This thesis has been performed at Clinical Chemistry at Sahlgrenska University Hospital in Gothenburg. The purpose of the project was to investigate new and alternative ways to determinate synthetic cannabinoids by gas chromatography-mass spectrometry. Currently, the possibilities to quantify synthetic cannabinoids are very limited. This can lead to an increased use of synthetic cannabinoids as the risk of detection is low, which may be known by drug users. The synthetic cannabinoids are sold mixed with different herbs and have varying names like Spice Gold, Spice Silver, K2, Smoke and Pot-pourri. The synthetic cannabinoids analyzed were JWH-018 and JWH-073, which are commonly found in seized Spice material. At intake of these drugs, usually through smoking, cannabis-like effects arise. This is because they bind to cannabinoid receptors in a similar way as THC does, which is the primary active cannabinoid of cannabis. For urine samples an analytical method would probably be the most sensitive if the major metabolite could be analyzed, as it is expected to be present in high concentrations in this sample type. Since information regarding the metabolism of synthetic cannabinoids is very limited there may be reasons to analyze the mother substance in urine. Further, in plasma and serum samples the mother substance is expected in high concentrations. Thus different ways to detect JWH-018 and JWH-073 directly were investigated in this project. Derivatization of JWH-018 and JWH-073 was the first step to get more selective and sensitive GC-MS analysis. Different derivatization-reagents were investigated, for example BSTFA and TFAA. The results show that the derivatization of JWH-018 with BSTFA after reduction and extraction was successful. To achieve this, samples had to be heated at 115°C for 1-3 hours, but still the samples were not completely derivatized. The results indicate that JWH-substances are difficult to derivatized, but they are possible to derivatize with BSTFA. This could mean that a GC-MS-method maybe could be established for these substances, preferably trough TFAA-derivatization.

The analysis of acute phase protein serum amyloid A (SAA) has recently been brought into clinical use in veterinary medicine. Some of the difficulties with incorporating the SAA method in clinical practice have been the expensive and rather large equipment required for the method. Due to these difficulties only larger clinics can afford to use the SAA analysis. The company Equinostic has recently developed a smaller instrument that costs one-tenth of a larger instrument. The instrument is named EVA1 and has so far only been used to analyze SAA in horses. The aim of this study was to investigate if the EVA1 instrument could be used to analyze SAA in cats. This study included 24 serum samples from cat, which were first analyzed twice on the EVA1 instrument and then sent to the Strömsholm Referral Animal Hospital in Sweden where they reanalyzed the samples using a validated reference method. Both instruments are based on an immunoturbidimetric assay. The correlation between the two instruments was good (r=0.97) but the EVA1 instrument showed constantly lower results than the reference method. The difference between the duplicates when analyzed on the EVA1 instrument was larger than expected. The conclusion is that EVA1 could be used to analyze SAA in cats. However, before it could be used clinically in veterinary practice an extended study is recommended.

Klinisk kemiska analyser har hög klinisk relevans. I serum/plasma kan olika parametrar kvantifieras. Dessa parametrar kan vara proteiner, enzymer, joner, metaller, lipider och kolhydrater. Med hjälp av referensintervall kan veterinärer ställa diagnos, följa behandling och sjukdomsförlopp. Parametrar detekteras med olika analysprinciper/metoder; kolorimetri, immunturbidimetri, enzymatisk metod och potentiometri. Djursjukhuset, AniCura, i Hässleholm mottagas både hundar och katter. Cirka 120 kemianalyser analyseras varje dag. AniCura har köpt in ett nytt våtkemiiinstrument, Indiko Plus, som ska ersätta Cobas C111. Med Indiko Plus tillkommer fler provpositioner, 8 nya analyser, ökad kapaciteten och underlättad användning.  Syftet med denna studie var att jämföra instrumenten och ta fram eget referensintervall som jämfördes med referensintervall framtaget av Thermo Fisher. Verifiering av det nya instrumentet genomfördes med precisionsstudie och linjäritetsstudie. Provtagning på hundar och katter utfördes av personal på AniCura. Jämförelsen gjordes med patientprover och referensintervall togs fram med hjälp av prover från friska hundar och katter. Jämförelsen visade att 9 av 13 analyser hade statistisk signifikant skillnad. Orsaken till det beror troligen på skillnaden av reagens, instrumentens ålder och tid mellan mätningar. Ett nytt referensintervall utarbetades och skiljde sig inte mycket från Thermo Fishers intervall. Vidare validering på grund av liten population rekommenderades. Precisionen för Indiko Plus blev godkänd. Linjäriteten blev icke linjär och berodde troligen på en dålig pipett och bör göras om. / Clinical chemical analyzes has high clinical relevance. In serum/plasma, different parameters can be quantified. Parameters can be proteins, enzymes, ions, metals, lipids, and carbohydrates. With reference intervals, veterinarians can set diagnosis, follow treatment and development of the disease. Parameters are detected with different analysis principles/methods; colorimetry, immunoturbidimetry, enzymatic method and potentiometry. The animal hospital, AniCura, in Hässleholm accept dogs and cats. About 120 chemical analyzes are analyzed every day. AniCura purchased a new instrument, Indiko Plus, which will replace Cobas C111. Indiko Plus provide, more sample positions, 8 new analyzes, increased capacity, and facilitated use. The purpose of this study was to compare the instruments and produce a new reference interval which was compared to the reference interval provided by Thermo Fisher. To verify Indiko Plus, a precision and linearity study were conducted. Blood sampling of dogs and cats was performed by staff at AniCura. The comparison was made with patient samples and the reference intervals were obtained using samples from healthy animals. The comparison showed 9 of 13 analyzes had a statistically significant difference. The reason for this is probably due to the difference in reagents, the age of the instruments and the time between measurements. A new reference interval was developed and did not differ much from the Thermo Fisher interval. Further validation due to low population was recommended. The precision for Indiko Plus was approved. The linearity study shows not linear trend but was likely due to a bad pipette and should be redone.

