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Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments. We assessed the performance of ten different 384- and 96-well ultra-low attachment (ULA) microplates in facilitating three-di

The human voice is notoriously variable, and conventional measurement paradigms are weak in terms of providing evidence for effects of treatment and/or training of voices. New methods are needed that can take into account the variability of metrics and types of phonation across the voice range. The “voice map” is a generalization of the Voice Range Profile (a.k.a. the phonetogram), with the potential to be used in many ways, for teaching, training, therapy and research. FonaDyn is intended as a proof-of concept workbench for education and research on phonation, and for exploring and validating the analysis paradigm of voice-mapping. Version 3.1 of the FonaDyn system adds many new functions, including listening from maps; displaying multiple maps and difference maps to track effects of voice interventions; smoothing/interpolation of voice maps; clustering not only of EGG shapes but also of acoustic and EGG metrics into phonation types; extended multichannel acquisition;24-bit recording with optional max 140 dB SPL; a built-in SPL calibration and signal diagnostics tool; EGG noise suppression; more Matlab integration; script control; the acoustic metrics Spectrum Balance, Cepstral Peak Prominence and Harmonic Richness Factor (of the EGG); and better window layout control. Stability and usability are further improved. Apple M-series processors are now supported natively., QC 20240214

In recent years great advances in the field of biomedical engineering and organic electronics have been achieved. One promising application would be the regulation of blood glucose concentration in type 2 diabetes patients. This application would eliminate medication and would improve the standard of life. To achieve this goal a system is needed which receives information about the glucose concentration and reacts upon it. This output reaction could then be used to stimulate the body's own glucose regulation mechanisms. This thesis combined a glucose sensor with an artificial neuron to take the first step towards such a system. Two different artificial neurons, the Axon-Hillock neuron and the astable multivibrator, were characterized and examined upon their usability. The Axon-Hillock, build with organic electrochemical transistors, revealed that it could be applied for both regulating high and low blood glucose concentrations. The astable multivibrator, build with silicon-based transistors, was not as functional as the Axon-Hillock neuron but with more development it could become as good. The placement of the glucose sensor in the astable multivibrator circuit is essential parameter to consider. The results demonstrate that the examined system is functional and could become a part of a larger closed-loop system. Future tests on an animal model may demonstrate its viability as a treatment for type 2 diabetes.

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishingmore robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, wedemonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximaloxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters oftwo or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their abilityto adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covarianceof size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covarieslognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to theirtissue- or organ-level metabolism and describing its size-related variability through scaling laws.

Periodontal disease ranks among the most expensive health conditions to treat, asreported by the World Health Organization (WHO). This is due to the fact thatdiagnosis is based on several specific clinical criteria that employ methods suchas inspection, palpation, probing, and interpretation of radiographic images.However, since these diagnostic tools do not provide information about patientsat risk of developing severe stage periodontal disease, patients are oftenovertreated. Porphyromonas gingivalis is a prevalent bacterium in thesubgingival crevice of patients with periodontal disease and has been termed akeystone pathogen in these conditions. P. gingivalis together with its enzymes,Rgp and Kgp, is therefore of interest as potential biomarkers on which to builddiagnostic tools based on artificial receptors. Firstly, molecularly imprintedpolymers using either the native enzymes or short sequence epitopes from themcan be used to determine the expression level of the enzymes in samples.Secondly, the enzymatic activity can be determined by recording changes inelectrochemical signals before and after hydrolysis of a specially designedpeptide sequence selective for one of the enzymes. Finally, reversible selfassembledmonolayers bearing ligands specific for bacterial adhesion throughmultivalent interactions can potentially be employed to selectively separate anddetect P. gingivalis. Together, they form the foundation for designing acommercially exploitable biosensor that combines detection methods to improvethe accuracy of diagnosis., Papers III and IV in dissertation are manuscripts.Paper III and IV is not included in the fulltext online

Polycystic ovary syndrome (PCOS) stands out as one of the most widespread endocrine disorders affecting women, often presenting as the primary reason for infertility due to anovulation. Individuals with PCOS typically exhibit clinical and biochemical markers aligning with ultrasound findings, predisposing them to challenges such as hyperandrogenism, impaired fertility, obesity, irregular periods, excess body hair, insulin resistance, and recurrent miscarriages. The diagnosis relies on the 2003/2004 Rotterdam criteria, which identify polycystic ovary syndrome (PCOS) through the presence of two out of three key features: anovulation, clinical or biochemical signs of hyperandrogenism, and polycystic ovarian morphology. The potential underlying causes of PCOS may include genetic factors, environmental influences, or maternal imprinting. The study investigated the effect of different concentrations of dihydrotestosterone (DHT) on the oogenesis of Drosophila melanogaster. Drosophila melanogaster, widely utilized in disease research, offers valuable insights into this syndrome. With a life cycle spanning approximately 12 days, these flies undergo metamorphosis and produce the steroid hormone ecdysone, which is analogous to human testosterone. The main aim of the study is to develop a model for studying polycystic ovarian syndrome (PCOS) through the Drosophila melanogaster as an animal model. The data was collected by measuring the fecundity, Triglyceride concentration, and RT-qPCR. The results revealed no significant difference between the different concentrations of DHT and the control group. The results from fecundity, triglyceride assay, and RT-qPCR were notable but not significantly different. These findings suggest that additional experiments are necessary to confirm Drosophila melanogaster as a good model for PCOS., Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.

Cryptosporidium is a parasite that causes gastrointestinal issues such as diarrhoea and stomach pain. The main transmission routes are through contaminated water or food, between humans and from animal to humans. Cryptosporidium infects through oocysts which contain four sporozoites that releases when entering a host and can continue to breed inside the body. Cryptosporidium can cause massive outbreaks if established in a water source used for drinking water. To prevent and detect an outbreak it´s important to trace the transmission back to the source. The GP60-gene is used to identify and subtype Cryptosporidium and is a very useful tool during contact tracing. The purpose of this study was to identify species and subtype of Cryptosporidium using nanopore sequencing. In this study the GP60-gene was amplified using a Nested PCR protocol and then sequenced using nanopore sequencing. The sequences acquired where then used to make a search in BLAST to identify the species. The GP60 subtyping method uses the hypervariable region on the GP60-gene. A series of tandem repeats are used to identify the subtype. In this study seven positive Cryptosporidium faeces samples were amplified and sequenced. Nanopore sequencing was possible for five of the seven samples with C. parvum identified in four of these samples. Targeting the GP60-gene to determine species and subtype works well for the most common human pathogen species of Cryptosporidium. Further optimization is required before the method can be implemented för diagnostic use.

Abalone is an edible snail that faces challenges from climate change, overfishing,and diseases such as Withering Syndrome and Abalone Viral Ganglioneuritis.Withering Syndrome affects digestive organ tissues due to Xenohaliotiscaliforniensis, while Abalone Viral Ganglioneuritis targets nervous tissues fromabalone herpesvirus. In this study, the aim was to investigate tissue damage in twospecies of abalone (Haliotis fulgens and Haliotis corrugata) to identify potentialimpacts of pathogens or environmental stress. Histological sections from six partsof each organism were analyzed, assessing damage on a scale from zero to three.The results revealed various tissue abnormalities, including edema, inflammation,and delamination. Yellow abalone in December showed a concentration of browncells around their gonads' hepatopancreas, possibly indicating post-spawningactivities. Differences in habitat preferences and temperature tolerance highlightvarying environmental responses between blue and yellow abalone. Blue abaloneare notably more susceptible to acidophilic Rickettsia-like organisms, leading todelamination and edema. However, this result does not match the recorded effectin the same area from previous studies, which may indicate environmental factorssuch as thermal stress or harmful algal blooms.

