Aripiprazol är den aktiva substansen i läkemedel för bipolaritet och schizofreni och metaboliseras av två enzym till den aktiva metaboliten dehydroaripiprazol. Till följd av interindividuella skillnader i aktiviteten hos enzymen samt att koncentrationen in vivo kan påverkas av andra läkemedel rekommenderas terapeutisk läkemedelsövervakning (TDM). Därmed har en selektiv och känslig vätskekromatografi- tandem masspektrometri (LC-MS/MS) validerats för kvantifiering av aripiprazol och dess aktiva metabolit dehydroaripiprazol i humant serum vid Specialkemi, Klinisk kemi och farmakologi vid Lunds universitetssjukhus utifrån interna kriterier byggda på riktlinjer från European Medicine Agency (EMA). LC-MS/MS analys utfördes på Tripple Quad 6500+ från AB Sciex med joniserande elektrospray (ESI) och multi reaction monitoring (MRM). Valideringen fastslog metodens mätområde till 4 – 2 500 nmol/L och kvantifieringsgräns till 4 nmol/L för respektive analyt. Metodens inomdags- och mellandags noggrannhet (variationskoefficient (CV%)) och riktighet (nominell differens) för kontrollprover (10 och 1 000 nmol/L) var mellan 3,5 – 9,1 % och mellan -6,8 – -13,0 för respektive analyt vilket var inom godkända kriterier. Innan metoden implementeras på kliniska prover bör framtida utvärdering undersöka om minimering av provsmittan är möjlig samt utvärdera långtidsstabiliteten av analyterna.

Overweight and obesity are a major problem today. It is a growing problem that harms health and is present in all ages. This study was designed to investigate the relationship between ISO-Body Mass Index in overweight children both boys and girls, and walking speed regardless of height. In addition, examine the relationship between weight, height and walking speed by using a six-minute walking test. The study included already collected measurements from the patients' first visit at the Energy Metabolic Laboratory, which is a research unit at the Department of Women's and Children's Health at the academic Hospital in Uppsala, between the years 2008–2021. Results were collected on 195 patients, 122 boys and 73 girls. Data collected were age, weight, height, walking speed, walking distance, heart rate, level of exertion and dyspnea. The results showed that the ISO-Body Mass Index affects walking speed in obese children. Regarding how walking speed differs between both genders of obese children, the results showed that higher ISO-Body Mass Index in boys leads to them walking more slowly compared to girls who are less affected. To be able to demonstrate how walking speed is affected by other continuous variables such as encouragement or motivation, more studies with greater focus on smaller age groups are needed to gain a better understanding of how the various variables affect walking speed.

Mitochondria are important cytosolic organelles present in nearly all eukaryotic cells. The main function of mitochondria are to generate the vast majority of ATP through the process of oxidative phosphorylation. Mitochondria have key roles regarding other systems in the body as well, such as regulation of apoptosis, calcium homeostasis, reactive oxygen production etc. Mitochondrial diseases are caused by impaired mitochondrial function, originating from mutations in either the mitochondrial DNA or the nuclear DNA. Epilepsy is a common symptom of mitochondrial disease, especially in children. The pathophysiology behind mitochondrial epilepsy is primarily based on ATP deficit, leading to a negative effect on a range of different nervous system related functions that in the end leads to seizures. The study aimed to investigate the effect on mitochondrial respiration of two commonly used antiepileptic drugs, namely valproic acid and Topiramate, and the anesthesic drug propofol, commonly used in case of refractory status epilepticus. The three drugs were titrated in different concentrations in a high-resolution respirometer from Oroboros Instruments (n=6). Propofol seemed especially inhibiting of mitochondrial function, and both propofol and topiramate had a significant decrease in mitochondrial respiration within the clinical concentrations. The result of the study supported research stating that propofol should be used with caution in patients with a mitochondrial disease, but further research should be done regarding all three drugs in order to draw definite conclusions.

Hudens mikrobiom utgör ett skydd mot patogener och domineras framförallt av grampositiva bakterier som Staphylococcus, där S. epidermidis är den mest förekommande arten. På huden är S. epidermidis en kommensal art och har en aktiv roll i att begränsa tillväxten och koloniseringen av patogener som S. aureus. S. epidermidis är en viktig art i att bibehålla stabiliteten av hudens mikrobiom och en reduktion av S. epidermidis innebär ökade risker för dysbios. S. aureus är en vanlig orsak till hudinfektioner och har upptäckts finnas i en hög utsträckning på patienter med atopisk dermatit samt hos diabetiker med risk för att utveckla fotsår. Syftet med denna studie var att optimera qPCR och undersöka om metoden kunde användas för att uppskatta förhållandet mellan S. aureus och S. epidermidis, vilket skulle kunna identifiera patienter med risk för hudsjukdomar. Tre primerpar undersöktes. Två var specifika mot S. aureus och en var specifik mot S. epidermidis. Flera tester genomfördes för att optimera qPCR och specificiteten av primerparen. Den annealingtemperatur och primerkoncentration som gav mest adekvata resultat användes vid den relativa kvantifieringen. Den utvecklade metoden kunde uppskatta förhållandet mellan S. aureus och S. epidermidis vid de lägre koncentrationerna (80–280 kopior/μl) bättre än vid de högre koncentrationerna (80 000–280 000 kopior/μl), men vidare optimering av metoden behövs.

