Caseous lymphadenitis (CLA) is a disease primarily in sheep and goats, caused by Corynebacterium pseudotuberculosis, characterized by abscess formation in external and/or internal lymph nodes and organs. Economic losses occur due to emaciation, reduced milk production and impaired growth in affected animals. At the Swedish Veterinary Agency, bacterial culture is used as a diagnostic method for detecting CLA, requiring puncture of clinical abscesses. This sampling method increases the risk for disease transmission and overlooks animals affected by internal abscesses. In this study, a commercially available enzyme-linked immunosorbent assay (ELISA)-kit, based on an indirect ELISA, was validated for detecting antibodies for CLA, aiming for a more cost-effective and safer method that also can detect animals with internal abscesses. Serum and milk samples were analyzed to investigate the concordance between the two sample types. A subset of serum and milk samples was sent to Norway for analysis to compare the ELISA results, and comparison with another laboratory's results was conducted. The ELISA-kit demonstrated high sensitivity (92% for serum, 100% for milk), crucial for controlling CLA at herd level, while the specificity was estimated at 68% for serum and 61% for milk. To avoid increased and unnecessary culling of animals, a confirmatory method should analyze positive individuals to increase the specificity. Pooled milk samples could reduce costs for herd-level analysis, followed by serum sampling for positive herds.

Bacterial bloodstream infections represent a severe and potentially life-threatening condition. Diagnostic tools, providing rapid species identification and antimicrobial susceptibility testing have a great impact on disease progression and treatment optimization. Typically, antimicrobial susceptibility testing involves a time-consuming process, and faster methods are likely to contribute in clinical settings. This study aimed to assess the reliability of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) rapid antimicrobial susceptibility testing (RAST) with a 16-20-hour incubation period for implementation in a clinical microbiology laboratory. This method was conducted alongside the standardized disk diffusion method from EUCAST, encompassing all bacterial species compatible with 16-20-hour RAST. The method was executed 80 times, and over 450 readings were performed. It confirms EUCAST's method validation, affirming 16-20-hour RAST as a swift and precise method for blood bacterial resistance determination. All inhibition zones were readable, with 89% of antibiotics tested falling within 1 mm of the set target value. Notable deficiencies were observed in antibiotic-species combinations, such as trimethoprim/sulfamethoxazole and gentamicin against E. coli, and erythromycin against S. pneumoniae. Accurate interpretation of inhibition zones demands precise reading practices as some zones were ambiguous. The study also underscores the importance of overnight cultures in antimicrobial susceptibility testing via standardized disk diffusion, accentuating 16-20-hour RAST's pivotal role in potentially accelerating resistance determination by up to a day. Given its commendable performance, 16-20-hour RAST is ready to be incorporated into the standard procedures at clinical microbiology laboratories, as demonstrated by its successful adoption at Unilabs, Skövde.

The ability of human pluripotent stem cells (hPSCs) to differentiate into different types of cells has been regarded as a significant discovery in the development of cell replacement therapy for type 1 diabetic patients. MicroRNAs can be transported to recipient cells via vesicles, so-called exosomes. Exosomal microRNA differ from those of the parent cells suggesting that cells possess an active selecting mechanism of exosomes and their contents. Therefore, microRNAs may directly or indirectly regulate the expression of pancreatic islet-specific transcription factors to control the differentiation and maturation of pancreatic islet cells. In this study, the dynamic expression of exosomal and intracellular microRNAs from human pancreatic islets were analyzed and were compared with that in stem cell-derived islet-like clusters. The study also aimed to analyze the expression levels of intracellular microRNA in human pancreatic islets and stem cell-derived islet-like clusters compared to exosomal microRNA extracted from human islet media and stem cell media, respectively. The primary method of exosome extraction was ultracentrifugation, followed by microRNA isolation using a kit. The exosomes were then characterized with NTA, and the isolated microRNAs were detected using RT-qPCR. The results showed that the expression of microRNAs was generally low in human islets compared to isolated exosomes. The microRNA expression levels in stem cell-derived islet-like clusters and their respective isolated exosomes were also analyzed and it showed that let-7a, miR-375 and miR-26a were more abundant in exosomes. The results can contribute to the generation of more functional stem cell-derived islet-like cell clusters prepared from hPSCs to some extent. However, continued research in this area is required.

