Paired box (PAX) gener kodar för en familj av nio stycken PAX transkriptionsfaktorer. PAX transkriptionsfaktorer är uppbyggda av en N-terminal DNA-bindande domän som utgörs av 128 aminosyror, en oktapeptid, och en C-terminal DNA-bindande domän. Studier har visat att PAX8 har en betydande roll vid utvecklingen av olika typer av tumörer. MRQ-50 är en antikropp som används inom Region Skåne för att påvisa PAX8 uttryck vid histopatologisk diagnostik. MRQ-50 riktas mot N-terminalen av PAX8 som är homolog med N-terminalen av PAX5. Detta kan leda till en korsreaktion i B-lymfocyter samt neoplasier, vilket inte är optimalt för diagnostik. Av den anledningen fortsätter forskning för att hitta en ny klon som kan ersätta MRQ-50. EP331 är en antikropp som är anpassad för detektion av det nukleära antigenet PAX8 och korresponderar till C-terminalen av human PAX8 protein. Syftet med arbetet är att optimera ett immunhistokemiskt färgningsprotokoll för detektion av PAX8 med hjälp av anti-PAX8 antikroppen EP331 och undersöka om godkända resultat kan erhållas. Detta gjordes för att ta reda på om det går att optimera ett protokoll med EP331, och om det erhålls lämpligare resultat än MRQ-50. Optimeringen gjordes på kontrollvävnader njure, tuba/äggledare och appendix och testades slutligen på patientprov med tumörvävnad. För protokolloptimering testades heat induced epitope retrieval, inkuberings tid av EP331, amplifiering, Ultra Wash i olika testomgångar. Resultatet visade att kärnor i epitelceller i proximala-, distala-njurtubuli och parietala epitelceller som klär Bowmans kapsel infärgades måttligt till starkt. De sekretoriska epitelen i tuba infärgades med stark intensitet och cilierade epitel måttligt. Infärgningen på patientprover visar godkänd specificitet, med en generell måttlig till stark intensitet. Med fler forskningsstudier av EP331 är det möjligt att primärantikroppen kan ersätta MRQ-50.

Vitamin K is a fat soluble compound which is synthesized by the gut microbiota and produced in many tissues within the body. Considering its role in the liver as a cofactor for gamma carboxylase enzymes, treatment of dark circles and pigments under the eye among others. It is clear that is some circumstances vitamin K has to cross biological barriers, particularly, when the vitamin is produced by microbiota in the intestine or applied topically on skin. Thus it is important to develop methods that allow studies of vitamin K permeability through the skin including its participation in redox reactions and transdermal permeability. Taking into account that transdermal permeability is strongly limited for high molecular weight compounds, i.e., compounds with higher than 500Da, the study was conducted with vitamin K of  lower molecular weight. Specifically vitamin K4 model, i.e., 1,4-dihydroxy-2 naphthoic acid, with molecular weight of 204g/mol. Vitamin K4 is suitable for this kind of study , because it can work as reducing (antioxidant) compound as well as has relatively beneficial physicochemical characteristics for transdermal permeability. Permeability studies were conducted with skin covered oxygen electrode and franz diffusion cell. Data from measurements were analyzed to estimate diffusion coefficients, apparent Michaelis-Menten constants and flux of a vitamin K4 model whilst contribution of different permeability pathways was determined theoretically.

Ensuring correct analytical responses is of the utmost importance in laboratory workand interferences with analyses can give incorrect results. Work to improve the precision of the analyses is an ongoing process. An interference in the analysis of Interleukin-6 (IL-6), a cytokine that reflects inflammatory processes in the body, is biotin. Biotin interferes with EnzymeLinkedImmunoSorbentAssay (ELISA) analysis by binding in to streptavidin thereby causing falsely low analytical responses. An attempt to reduce that interference is to raise the threshold for biotin by introducing a new reagent. The aim of this project was to introduce a new reagent with elevated biotin interference threshold and carry out a precision measurement as well as a patient comparison. In addition, a sustainability study was carried out to investigate possible changes in IL-6 concentration when samples are stored in room temperature with open or closed vessels. Control material was analysed during three consecutive days. The patient comparison was performed by analysing 20 patient samples with both the older reagent and the new one. The sustainability study was preformed last and here were 7 patient samples used to perform this part of the study. The control material was analysed with good results, a total coefficient of variation (CV%) was calculated at 1.6% for the lower control and 3.0% for the higher one. The comparison showed good correlation with only a minor negative bias for the new reagent. The sustainability study showed, in line with what the supplier has indicated, a sample shelf life of 6 h at room temperature without sealing. Interesting to mention is that samples over 50 pg/mL showed a shelf life more than 24 h at room temperature without sealing. The decision was made by the medically responsible doctor that the reagent was approved for use in the laboratory.