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. TF is over-expressed on many malignant cells and apart from increasing the risk of thrombosis, the presence of TF/FVIIa also promotes the progression of cancer and metastasis by intracellular signaling. TF expressing microparticles (MP) are, moreover, often found in the circulation of cancer patients. Aim: The aim of this thesis was to study different aspects of TF activity, e.g. the importance of procoagulant MP and TF-induced intracellular signaling pathways, with focus on cell migration (chemotaxis) and apoptosis. Results: The TF signaling complexes were shown to prevent apoptosis induced by serum starvation and TRAIL in cancer cells by reduced activation of caspase-8 in a PI3k/AKT-dependent manner. FVIIa also decreased transcription of pro-apoptotic genes in cancer cells treated with TRAIL. Simvastatin triggered apoptosis by transcriptional reduction of BCL-2 due to cytosolic retention of NFκB. Simvastatin also inactivated the PI3k/AKT pathway and reduced the production of the MP-like prostasomes which, respectively, impaired the anti-apoptotic signaling by TF and reduced the procoagulant activity in the vicinity of prostate cancer cells. Intracellular events conducted by the TF/FVIIa complex selectively enhanced PDGF-BB induced chemotaxis which was partly explained by the TF/FVIIa-induced transactivation of the PDGFβ-receptor. This was dependent on Src-family members and engagement of PAR2. Conclusions: The results presented in this thesis extend the current knowledge of TF-mediated signaling. We report the TF complexes to govern the extrinsic pathway of apoptosis, present data on FVIIa-dependent regulation of apoptosis-related genes, and exclude known surface proteins as transmitters of the anti-apoptotic signals. We moreover describe TF/FVIIa to transactivate the PDGFβ-receptor and play a decisive role in the potentiated chemotaxis toward PDGF-BB in a number of cell types. Finally, we explain the mechanism behind simvastatin-induced apoptosis in cancer cells and how statins interfere with TF-dependent signaling and coagulation.

Laboratory analyses, essential in screening, diagnosis, treatment, and monitoring of disease, are indispensable in health care, but appropriate utilization is intricate. The overall aim of this thesis was to study the use of laboratory tests in Sweden with the objective to evaluate and optimize test utilization. Considerable inter-county variations in test utilization in primary health care in Sweden were found; variations likely influenced by local traditions and habits of test ordering leading to over- as well as underutilization. Optimized test utilization was demonstrated to convey improved quality and substantial cost savings. It was further established that continuing medical education is a suitable means of optimizing test utilization, and consequently enhancing quality and cost-efficiency, as such education was demonstrated to achieve long-lasting improvements in the test ordering habits of primary health care physicians. Laboratory tests are closely associated with other, greater, health care costs, but their indirect effects on other areas of medicine are rarely evaluated or measured in monetary terms. In an illustrative example of the effects that optimal test utilization may have on associated health care costs it was demonstrated that F-calprotectin, a fecal marker of intestinal inflammation, has the potential to substantially reduce the number of invasive investigations necessary in, and the costs associated with, the diagnosis of Inflammatory Bowel Disease. Information on trends in test utilization is essential to optimal financial management of laboratories. A longitudinal evaluation revealed that test utilization had increased by 70% in 6 years, and even though the selection of tests more than doubled, a very small number of tests represented a stable, and disproportionally large, share of the total number of tests ordered. The study defines trends and thus has potential predictive values. In summary, appropriate utilization of laboratory analyses has both clinical and economical benefits on all levels of health care.