Langerhansceller (LC) är en specifik typ av antigenpresenterande celler och är deenda residenta immuncellerna i epidermis. LC har likheter med både dendritiska celler och makrofager men kännetecknas av sitt uttryck av lektinreceptorn langerin (CD207) och lipid-antigenpresenterande komplexet CD1a. En rekonstruerad human epidermismodell är ett mycket användbart verktyg för olika typer av studier om epidermis, och integrering av LC i modellen ger även möjlighet till att studera immunologiska funktioner in vitro. Det har tidigare visatsatt perifera monocyter kan in vitro differentieras till LC med hjälp av granulocyt-makrofagkolonistimulerande faktor (GM-CSF), transformerande tillväxtfaktor beta1 (TGF-β1) och interleukin 4 (IL-4), men det finns olika beskrivningar på själva metoden. Syftet med den här studien är att etablera en metod för att differentiera perifera monocyter till LC, så att dessa sedan kan integreras i en epidermismodell. Resultatet från studien visar att huvuddelen av de differentierade cellerna befinner sig i suspension och behöver därför återföras till odlingen dag två vid byte av medium. Ingen väsentlig skillnad kan dock detekteras mellan de adherenta- och suspensionscellerna avseende andelen erhållna LC, vilket gör att suspensionscellerna kan med fördel användas för vidare studier. Det finns stor variation i utfallet av erhållna LC mellan blodgivare, men utbytet av LC kan förutses redan dag fyra. Sammanfattningsvis, i den här studien fastställdes en metod där monocyter från perifert blod differentieras till LC under sex dagar genom stimulering med GM-CSF, TGF-β1 samt pulsning med IL-4 detvå första dagarna, men vidare optimering är nödvändig innan dessa LC kanintegreras i en epidermismodell.

Cancer is one of the leading causes of death in the world, often due to failed treatments because of drug resistance. Treatment is difficult as resistance is hard to detect before treatment and can develop during treatment. The fluorometric microculture cytotoxicity assay (FMCA) is a reliable, rapid method for testing drug cytotoxicity but requires large cell samples, which can be challenging to obtain. Patient-derived cancer cells (PDC) have proven challenging to culture in monolayer models, but recent studies have shown the possibility of using tumoroids. Tumoroids are three-dimensional models where cells are grown in basement membrane matrix hydrogel, allowing scaffold growth like in vivo tumors. This study aimed to culture colorectal PDC in the form of tumoroids, transfecting them, and examine cell cycle and tumor resistance for 5-Fluorouracil, Oxaliplatin and Irinotecan. Cells were deposited in gels with medium mimicking in vivo conditions, supporting growth and allowing extracellular signaling. The study succeeded in culturing both untransfected and transfected cells, resulting in cells expanding 48 and 42 times, respectively. Cell cycle remained unchanged. No changes were observed in 5-Fluorouracil, but a change was seen in transfected cells at passage 3 with oxaliplatin. The cells showed a 22% difference in survival indexes compared to naïve cells. Changes were seen in Irinotecan’s half maximal inhibitory concentration (IC50); all cell passage IC50 values differed >15.17 µM (p-value 0.0184). In conclusion, PDC can be cultured as tumoroids, but more studies are needed to determine if the model can generate reliable results representing PDC regarding tumor resistance.

Background: Pulmonary Embolism (PE) is a potentially life-threatening condition that is characterized by one or several blood clots blocking the arteries in the lungs. The existing diagnostic tools for PE have their shortcomings, highlighting the importance of investigating new diagnostic methods. The development of non-invasive methods to collect microparticles from exhaled breath has opened possibilities to explore new potential biomarkers. SensAbues® is a sampling device that utilizes electrostatic filters to capture microparticles from the exhaled breath. The objective of this project was twofold: firstly, to assess the suitability of SensAbues® sampling device for a future proteomics study where the goal is to identify biomarkers for PE; and secondly, to evaluate the efficacy of various extraction solutions in retrieving proteins from the electrostatic filters. Materials and methods: Samples were collected from three healthy volunteers using the SensAbues® device. The electrostatic filters were then extracted using either PBS or 15% ethanol and the protein content was then estimated using a modified Bradford method. Additionally, two blank SensAbues® filter extracts, from PBS and 15% ethanol were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: The attempts to evaluate extraction solutions using the Bradford method were unsuccessful, as all the samples yielded negative values. The filter-blank extracts analyzed with LC-MS/MS contained a significant amount of polyethylene glycols of varying sizes. Conclusion: The polyethylene glycols from the SensAbues® filters may have interfered with the Bradford method. Polyethylene glycols can also interfere with proteins, making the SensAbues® sampling device unsuitable for the prospective proteomics study.

