Cytopathology plays a significant role in cancer diagnostics. One of the ancillary methods used for prognostication is immunocytochemistry (ICC). Formalin pre-fixed tissue blocks are often used as validated immunohistochemical controls for cytology samples. However, those are not treated as clinical cytology specimen, which are furthermore commonly fixed in alcohol. As cell structure might be affected by added fixatives and block materials, the aim of this project was to compare the effects of fixation in formalin and PreservCyt®, a methanol-based fixative, and the two cell block materials, plasma-thrombin and agar, on the ICC results. The material used in the study consisted of several cell lines provided by the Technological University Dublin, which were chosen to be representative of the clinical cytology samples. The cells were fixed and put into cell blocks for each cell type or as a mixture of different cell types – a composite cell block. All cell blocks were then stained with suitable antibodies. Composite cell blocks could be tested as simultaneously positive and negative ICC control material. The results showed that in general, PreservCyt®-fixed cell blocks preserved sharper cellular morphology compared to those fixed in formalin. However, PreservCyt® also gave unexpected negative results for some antibodies, including some nuclear markers. Plasma-thrombin cell block method showed to have more advantages than the agar method.This study suggests, that with further optimization and validation of both fixative methods and cell blocks, composite cell block has good potential to be used as source of control blocks for cytology samples.

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Tripeptidyl peptidse II (TPPII) is an enzyme forming a complex of 4,5 MDa. It is located in the cytosol and participates in protein turnover where it degrades oligopeptides to smaller peptides and tripeptides. Moreover, some of its products might take part in antigen presentation through MHC-class 1. High expression of the enzyme is believed to correlate with tumor progression. A decreased concentration seems to contribute to increased susceptibility to infection. The aim of this project was to develop an enzyme linked immusorbent assay (ELISA) specific for TPPII and compare it to two commercial kits. TPPII was detected through western blot and activity assay to ensure which samples were containing the enzyme. Samples with purified enzyme from different species, erythrocyte lysate and plasma were used to develop an indirect ELISA as well as compare all the assays and evaluate which one performed the best. The commercial kits were based on sandwich and competitive techniques. It was observed that the developed ELISA was able to detect the purified enzyme of different concentrations whereas the commercial kits could not. On the other hand, the commercial kits were able to generate their own standard curve which leads to suspicion that these might be non-specific to TPPII. Though, all assays did detect the enzyme in erythrocyte lysate. This study presents that it is possible to develop an ELISA specific for TPPII using an indirect technique which also performed better than commercial kits.

Fungals are opportunistic pathogens that can cause invasive infections primarily among immunocompromised individuals. Traditionally, fungi have been identified by culturing on agar plates and using morphological criteria. Molecular detection methods such as polymerase chain reaction (PCR) and sequencing is faster and superior at identifying fungi at species level compared to culturing and microscopic examination. The aim of this project was to identify fungi at the species level by sequencing the internal transcribed spacer (ITS) region of different control strains and patient samples. This was accomplished using a nested PCR followed by Nanopore sequencing. Two different workflows using different databases were created. One workflow created a consensus sequence which was followed by a BLAST search. The other workflow used an Emu program and mapped reads against the UNITE database. The result was compared to identification previously achieved. Amplification was successful in 28 out of 30 positive samples. The BLAST workflow managed to identify nine out of eleven samples, but was difficult to interpret. The Emu workflow was only concordant with previous identification in 20 out of 28 species in sequenced samples and failed to identify 4 previously identified fungi at the species level, but was easier to interpret and could identify several species from a mixed culture. Amplifying and sequencing of the ITS region can in several cases provide accurate identification, but the method of extraction, choice of DNA polymerase and choice of database need to be considered for successful amplification and accurate species identification before implementation as a diagnostic method.

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.

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.

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.

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.

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.

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.