The genetic difference between humans is only 0.1%. The variation that is most common is single nucleotide polymorphisms (SNPs) where only one base in a sequence differs between individuals. SNPs is associated with many different diseases which are diagnosed using different types of analyses. Some known conditions that SNP assays can detect are hereditary hemochromatosis, methylenetetrahydrofolate reductase deficiency, lactose intolerance, and elevated risk of venous thrombosis due to defective coagulation factor V or prothrombin. In this project, new SNP variation kits from Thermo Fisher Scientific for the conditions mentioned above were evaluated using Real-time PCR. The purpose of this investigation was to compare the ready-made reagent kits with the in house developed reagents that have so far been used at the department of Clinical Chemistry, Uppsala University Hospital for the analysis of SNPs, as well as to compare the analysis results with and without albumin present in the PCR reaction. The results of the evaluation showed complete agreement with previous results obtained with in- house developed reagents for most of the assays. The results with new reagents regarding hereditary hemochromatosis 187 were not disturbed by the SNP hereditary hemochromatosis193. In addition, the results of analyzes with and without bovine serum albumin (BSA) were in complete agreement for all analyzes except for lactose tolerance, where the analysis showed deviant results for two samples. In general, a concordance was shown between the results of new kit reagents and self-developed reagents. This means that the reagent kits were as reliable as the previously used reagents.

Blood transfusions and access to qualitative blood units are crucial to patients in urgent need. When blood donors donate blood, the whole blood is separated into blood components: erythrocytes, plasma, and platelets, and after that, leucocyte-reduced. The Swedish National Board of Health and Welfare regulations describe threshold requirements for the finished component units regarding cell content and volume. The method for analyzing component quality controls today in the clinical chemistry laboratory in Kalmar is to analyze hemoglobin and platelet content on Sysmex XN-9100. At the same time, leucocyte count is performed at an external laboratory on the flow cytometer BD FACSLyric™. The study aimed to evaluate the software Blood Bank mode on Sysmex XN-1000 to analyze all parameters in component quality controls on the same instrument instead of the time-consuming and expensive process with FACSLyric™. Analyses of 25 erythrocyte units and 25 platelet units and correlation studies between Blood Bank mode on Sysmex XN and leucocount kit on FACSLyric™ were made. Repeatability studies were performed, and the stability of the controls was investigated. The result showed a high correlation between platelet counting methods and hemoglobin content analysis, r=0,9696 resp. r=0,9779 (p <0,0001), and the repeatability for Blood Bank mode was good. The weakness of the Blood Bank mode was leukocyte counting (r=0,4310 resp. r=0,08597, p=0,0315 resp. p=0,6828). Also close to the threshold value of 1 x 106 leukocytes/unit (clinically relevant levels), the coefficient of variation (CV%) and standard deviation (SD) were relatively high in the repeatability study (CV 19,58% SD 0,67 x106/L in erythrocyte units, CV 24,43% SD 1,01 x106/L in platelet units). More data needs to be collected regarding the high leukocyte levels to further investigate the reliability of Blood Bank mode., Blodtransfusioner kan rädda liv, särskilt vid akuta medicinska tillstånd. Därför är det avgörande att ha tillgång till kvalitativa blodkomponenter vid behov av transfusion. Framställningsprocessen för dessa blodkomponenter är därför strikt reglerad. När blodgivare tappat blod separeras helblodet i tre huvudsakliga blodkomponenter: erytrocyter, plasma och trombocyter. De färdigseparerade komponenterna leukocytreduceras. Socialstyrelsens föreskrifter beskriver gränskrav för de färdigställda blodkomponentsenheterna gällande cellinnehåll och volym samt krav på hur många av utförda komponentkontroller som ska uppnå gränskraven. Komponentkvalitetskontroller utförs idag vid avdelningen för klinisk kemi och transfusionsmedicin, Region Kalmar län genom att analysera hemoglobin- och trombocytinnehåll på ett internt instrument: Sysmex XN-9100. Men, leukocytmätningen utförs vid ett externt laboratorium och då används det flödescytometriska instrumentet BD FACSLyricTM. Syftet med studien var att utvärdera programvaran Blood Bank mode på Sysmex XN-1000 som en metod för att analysera alla parametrar i komponentkvalitetskontroller på samma instrument. Detta är tänkt att ersätta den nuvarande metoden som använder FACSLyricTM, vilken är både tidskrävande och kostsam. Studien innefattade 25 erytrocytenheter och 25 trombocytenheter, som analyserades för att utföra en korrelationsanalys mellan Blood Bank mode på Sysmex XN och leukocount kit på FACSLyricTM. Därtill utfördes repeterbarhetsstudier och hållbarhetsstudier på kontrollerna. Trombocyträkning och analys av hemoglobininnehåll gav en hög korrelation, r = 0,9696 respektive r = 0,9779 (p <0,0001), mellan metoderna och repeterbarheten för Blood Bank mode var god. Svagheten för Blood Bank mode var leukocyträkningen (r = 0,4310 respektive r = 0,08597, p = 0,0315 respektive p = 0,6828). Även vid kliniskt relevanta leukocytnivåer, nära gränsvärdet 1 x106 leukocyter/enhet, var CV(%) och standardavvikelse (SD) relativt höga i repeterbar

As pollinators bumble bees play the crucial role of contributing to propagation of flowering plants in favour of food production as well as biodiversity. Over the course of a few decades bumble bees have seen a remarkable decline, with contributing factors being climate change, pesticides and pathogens such as viruses and parasites. In Sweden, commercially reared bumble bees are bought for the purpose of pollination in fruit and berry plantations. However, these reared bumble bees are a suspected contributor to a spillover of pathogens to wild bees in the same area. The aim of the study was to determine the prevalence of five viruses and five parasites in wild bumble bees nearby commercially reared bumble bees and to determine seasonal variation in pathogen distribution. qPCR was used for analysis of Acute bee paralysis virus, Deformed wing virus, Slow bee paralysis virus, Black queen cell virus and Sacbrood virus as well as the parasites Crithidia bombi, Apicystis bombi, Nosema bombi, Sphaerularia bombi and Locustacarus buchneri. The results showed a statistically significant, 4,8 times higher prevalence of A. bombi nearby commercially reared bumble bees in greenhouses compared to control landscapes. The results were also compared to pathogen prevalences in bumble bees caught in June the same year, showing a significantly higher prevalence in a majority of the parasites. It also showed a decrease in all viruses except Black queen cell virus, where the decrease might be explained by RNA degradation.

Legionella omfattar bakterier som kan orsaka allvarliga lunginflammationer, såsom legionärsjuka hos människor. Den mest kända sjukdomen som kan orsakas av Legionella-bakterier är legionärsjuka, en allvarlig typ av pneumoni, vilket utgörs oftast av Legionella pneumophila (L. pneumophila). L. pneumophila kan delas in i olika serogrupperbaserat på variationer i dess yttre strukturer. Majoriteten av L. pneumophila framkallas av serogrupp 1. I Region Skåne utgörs diagnostiken med PCR analys för att detektera Legionella bakterier, som utförs på nedre luftvägsprov (Bronkoalveolärt lavage, förkortad BAL, och sputum). PCR-positiva prover odlas för att artbestämmas genom riktad odling på fasta substrat för att därefter försöka matcha Legionella stammen som isolerats från miljöer med patienten genom genetisk typning. Då odling av Legionella stammen för artbestämning vanligtvis ej svaras ut förens efter 10 dygn kan utvärdering av en PCR förenkla särskiljning av serogrupp 1 och serogrupp 2-14 för att kunna behandla patienten i tid. Wzm-PCR:en anses kunna särskilja serogrupp 1 från serogrupp 2-14. Genom att odla fram L. pneumophila serogrupp 1 isolat och därefter utföra en spädningsserie kunde PCR-effektiviteten med hjälp utav qPCR beräknas vara 96,5 %. L. pneumophila serogrupp 2-14 odlades även ut för att kunna avskilja serogrupp 1 från serogrupp 2-14 vilket gavs till resultat, men vidare testning av metoden behövs., Legionella is considerable bacteria that can cause serious lung infections, such as Legionnaires' disease in humans. The most well-known disease caused by Legionella bacteria is Legionnaires' disease, a severe type of pneumonia, most commonly caused by L. pneumophila. L. pneumophila can be divided into different serogroups based on variations in its outer structures. The majority of L. pneumophila cases are caused by serogroup 1. In Region Skåne, diagnosis involves PCR analysis to detect Legionella bacteria, performed on lower respiratory tract samples (Bronchoalveolar lavage, abbreviated BAL, and sputum). PCR-positive samples are cultured to identify the species through directed culture on solid substrates, and then attempts are made to match the Legionella strain isolated from the patient's environment through genetic typing. Since culture of the Legionella strain for species identification usually takes up to 10 days, evaluation of a PCR can simplify the differentiation between serogroup 1 and serogroups 2-14 to enable timely treatment of the patient. Wzm-PCR is considered capable of distinguishing serogroup 1 from serogroups 2-14. By culturing L. pneumophila serogroup 1 isolates and then performing a dilution series, the PCR efficiency with the help of qPCR was calculated to be 96,5 %. L. pneumophila serogroups 2-14 were also cultured to differentiate serogroup 1 from serogroups 2-14, which yielded results, but further testing of the method is needed.

