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SMARCA4 är en gen som kodar för ett protein vid namn Brahma-related gene 1 (BRG1). BRG1 utgör en väsentlig katalytisk subenhet av SWI/SNF-komplexet, vilket är ett proteinkomplex utvecklat för kromatinremodellering. Att modulera kromatinstrukturen bidrar till att viktiga funktioner, såsom transkription och celldifferentiering, kan utföras. En mutation i SMARCA4-genen kan ge upphov till en lungtumör med benämningen Thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT), vilken kännetecknas av en förlust av uttrycket BRG1 i de maligna cellerna. Då tumören kan variera i morfologiskt utseende och i många fall påminna om andra tumörtyper, kan tillämpning av immunhistokemiska metoder vara till stor hjälp vid den medicinska diagnosticeringen. Syftet med denna studie var att vidareutveckla ett befintligt protokoll för att uppnå en optimal immunhistokemisk infärgning av SMARCA4 (BRG1), för att diagnostiskt kunna differentiera mellan olika lungtumörer. För detta ändamål testades flera olika infärgningsprotokoll och kontrollvävnader, samt två antikroppar av olika kloner från företagen Abcam och Santa Cruz. Infärgning med Abcam-antikroppen (klon EPNCIR111A) uppvisade intensiva och specifika resultat, medan Santa Cruz-antikroppen (klon G-7) gav upphov till negativa infärgningar, trots flera försök till optimering. I samråd med en patolog fastställdes det infärgningsprotokoll som gav bäst resultat, vilket inkluderade en förbehandling bestående av buffert CC1 (64 min, 95° C), antikroppsinkubation med Abcam-antikroppen (32 min, 36° C, spädning 1:100), detektion med ultraView Universal DAB Detection Kit, samt kontrastfärgning med hematoxylin (8 min) och blåning (4 min). Denna immunfärgning kommer att införas som ny metod och användas inom den kliniska verksamheten. Däremot kommer metoden att fortsätta prövas under en tid framöver, för att helt säkerställa dess pålitlighet och kunna valideras inför framtida bruk.

Diabetes mellitus is one of the most common diseases worldwide. The continued prevalence increase, together with the complexity of the disease with severe long-term complications makes efficient diagnostical and monitorial methods very important. The determination of glycated hemoglobin, HbA1c, plays an important role in both, underlying the importance of high-quality analytical instruments. Whenever a new instrument is introduced into routine clinical use an evaluation is needed. This study evaluated the agreement between the new Tosoh G11 HbA1c high performance liquid chromatography system as a replacement for the Bio-Rad D-100 at the Chromatographic Department of the Institute of Laboratory Medicine, Faculty of Medicine, University of Debrecen. In total, 66 samples were analyzed using both analyzers and a Bland-Altman comparison was performed together with a correlation study. Additionally, a linearity check, optimal error and routine error testing was carried out specifically for the Tosoh G11. The results showed high agreement between measurements, r=0.9975 (p<0.001) for HbA1c mmol/mol and r=0.9971 (p<0.001) for HbA1c %. High repeatability and accuracy were observed of the Tosoh G11 with the highest variation coefficient of 1.50 % and linearity r =0.9999. The conducted study supports the replacement of the Bio-Rad D-100 to the Tosoh G11. / Diabetes mellitus är en av de vanligaste världsomfattande sjukdomarna. Fortsatt prevalensökning, tillsammans med den komplexa sjukdomsbilden innefattande allvarliga långsiktiga komplikationer gör effektiva diagnostiska och övervakande metoder väldigt viktiga. Bestämning av glykerat hemoglobin, HbA1c, spelar en viktig roll i båda, vilket kräver att analysinstrument av hög kvalitet finns. När ett nytt instrument introduceras inom kliniska rutinanalyser krävs utvärdering. Denna studie utvärderade överrensstämmelsen mellan det nya Tosoh G11 HbA1c högupplösande vätskekromatografisystemet som en ersättning för Bio-Rad D-100 på den kromatografiska enheten av laboratoriemedicin på Debrecens universitet. Totalt analyserades 66 prov med respektive instrument och Bland-Altman jämförelse genomfördes tillsammans med en korrelationsstudie. Dessutom utfördes ett linjäritetstest, optimal- och rutinfeltest specifikt för Tosoh G11. Resultaten visade en hög överrensstämmelse mellan mätningarna, r=9975 (p<0.001) för HbA1c mmol/mol och r =9971 (p<0.001) för HbA1c %. Tosoh G11 visade hög repeterbarhet och noggrannhet, med den högsta variationskoefficient på endast 1.50 % och en linjäritet på r=0.9999. Den genomförda studien stödjer Tosoh G11 som ersättning för Bio-Rad D-100.

