Procalcitonin (PCT) is a precursor protein of the hormone calcitonin and is encoded by the gene Calcitonin-1. In the Blekinge Regional, P-PCT is only analyzed in Karlskrona. The analysis is performed in the department of clinical chemistry on the Alinity i-series instrument. PCT indicates bacterial infections and therefore, it is important to have a backup method for the analysis when the instrument in the city of Karlskrona is out of order. The aim of this work was to verify the analysis method of P-PCT on the instrument Alinity i-series in the city of Karlshamn. Analysis method verification means to confirm and prove that the method meets the specified requirements. Verification was performed by analyzing 35 samples with different concentration of PCT on the master instrument in Karlskrona and on “Alinity 1” and “Alinity 2” in Karlshamn. The method was compared by studying correlation coefficient and bias. The precision was measured only on “Alinity 1” which would be the master instrument in Karlshamn. Precision was measured by analyzing 25 replicates at two control levels and then was 5 replicates of each control level analyzed over 5 days. The correlation was good and no significant bias between results from Karlskrona and “Alinity 1” and between results from “Alinity 1” and “Alinity 2”. Precision on “Alinity 1” meets the requirements. The conclusion was that verification of PCT on master instrument “Alinity 1” and slave instrument “Alinity 2” was approved and the backup method for the PCT analysis in Karlshamn was verified. / Prokalcitonin (PCT) är en peptidprekursor till hormonet kalcitonin och kodas av genen kalcitonin-1 (CALC-1). PCT används som biomarkör för bakteriella infektioner och sepsis. I Region Blekinge analyseras P-PCT bara i Karlskrona. Analysen utförs på avdelningen för Klinisk kemi på instrumentet Alinity i-serie Abbot. PCT indikerar bakteriella infektioner och av denna anledning efterfrågas av flera avdelningar. Därför är det viktigt att verifiera en analysmetod för P-PCT i Karlshamn, ifall instrumenten i Karlskrona är ur funktion. Syftet med arbetet var att verifiera analysmetoden för P-PCT på instrumentet Alinity i-serie Abbot i Karlshamn. Metodverifiering innebär att bekräfta och bevisa att metoden uppfyller specificerade krav. Metodverifiering utfördes genom att analysera 35 prover med olika PCT-koncentrationer på masterinstrument ”Shrek” i Karlskrona och på ”Alinity 1” och ”Alinity 2” i Karlshamn. Metoderna jämfördes genom att studera korrelationskoefficient och bias. Inom och total serie-precision mättes bara på ”Alinity 1” som ska vara masterinstrument i Karlshamn. Inom serie-precision mättes genom att analysera 25 replikat av två kontrollnivåer och total serie-precision genom att analysera 5 replikat/dag under 5 dagar. Resultaten blev att metoden på ”Alinity 1” hade en god korrelation med metoden på masterinstrument ”Shrek” och ingen signifikant bias observerades mellan metoderna. Inom och total serie-precision på ”Alinity 1” uppfyller kraven. Metoden på ”Alinity 2” hade en god korrelation med metoden på masterinstrument ”Alinity 1” och ingen signifikant bias observerades mellan metoderna. Slutsatsen var att verifiering av P-PCT på masterinstrument ”Alinity 1” och slavinstrument ”Alinity 2” var godkänd och därmed verifierades en backupmetod för analysen P-PCT i Karlshamn.

It is important to have well-trained and qualified laboratory professionals. Seventy percent of patient care is based on decisions made from laboratory results, yet there is a growing shortage of medical technologists. Although some baby boomers are delaying retirement, worsening of the shortage crisis is inevitable. Retention of medical technologists has become more important than recruitment. The purpose of this study was to identify and evaluate innovative retention strategies used by clinical laboratory managers throughout the United States. A significant finding of this study was the lack of qualified medical technologists entering the ranks of laboratory managers. This study identified a need for a more defined career path and more recognition of the importance of laboratory scientists. It is recommended that studies be undertaken to examine the opinions of hospital and medical group practice administrators as well as the view of medical technologists regarding retention strategies that are proven to be effective.