Legionella pneumophila is a bacterium which can be found in fresh water and causes Legionnaires’ disease, which can be deadly for humans depending on the condition of the infected individual. The bacterium is a gram-negative rod and can withstand severe conditions such as high temperature. Therefore, various treatments including heat and acid treatment are performed on the water to inhibit interfering microorganisms. However, to examine a larger volume of water, the water needs to pass through a filter, which can be very time consuming, and there are various variables that have a negative impact on the filtration speed. The aim of this study was to examine these variables and find the fastest setup for detection of L. pneumophila. To filtrate the water, a manifold with funnels, where you put the water, is used, and the manifold is connected to a pump. Under the funnels, steel frits are placed, and the filter is placed on the steel frits. To examine the fastest setup, different manifolds, pumps, filters, and settings were investigated by timing the water running through in the different settings. A new way of sterilization, that does not damage the steel frits was tested, and the recovery of bacteria was examined on the filters with the top filtration speed. In conclusion, the most efficient setup is the Cyclopore (GE Healthcare Life Sciences) filter, the pump from KNF and the manifold MBS1 (Whatman), and the new way of sterilizing should be used to reduce the damage of the steel frits.

Blood transfusion is a very common procedure. Before a blood transfusion it is important to find compatible blood for the patient. If the blood is incompatible with that of the patient a transfusion reaction may occur, which can be mortal. It is also important to avoid alloimmunisation. Alloimmunisation occurs when antibodies are produced against a specific antigen. These antibodies are called irregular antibodies and they can be produced after a transfusion, pregnancy, or transplantation. Alloimmunisation makes it harder to find compatible blood for patients in the future and it is a major concern for patients who require blood transfusions repeatedly. By phenotyping erythrocyte antigens, it is easier to find compatible blood for patients before a blood transfusion. Until now, a manual method has been used for phenotyping erythrocyte antigen at the Sundsvall County hospital and an automatization of this method was desired. For this study, 99 anonymised blood doner tests were used. The erythrocyte antigens M, Jka, Jkb, Fya, Fyb, S and s were phenotyped both manually and automated with ORTHO Vision Analyzer. A Clopper Pearson test was used to evaluate the accordance between the methods. A comparison was also made in regard totime and cost. The results showed a good accordance between the methods. The automated procedure using ORTHO Vision has several advantages over the manual procedure using gel cards. The risk of errors is reduced, there are fewer manual steps, it is faster and the personnel can do other tasks while the instrument is processing the tests.

Tuberkulos orsakad av Mycobacterium tuberculosis, är en av de ledande dödsorsakerna globalt sett enligt världshälsoorganisationen (WHO). M. tuberculosis ingår i Mycobacterium tuberculosis komplex (MTBC) där bland annat Mycobacterium bovis, M. bovis BCG (Bacillus Calmette-Guérin) och Mycobacterium africanum också ingår. Förutom MTBC ingår även icke-tuberkulösa mykobakterier (NTM) i genuset Mycobacterium. Fall av M. tuberculosis-infektioner rapporteras vanligtvis i utvecklingsländer medan NTM-infektioner är vanligare i västvärlden. Idag använder man sig av sputumprover för att diagnostisera vid misstanke om tuberkulos men bakterierna detekteras också från vätskor, sekret, exkret och vävnader. Odling är den vanligaste diagnostikmetoden men även mikroskopi används och molekylärbiologiska tekniker som realtids-PCR (polymerase chain reaction) vilken kan amplifiera och detektera M. tuberculosis för en snabb och säker analys. Syftet med denna studie var att utveckla en realtids-PCR-metod för att detektera NTM och särskilja dem från MTBC i formalin-fixerade paraffininbäddade vävnadsprover (FFPE). Fyra tidigare publicerade primerpar, senX3, MTC, IS6110 och IS1081, specifika för MTBC utvärderades med realtids-PCR parallellt med det kommersiella kittet AnyplexTM MTB/NTMe real-time detection med dual priming oligonukleotider (DPO). Resultaten visade att de fyra primerparen inte var specifika nog för att kunna särskilja MTBC- och NTM-stammar utan PCR-produkter av olika storlek erhölls även från NTM-stammar. Anyplex-kittet visade däremot hög specificitet och kunde gruppera samtliga testade stammar korrekt som MTBC eller NTM. Samma goda resultat erhölls vid analys av DNA-extrakt från 21 FFPE-prover. Sammanfattningsvis visar denna studie att AnyplexTM MTB/NTMe-kittet uppfyller kraven för att särskilja MTBC från NTM från FFPE-material vilket inte realtids-PCR med primers senX3, MTC, IS6110 och IS1081 gör. / Tuberculosis mainly caused by Mycobacterium tuberculosis, is one of the leading causes of death globally according to the World Health Organization (WHO). M. tuberculosis is included in the Mycobacterium tuberculosis complex (MTBC) which contains also Mycobacterium bovis, M. bovis BCG (Bacillus Calmette-Guérin) and Mycobacterium africanum. In addition to MTBC, non-tuberculous mycobacteria (NTM) are also included in the genus Mycobacterium. Cases of M. tuberculosis infections are usually reported in developing countries while NTM infections are more common in the western world. Today, sputum samples are used to diagnose tuberculosis but the bacteria can also be detected from analysis of fluids, secretions, excreta and tissues. Cultivation is a common diagnostic method but also use of microscopy and molecular biology techniques such as real-time PCR (polymerase chain reaction) which can amplify and detect M. tuberculosis for a rapid and specific analysis. The purpose of this study was to develop a real-time PCR method for detecting MTBC and distinguishing them from NTM in formalin-fixed paraffin-embedded tissue samples (FFPE). AnyplexTM MTB/NTMe real-time detection kit was compared with previously published primers senX3, MTC, IS6110 and IS1081. 21 FFPE samples were processed by DNA extraction for further analysis with real-time PCR for amplification and detection and fragment analysis for size determination. The results show that the kit meets the requirements for distinguishing MTBC from NTM both with purified strains and samples from FFPE material while the different primer combinations senX3, MTC, IS6110 and IS1081 don't. To conclude, the AnyplexTM MTB/NTMe kit can be used for diagnostics of MTBC and NTM from FFPE samples.