Background: Resistance against antibiotics is a global problem that can lead to serious complications. Many bacteria have developed resistance against several antibiotics which causes infections that are hard to treat. Resistant bacteria such as Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus (VRE) are mainly spread within healthcare. To combat these infections, it is important to have reliable detection methods. Purpose: To qualitatively compare the Copan Transsystem 114c with theESwab-system for the detection of MRSA and VRE. Methods: For the study, Polymerase chain reaction (PCR) methods, Matrix-assisted laser desorption/ionization Time of Flight (Maldi-Tof), and microbiological cultivation methods was used on simulated and clinical samples. Results: For the VRE-samples no difference in results were identified. The MRSA samples showed significant differences in PCR-results where the Transsystem 114c detected 16/60 positive samples whereas the ESwab-system detected 12/60. But the cultivation results showed opposite results where the Transsystem 114c detected three positive samples and the ESwab-system detected four. Conclusion: The results show that both the Transsystem 114c and ESwab show similar results on simulated samples but differ on clinical samples. Further comparative tests need to be carried out to validate the methods before they can be used in routine operation thus no real conclusion can be drawn from this study.

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.

Sepsis, a life-threatening condition triggered by infection, has a long history evolving from ancient references to modern understanding. Despite advancements, its symptoms often resemble those of other illnesses until late stages, delaying recognition. The blood culture method, currently used for diagnosing sepsis, is not entirely reliable and often time-consuming, posing challenges for timely intervention in many cases. Recently, miRNAs have emerged as promising biomarkers for a variety of infectious diseases, prompting the suggestion of a new multi-marker panel for sepsis diagnosis. However, the lack of optimized methods for miRNA extraction and quantification necessitates investigation before constructing such a panel. This project aimed to examine and compare manual and robotic methods to determine which yielded superior quality, quantity, time management, and results when employing two-tailed RT-qPCR. Following experimentation, it was observed that both methods provided satisfactory purity and concentration, yet the processing time was notably shorter with the robotic method. The twotailed RT-qPCR method successfully detected DNA with copy numbers as low as 10^5 when spiked-in, although the candidate miRSeps-6 was not detectable in healthy donor plasma. Despite the unsuccessful detection of the candidate miRNA, positive controls demonstrated that processes such as manual and robotic extractions, along with two-tailed RT-qPCR, could be applied to other candidates to identify suitable matches for the kit.

Since the 1960s, biosensors have been used in various fields of society including medicine. Many biomarkers are in low concentrations in bodily fluids, hence, the development of sensitive biosensors is necessary. Biosensors used in medicine can utilise various physical phenomena e.g. optical, electrochemical, and electrokinetics. In this study, the project will assess the use of electrokinetic phenomena streaming current to detect single-stranded DNA (ssDNA). In the study, a microchip streaming current analyzer with parallel microfluidic channels was utilized to detect single-stranded DNA. The walls of the microchannels were coated with peptide nucleic acid (PNA) as a capture probe for the detection. Additionally, the experimental setup and electrical parameters were analyzed. The project results showed a deviation of the flow rate between the theory and experimental result caused by the inwards bending of polydimethylsiloxane (PDMS). Platinum electrodes worked according to theory and the system operated leakfree. Detection of target DNA was successful and selectivity of the system was proven. The microfluidic biosensor showed positive results that are significant for clinical application, particularly in the non-invasive detection of biomarkers through liquid biopsy. The high sensitivity, precision, and dependability of the biosensor are an indication of a valuable instrument opening doors to numerous biomedical settings such as early cancer detection and monitoring.

Sepsis is a life-threatening condition, which in time damages the patient’s organs. The quick sequence of events that could let a patient’s condition go from stable to severe in a few hours requires physicians to establish an early and accurate diagnosis. The research on new potential biomarkers is in full throttle. miRNA has elevated its potential as a diagnostic biomarker and may be included in a multiple-biomarker panel for sepsis diagnosis. In this project, the aim was first to evaluate if miRSeps-7 could be detected and quantified in human plasma by using the Two-tailed RT-qPCR method. Secondly, to compare the efficiency of two total RNA extraction methods (manual and robotic (Qiacube) extraction). This comparison was based on measuring the Hands-On Time (HOT) and Turnaround Time (TAT) during the extraction processes, along with analyzing the Cq values obtained from the qPCR reactions. Additionally, a QC check was performed on all samples after RNA extraction. No significant differences were discovered between the two extraction methods in terms of time management (e.g. HOT & TAT) nor the quality assessment, with quantification cycles (Cq). The small sample size was a major disadvantage. Amplification was achieved on 7/20 samples with the Two-tailed RT-qPCR. However, it has yet to be confirmed that the amplified product was miRSeps-7.

Tularemi är en zoonos som orsakas av den gramnegativa bakterien Francisella tularensis. Sjukdomen förekommer främst i norra hemisfären och ger relativt allmänna symtom som varierar beroende på smittväg. Diagnostik av tularemi sker främst med serologiska metoder då renodling av bakterien innebär risk för laboratoriesmitta. En av de vanligaste serologiska metoderna är enzyme-linked immunosorbent assay (ELISA), där detektion av antikroppar mot F. tularensis kan ske med hjälp av enzymer. Chemiluminescent immunoassay (CLIA) är en serologisk metod som bygger på ELISA och används av instrumentet VirClia för detektion av antikroppar. SERION ELISA classic Francisella tularensis IgG respektive IgM är kommersiella kit som kan användas för diagnostik av tularemi genom att påvisa antikroppar mot F. tularensis. Detta arbete syftar till att utvärdera och verifiera kitet utifrån VirClia, för att kunna svara på frågan om SERION ELISA classic Francisella tularensis IgG respektive IgM kan ersätta eller komplettera VirClia på Örebro Universitetssjukhus. 146 prover kördes i båda analyserna och gav index som tolkades som negativa eller positiva utifrån ett cut off. Kiten fick en sensitivitet på 100% och en specificitet på 90% med VirClia som referensstandard. Utifrån resultatet skulle kiten kunna ersätta eller komplettera VirClia i klinisk användning., Francisella tularensis is a gram-negative bacterium that exists in the northern hemisphere and causes a zoonitic disease called tularemia. The symptoms of the disease are vague and varies depending on how infection occurred. Because of the high risk of laboratory infection associated with isolation of the bacterium the most common method for diagnosing the disease is with serological assays. Enzyme-linked immunosorbent assay (ELISA) is one of the more common used methods and uses enzymes to detect antibodies against F. tularensis. VirClia is an automated instrument that uses Chemiluminescent immunoassay (CLIA), a method based on ELISA. SERION ELISA classic Francisella tularensis IgG respektive IgM are commercial kits that can be used to detect antibodies against F. tularensis. The aim of this study was to evaluate and verify theses kits to answer the question whether they can replace or complement the now used VirClia at Örebro University Hospital. 146 samples of sera were analyzed in both methods and the results were given in indexes which were labeled either positive or negative depending on a set cut off. The sensitivity was calculated to 100% and the specificity 90% for the kits with VirClia as reference standard. The kits could replace or complement VirClia.