Neuroblastoma constitutes roughly 8% of all childhood cancers where 95% of all neuroblastoma cases occur before the age of 10. The survival rate of infants and young children is very poor, which alone contributes to research novel biomarkers for classification methods, improved diagnosis and better anti-tumor therapies. The aim of this meta-analysis was to identify dysregulated miRNAs in neuroblastoma that has the potential to be used as antioncogenic biomarkers for diagnostic interventions. Additionally, explore miRNA interconnectedness on a systemic level and conversely extend the support of using miRNAs as biomarkers. A comprehensive literature search was performed within NIH-PubMed, NCBI-PMC and in the reference list of already reviewed publications, which yielded 9 eligible publications. Quality of evidence was assessed according to the guidelines adapted from MIAME, MINSEQE and MIQE. miRNet 2.0 was used to find the most significantly enriched annotations linked to neuroblastoma. A total of 251 samples (Cancer: 141; Control: 110) was reported by the 9 studies. These involved 66 dysregulated miRNAs (Up-regulated: 43; Down-regulated: 23) which was used for enrichment analysis. Four miRNAs (miR-17-5p, -92a-3p -421, -125b) were significantly linked to neuroblastoma, and associated secondary diseases; medulloblastoma (-92a-3p, -125b), bladder cancer (-17-5p, -125b), acute myeloid leukemia (-92a-3p, -125b) and cardiac hypertrophy (- 125b). miR-125b showed exceptional interconnectivity with these diseases and a multidimensional potential in neural tumorigenesis. This study showed that dysregulation and biological processes of these miRNAs were concurrent with the original studies, endorsing that these miRNAs have potential as diagnostic indicators or classifiers of such diseases. / Popular scientific summary Neuroblastoma (NB) is one of the most common types of pediatric neurological cancers in children and constitutes roughly 8% of all childhood cancer types, in which 95% of all NB cases occur before the age of 10. Even with frequent advancements in medical diagnosis and anti-tumor therapies, the current treatment options for patients with NB offers a survival rate that is very poor. This alone is a reason to pursue developing novel classification methods, improve diagnosis and research better anti-tumor therapies. Micro Ribonucleic Acids (miRNAs) are small non-coding single stranded biomolecules that have gotten a lot of attention in recent years due to their ability to regulate genes involved in various biological cancer processes, such as; tumor growth and development. miRNAs regulate these processes by altering the function of messenger RNAs (mRNAs), which are single-stranded biomolecules that resembles a piece of genetic code from the DNA of an organism cell. When these mRNAs become dysregulated, their cancer-promoting genes are disrupted which prevent them from working properly, leading to tumor regression or termination. The effect of this biological event is then objectively measured by using the miRNA as an indicator, also known as a biomarker. miRNA biomarkers have massive potential to improve various medical applications, such as; faster and more accurate diagnosis, detailed disease-classification and more precise drug trial predictions. However, a lot of individual studies have been published about the same miRNAs, which report a variation of conclusions. This makes it more difficult to determine the true nature of miRNAs. This issue can be addressed with systematic reviews and meta-analyses, which could yield additional support and give a broader picture of how miRNAs regulate different biological processes in NB. A meta-analysis is a scientific statistical process that combines the results of many research publications associated with the same scientific question and presents the best collective estimate of truth with increased precision than what could be achieved from individual studies alone. Thus, meta-analysis is an important tool in research which makes sure that the most trustworthy effect estimate can be achieved among many similar answers. The aim of this meta-analysis was to identify dysregulated miRNAs in NB that has the potential to be used as anti-cancer promoting biomarkers for diagnostic interventions. Additionally, explore how different miRNAs are connected to NB and conversely extend the support of using miRNAs as biomarkers. The end goal of this meta-analysis is to provide more reliable evidence for further research that can improve the life expectancy of NB patients in the future. In this study, 4 miRNAs (miR-17-5p, -92a-3p -421 and -125b) were identified to be significantly linked to NB, and associated secondary diseases; medulloblastoma (-92a-3p & -125b), bladder cancer (-17-5p & -125b), acute myeloid leukemia (-92a-3p & -125b) and cardiac hypertrophy (- 125b). Specifically, miR-125b showed exceptional interconnectivity for these diseases and potential to indirectly down-regulate n-Myc in NB, a gene that promote cancer cell proliferation. miR-125b was also found to be a significant sole regulator and effector of the CDX2 gene responsible for cancer cell differentiation in acute myeloid leukemia, a relationship that has been supported by other publications. This meta-analysis showed that the reported dysregulation and biological processes of these miRNAs were concurrent with the original studies, endorsing that these miRNAs have potential as diagnostic indicators or classifiers of such diseases while warranting that the gene regulatory function of miRNAs are becoming more intricate than previously thought.