No description available.

Xylen är ett starkt toxiskt ämne, som trots att dess hälsofarliga egenskaper länge varit kända, fortsatt har använts som klarningsmedel i den histologiska processen. Syftet med den här studien var att undersöka om det betydligt mindre hälsofarliga lösningsmedlet, Histolab Clear, kunde substituera xylen som klarningsmedel, utan att kompromissa med kvalitén på de histologiska glas som laboratoriet lämnar ut för granskning. Två serier med samma uppsättning vävnadsbitar preparerades och dehydrerades med var sitt klarningsmedel. Klossarna granskades och bedömdes utifrån snittbarhet, färgkvalité och snittkvalité. Vid jämförelse av de två serierna visades inga betydande skillnader inom något av bedömningsområdena. Att resultatet är likvärdigt inom alla tre bedömningsområden stärker slutsatsen att Histolab Clear är ett bra alternativ som xylensubstitut. Däremot visar studien även att klossarnas snittbarhet tydligt påverkas av tjockleken på den utskurna vävnadsbiten. Tjocka vävnadsskivor ger en sämre snittbarhet vilket kan påverka snittets kvalité. För att säkra snitt av hög kvalité med välbevarad morfologi, bör det vid utskärningen inte tas vävnadsskivor som är tjockare än fyra millimeter. / Xylene is a highly toxic substance that, despite its long-known hazardous characteristics, still has been used as a clearing agent in the histological process. The purpose of this study was to investigate whether the significantly less toxic solvent, Histolab Clear, could substitute xylene as a clearing agent, without compromising the quality of the histological slides released by the laboratory for examination. Two series with the same set of tissue pieces were prepared and dehydrated with a clarifying agent each. The blocks were examined and assessed based on sectionability, quality of color and quality of section. When comparing the two series, no significant differences were shown in any of the assessment areas. The fact that the result is equivalent in all three assessment areas reinforces the conclusion that Histolab Clear is a good alternative to substitute xylene. However, the study also shows that the sectionability of the blocks is clearly affected by the thickness of the tissue. Thick tissue slices result in poorer sectionability, which can affect the quality of the slide. To ensure high quality sections, with well-preserved morphology, tissue slices that are thicker than four millimeters should not be cut during grossing.

Laing distal myopathy is inherited in an autosomal dominant manner usually before the age of five that initially involves the dorsiflexion in the ankles’ and in big toes to the finger extensors. Weakness of the flexor muscles in the neck is seen in most affected individuals and mild facial weakness is also often present. Hypertrophic or dilated cardiomyopathy, starting at birth to respectively second or third decade of life, is the symptom in the affected humans.This study performed on Drosophila melanogaster, has evaluated whether feeding MuRF1 enzyme (which has a similar role as ABBA enzyme) to Drosophila larvae, in different concentrations, will have a positive effect on the larvae’s muscular abilities through an analysis of their manifestation, the distance they manage to crawl and the time it takes for them to turn from a ventral up to dorsal up position.The result show no significant impact on larvae ability to turn or crawl between different groups fed with MuRF1 enzyme, nor between the two control groups, wild larvae and mutated larvae. Other studies have proven that there is a significant difference in muscular ability between wild and mutated larvae, so explanations to why this study did not manage to replicate these results were evaluated. The study found that how many days has passed since hatching has a significant impact on performance of turning and crawling for wild larvae that are not treated with enzyme.There are a number of improvement suggestions to the experimental design and the methodology to enable a proper evaluation of the research aim of this thesis. Future research on the topic should implement these and redo the experiments and measurements of this study. In addition, the quantity of larvae that reaches pupa stage should be captured to evaluate whether the MuRF1 enzyme has a positive impact on mutated larvae reaching pupation stage. The most important parts of the improvement proposals to measure the ability of larvae when they are about the same age, as this was proven with statistical significance to have an impact on crawling and turning.