Bakgrund: Kallvattenimmersion är en vanlig förekommande metod som är populär inomtränings- och tävlingsvärlden samt terapimiljöer och kan resultera i förbättrat humör. Vidsådana tillfällen kan akuta fysiologiska effekter inträffa, såsom ökad kardiovaskulärarbetsbelastning vilket kan leda till höjt blodtryck och hjärtfrekvens. Syftet: Syftet med denna studie är att analysera de akuta effekterna avkallvattenimmersion på blodtryck och hjärtfrekvens hos både vana och ovana individersamt att undersöka eventuella skillnader mellan grupperna. Metod: En kvantitativ studie utfördes på 40 deltagare, varav 20 ovana och 20 vana.Deltagarna inkluderade både män och kvinnor i åldersgruppen 18 till 70 år. Ovanadeltagare exponerades för kallvatten i 30 sekunder medan vana deltagare utsattes i 2minuter. Resultat: Resultaten visade signifikanta förändringar i både systoliskt och diastolisktblodtryck samt pulstryck hos båda grupperna. Dock observerades ingen signifikantskillnad i hjärtfrekvens hos de vana, utan snarare hos de ovana. Ingen statistisktsignifikant skillnad observerades mellan grupperna vad gäller blodtrycket, däremotnoterades en skillnad i hjärtfrekvensen. Slutsats: Både vana och ovana deltagare uppvisade ökning i blodtrycket, medan endastde ovana deltagarna uppvisade en ökning i hjärtfrekvens., Physiological reactions during cold-water immersionBackground: Cold water immersion is a common method popular in training,competition, and therapeutic settings and can result in improved mood. During suchoccasions, acute physiological effects can occur, such as increased cardiovascularworkload, leading to elevated blood pressure and heart rate. Aim: The purpose of this study is to analyze the acute effects of winter swimming onblood pressure and heart rate in both experienced and inexperienced individuals andinvestigate any differences between the groups. Method: A quantitative study was conducted with 40 participants, 20 inexperienced and20 experienced. The participants included both men and women aged 18 to 70 years.Inexperienced participants were exposed to cold water for 30 seconds, while experiencedparticipants for 2 minutes. Results: The results showed significant changes in both systolic and diastolic bloodpressure and pulse pressure in both groups. However, no significant difference in heartrate was observed in the experienced group, but in the inexperienced group. Nostatistically significant difference was observed between groups regarding blood pressurehowever, a difference in heart rate was noted. Conclusion: Both experienced and inexperienced participants showed changes in bloodpressure, while only the inexperienced participants exhibited an increase in heart rate.

Equine Coronavirus (ECoV) is a virus that affects horses. Symptoms of ECoV are often gastrointestinal, including fever, lethargy, anorexia, and changes in fecal consistency. While the disease is usually mild, it can lead to more severe symptoms where death has occurred. Diagnosis of ECoV is typically confirmed through Polymerase Chain Reaction (PCR) testing of fecal samples. Initial studies have shown that fecal samples from horses can inhibit PCR analysis for ECoV, other PCR systems and buffers may have a better effect on inhibition in fecal samples then the current method used at Swedish Veterinary Agency (SVA) today, leading to false negative results. To avoid false negative results due to inhibition, the option of using an internal control exists. If the internal control yields a negative result, it suggests that the PCR reaction did not function as intended and a negative result for ECoV would therefore not be reliable. The purpose of this study was to gather more information about the current method's performance and possibly optimize its sensitivity. Seven different buffers were tested to see which works best for horse feces and the current method was compared with other PCR methods in order to choose the method with the highest sensitivity. The results indicated a statistically significant difference between PB and PVP buffers. Additionally the comparison between the PCR systems in the study showed a significant difference, indicating that the new system for ECoV detection has the potential to improve upon the current routine method.

Diabetes, a prevalent global group of metabolic diseases, is characterized by hyperglycemia, which can lead to potential complications with prolonged exposure. Treatment goals involve lowering blood sugar levels and preventing future complications, with glycated hemoglobin, HbA1c serving as a marker for treatment control. HbA1c, forms when glucose binds to hemoglobin, aids in treatment monitoring. This study aimed to validate capillary HbA1c analysis on the Cobas Pro analyzer to comply with new in vitro diagnostic (IVD) regulations and streamline processes. Unlike the high-performance liquid chromatography method, (HPLC) to be replaced, all reagents used in the Cobas Pro are IVD-marked. Transitioning to capillary HbA1c analysis involves a change in measurement principle. The current method utilizes ion exchange chromatography with photometric detection, while the Cobas Pro employs turbidimetric inhibition immunoassay. The correlation coefficient for 31 capillary blood samples between the methods was 0.97, with most data points falling within the confidence interval. Despite obtaining a statistically significant difference from the paired ttest, the difference between the measurement values did not exceed 3 mmol/mol, which is the proposed clinical threshold. Comparison of capillary and venous blood analyzed with Cobas Pro yielded a linear correlation of 0.99, with no significant difference found by the paired ttest. A coefficient of variation (CV) under 4% was achieved for controls on the Cobas Pro. The primary advantage of transitioning the method was that it complied with IVDR regulations and enhances patient safety. Based on these results, the method is deemed safe for implementation.

Parodontit är en inflammatorisk tandsjukdom som tar sin utgångspunkt i tandköttsinflammation (gingivit). När inflammationen sprider sig till tandköttsskåran bryts stödjevävnaden ned och en tandköttsficka bildas. Skillnader har påvisats i den subgingivala mikrobiotans storlek och komplexitet mellan parodontitpatienter och friska personer. Etiologin bakom parodontit är inte fullständigt klarlagd, men fynd som dessa ger stöd åt teorin om att sjukdomsförloppet initieras genom uppkomsten av obalans i mikrobiotans sammansättning. Porphyromonas gingivalis är en gramnegativ, obligat anaerob, stavformad bakterie som framför allt återfinns som en del av biofilmen i de djupare delarna av tandköttsskåran. Den är en proteolytisk bakterie som utnyttjar fermentering av aminosyror för sin energiproduktion. För detta ändamål uttrycker P. gingivalis flera olika proteaser. Förutom att proteaserna som P. gingivalis uttrycker är essentiella för dess näringstillförsel utgör de betydande virulensfaktorer. Tillväxt och överlevnad hos P. gingivalis är delvis beroende av dess förmåga att tillägna sig järn, vilket upptas från omgivningen. Nedbrytning och upptag av hem och hemliknande molekyler förmedlas av trypsin-liknande proteaser kända som gingipains. För att klargöra sambandet mellan järntillgång, tillväxt och proteolytisk aktivitet odlades P. gingivalis W50-d under 72 timmar i Brain heart infusion (BHI) med olika hemintillsatser. Den optiska densiteten vid 600 nm och antalet colony forming units (CFU) användes för att mäta tillväxten medan proteolytisk aktivitet mättes med två olika fluorescerande substrat. Mätningarna visade konsekvent störst tillväxt och proteolytisk aktivitet vid låga heminhalter. Resultaten kan förklaras av att bakteriestammar med hög patogenicitet har ett lägre krav på hemintillgång. Det är sannolikt att tillväxt och proteolytisk aktivitet hos Porphyromonas gingivalis påverkas av hemintillgång, men för att med tillförlitlighet kunna hävda på vilket sätt krävs vidare, Periodontitis is an inflammatory periodontal disease that originates from inflammation of the gums (gingivitis). The spreading of inflammation to the gingival crevice may lead to tissue destruction and formation of a periodontal pocket. Differences have been observed in the size and complexity of the subgingival microbiota between patients with periodontitis and healthy individuals. The etiology behind periodontitis is not fully understood, but evidence suggests that the disease may be initiated by the emergence of dysbiosis. Porphyromonas gingivalis is a gram-negative, obligate anaerobic rod primarily found as part of the biofilm of the gingival crevice and in the deep periodontal pocket. Being a proteolytic bacterium, it utilizes the fermentation of amino acids for energy production. For this purpose, P. gingivalis expresses various proteases. In addition to the proteases expressed by P. gingivalis being essential for its nutrition, they constitute important virulence factors. The growth and survival of P. gingivalis are, to some extent, dependent on its ability to acquire iron from the environment. Degradation and uptake of heme and heme-like molecules are facilitated by trypsin-like proteases known as gingipains. To elucidate the relationship between iron availability, growth, and proteolytic activity, P. gingivalis W50-d was cultured for 72 hours in Brain Heart Infusion (BHI) supplemented with various hemin concentrations. Optical density at 600 nm and the number of colony-forming units (CFU) were measured to assess growth, while proteolytic activity was determined using two different fluorescent substrates. Analyses consistently showed increased growth and proteolytic activity at low hemin concentrations. The results can be explained by bacterial strains with high pathogenicity having a lower requirement for hemin availability. It is likely that the growth and proteolytic activity of P. gingivalis are influenced by hemin availability, but further studies are r