Crohns sjukdom är en kronisk inflammatorisk sjukdom som påverkar hela gastrointestinala kanalen, från mun ner till ändtarmen. Sjukdomen är progressiv och innefattar olika skov av symptom, som till exempel diarré, magont, rektalblödning, viktminskning, feber och trötthet. Den exakta patofysiologiska orsaken är ej klargjord, men tros bero på olika faktorer som till exempel genetiska faktorer, miljöfaktorer samt immunologiska faktorer. Det är känt att individer med inflammatorisk tarmsjukdom (IBD) uttrycker en större population av mastceller och eosinofiler i tarmsubmukosa, och kan orsaka diverse symptom. Histamin-4 receptorer är G-proteinkopplade receptorer som har en nyckelroll i att förmedla inflammatoriska svar, och spelar en roll i immuncellsmigration till inflammerade vävnader. Syftet med projektet är att studera uttrycket av histaminreceptorn H4 på eosinofiler och mastceller i inflammerad tarmvävnad från patienter med Crohns sjukdom. Det medverkade 16 patienter, varav 8 hade Crohns sjukdom, och 8 var friska kontrollpatienter som opererats för tarmcancer. Uttrycket av eosinofiler, mastceller och Histamin-4 receptorer studerades med hjälp av immunohistokemisk metod. Resultatet visar statistisk signifikanta skillnader mellan patient och kontrollgruppen när det kommer till eosinofiler och mastceller, med ett p-värde <0,05. Det uppvisades ingen signifikant skillnad mellan grupperna när det kom till icke inmärkta celler som uttrycker histamin-4 receptorer, eosinofiler som uttrycker receptorn samt mastceller som uttrycker receptorn. Sammanfattningsvis finns det ett behov att studera detta område igen, med större urval, och möjligtvis förbättrade metoder för att komma fram till säkerställda slutsatser. / Crohn's disease (CD) is a chronic inflammatory disease that affects the entire gastrointestinal tract, from the mouth down to the rectum. The disease is progressive and involves flare-ups of symptoms such as diarrhea, abdominal pain, rectal bleeding, weight loss, fever, and fatigue. The exact pathophysiological cause is unclear but is believed to be related to various factors, including genetic, environmental, and immunological factors. It is known that individuals with inflammatory bowel disease (IBD) express a higher population of mast cells and eosinophils in the intestinal submucosa, which can cause diverse symptoms. Histamine-4 receptors are G-protein-coupled receptors that play a key role in mediating inflammatory responses and are involved in immune cell migration to inflamed tissues. The aim of the project is to study the expression of the histamine receptor H4 on eosinophil granulocytes and mast cells in inflamed intestinal tissue from patients with CD. Sixteen patients participated, including 8 with CD and 8 control patients who underwent surgery for colon cancer. The expression of eosinophils, mast cells, and histamine-4 receptors was studied using immunohistochemical methods. The results show statistically significant differences between the patient and control groups in terms of the number of eosinophils and mast cells, with a p-value <0.05. There was no significant difference between the groups in terms of un-stained cells expressing histamine-4 receptors, eosinophils and/or mast cells expressing the receptor. In conclusion, there is a need to further study this area with larger sample sizes and possibly improved methods to arrive at conclusive findings.