Introduction: Sleep apnea is a pathological health condition with repeatedly paused breathing during sleep. The condition can cause serious health problems and decrease quality of life. Offering a fast diagnosis and treatment could prevent further progress of the condition. The severity of sleep apnea is indicated by an apnea hypopnea index (AHI), which is scored based on a home sleep apnea test (HSAT). The purpose: This study compared the differences between manually and automatically scored AHI, to examine if the automatic scoring is an acceptable singular method for sleep apnea diagnostics. This study also examined if AHI differences could be predicted by HSAT airflow signal quality and the degree of central or mixed apneas. Methods: Sleep apnea patients were instructed by the author how to use the HSAT equipment, data of 182 one-night HSAT recordings were then collected. Each recording was analyzed automatically and manually by a sleep specialist, using the software Noxturnal 6.3. Results: There was a great correlation between the two AHI scoring methods (Spearman’s r 0,97), but a statistically significant difference was found. The positive predictive value (PPV) and negative predictive value (NPV) of the automatic method were 96% and 97%, respectively, sensitivity was 99% and specificity 84%. A moderate, negative correlation between signal quality and AHI differences (Pearson’s r -0,31) was found, but none with central apneas. Conclusion: The results were contradictory, but considering a low Cohen’s d, this study still concludes that clinical use of automatic AHI scoring should be sufficient if AHI > 15.

Extracellular vesicles (EV) originate from various cell types and reflect the contents of the originating cells. EVs are ubiquitous in nearly all body fluids, including blood plasma, and exhibit significant potential as biomarkers in disease diagnostics. However, isolating EVs from blood plasma remains challenging due to the lack of a standardised method. This study aimed to compare and optimize a density gradient ultracentrifugation workflow (DUC) against size exclusion chromatography-cation exchange chromatography (SEC-CEC) and evaluate SEC versus SEC-CEC. Common contaminants during isolation include lipoproteins (LP); previous studies have shown that lowering the pH of blood plasma can precipitate LP, enhancing isolation efficiency. Acidified blood plasma was compared with neutral plasma for EV isolation using all above mentioned methods. To assess the ability of the isolation methods to purify contaminants while retaining maximal EV yield, samples were analysed using multiple techniques, including particle quantity, free proteins, LP-associated apolipoprotein B, purity index (μg protein/particle), and EV-associated surface markers. The results indicate potential for DUC, but further optimization is necessary to improve the method and its isolation of EV. SEC-CEC emerged as an effective method, reducing contaminants by 71% (SEC) to 99% (SEC-CEC), increasing purity by 80%, and yielding positive signals from EV markers (SEC-CEC). The effect of acidification was ambiguous, it reduced apolipoprotein-B levels in plasma pre-isolation. However, post- isolation, neutral plasma exhibited significantly lower contaminations, albeit at the expense of total particle content and risking EV loss. The study underscored several advantages of SEC-CEC but indicated that acidification did not optimise isolation efficiency.

Diabetes is a significant health burden worldwide. The most common types of diabetes, type 1 and type 2 diabetes are typically characterized by complete insulin deficiency and varying degrees of insulin deficiency, respectively. Glycated hemoglobin (HbA1c), formed when hemoglobin and glucose combine through glycation, can be used to monitor treatment in diabetic patients, and thus prevent future complications of the disease. HbA1c can be measured using point-of-care (POC) instruments, providing rapid results and immediate feedback on HbA1c levels. Measurement with POC instruments can reduce the need for additional visits for blood sampling, thereby lowering costs for both patients and healthcare systems. The main purpose of this study was to verify HbA1c on the POC instruments QuikRead Go and DCA Vantage by comparing the results with the reference method Cobas Pro and by comparing capillary and venous blood samples. The study utilized 30 venous patient samples, including 20 samples already analyzed on Cobas Pro and 10 samples collected venously and capillary from volunteer individuals. The coefficient of variation (CV) for QuikRead Go fell within the quality goal, while DCA Vantage exceeded the goal. The results demonstrated good agreement between capillary blood samples analyzed on POC instruments and venous samples analyzed on Cobas Pro. However, a statistically significant difference was found comparing venous samples analyzed on POC instruments and Cobas Pro. The results suggest that capillary sampling should be used for analysis on POC instruments. Certain limitations of the study should be considered when using QuikRead Go and DCA Vantage in practice.

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