Järn är ett livsviktigt spårämne som vid både brist och överskott kan leda till sjukdom. Järnmetabolismen är komplex och det finns flera biomarkörer som används för att utvärdera järnstatus, till exempel transferrinbundet järn. På grund av detta är det avgörande att den analytiska säkerheten för järnanalys bibehålls. Syftet med studien var att verifiera ett nytt reagens, Iron2 för att ersätta det nuvarande reagenset, Iron för kvantitativ järnanalys på Abbott® Alinity ci-series. Den beräknade precisionen förväntades vara jämförbar med tillverkarens specifikationer, samt visa en stark korrelation med nuvarande reagens. Inom- och mellanserieprecisionen bedömdes genom upprepad analys av kontrollmaterial. Metodjämförelsen genomfördes genom att jämföra resultaten från 35 patientprover analyserade med båda reagensen. Precisionen vid inomserieprecisionsskattningen hade en beräknad variationskoefficient (CV) på 0,28 % för lågnivåkontrollen och 0,25 % för högnivåkontrollen. För den mellanliggande precisionen var resulterande CV 1,21 % respektive 0,55 %. Korrelationsanalysen visade ett linjärt samband, men en tendens för Iron2 att mäta högre värden vid högre analytkoncentrationer. Sammanfattningsvis fann studien att precisionen för Iron2 var bättre eller jämförbar med tillverkarens specifikationer och nuvarande reagens. Det fanns en stark linjär korrelation mellan reagensen utan någon signifikant skillnad mellan resultaten. Iron2 bedömdes som lämplig för implementering. / Iron is a vital trace element that can lead to issues both in deficiency and excess. The metabolism of iron is complex and there are multiple biomarkers used to evaluate the iron status, for example, quantification of transferrin-bound iron. Because of this, it is imperative that the analytical certainty of iron analysis is maintained. The study aimed to verify a new reagent, Iron2 to replace the current reagent, Iron for quantitative iron analysis on Abbott® Alinity ci-series. The calculated precision was expected to be comparable to the manufacturer’s specifications and show a strong correlation with the comparison method. Within-series and intermediate precision were assessed by repeated analysis of control material. The method comparison was carried out by comparing the results of 35 patient samples analysed with both reagents. The within-run precision had a calculated coefficient of variation (CV) of 0,28% for the low-level control and 0,25% for the high-level control. For the intermediate precision, the resulting CVs were 1,21% and 0,55% respectively. The correlation analysis showed a linear relationship, but a tendency for Iron2 to measure higher values at higher analyte concentrations. In conclusion, the study found that the precision of Iron2 was better or comparable to the manufacturer's specifications and comparison method. There was a strong linear correlation between the reagents with no significant difference between the results. The reagent was deemed satisfactory for implementation.

Background: Platelets are the smallest cells in the blood and are involved in the wound healing process. Platelet transfusion is an important part of treating illnesses such as thrombocytopenia and to prevent and stop major bleedings. Platelets for transfusion are received after processing of whole blood through automated or semi-automated systems and pooling of interim platelet units received after the processing. A limit is set for the minimum total estimated platelet yield in a pool to ensure that the final product consists of enough platelets. Aim: The aim of this study was to evaluate the platelet yield index received from Reveos automatic blood processing system by comparing it to the fluorescent flow cytometry method on Sysmex cell counter.  Material and method: Whole blood from 147 donors were processed in the Reveos automated blood processing system into the separate components. All the interim platelet units received a platelet yield index from Reveos. A sample from each interim platelet unit was collected and analyzed on Sysmex in the channel for fluorescent flow cytometry. Result: The difference between the platelet count from the two methods was 45 ±26 x109/unit (p<0,001). Platelet yield index from Reveos was 67 ±17 x109/unit and the platelet count from Sysmex was 112 ±35 x109/unit. The r2 from the scattergram was 0,49. Conclusion: The platelet yield index from Reveos underestimates the platelet yield compared to fluorescent flow cytometry from Sysmex. The limit for the total platelet yield in a pool of interim platelet units could therefore be decreased.

Introduction: Effusion cytology can be performed for the purpose of diagnosis, treatment, and prognosis of malignant disease. A common analysis of effusion cytology samples is the May Grunwald Giemsa stain.    Aim: The aim of the study was to compare May Grunwald Giemsa stain and May Grunwald Giemsa Quick Stain in order to determine the best quality stain and suggest ways to improve the current staining protocol.     Materials and Methods: The methods used in this study are the routine laboratory’s standard procedures for  May-Grunwald Giemsa stain and May-Grunwald Giemsa Quick Stain but with adapted washing steps that investigates the effect of tap water, distilled water, and phosphate buffer on stain quality. Two pleural effusion samples were stained in the initial experiment and two pleural effusions and one ascites sample in the second experiment.    Results and Conclusion: All samples gave a greater score when stained with May-Grunwald Giemsa Quick Stain compared to traditional May-Grunwald Giemsa stain. For the traditional May-Grunwald Giemsa, the use of any of the three phosphate buffers scores higher than the routine washing where tap water is used. In conclusion, it would be of benefit to further investigate and implement phosphate buffer in traditional staining or proceed with the May-Grunwald Quick Stain for all pleural and ascites effusions.