Type-2 diabetes has become a common disease. The complications associated with thedisease are often cardiovascular. Platelets are important components in hemostasis, one function is platelet adhesion. Platelet adhesion is the first step to form a platelet plug, together with subendothelial proteins. Blood lipids contribute to membrane structure of cells and has an important role in different signaling pathways. They have a key role in platelet activation and is associated with the changes in membrane lipids. C-reactive protein (CRP) is an acute phase protein and increases during inflammation. The physiological role is not fully known but increasing levels of CRP increases the risk for cardiovascular complications. The aim of this study was to analyze the correlation between platelet adhesion with blood lipids and high sensitivity CRP in type-2 diabetics. 69 patients with type-2 diabetes participated in this study. Lipids, CRP and platelet adhesion were analyzed, the values were used to perform correlation analysis. The results show statistically significant positive correlations between ApoA-1, HDL and platelet adhesion as well as significant negative correlations between LDL, ApoB/ApoA-1-ratio, LDL/HDL-ratio and platelet adhesion. ApoA-1 and platelet adhesion with collagen showed statistically significant correlations with r-values 0,299-0,436. ApoA-1/ApoB-quota showed statistically significant correlations with r-values from -0,492 to -0,268. Majority of correlations between totalcholesterol, ApoB, triglycerides, hsCRP and platelet adhesionshowed no statistically significant correlations. / Typ-2 diabetes har blivit en vanligare sjukdom. De komplikationer som kan uppstå vid diabetes, särskilt obehandlad diabetes, är ofta kardiovaskulära komplikationer. Trombocyter är viktiga komponenter i hemostasen, en av deras funktioner är trombocytadhesion. Trombocytadhesionen är första steget vid bildandet av en trombocytpropp, tillsammans med subendoteliala proteiner så kan trombocyterna fästa till kärlväggen och hemostasen påbörjas. Blodlipider bidrar till cellmembraners struktur, energiförvaring och har en viktig roll vid olika typer av signalering. De har en nyckelroll vid trombocytaktivering eftersom trombocytaktivering är associerad med förändringar som sker hos membranlipiderna. C-reaktivt protein (CRP) är ett akutfasprotein och ökar vid inflammation. Fysiologiska rollen är inte helt känd men stegring av CRP ökar risken för kardiovaskulära komplikationer. Syftet med denna studie är att analysera korrelationen mellan trombocytadhesion med blodlipider och högkänsligt CRP hos typ 2 diabetiker. Det medverkade 69 patienter med typ-2 diabetes i studien. Blodlipider, högkänsligt CRP och trombocytadhesion analyserades ochvärdena för respektive analyt användes för korrelationsanalyser.Resultaten visar statistiskt signifikanta positiva korrelationer mellan ApoA-1, HDL och trombocytadhesion samt signifikanta negativa korrelationer mellan LDL, ApoB/ApoA-1-kvot, LDL/HDL-kvot och trombocytadhesion. ApoA-1 och trombocytadhesion med kollagen som proteinyta uppvisade statistiskt signifikanta korrelationsintervall med r-värden på 0,299-0,436. ApoA-1/ApoB-kvoten visade statistiskt signifikanta korrelationsintervall med r-värdenmellan -0,492 till -0,268. Majoriteten av korrelationerna för variablerna: totalkolesterol, ApoB, triglycerider, högkänsligt CRP och trombocytadhesion visade inga statistiskt signifikanta korrelationer.

