade efter transport i rörpost. Medelvärdet för halten natriumjoner minskade efter transport i rörpostsystemet vilket innebar att hemolys inte förekom. Slutsatsen var att det finns en signifikant skillnad mellan blodgassprutor transporterade i rörpostsystem och blodgassprutor transporterade manuellt på vissa analyser, men skillnaden har ingen klinisk betydelse. / Blood gas syringe are ordered to examine the patient´s acid-base status, lactate concentration and electrolyte concentration. Some causes for imbalance could be trauma, lack of oxygen, infection, poisoning or starvation. In 2018, a new pneumatic tube transport system was installed at Västervik´s hostpital and the purpose of this study was to investigate if it is possible to transport samples for blood gas analyses with the new pneumatic tube transport system without affecting the test results. The analyses which were investigated were pH (power of hydrogen), oxygen tension, carbon dioxide tension, saturation, sodium ions, potassium ions, free calcium ions, standard bicarbonate, base excess and lactate. The study included 27 arterial double samples, one samples was transported in the pneumatic tube transport system and the other was manually transported to the laboratory. The samples were analysed within 30 minutes after the sampling, on the ABL 800 Flex instrument, using the methods potentiometry, amperometry and spectrophotometry. The results were compared using a correlation diagram with a regression line to study the relationship between the parameters. The correlation diagram shown a positive linear correlation and a relationship could be demonstrated for all the parameters (r = 0,929-0,998). If the results from the samples transported manually increased, the results also increased from the samples transported in the pneumatic tube transport system and vice versa. A bar chart was created to visualize differences in the mean values. A difference could be seen in base excess and the mean value increased after transport in the pneumatic tube transport. A two-sided paired t-test was performed to demonstrate any significant difference between the parameters. The t-test demonstrated a significant difference in the oxygen tension (p = 0,04), oxygen saturation (p = 0,04), base excess (p = 0,001) and the standard bicarbonate (p = 0,006) and statistically the values was higher after transport with the pneumatic tube transport system. The mean value for sodium ions decreased after transport in the pneumatic tube transport system and that indicate that hemolysis did not occur. The conclusion of the study was that there is a significant difference between blood gas syringes transported with pneumatic tube transport system and blood gas syringes transported manually, but the differences are not clinically relevant.

Akut- och psykiatriska avdelningar på länssjukhuset Ryhov i Jönköping använder sig av snabbtest för drogscreening med varierande kvalitet under de tider då analysinstrumentet Konelab Prime 30i inte är bemannat. Syftet med studien var att utvärdera sensitivitet och specificitet hos Multitest 15 från tillverkaren Acro Biotech, och jämföra resultat från två olika avläsningstider. Antalet urinprover som samlades in för analys uppgick till 272. Positiva och negativa urinprover med drogkoncentrationer inom ±50% från varje drogs gränsvärde insamlades. Senare inkluderades drogkoncentrationer utanför detta intervall. Proverna testades med Multitest 15 vid laboratoriet för klinisk kemi på Ryhov efter utförd analys med Konelab Prime 30i, vars analysresultat utgjorde referens. De droger som testades var amfetamin, metamfetamin, ecstasy, bensodiazepiner, buprenorfin, kokain, metadon, morfin, THC, oxykodon och tramadol. För alla droger sammantaget var sensitiviteten 86,7% - 100%, specificiteten 33,3% - 100% och träffsäkerheten 71,4% - 94,7%. Provurvalet inom intervallet ±50% från gränsvärdet var begränsat, vilket avsevärt påverkat dessa beräkningar, och Konelab Prime 30i använder semikvantitativ metod vilken endast ger approximativa koncentrationsvärden som referens. / The emergency and psychiatric wards on the county hospital Ryhov in Jönköping utilize onsite drug testing with varying quality during evenings and night-time when no staff are operating the chemistry analyzer Konelab Prime 30i. The aim of the study is to evaluate the performance of sensitivity and specificity of Acro Biotech Multitest 15 and comparing results from two different reading-times. The number of urine samples collected for analysis was 272. Positive and negative urine samples with drug concentrations within ± 50% from cut-off were collected. Later, concentrations outside of this range was included. The samples were tested with Multitest 15 at the laboratory for clinical chemistry at Ryhov after analysis with Konelab Prime 30i providing reference results. The drugs tested were amphetamine, methamphetamine, ecstasy, benzodiazepines, buprenorphine, cocaine, methadone, morphine, THC, oxycodone and tramadol. All drugs included, the sensitivity was 86.7% - 100%, the specificity 33% - 100% and the accuracy 71.4% - 94.7%. The sample selection within the range ±50% from the cut-off value was limited, which significantly affected these calculations, and Konelab Prime 30i uses a semi-quantitative method only providing approximate concentration values for reference.