Heparin är ett läkemedel som används för att förebygga och behandla trombos. Läkemedlet, som finns i ofraktionerad (UFH) och lågmolekylär form (LMWH), verkar blodförtunnande genom indirekt inhibering av aktiverad faktor X (FXa). För att styra behandling med heparin kan det vara nödvändigt att monitorera läkemedelsnivån i blodet. Detta kan göras med den kromogena analysmetoden anti-FXa (heparin). Syftet med examensarbetet var att verifiera en metod för heparin-beroende anti-FXa på koagulationsinstrumentet Sysmex CS-2500 på Klinisk Kemi vid Centrallasarettet i Växjö. Två identiska instrument, master och slav, ingick i verifieringen. Som del i verifieringsarbetet utfördes en metodjämförelse mot ett referenslaboratorium nyttjandes samma analysprincip, med 21 patientprover av citratplasma innehållande heparin. Vidare utvärderades inomserie- och total-precisionsanalys. En linjäritetsstudie för läkemedlen Fragmin [12500 IE/ml] och Heparin APL [100 IE/ml] utfördes även. Metodjämförelse mellan master- och referensinstrumentet (Sysmex CS-5100) gav korrelationskoefficienten 0,952. Metodjämförelsen mellan slav- och masterinstrumentet gav korrelations-koefficienten 0,992. Inget signifikant systematiskt mätfel förelåg för något av instrumenten. Inomserieprecision för master- och slavinstrumentet gav variations-koefficienten (CV) 2,21% respektive 1,33% för plasmapoolnivå 1 [0,45 IE/ml] och CV 2,03% respektive 1,39% för plasmapool nivå 2 [0,70 IE/ml]. Totalprecision för master- och slavinstrumentet gav CV 4,64% respektive 2,49% för kontrollnivå 1 [0,36 IE/ml] och CV 3,70% respektive 1,60% för kontrollnivå 2 [0,91 IE/ml]. Linjäritets-studien för Fragmin och Heparin APL gav för båda determinationskoefficienter (r2) >0,99. Samtliga resultat från metodjämförelser, precisionsmätningar och linjärtitets-studierna bedömdes, utifrån de kliniska och metodspecifika krav som finns, som godkända. Analysen kan därmed tas i kliniskt bruk på Klinisk Kemi vid Centrallasarettet i Växjö., Heparin is a pharmaceutical drug used for the prevention and treatment of thrombosis. The drug, which is available in both unfractionated (UFH) and low molecular weight (LMWH) forms, acts as a blood thinner by indirectly inhibiting activated factor X (FXa). Monitoring heparin blood levels may be necessary to correctly regulate treatment with heparins. This can be done using the chromogenic assay method anti-FXa (heparin). This study aimed to verify a heparin-dependent anti-FXa method on the coagulation instrument Sysmex CS-2500 at the Clinical Chemistry laboratory at the Central Hospital in Växjö, Sweden. Two identical instruments, master and slave, were included in the verification. As part of the verification, a method comparison was performed with 21 patient samples of citrate plasma containing heparin. Furthermore, within-run and overall precision were evaluated. A linearity study for Fragmin [12500 IU/ml] and Heparin APL [100 IU/ml] was also performed. From the method comparison between the master and reference instrument (Sysmex CS-5100), a correlation coefficient of 0,952 was obtained. Between the slave and master instrument, a correlation coefficient of 0,992 was obtained. No significant systematic errors were present for any of the instruments. Within-run precision for master and slave instruments yielded a coefficient of variation (CV) of 2.21% and 1.33%, respectively, for plasma pool level 1 [0.45 IU/ml] and a CV of 2.03% and 1.39% for plasma pool level 2 [0, 70 IU/ml]. Master and slave instrument overall precision yielded a CV of 4.64% and 2.49%, respectively, for control level 1 [0.36 IU/ml] and a CV of 3.70% and 1.60% for control level 2 [0.91 IU/ml]. The linearity study for Fragmin and Heparin APL obtained a coefficient of determination (r2) > 0.99. All results from the method comparisons, precision measurements, and linearity studies were approved, based on clinical and method-specific requirements The anti-Xa method can thus be put into clinical

The prevalence of diabetes is sufficiently high to be classified as an epidemic, and 20-40% of these patients are expected to develop diabetic nephropathy, a leading cause of end-stage renal failure. Studies have identified a correlation between diabetic nephropathy and hypoxia in renal tissue in human studies. Increased oxygen consumption has been associated with the proton transport protein, uncoupling protein-2 (UCP-2), which uncouples the mitochondria. Previous research has reported elevated levels of UCP-2 in diabetic renal tissue. Consequently, it is crucial to determine how different preservation methods affect the detectability of UCP-2 in renal tissue for clinical applications. This study aimed to evaluate the effectiveness of Western blotting for detecting UCP-2 in snap frozen, fresh untreated, formalin-fixed, methyl carnoy-fixed, and RNA later-preserved rat kidneys. Preliminary trials were conducted to identify the optimal antibody combinations, followed by testing on various preserved tissues. The antibodies produced non-reproducible, unspecific, and unselective results. Additionally, technical challenges, such as gels adhering to membranes and low protein concentrations in some samples, rendered the results inconclusive. Further investigations are necessary to explore additional antibodies and variables that may influence the detection of UCP-2 in differently preserved tissues. Overall, this study highlights the complexity and challenges in developing reliable protocols for UCP-2 detection in preserved renal tissue, indicating that significant optimization is still required for consistent results.