Standardbikarbonat innebär koncentrationen bikarbonat i plasmadelen av helblod under standardiserade förhållanden. Analysen baseras på uppmätta värden av partialtryck för koldioxid (CO2), pH, syrgasmättnad och total-hemoglobin samt en beräkning. Bikarbonat (HCO3-) har tillsammans med CO2 en central roll i syra-basregleringen där njurarna kan styra HCO3--koncentrationen och lungorna kan styra CO2-koncentrationen. Indikationer på att analysera standardbikarbonat är exempelvis misstanke om acidos eller alkalos samt hos personer med njur- och eller lungsjukdom. Syftet med examensarbetet var att studera den preanalytiska hållbarheten för vB-Standardbikarbonat för att om möjligt förlänga tiden till analys inom region Kalmar län. Analysen genomfördes på blodgasinstrumentet ABL 825 FLEX. Provmaterialet bestod av venöst helblod i mörkblå Natrium-heparin-rör från 68 blodgivare och 7 dialyspatienter. De provrör som förvarades i kylskåpstemperatur (+2 till +8 °C) analyserades efter 0, 2, 3, 4 och 5 timmars förvaring. De provrör som förvarades i rumstemperatur (+18 till +25 °C) analyserades efter 0, 1, 2, 3 och 5 timmars förvaring. Resultatet visade att hållbarheten var bättre vid förvaring i kylskåpstemperatur än vid förvaring i rumstemperatur. Vid förvaring i kylskåpstemperatur erhölls en genomsnittlig förändring på -0,14 mmol/L (-0,6 %), -0,44 mmol/L (-1,8 %), - 0,45 mmol/L (-1,8 %), -0,58 mmol/L (-2,4 %) vid respektive förvaringstid 2, 3, 4 och 5 timmar. Vid förvaring i rumstemperatur erhölls en genomsnittlig förändring på -0,36 mmol/L (-1,5 %), -0,65 mmol/L (-2,7 %), -0,98 mmol/L (-4,0 %), -1,57 mmol/L (-6,4 %) vid respektive förvaringstid 1, 2, 3 och 5 timmar. Efter genomgång av resultatet med medicinskt ansvariga läkare framkom det att två timmars förvaring i rumstemperatur och fem timmars förvaring i kylskåpstemperatur var godtagbara förvaringstider. Utifrån resultatet i denna studie föreslås därför en uppdatering av hållbarheten på analysen vB-Standardbikarbonat till två timmar i rumstemperatur och fem timmar i kylskåpstemperatur inom region Kalmar län. / Standard bicarbonate is the measured concentration of bicarbonate in the plasma-part of whole-blood under standardized conditions. The analysis is based on measurement of partial pressure of carbon dioxide, pH, oxygen saturation, and total hemoglobin as well as a calculation. Bicarbonate (HCO3-) and CO2 are particularly important components of the acid-base-regulation through the kidneys’ ability to manage the HCO3--concentration and the lungs’ ability to manage the CO2-conentration. Analysis of standard bicarbonate is indicated by, for example, a clinical suspicion of acidosis or alkalosis and in patients with renal- and or pulmonary-disease. The aim of this study was to evaluate the time from sampling to analysis regarding the analysis of vB-standard bicarbonate and if possible, upgrade the pre analytical handling within region Kalmar County. The analysis was performed with the blood gas-instrument ABL 825 FLEX. The sampling in this study consisted of venous whole-blood in dark blue sodium-heparin-test tubes from 68 blood donors and 7 dialysis patients. The test tubes were either placed at refrigerator-temperature (+2 to +8 °C) or at room-temperature (+18 to +25 °C). Test tubes stored at refrigerator-temperature were stored for 0, 2, 3, 4 and 5 hours before analysis and the test tubes stored at room-temperature were stored for 0, 1, 2, 3 and 5 hours before analysis. The results show that storage at refrigerator-temperature was better in comparison to storage at room-temperature. The mean change at refrigerator-temperature was -0,14 mmol/L (-0,6 %), -0,44 mmol/L (-1,8 %), - 0,45 mmol/L (-1,8 %), -0,58 mmol/L (-2,4 %) for each storage time 2, 3, 4 and 5 hours respectively. The mean change at room-temperature was -0,36 mmol/L (-1,5 %), -0,65 mmol/L (-2,7 %), -0,98 mmol/L (-4,0 %), -1,57 mmol/L (-6,4 %) for each storage time 1, 2, 3 and 5 hours respectively. After conversation with the medically responsible doctor about the acceptable storage time, the conclusion was that two hours at room-temperature and five hours at refrigerator-temperature were acceptable storage times. Therefore, based on the results of this study, a proposal was made to change the time of storage to two hours at room-temperature and five hours at refrigerator-temperature regarding the analysis vB-standard bicarbonate in region Kalmar County.