Pollination by insects is of great importance for the global food production. There is a specific need for pollination by bees in greenhouses and tunnel cultivations to increase the quantity, quality and market value of the crops. Imported bee colonies from central Europe are used for pollination of Swedish crops and have a great economic importance but are also a threat to wild Swedish bees by posing a risk of pathogen transmission between the bee species. The aim of this study was to investigate how imported bees affect the prevalence of pathogens amongst wild bees.  Analysis was performed on 236 wild bees collected in near proximity to tunnel cultivation, greenhouse cultivation and collected from two control landscapes. The abdomen of the bees was used to extract RNA/DNA for further detection and quantification of ten pathogens using qPCR. The proportion of infected bees within each group was calculated based on the results from the qPCR analysis. A two-proportion z-test was used to determine whether the difference in pathogen prevalence between the four groups was of statistically significant at α = 0.05. The results show that there was no significant difference when comparing the presence of all pathogens between bees in the test groups and the bees in the control groups (p= 0,29- 0,33). However, the prevalence of three viruses was significantly higher among bees collected in the near proximity of a greenhouse compared with bees collected from the near proximity of a tunnel cultivation (p< 0,003). For Slow bee paralysis virus the prevalence was 2,5 times higher and for Deformed wing virus and Black queen cell virus the prevalence was 3,5 and 1,3 times higher among bees collected near a greenhouses compared to near a tunnel cultivation.

Syftet med arbetet var att försöka utveckla en metod för att isolera lipoprotein(a), ett lipoprotein som korrelerar i hög grad till kardiovaskulär sjukdom, från plasma. Det har varit problematiskt att på ett enkelt sätt separera lipoprotein(a) från LDL på grund av dess strukturella likheter. Som inspiration användes en artikel där separationen utfördes med anjonbyteskromatografi-HPLC (high performance liquid chromatography). Målet var att applicera metoden på ett FPLC-system (fast protein liquid chromatography) med en gelfiltreringskolonn och en anjonbyteskolonn där det kaotropiska saltet natriumperklorat användes som elueringsmedel under anjonbyteskromatografin. Resultaten kontrollerades med DLS (dynamic light scattering), SDS-PAGE och Western blot med antikroppar riktade mot lipoprotein(a). Resultaten visade på goda möjligheter till en bra separation vid fortsatta studier.

Background: Neuronal plasticity and regeneration in cancer are understudied aspects of cancer research. Studies have shown that neurogenesis and axonogenesis are associated with cancer progression and metastatic potential. Purpose: The purpose of this project was to optimize an immunofluorescence stain to observe nerve development and regeneration in cancer tissue, with the use of antibodies against neurofilament light chain (Nf-L), growth associated protein 43 (gap-43), and doublecortin X (DCX). Material and method: Staining optimization included evaluations of antigen retrieval, tissue permeabilization, antibody dilution, and duration of primary antibody incubation. The analyses were tested on colorectal- and lung cancer tissues. Results: The detection of Nf-L was not successful in any combination of the analyses or on ether tissue. The staining Gap-43 showed the best results using antigen retrieval with pepsin in HCl and primary antibody dilution 1:500 combined with incubation overnight at 4 °C. Staining for DCX needs more evaluation due to non-specific binding in lung cancer tissue. The stain showed the best results with antigen retrieval performed with pepsin in HCl, primary antibody dilution 1:250 combined with 1 hour incubation at room temperature of the primary antibody. Permeabilization has to some degree shown good results in combination with antigen retrieval with pepsin in HCl. Conclusion: A good protocol was established for Gap-43 detection, but the procedures for Nf-L and DCX detections need to be optimized.

Activated Partial Thromboplastin Time (APTT) is a screening method used to detect defects in the intrinsic pathway. Different preanalytical variables such as centrifugation and thawing can affect the given test results from APTT analysis. The purpose of the study was to evaluate how the results for APTT were affected when frozen citrate plasma was thawed in a heating cabinet or a water bath, and how the results for APTT were affected if the samples were single or double centrifuged before freezing. A total of 40 fresh samples were collected for each study purpose and analyzed. To study how thawing conditions affected frozen samples, aliquoted plasma was frozen at –20°C for a week and then thawed in either a water bath or in a heating cabinet. To study how single and double centrifugation affected the test results, one of the two aliquots per sample was centrifuged again while the other remained single centrifuged. The samples were then stored frozen for one week and afterwards thawed. The results showed statistically significant differences between fresh samples and frozen samples thawed in a heath incubator or water bath. Regarding centrifugation conditions, both single and double centrifugation yielded statistically significant differences in comparison to fresh plasma. All the evaluated sample treatments showed statistically significant differences in comparison to fresh samples. However, in all cases the mean percentage change was ≤10%, which indicates no clinical significance. In conclusion APTT-samples seem to be sensitive to preanalytical variables and further studies are needed to evaluate their effects.