ABO- och Rh-systemet är de mest kliniskt betydelsefulla blodgruppssystem för nutidens serologiska diagnostik. En viktig analysmetod inom detta område är kvantifiering av blodgruppsantikroppar då mängden antikroppar i plasma bestäms. Denna metod har flera olika användningsområden, bland annat för att bestämma mängden av irreguljära antikroppar för gravida vid immunisering då mamman har bildat antikroppar mot ett antigen som fostret har. Metoden utförs även i samband med transplantationer och för trombocytgivare. Kvantifiering av blodgruppsantikroppar kan utföras både manuellt och automatiskt. Den manuella metoden utförs med diluent pH7, en form av fosfatbuffrad saltlösning (PBS), som spädningsvätska, medan den automatiska utförs med en dyrare titreringslösning. Ett mer kostnadseffektivt val skulle vara att använda diluent pH7 som spädningsvätska för den automatiska metoden. Denna studie är en metodoptimering för automatisk titrering av blodgruppsantikroppar med IH-500 instrument. Plasmaprover från tre olika kategorier av individer (transplantationspatienter, trombocytgivare och gravida kvinnor) analyserades på IH-500 instrument för både ABO- och andra blodgruppsantikroppar. I maskinen utförs en spädningsserie av plasma från patienter och efter tillsättning av testerytrocyter observeras var det skett en agglutination och hur starkt den var. Det första spädningssteget som graderas som 1+ reaktion räknas som antikroppstitern. Resultat redovisades i form av stapeldiagram som visar skillnaden i antikroppstiter med både titreringslösning och PBS som spädningsvätska. Samtliga resultat ligger inom ramen för vad som definierats som ett godkänt resultat. Konklusionen av studien är att PBS är ett effektivt och billigare alternativ som kan användas som spädningsvätska vid automatisk titrering av ABO- och andra blodgruppsantikroppar på IH-500 instrument., The ABO and Rh blood group systems are the most clinically significant blood group systems for contemporary serological diagnostics. A vital analysis method in this area is the quantification of blood group antibodies, where the amount of antibodies in plasma is determined. This method has several different uses, including the determination of irregular antibodies in pregnant women suspected of immunization if the mother has developed antibodies against an antigen that the fetus possesses. The method is also performed in connection with transplants and for platelet donors. Quantification of blood group antibodies can be performed both manually and automatically. The manual method utilizes diluent pH7, a form of phosphate-buffered saline (PBS), as the diluent, while the automatic method is performed with a more expensive titration solution. A more cost-effective choice would be to use diluent pH7 as the diluent for the automatic method. This study aims to optimize the method for automatic titration of blood group antibodies using the IH-500 instrument. Plasma samples from three different categories of individuals (transplant patients, platelet donors and pregnant women) were analyzed on the IH-500 instrument for both ABO and other blood group antibodies. From patient plasma samples a dilution series was made, and after adding test erythrocytes the level of agglutination was observed. The first dilution step graded as 1+ reaction is counted as the antibody titer. Results were presented in the form of bar graphs illustrating the difference in antibody titers with both titration solution and PBS as the diluent. All results fall within the range of what has been defined as an acceptable result. The conclusion of the study is that PBS is an effective and inexpensive alternative that can be used as the diluent for automatic titration of ABO and other blood group antibodies on the IH-500 instrument.

Addressing the formidable challenges presented by diffuse midline glioma (DMG), a notably aggressive pediatric brain tumor with limited therapeutic options, this study investigates the oncogenic roles of MYC and PDGFB and evaluates SOX9's contribution to therapeutic resistance. Utilizing advanced transgenic mouse models and the RCAS virus system, our research effectively simulates the progression and treatment responses characteristic of DMG. In our approach, we established a DMG tumor model by manipulating Tumor Virus A receptor (TVA)-expressing embryonic neural stem cells sourced from the E12.5 hindbrain in vitro. Validation of this model and its genetic perturbations was achieved through detailed Western blot analyses. Results indicate that DMG cells overexpressing MYC tended to be significantly more sensitive to chemotherapy compared to PDGFB cells that do not overexpress MYC, positioning MYC as a crucial therapeutic target. On the other hand, increased expression of SOX9 was linked to a slight increase in resistance to conventional treatment modalities, highlighting its role in promoting adaptive resistance mechanisms within the tumor microenvironment. This research emphasizes the critical importance of merging molecular biology techniques with comprehensive in vivo modeling to elucidate DMG's pathophysiology and identify actionable therapeutic targets. Our findings offer significant insights into the molecular dynamics of DMG and suggest novel targets for therapeutic intervention that could substantially improve clinical outcomes in this challenging pediatric malignancy. Future research should aim to broaden the genetic profiling of DMG, and tailor therapeutic approaches based on specific molecular profiles to optimize treatment efficacy and precision.

Gomoris trichrome infärgning är en histologisk infärgning som används för bland annat morfologisk visualisering och differentiering av muskler och deras strukturer. Infärgningsmetoden består av en plasmatisk färg Chromotrope 2R, som infärgar mitokondriellt och endoplasmatiskt retikulärt membran samt avvikande strukturer som exempelvis inklusionskroppar, röda. Fiberfärgen Fast Green FCF infärgar myofibrill och kollagen grönblått och Harris hematoxylin infärgar cellkärnor blått/lila. Arbetet syftar till jämförelsen av egentillverkad och inköpt In vitro medical device regulation märkt Gomoris trichrome färg. Metoden bygger på fryssnittning av tio skelettmuskelbiopsier i kryostat där varje biopsi tilldelades två mikroskoperingsglas med två vävnadssnitt på varje. Tio vävnadspreparat färgades med både den nuvarande Gomoris trichrome metoden och nya metoden. De största skillnaderna mellan metoderna är tiden i Gomoris trichrome lösning: ättiksyra och Harris hematoxylin, samt att den nya metoden innehåller en blåning och tvättning. Resultatet av de bedömningsbara snitten blev att samtliga vävnader infärgade med den nya metoden gav blekare snitt, både makro- och mikroskopisk, sämre visualisering av cellkärnor samt total avsaknad av plasmatisk infärgning. Inga vävnader infärgade med den nya metoden kunde godkännas. Slutsatsen blev att den nyinköpta Gomoris trichrome färgen ej är bättre än, eller likvärdig, den nuvarande och kan därför inte inrättas på patologavdelningen på Universitets Sjukhuset Örebro., Gomori's trichrome is a histological stain used for morphological visualization and differentiation of muscles. The staining method consists of a plasmatic stain Chromotrope 2R, which stains mitochondrial and endoplasmic reticular membranes as well as abnormal structures such as inclusion bodies red. The fiber stain Fast Green FCF stains myofibrils and collagen green-blue. Harris hematoxylin stains cell nuclei blue/purple. This study’s aim is the comparison of self-made and purchased In-vitro medical device regulations labeled Gomori's trichrome. The method is based on frozen sectioning of ten skeletal muscle biopsies in a cryostat, each biopsy received two microscope slides with two sections on each. Ten tissues were stained according to the old method and ten to the new method. The biggest differences between the methods were the time in Gomori's trichrome solution, acetic acid, and Harris Hematoxylin. The new method also has bluing. The result was that all tissues stained with the new method gave paler sections, both macro- and microscopically, worse visualization of cell nuclei and absence of plasmatic staining. No tissues could be approved, and the conclusion was that the new stain was not better, or equivalent to the current one and therefore should not be used in the pathology department.