Giardia is a parasite that causes disease in both humans and animals all around the world. Approximately 200 million people are infected each year. The parasite is spread mainly through contact with contaminated feces but can also be spread through food and water contaminated with infectious cysts. Symptoms that may occur during infection could be diarrhea and abdominal pain. Not all host organisms get symptoms and there are lots of asymptomatic carriers. The aim of this report was to compare and evaluate the performance of three different methods used to detect Giardia in dogs and cats. Today the gold standard is detecting cysts with immunofluorescence, which was one of the methods evaluated. One cyst visible was enough to say the test was positive. The other two methods were an ELISA and a rapid test, and both detected soluble Giardia antigen. For both methods, color development occurred in case of positive results. The ELISA wells were read spectrophotometrically at 450 nm. During this project, 294 tests from randomly chosen dogs and cats were analyzed. Of all these tests, 103 were analyzed with all three methods, whereas 191 tests were only analyzed with immunofluorescence and ELISA. When analyzing the results, the number of positive test results was very similar between the three methods and there was no statistically significant difference between them. Therefore, when price, time and results were taken into consideration, the method recommended was ELISA.

Multiple myeloma, or myeloma, is a bone marrow cancer which characterizes by uncontrolled proliferation of mutant plasma cells. It is a disease that claims many lives every year, mostly due to the absence of curative treatment. Finding a suitable treatment is therefor of great importance. One way to study different diseases is to use a gene editing method for knockdown or knockout of specific genes. The main aim of this project was to design guide RNAs, to be able to use CRISPR/Cas9 for knockout of the two genes BMPR1A and BMPR2 in different myeloma cell lines (KJON, INA-6 and IH-1). This, to be able to study the expression and function of these genes. Further aim of the project was to investigate potential SMAD activation by treatment with different bone morphogenetic proteins (BMPs). However, due to limited time this could not be carried through. Six guide RNAs were designed and ligated into pLentiCRISPRv2. Plasmid amplification was done by transformation of Escherichia coli. To check the quality of the plasmids, PCR, gel electrophoresis and Sanger sequencing was performed. The results from the gel electrophoresis showed that nine of the twelve samples for BMPR1A and seven of the thirteen samples for BMPR2, that were tested, were positive. The results from the Sanger sequencing confirmed that all guides that were tested (BMPR1A 3.2.3, BMPR1A 4.2.2, BMPR2 1.1.4 and BMPR2 2.1.2), were properly ligated into the plasmids. The main aim of the project was successfully accomplished, but additional work is needed for any further conclusions.

Levels of Immunoglobulin G (IgG)-subclasses are analyzed when patients have reoccurring infections and in order to follow the treatment of IgG4 related disease. The measured quantity of IgG-total can be within the reference interval even if the patient has a deficiency in one of the IgG-subclasses. Due to this Sundsvall’s hospital plans to begin analyzing IgG-subclasses. The aim was to verify the performance level of the analysis IgG-subclasses (IgG1-4) with Siemens Atellica NEPH 630. The method was verified by evaluating the method’s precision and by comparing the sum of IgG-subclasses with the quantity of IgG-total analyzed on Cobas c502. In addition, the reference intervals provided by Siemens were evaluated using samples from blood donors. The study evaluated whether there was a correlation between infection with SARS-CoV2 and a deficiency in IgG-subclasses. The verification began by performing quality control twice daily over a period of four weeks. The IgG-subclasses test was performed on blood donor samples with Siemens Atellica NEPH 630 for evaluation of the reference values. The coefficient of variation was less than 6 % for all four subclasses. The reference values for IgG1, IgG2, IgG3, and IgG4 are all in alliance with the reference values provided by Siemens. The sum of IgG-subclasses corresponded well with IgG-total with a correlation value (R) 0.82. No correlation was found between IgG deficiency and infection with SARS-CoV2. The validation of the analysis of IgG-subclasses was successful with quality measurements within the supplier’s intervals. No adjustments will be needed for the reference intervals.