Through significant federal investment and incentives, Electronic Health Records have become ubiquitous in modern hospitals. Over the past decade, these computer support systems have provided healthcare operations with new safety nets, and efficiency increases, but also introduce new problems when they suddenly go offline. These downtime events are chaotic and dangerous for patients. With the safety systems clinicians have become accustomed to offline, patients are at risk from errors and delays. This work applies the Macroergonomic methodology to facilitate an exploratory study into the issues related to patient care during downtime events. This work uses data from existing sources within the hospital, such as the electronic health record itself. Data collection mechanisms included interviews, downtime paper reviews, and workplace observations. The triangulation of data collection mechanisms facilitated a thorough exploration of the issues of downtime. The Macroergonomic Analysis and Design (MEAD) methodology was used to guide the analysis of the data, and identify variances and shifts in responsibility due to downtime. The analysis of the data supports and informs developing potential intervention strategies to enable hospitals to better cope with downtime events. Within MEAD, the assembled data is used to inform the creation of a simulation model which was used to test the efficacy of the intervention strategies. The results of the simulation testing are used to determine the specific parameters of the intervention suggestions as they relate to the target hospitals. The primary contributions of this work are an exploratory study of electronic health record downtime and impacts to patient safety, and an adaptation of the Macroergonomic Analysis and Design methodology, employing multiple data collection methods and a high-fidelity simulation model. The methodology is intended to guide future research into the downtime issue, and the direct findings can inform the creation of better downtime contingency strategies for the target hospitals, and possibly to offer some generalizability for all hospitals. / Ph. D. / Hospitals experience periodic outages of their computerized work support systems from a variety of causes. These outages can range from partial communication and or access restrictions to total shutdown of all computer systems. Hospitals operating during a computerized outage or downtime are potentially unable to access computerized records, procedures and conduct patient care activities which are facilitated by computerized systems. Hospitals are in need of a means to cope with the complications of downtime and the loss of computerized support systems without risking patient care. This dissertation assesses downtime preparedness and planning through the application of Macroergonomics which has incorporated discrete event simulation. The results provide a further understanding of downtime risks and deficiencies in current planning approaches. The study enhances the application of Macroergonomics and demonstrates the value of discrete event simulation as a tool to aid in Macroergonomic evaluations. Based on the Macroergonomic Analysis and Design method, downtime improvement strategies are developed and tested, demonstrating their potential efficacy over baseline. Through this dissertation, the deficiencies in current contingency plans are examined and exposed and further the application of Macroergonomics in healthcare.