Extended Spectrum β-Lactamases (ESBLs) are produced by the Enterobacteriaceae bacterial family, mainly by E. coli and K. pneumoniae. As these species are some of the main causes of urinary tract infections and sepsis, ESBL-production is of major concern. Occurrence of ESBLs also gives rise to concern as it is increasing epidemically. This because the genes coding for ESBLs (i.e. bla-genes) are located on plasmids replicating and spreading the replicated copies independently. Plasmids replicate by replicons. Plasmids with the same replicon variant are grouped into the same plasmid family. The aim of this study was to detect plasmid families carrying bla-genes in E. coli and K. pneumoniae from clinical (n = 6) and environmental water (n = 22) isolates. Plasmid family prevalence was examined. Association between plasmid families and bla-genes was also examined. Plasmid families were detected by a PBRT kit (PCR Based Replicon Typing), a multiplex PCR kit that detected 30 replicons, whereof 27 replicons representing the 27 plasmid families in Enterobacteriaceae, and three novel replicons. The IncF plasmid family was the most prevalent for both species in both clinical and environmental isolates. IncF seemed to be prevalent for all examined ESBLs, but it was difficult to associate one bla-gene with one plasmid family as most isolates carried several bla-genes and several plasmid families. / Extended Spectrum β-Lactamases (ESBLs) produceras av bakteriefamiljen Enterobacteriaceae, främst av E. coli och K. pneumoniae. Eftersom dessa arter är bland de vanligaste orsakerna till urinvägsinfektioner och sepsis är ESBL-produktion ett allvarligt problem. ESBL är också oroande eftersom det sprids epidemiskt. Detta möjliggörs av att generna som kodar för ESBLs (s.k. bla-gener) ligger på plasmider, som replikerar och sprider de replikerade plasmidkopiorna självständigt. Plasmider replikeras som s.k. replikon. Plasmider med samma replikonvariant tillhör samma plasmidfamilj. Syftet med detta arbete var att detektera plasmidfamiljer som bär bla-gener i E. coli och K. pneumoniae isolerade från kliniska prov (n = 6) och miljöprov (n = 22) från Helge Å. Plasmidfamiljernas prevalens undersöktes, liksom sambandet mellan plasmidfamiljer och bla-gener. Plasmidfamiljerna detekterades med ett PBRT-kit (PCR Based Replicon Typing), ett multiplext PCR-kit som detekterade 30 replikon varav 27 replikon som representerar de 27 plasmidfamiljer som finns i Enterobacteriaceae och tre nya replikon. Plasmidfamiljen IncF var vanligast förekommande i båda arter i både kliniska isolat och miljöisolat. IncF verkade förekomma för alla undersökta typer av ESBL, men det var generellt svårt att förknippa en bla-gen med en plasmidfamilj, eftersom de flesta isolaten bar flera bla-gener och flera plasmidfamiljer.

<p>Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. </p><p><i>In vitro</i> tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary.</p><p>Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD).</p><p>In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (<i>Phleum pratense</i>) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD.</p><p>Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp <i>Penaeus aztecus</i> (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach.</p><p>In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.</p>

Immunoglobulin E (IgE)-mediated allergy occurs when our immune system causes a reaction to otherwise harmless substances (allergens). Allergens are predominantly proteins present in biological materials such as pollens, mites, animal epithelia, moulds and foods. In vitro tests for specific IgE antibodies usually employ an allergen source extract as an antibody capturing reagent. The proportion of allergenic molecules in these biochemically complex extracts may vary. Recombinant allergens may be obtained in large quantities with biotechnological techniques. These proteins can be characterized biochemically and immunologically, resulting in tests with minimal batch-to-batch variation. This thesis describes different uses of recombinant allergens in component-resolved diagnostics (CRD). In CRD, single allergenic proteins are used to establish a sensitization profile of the patient. Two timothy grass (Phleum pratense) pollen allergens, Phl p 11 and Phl p 4, were cloned and expressed as recombinant proteins. They were subsequently characterized and can, for example, be used in a panel for grass pollen CRD. Single allergens may be useful as diagnostic markers for allergic sensitization. This phenomenon was studied using tropomyosin, a major allergen from the shrimp Penaeus aztecus (Pen a 1). The characteristics of the recombinant and natural proteins were compared. The recombinant tropomyosin was then extensively tested using specific competition for IgE binding against extracts of other crustacean species, house dust mite and cockroach. In cases when an important allergen is missing or underrepresented in a natural extract, the corresponding recombinant allergen may be added to the extract as a spiking reagent. Previous studies have shown that latex extracts for diagnostic testing may lack the allergen Hev b 5. Recombinant Hev b 5 was expressed from a synthetic gene construct, incorporating several adaptations to enable efficient large scale production of the recombinant protein, to be used as a spiking reagent.

One-lung ventilation (OLV) as an established procedure during thoracic surgery may be injurious in terms of increased mechanical stress characterised by alveolar cell stretch and overdistension, increased cyclic tidal recruitment of alveolar units, compression of alveolar vessels and increased pulmonary vascular resistance. This may result in ventilation-induced lung injury with pro-inflammatory cytokine production, leukocyte recruitment and neutrophil-dependent tissue destruction. Despite the consequences of delivering the whole tidal volume (VT) to only a single lung, relatively high VT are used during OLV to maintain arterial oxygenation and carbon dioxide elimination. However, this may increase mechanical stress in the dependent lung and may aggravate alveolar injury. There is a lack of data on the alveolar immune consequences of OLV. Therefore, the present studies investigate the epithelial damage and pro-inflammatory response induced by mechanical ventilation and OLV. OLV induced pulmonary injury, but alveolar damage in the ventilated lung decreased by reduction of the tidal volume in patients scheduled for thoracic surgery (study I). The use of the volatile anaesthetic desflurane in OLV patients attenuated the OLV-induced alveolar immune response (study II). Furthermore, an experimental model of thoracic surgery was established to investigate the systemic and pulmonary consequences of OLV and thoracic surgery in comparison with the effects of conventional two-lung ventilation and spontaneous breathing. The experimental data indicate that beside the pulmonary immune response volatile anaesthetics have also modulated the plasma concentrations of cytokines during and after OLV (study III). In contrast, OLV and thoracic surgery increased the expression of pro-inflammatory mRNA in BAL cells and lung tissue samples. General anaesthesia did not affect this response (study 4). The results of the present studies indicate that OLV and thoracic surgery may be injurious to the lung tissue to a similar degree. The recruitment and activation of alveolar granulocytes characterise the alveolar damage. The administration of different anaesthetics modulates the activation of alveolar cells, specified by decreased inflammatory mediator release in subjects that receive desflurane anaesthesia, which does not affect the expression of cytokine mRNA in alveolar cells and lung tissue samples.