Efter upptäckten av penicillinet observerades en ökad resistens hos S. aureus vilket ledde till att alla stammar ansågs vara resistenta. Dock har många stammar enligt senare studier visat sig vara känsliga vilket har öppnat upp möjlighet att åter använda benzylpenicillin som ett behandlingsalternativ eftersom det kan ges i lägre koncentration jämfört med andra alternativ. Metoden för att upptäcka penicillinresistens hos S. aureus enligt EUCAST har bedömts svår att tillämpa på grund av subjektivitet vid bedömning av zonkanten. Studiens syfte var att utvärdera metoden för senare implementering på Klinisk Mikrobiologi Unilabs i Skövde. Metodens användbarhet utvärderades genom resistensbestämning av en referensstam samt tio stammar från ett referenslaboratorium för EUCAST. Bedömningen av de tio stammarna från referenslaboratoriet stämde vid jämförelse överens med deras resultat förutom för en stam som bedömdes som resistent i stället för känslig. Repeterbarheten var hög då ingen skillnad (p=0,156) uppvisades för referensstammen. Resultatet tyder på att det kan vara svårt att göra en korrekt tolkning av zonkantens utseende då det är en subjektiv bedömning. För att uppnå en högre säkerhet kan det vara fördelaktigt att mer än en person gör en bedömning. Slutsatsen var att metoden kan implementeras på laboratoriet., Following the discovery of penicillin, resistance rates increased until all strains of S. aureus were considered resistant. Recent studies have shown that many strains are susceptible, making it possible to again use penicillin as an alternative for treatment since it can be used in lower concentrations compared to alternatives. The EUCAST method for discovering penicillin resistance in S. aureus has been judged difficult due to the subjectiveness of assessing the zone edge. The aim of this study was to evaluate the method to implement it in the Section for Clinical Microbiology Unilabs Skövde. The useability of the method was evaluated by resistance testing of a reference strain and ten strains from a EUCAST reference laboratory. The assessment of the strains from the reference laboratory equaled their results except for one strain that was assessed resistant rather than sensitive. The repeatability was high since no difference (p=0,156) was found for the reference strain. The results show how difficult it is to correctly assess the zone edge due to its subjectiveness. It could be beneficial with more than one person assessing the zone edge to achieve a higher security. In conclusion the method is possible to implement in the laboratory.

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.

The zone of Ranvier has been identified as a stem cell niche located directly next to the epiphyseal growth plate in the knee structure, which has drawn attention as a potential area of study in stem cell therapy treatments for degenerative cartilage diseases, such as osteoarthritis. Cathepsin K, a cysteine protease, is a stem cell marker used to identify stem cells and the niches they reside in. This research aims to analyse Cathepsin K expression in the zone of Ranvier in 10 day postnatal mice, as well as zebrafish of three ages (3 and 5 days post fertilisation, and adult). Staining through immunohistochemistry, and imaging with fluorescent microscopy were the methods chosen to visualise the expression of each marker as they allow a preservation of the structural integrity of the sample. The images showed a nuclear expression of Cathepsin K in the zone of Ranvier. Meanwhile in zebrafish, it was concentrated in both nuclei and the extracellular matrix of jaw bones. There were no compelling differences in the expression between the developmental stages. The counterstaining performed with known stem cell niche markers helped identify both the location of the stem cell niches where Cathepsin K would be expected and the differences of expression between murine and fish tissues. The findings of this research showed Cathepsin K as one of the markers expressed in the zone of Ranvier, which gives further insight into this stem cell niche and its possible use in the treatment of osteoarthritis and other cartilage degenerative diseases.

Cancer relapse in myelodysplastic syndrome (MDS) patients receiving bone marrow transplantation can be predicted with measurable residual disease (MRD), by droplet digital polymerase chain reaction (ddPCR). ddPCR quantifies genomic DNA molecules in an absolute manner using end-point amplification. This work aims to demonstrate that ddPCR assay evaluation can be conducted with fewer healthy donor controls compared to methods for relative quantification. The hypothesis is further studied by applying the total error computed in the ddPCR system as a threshold for background noise in personalized assays. Ten assays for detecting MRD markers were evaluated in an optimized PCR-plate setup for accuracy and reproducibility of background in negative controls. Additionally, data analysis of negative controls collected from patient tests complied to the empirical limit of blank based on false - positive counts, in each assay. The findings indicate that the optimized setup accurately determines background noise, and empirical cutoffs for individualized assays are reliable for performance evaluations. This study supports ddPCR integration into clinical settings for personalized mutation analyses in MDS, providing an optimized setup and alternative metrics of evaluating assay performance in respect to the absolute quantification methodology.

Autoimmunitet är ett tillstånd där kroppens immunsystem felaktigt attackerar och skadar sina egna vävnader och celler. HLA-B27 är en genvariation som kan kopplas till autoimmun sjukdom som ankyloserande spondylit med en prevalens på 2-4% i världens befolkning. Denna studie syftade till att utvärdera och jämföra två kit för HLA-B27 alleler mot den nuvarande metoden på Länssjukhuset Ryhov i Region Jönköpings län. De metodprinciper som användes var realtids-PCR samt LAMP. Totalt analyserades 37 avidentifierade blodprover med vardera av kiten samt med nuvarande metod. Resultatet visade en överensstämmelse med avseende på förväntade positiva och negativa resultat för HLA-B27 för de två kiten jämfört med nuvarande metoden. De tre metoderna/kiten detekterar de vanligaste HLA-B27 allelerna. Utifrån studiens resultat visade sig båda kiten vara effektiva, lättanvända samt ha stabila reagenser. Dessutom uppnådde båda kiten de IVD-krav som ställs inom EU., Autoimmunity is a condition where the body's immune system mistakenly attacks and damages its own tissues and cells. HLA-B27 is a genetic variation that can be linked to autoimmune diseases such as ankylosing spondylitis, with a prevalence of 2-4% in the world’s population. This study aimed to evaluate and compare two kits for HLA-B27 alleles against the current method at Ryhov County Hospital in Region Jönköping County. The methodological principles used were real-time PCR and LAMP. A total of 37 anonymized blood samples were analyzed using each of the kits and the routine method. The results showed concordance with the expected positive and negative results for HLA-B27 between the two kits compared to the current method. The three methods/kits detect the most common HLA-B27 alleles. Based on the study’s results, both kits proved to be effective, user friendly, and have stable reagents. Additionally, both kits met the IVD requirements set within the EU.

In this study, the efficacy of MarrowMAX, a new IVD-certified culture medium for bone marrow samples, was evaluated and compared to the in-house modified culture medium RPMI-1640, which is routinely used at the Academic Hospital in Uppsala for chromosomal analysis. The hypothesis was that the new culture medium MarrowMAX would lead to an increased number of metaphases, resulting in a higher mitotic index, better spreading, and improved metaphase morphology. Twenty patient samples were cultured in parallel using both media and subjected to identical methods for sample preparation and staining for chromosomal analysis. The results demonstrated significant differences between MarrowMAX and RPMI-1640 for one-day, two-day, and three-day cultures. However, no significant differences were observed for directly harvested cultures and overnight colcemid cultures. These findings suggest that MarrowMAX is more effective than RPMI-1640, resulting in a higher frequency of metaphases, which is crucial for detailed chromosomal analysis. This was further confirmed by flow cytometry analysis, showing a higher number of proliferated cells in the MarrowMAX culture compared to the RPMI-1640 culture, in one representative sample. MarrowMAX's enhanced capacity to promote cell growth and structure in a laboratory setting indicates its potential as a promising alternative to RPMI-1640 for future diagnostic applications, potentially leading to faster diagnoses and improved treatment efficacy for patients with hematological conditions.

Polycystic ovary syndrome is a common endocrine disorder that hinders women’s metabolism and reproductive health characterized by elevated androgen levels, polycystic ovaries, infertility, hirsutism, inflammation, obesity and insulin resistance. Research suggests mitochondrial dysfunction as an underlying cause of PCOS. This study aimed to develop a PCOS model of Drosophila melanogaster by induction of androgens (testosterone). The eggs of the wild-type stock flies were exposed to ethanol (control) and varying doses of testosterone (1-10mg/kg) and examined throughout development. Androgen and ethanol exposure caused alterations in the external morphology and ovarian appearance suggesting a link to PCOS-related symptoms. Fertility measured by fecundity rates decreased by 32.6% in androgen treated flies, though statistical tests revealed no significant differences between groups. Androgen exposure altered the mitochondrial gene expression but with no statistical difference between conditions in all genes (p values: MTCO1 = 0.368; SOD1 = 0.276; ATPsynthase = 0.102). MTCO1 showed approximately a 99% difference in low and high testosterone groups from control. SOD1 increased by 7645.72% and 98.8%, and ATPsynthase by 319.9% and 703.4%in low and high testosterone conditions, respectively. Triglyceride levels were elevated in androgen groups across F1 and F2 generations, indicating potential metabolic disruptions. Significant differences revealed between control and high testosterone (F1, p = 0.04; F2, p = 0.006) but not in low testosterone (F1, p = 0.402; F2, p = 0.328). Lack of significant findings renders the results unreliable. Future research with broader gene panels and improved methologies are required to validate the model's applicability for PCOS research., Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.