Leukocyte extravasation is mediated in part by tissue specific chemotactic cytokines (chemokines) and specific chemokine receptors expressed on the surface of circulating cells. C-C chemokine ligand CCL25 is expressed exclusively in the intestine and thymus and mediates chemotaxis by cells expressing receptor CCR9. This chemokine and receptor pair may be relevant in the pathogenesis of intestinal inflammation, in diseases such as Crohn’s disease (CD) and coeliac disease. In this thesis I investigated CCR9 expression in situ, in tissues affected by intestinal inflammation, and also examined the effects of CCR9 antagonist treatment in patients. In vitro I investigated CCR9 function using human peripheral blood T cells enriched for CCR9 by cell sorting or all-trans retinoic acid treatment. Using tissues collected as part of a clinical trial in CD testing CCR9 antagonist, CCX282-B, I investigated ways of measuring if treatment reduced the number of CCR9 expressing cells in the intestinal mucosa. However, in situ staining for CCR9 by immunohistochemistry was unsuccessful, and in this thesis, I explored reasons why this might be the case. Treatment with CCX282-B did however, show a tendency to reduce T cell density in the intestinal mucosa, although results were highly variable between individuals. In an examination of human CCR9 function in vitro I demonstrate for the first time that CCL25 stimulates CCR9 surface internalization. These data clarify the observation that CCR9 staining by IHC produces poor results in tissues where ligand is abundant, such as the intestine and thymus. I describe a novel technique for measuring calcium flux in two populations simultaneously by flow cytometry, which confirmed that in a heterogeneous population of cells, only CCR9 expressing cells respond to CCL25 by calcium flux. Variability in clinical trials is partly created by the use of concomitant medications, and in CD, corticosteroids are widely used. For the first time I show that glucocorticoids (GC) impair CCR9 mediated chemotaxis, calcium flux and intracellular signalling without changes to CCR9 mRNA and surface protein expression. Reduced CCR9 mediated signalling was accompanied by an enhanced expression and function of co-expressed CXCR4, demonstrating that the effects of GC were receptor-specific and not mediated by non-specific toxicity or inhibition of cell signalling. In a second study CCX282-B was tested in patients with coeliac disease, and in this trial, there was no reported concomitant use of GCs. It was confirmed that dietary gluten stimulates significant T cell recruitment to the intestinal mucosa with a pronounced accumulation of intraepithelial lymphocytes (IEL) and a rise in the frequency of FoxP3 expressing cells. Patients on CCX282-B had lower IEL counts, and an equivalent proportion of FoxP3 expressing T cells, suggesting that CCR9 blockade restricted the recruitment of effector T cell subsets. This thesis confirms that the accumulation of T cells is central to inflammation in the intestine and that modulating chemokine receptor function may affect this. Furthermore, this thesis demonstrates that the function of CCR9 is suppressed by GCs, which are widely used therapeutically and therefore could identify a novel mechanistic basis for their activity in CD.

Treatment of lower respiratory tract infection (pneumonia and pleural infection) relies on the use of empirical broad spectrum antibiotics, primarily because reliable pathogen identification occurs infrequently. Another consequence of poor rates of pathogen identification is that our understanding of the microbiology of these infections is incomplete. This thesis addresses some of these issues by combining the acquisition of high quality lower respiratory tract samples, free from nasooropharyngeal contamination, with novel molecular microbiological techniques in an attempt to increase rates of pathogen identification. Four main areas are examined: (i) The role of so-called ‘atypical pneumonia’ bacteria in causing pleural infection. These pathogens have been previously identified in the pleural space infrequently and routine culture usually fails to isolate such bacteria. High sensitivity nested polymerase chain reaction (PCR) is a culture-independent technique which is used to undertake a systematic evaluation for these pathogens in pleural infection samples. (ii) The role of Pneumocystis jirovecii in pleural infection, either as a co-infecting pathogen or in monomicrobial infection. This fungus causes severe pneumonia, particularly in the immunosuppressed, but is increasingly recognised as a co-pathogen in community-acquired pneumonia, and is frequently isolated in the upper and lower respiratory tract in health. A high sensitivity real-time PCR assay is used to examine for this fungus. (iii) Ultra-deep sequencing of the 16S rRNA gene is used to perform a comprehensive microbial survey in samples taken from the multi-centre MIST2 study of pleural infection. The techniques employed allow analysis of polymicrobial samples and give very high taxonomic resolution, whilst incorporating methods to control for potential contamination. Further, these techniques provide confirmation of the results from the ‘atypical’ bacteria nested PCR study. (iv) Bedside ultrasound-guided percutaneous transthoracic needle aspiration (TNA) of consolidated lung is undertaken in patients with pneumonia, as part of the PIPAP study. An evaluation is undertaken of the efficacy and acceptability of TNA. Aspirate samples acquired are also processed using ultra-deep sequencing of the 16S rRNA gene. Other samples obtained as part of the PIPAP study, such as ‘control’ lung aspirates and ‘control’ pleural fluid samples, are similarly processed to enable calculation of sensitivity and specificity of the sequencing methodology.