Infertility is a problem that could be treated by using In Vitro Fertilization (IVF). This procedure starts by increasing estrogen levels and then retrieving oocytes. However, studies have showed an increased risk for venous thromboembolism (VTE) and lung embolism that is correlated with the rising levels of estrogen. Microvesicles, vesicles formed from the cell membrane, increases in number in connection with IVF and diseases such as thrombosis. These vesicles can transport proteins, one of them is fetuin-B. Fetuin-B plays a role in fertilizing the egg by inhibiting ovastacin, a protein that leads to hardening of zona pellucida. Phosphatidylserine, a component of the cell membrane with strong binding capacity to annexin V, has also been studied while investigating VTE. The aim of this project was to analyze the amount of fetuin-B, ovastacin and phosphatidylserine in women undergoing IVF. The study included 55 women where three blood samples were collected from them at different time points. The tests were analyzed using flow cytometry and fluorescent antibodies targeting the proteins in question. The results showed a significant increase in fetuin-B and ovastacin after the treatment where double positive microvesicles (fetuin-B+ ovastacin+) had the highest significant difference, a rise of 78% after the hormone increase. (Fetuin-B+ annexin V+) increased over time, 205% after increasing estrogen levels, while no difference was seen for (ovastacin+ annexin V+). This thesis suggests that fetuin-B is strongly related to fertility and coagulation in women undergoing IVF. The protein has also a noticeable relationship to ovastacin. Most importantly, (fetuin-B+ ovastacin+) was suggested to be a better marker for women’s fertility than the other analyzed parameters.

Meticillinresistent Staphylococcus aureus (MRSA) tillhör multiresistenta bakterier genom mecA-genen vars produkt orsakar resistens mot β-laktamantibiotika. Screening av MRSA i sjukvården sker ofta med kromogena medier som CHROMagar MRSA (C-MRSA). Målet med screeningen är att minska risk för smittspridning när ökad risk för bärarskap av MRSA kan förväntas. Det gäller exempelvis efter utlandsresa till land med hög MRSA-förekomst eller vid arbete där MRSA finns. Den nuvarande MRSA-screeningmetoden baseras på anrikning i en selektiv buljong innan odling på C-MRSA och efterföljande artidentifiering, genotypning samt resistensbestämning. Examensarbetets syfte var att utvärdera en metod för detektion av MRSA som utesluter den nuvarande selektiva anrikningbuljongen för att minska det laborativa arbetet och förkorta svarstid och därmed öka kostnadseffektiviteten. Godkänt resultat skulle vara samma eller ökad sensitivitet och specificitet jämfört med ordinarie metod. Metoder som användes var laboratoriets ordinarie metod för MRSA-screening samt en direkt metod utan anrikningsbuljong där prover från 318 konsekutiva kliniska screeningprover analyserades. Huvudsakligen var provtyperna från svalg, näsa och perineum. Ytterligare användes odling av MRSA i mono- och multikultur där provmaterialet var tre referensstammar respektive 50 kliniska MRSA-isolat. Den direkta metoden visade en lägre sensitivitet jämfört med ordinarie metod då endast 42,1% detekterades. Känsligheten var ökad efter två dygns inkubering (80%) och specificiteten var 100% oberoende inkuberingstid. MRSA-odlingen i både mono- och multikultur på C-MRSA uppvisade högst sensitivitet vid högre koncentrationer MRSA och längre inkubationstid jämfört med ordinarie metod. Baserat från resultaten från arbetet bör anrikningsbuljongen inte exkluderas utan vidare justering av metoden, genom exempelvis förändring av inkubationstid eller provvolym på C-MRSA.

The manual differential count of leukocytes is a common analysis in the hematological laboratory. It is used for morphological assessment of the blood cells and can get valuable information according to diagnosis of hematological diseases. The microscopy assessment is dependent of a good staining result of the blood smear in order to get the best conditions to differentiate the different cell types and detect morphological or pathological findings.The aim of this study was to determine if it was possible to change the staining method without any compromises to the quality of the staining results. For this study 25 whole blood samples were collected, and blood smears was made and stained with four different staining protocols, including the one currently used in the routine practice at laboratoriemedicin Sundsvall. The samples were examined by three biomedical scientists and the staining quality of the cells was graded on a four-point scale. The statistical results with Friedmans and Wilcoxons signed-rank test showed differences between the methods on the nuclear and cytoplasm of lymphocytes and the nuclear of monocytes and neutrophils. The recommended staining protocol from the manufacturer was the method that had highest frequency of statistically significant differences compared to the other methods for those cell types. The differences were in favour for the other methods, and the current method showed the best performance. In conclusion it’s not recommended to continue the study with the manufacturer´s staining protocol, but its valuable to continue compare the best performed staining protocols.