ANA-buljongen är utvecklad för att kunna anrika både aeroba och anaeroba bakterier vid inkubering i aerob miljö. Odlingsbuljongen innehåller antioxidanter som skyddar anaeroberna mot reaktiva syreradikaler, vilket främjar anaerob anrikning. Syftet med arbetet var att utvärdera om ANA-buljongen kan ersätta de två befintliga odlingsbuljongerna som idag används vid anrikning av bakterier på avdelningen för klinisk mikrobiologi, Kronoberg Blekingesjukhuset. Elva bakteriearter och fyra blandfloror användes i utvärderingen av ANA-buljongen. En mikroliter (µl) inokulat som innehöll ca 100 colony forming units tillsattes till ANA-buljong samt aeroba buljongen Brain Heart Infusion (BHI) eller den anaerobbuljongen Fastidious Anaerobe Broth (FAB). Prov från buljongerna odlades ut efter två, fyra och sju dygns inkubering. Tillväxtsgrad vid olika inkubationstider jämfördes mellan ANA-buljongen och BHI-buljongen/FAB. ANA-buljongen utvärderades även för odling av kliniska prov. De kliniska proven odlades ut på agarplattor samt inokulerades till ANA-, BHI-buljong och FAB. Vid utodling från buljongerna jämfördes fynden från BHI-buljongen och FAB med fynden från ANA-buljongen. Tillväxten verkade vara snabbare i ANA-buljongen jämfört med BHI-buljong och FAB. Vissa arter, exempelvis Cutibacterium acnes, uppvisade en betydligt snabbare tillväxt i ANA-buljongen. Vissa bakteriearter tillväxte men överlevde inte längre inkubationstider och kunde efter dessa inte detekteras. Anrikningen av blandfloror i ANA-buljongen resulterade i överväxt av en art vilken konkurrerade ut de övriga arterna. Detta fenomen uppstod inte i FAB. Att ersätta de två kliniska buljongerna med ANA-buljongen hade varit fördelaktigt både praktiskt, ekonomiskt och ur ett hållbarhetsperspektiv. Eftersom ANA-buljongen enbart visade sig användbar vid odling av renkulturer men inte vid odling av blandfloror är den inte optimal för klinisk diagnostik. Men den skulle eventuellt kunna nyttjas vid ortopediska Kamme-Lindberg-odlingar då det oftast är fråga om renkulturer. / ANA-broth is developed to enrich both aerobic and anaerobic bacteria by incubation in an aerobic environment. The broth contains antioxidants that protect anaerobic bacteria from reactive oxygen species and make it possible for anaerobic bacteria to grow in an oxygenated environment. The purpose of the study was to evaluate the ANA-broth and see if the broth could replace the two broths that are being used clinically today.  Eleven bacterial species and four mixed floras were used to evaluate the ANA-broth. One microliter (µl) inoculum with about 100 colony forming units was used to inoculate ANA-broth, Brain Heart Infusion (BHI) broth and Fastidious Anaerobe Broth (FAB), respectively. Samples from the broths were then used to inoculate agar plates after two, four and seven days of incubation, to evaluate the difference in enrichment effectiveness at different incubation times in relation to the clinically used broths. The ANA-broth was also evaluated by using clinical samples from patients. The findings from the ANA-broth were compared with the findings from the BHI-broth and FAB. The growth in the ANA-broth was quicker compared to the BHI-broth and FAB. Some bacterial species grew but became non-viable in the ANA-broth after a longer period of incubation. Bacterial species within mixed floras were not able to grow in coexistence in the ANA-broth. One bacterial species took over and competed out the residual bacteria. This phenomenon did not occur in FAB. If the ANA-broth could replace the broths now used clinically then that would be advantageous both in terms of practicality, economy and from a sustainability perspective. Though, the ANA-broth is not optimal for clinical use considering that only one bacterial species within a mixed flora was able to grow. As the broth has greater potential for cultures containing only one bacterial species it could possibly be suited for orthopaedic Kamme-Lindberg cultivation.