Aim: To study the possibility of detecting lymph node metastasis in locally advanced urinary bladder cancer (UBC) treated with radical cystectomy (RC) by using preoperative positron emission tomography/computed tomography (PET/CT) and peroperative sentinel node biopsy (SNB) technique. We also investigate the clinical significance of macrophage traits expression by cancer cells, M2-macrophage infiltration (MI) in tumor stroma and the immunohistochemical expression of biomarkers in cancer cells in relation to clinicopathologic data. Patients and Methods: We studied prospectively 122 patients with UBC, pathological stage pT1–pT4 treated with RC and pelvic lymph node dissection (PLND) during 2005–2011 at the Department of Urology, Linköping University Hospital. In the first study, we compared the results of preoperative PET/CT and conventional CT with the findings of postoperative histopathological evaluation of lymph nodes (LNs). In the second study we investigated the value of SNB technique for detecting pathological LNs during RC in patients with UBC. W also examined the significance of the primary tumor location in the bladder in predicting the site of LN metastases, and the prognostic significance of lympho-vascular invasion (LVI) and lymph node metastasis density (LNMD) on survival. In the third study, we investigate the clinical significance of macrophage infiltration (MI) in tumor stroma and macrophage-traits expression by tumor cells. In the fourth study, we investigate the cell cycle suppression proteins p53, p21, pRb, p16, p14 ARF as well as tumors proliferative protein Ki67 and DNA repair protein ERCC1 expression in cancer cells. The results were compared with clinical and pathological characteristics and outcome. Results: Prior to RC, PET/CT was used to detect LN metastasis in 54 patients. PET/CT had 41% sensitivity, 86% specificity, 58% PPV, and 76% NPV, whereas the corresponding figures for conventional CT were 41%, 89%, 64%, and 77%. SNB was performed during RC in 103 patients. A median number of 29 (range 7–68) nodes per patient were examined. SNs were detected in 83 out of 103 patients (81%). The sensitivity and specificity for detecting metastatic disease by SNB varied among LN stations, with average values of 67% -90%. LNMD or ≥8% and LVI were significantly related to shorter survival. In 103 patients, MI was high in 33% of cases, while moderate and low infiltration occurred in 42% and 25% of tumors respectively. Patients with tumors containing high and moderate compared to low MI had low rate of LN metastases (P=0.06) and improved survival (P=0.06), although not at significant level. The expression of different tumor suppression proteins was altered in 47-91% of the patients. There were no significant association between cancer specific survival (CSS) and any of the studied biomarkers. In case of altered p14ARF, ERCC1 or p21, CSS was low in case of low p53 immunostaining but increased in case of p53 accumulation, although not at a significant level, indicating a possible protective effect of p53 accumulation in these cases. Conclusion: PET/ CT provided no improvement over conventional CT in detection and localization of regional LN metastases in bladder cancer. It is possible to detect the SN but the technique is not a reliable for perioperative localization of LN metastases; however, LVI and LNMD at a cut-off level of 8% had significant prognostic values. MI in the tumor microenvironment but not CD163 expression in tumor cells seems to be synergistic with the immune response against urinary bladder cancer. Our results further indicate that altered p53 might have protective effect on survival in case of altered p14ARF, p21, or ERCC1 indicating an interaction between these biomarkers.