Amyloidosis is a disease caused by misfolding of proteins that accumulate in different organs in the body. One of the most common forms of amyloidosis is Transthyretin amyloidosis (ATTR-amyloidosis). Diagnostic of amyloidosis is done by Congo red staining and immunohistochemistry where high-affinity monoclonal antibodies are preferred. For treatment to be effective it is necessary to obtain the diagnosis at an early stage. The aim of this study was to produce monoclonal antibodies against transthyretin for diagnostic purposes of ATTR-amyloidosis. To produce monoclonal antibodies, the spleen from a mouse immunized with transthyretin was collected. Antibody producing cells from the spleen were fused with myeloma cells deficient of their own antibody production. After 2 weeks culture in HAT selection media cells were screened for antibody production with ELISA and immune-histochemistry. Recombinant transtyretin (TTR) was produced and used as antigen in the ELISA. All 65 hybridomas tested were negative on ELISA. Three out of 25 hybridomas tested in immunohistochemistry showed visible staining against muscle cells in tissue from patients with TTR amyloid deposits. No antibody could be produced that detected ATTR-amyloidosis deposits.

To reduce the risk of transfusion related complications, blood components should, according to European guidelines, contain less than 1 x 106 leukocytes per unit. To verify that these guidelines are upheld, residual leukocytes are measured in randomly selected blood components as means of quality control. At Uppsala University Hospital, the method currently used for this is flow cytometry (FCM). However, the hospital recently purchased a new instrument, ADAM rWBC2, for this purpose. The aim of this study was to verify ADAM rWBC2 as a replacement method for FCM and investigate whether the type of test tube chosen for the instrument (EDTA or micro test tube) would affect the leukocyte concentration. To conduct the study, 30 red blood cell units (RBCs), 30 platelet units (PLTs) and 30 plasma units were analyzed on both the ADAM rWBC2 instrument and with FCM. In addition to this, each RBC and PLT unit was allocated into both an EDTA tube and a micro test tube before analysis on the ADAM rWBC2 instrument. Results from both methods and tubes were compared using statistical analysis. The results from ADAM rWBC2 tended to be higher than the results from FCM, and the difference turned out to be statistically significant (p<0,001). No significant difference could be detected between the results from the different test tubes. The assessment is that ADAM rWBC2 will replace FCM for quality control of residual leukocytes in blood components. According to the results, the type of test tube used does not affect the leukocyte concentration.

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.

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 antiseizure medication levetiracetam is used to treat epilepsy with significant success, the medication concentration in serum may be affected by other co-administered medication. Levetiracetam is excreted renally, and the halftime is depending on the renal function which is often correlated to age. The clinical chemistry laboratory at Sundsvall hospital did previously send samples for levetiracetam analysis to Karolinska University Hospital in Stockholm. There was a wish to start analysing the medication locally to reduce analysis time and thereby increase patient safety. ARK Diagnostic markets a kit for quantitative analysis of levetiracetam on automated chemistry analysers. The sample may be taken in a few different test tubes with slightly different characteristics. The aim of this study was to verify the ARK kit on Cobas Pro c503 at Sundsvall hospital and investigate the effects of different test tubes. To assess the accuracy and precision of the method, serum was spiked with levetiracetam from a liquid solution to construct dilution series for testing linearity and sample materials, and by running internal controls to assess the repeatability and reproducibility of the method. The results show that the method does in fact give accurate measurements of the levetiracetam concentration and the results does not vary more than acceptable between measurements nor over time and that different sample type does not show a clinically important difference in result.

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.

Staphylococci are bacterial species where some strains can be found in the human microbiome. If the bacteria infect other body parts it could cause mild to life-threatening infections. The infection should be treated with an effective antibiotic where the bacteria strain’s antibiotic sensibility should be tested. One of the most trustworthy laboratory methods is the broth microdilution (BMD) method. BMD is based on exposing bacteria to different concentrations of different antibiotics to determine the minimum inhibitory concentration (MIC) a specific bacteria strain has, and thus the kind of antibiotic the bacteria strain is most susceptible to. A sensititre plate with 96 wells is used containing freeze-dried antibiotics in varied concentrations. Therefore, the microbiologic laboratory at Gävle Hospital wants to implement BMD as a routine analyzing method where a so-called SEMSE7 sensititre plate verification is needed. The verification is essential to ensure the plate’s performance and ability to give reliable results which was the purpose of this study. Obtained results were compared to reference laboratory values, where 100 % accordance regarding ± one dilution step was obtained of all MIC-values (90 % limit) and 94,3 % accordance regarding the SIR-system (90 % limit). Contrariwise, one very major error, and one major error were obtained which means 5,7 % where the limit was at 3 %. Furthermore, concerning the plate’s performance 97,3 % accordance was obtained regarding ± one dilution step (90 % limit). Most verification requirements were fulfilled but completion is needed before implementing BMD as a routine analysis method.

Myokardscintigrafi är en nuklearmedicinsk metod som används för att kartlägga och visuellt bedöma blodflödet till hjärtat. Vid undersökningen används radiofarmakan Myoview, som består av radionukliden 99mTechnetium som vid sönderfall avger gammastrålning som kan registreras med en gammakamera. StarGuide är en modern gammakamera som implenterades i verksamheten på Skånes universitetssjukhus år 2022. Skillnaden mellan konventionella kamerasystem och StarGuide är främst utformningen och uppbyggnaden av detektorerna, vilket även påverkar hur bilderna framställs. Det här gör att det inte finns några riktlinjer på kliniken gällande kvalitetssäkring i myokardscintigrafi-bilder tagna med StarGuide. Syftet med studien var att undersöka om det finns korrelation mellan counts och kroppsvikt i myokardscintigrafibilder tagna med StarGuide. Retrospektiva data från totalt 60 patienter användes till studien. Medelcounts/s/MBq sattes mot vikt i en scatterplot samt utfördes även en korrelationsanalys. Korrelationsanalysen för hela populationen gav ett värde på -0,790. Könsfördelade korrelationsanalyser utfördes även och gav värdena -0,784 för kvinnor och -0,706 för män. Slutsatsen är att det finns en märkbar korrelation mellan counts och vikt tagna på StarGuide men att det behövs vidare arbete för att stärka studiens fynd. Framför allt behövs insamling av mer data från patienter med en lägre vikt då de var underrepresenterade i studien., Myocardial perfusion SPECT is a nuclear test used to map and visually assess blood flow to the heart. In the test, the radiopharmaceutical Myoview is used, which consists of the radionuclide 99mTechnetium, which when decaying emits gamma radiation that can be registered with a gamma camera. StarGuide is a modern gamma camera that was implemented in the clinics at Skåne University Hospital in 2022. The difference between conventional camera systems and StarGuide is primarily the design and construction of the detectors, which also affects how the images are produced. This means that there are no guidelines in the clinic regarding quality assurance in myocardial scintigraphy images taken with StarGuide. The purpose of the study was to investigate whether there is a correlation between counts and body weight in myocardial scintigraphy images taken with StarGuide. Retrospective data from a total of 60 patients were used for the study. Mean counts/s/MBq were plotted against weight in a scatter plot and a correlation analysis was also performed. The correlation analysis for the whole population gave a rsvalue of -0.790. Gender-distributed correlation analyzes were also performed and yielded rsvalues ​​of -0.784 for women and -0.706 for men. The conclusion is that there is a noticeable correlation between counts and weight taken on the StarGuide, but that further work is needed to strengthen the study's findings. Above all, the collection of more data is needed from patients with a lower weight as they were underrepresented in the study.