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Background: Clinical procedural packs were identified as an area of healthcare where an enhancement in design and implementation could improve practitioner performance and associated service user outcomes. Objectives: This study assessed the clinical impact of two interventions, (1) procedural packs with an enhanced training programme and (2) feedback system, on both blood culture sampling and PVC insertion procedures within Antrim Area Hospital. Study Design: A time series design study, using retrospective and prospective data, was used to evaluate impact of two interventions on complication rates associated with both procedures. Impact of the interventions was evaluated by comparing pre- and post- mean complication rates calculated through statistical process control p-chart analysis. Qualitative data gathered from practitioner focus group discussion and quantitative data from patient questionnaires further facilitated exploration of underlying issues. Results: A non-significant reduction in mean blood culture contamination rate was recorded from a Phase A baseline rate of 2.5%, with a reduction to 1.89% from Phase B intervention and to 1.47% from Phase C intervention, over a 30 month study, p = 0.066. However, regression analysis did show a significant downward trend of mean blood culture contamination rate over the 30 month study period, p = 0.015. A significant decrease in mean PVC clinical adverse event rates were recorded from a Phase A baseline rate of 12.84%, with a reduction to 9.48% from Phase B intervention (p = 0.008) and a further drop to 5.96% from Phase C intervention, over a 72 week study (p < 0.001). However, a high rate of PVC failures due to non-clinical adverse events was recorded through Phase A (22.30%), Phase B (31.80%) and Phase C (25.66%) of the study. Conclusions: The combination of best practice guidelines, procedural pack with an enhanced training programme and feedback system achieved and maintained optimal procedural practice.

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1. The first studies of this thesis concerned human phagocytic cells in vivo as they responded to physical trauma in an experimental skin abrasion. This showed the sequence of cellular responses during acute inflammation and provided quantitative data about the phagocytic cell concentrations at each stage. 2. In an identical skin abrasion several antibiotics, administered systemically, were assayed. This provided information about the approximate concentration of each drug in acute inflammatory exudates. 3. A series of in vitro tests were performed to quantify the rate at which phagocytic cells, or the various antibiotics, could inactivate Staphylococcus aureus type 42b. The concentrations used were based on concentrations measured in vivo (paragraphs 1 and 2 above). These studies showed that phagocytosis by human granulocytes is rapid (T-§- = 5.6 minutes), but subsequent intracellular killing is much slower. In a cell-free system different antibiotics had variable rates of bacterial inactivation but, in general, these rates were of the same order as the rate of intracellular killing by normal/ normal human granulocytes. All these experiments utilised phagocytic cells and antibiotics separately. 4. The most important findings relate to the combined action of phagocytic cells and antibiotics (particularly ampicillin). The first clinical studies showed that during antibiotic therapy the rate of intracellular killing usually decreased. This effect could not be demonstrated by adding ampicillin in vitro to granulocytes during their phase of intracellular killing. However, when ampicillin was administered orally to 4 healthy subjects there was a decrease of intracellular killing at 1 and 2 hours. The exact cause has not been established, but collaborative work indicates that this may be the result of inhibition by ampicillin of leucocyte myeloperoxidase and interference with other enzymes involved in the production or breakdown of intravacuolar hydrogen peroxide. 5. The importance of these findings is discussed in the light of a growing awareness that antibiotic therapy has considerable limitations and may have serious adverse effects.

Glutathione and glutathione-dependent enzymes play a central role in the protection of cells from cytotoxic chemicals. In particular, reduced glutathione (GSH) and glutathione S- transferases (GST's) have been studied in relation to intrinsic and acquired resistance of tumours to cytotoxic drugs. There are however, other glutathione-dependent enzymes which may also be involved in drug resistance: these include glutathione perox-idase (GPX) and the enzymes, fglutamyltranspeptides (fGT), fglutamyl cysteinyl synthetase ('yGCS) and glutathione reductase (GRD). The latter enzymes are involved in the maintenance of reduced GSH levels within cells. GSH and the above glutathione-dependent enzymes have been studied in a series of drug resistance models, a) drug resistant tumour cell lines generated in vitro and in vivo; b) chemotherapeutic agent induced resistance in normal bone marrow cells; c) in oxygen resistant lung cells and d) in preneoplastic foci, in order to evaluate whether GSH and associated glutathione-dependent enzymes form part of an adaptive response involved in protection against the environment. Several models of drug resistance involving cell lines in culture were taken for study. A CHO cell line resistant to chlorambucil, an ovarian cell line generated in vivo, resistant to cis-Platinum and chlorambucil, and a sarcoma cell line resistant to adriamycin. In all these cell lines GST, GSH and 7GT were significantly elevated. In the latter two cell lines selenium dependent GPX was also induced. In the CHO line the elevated GST activity was explained by a 40 fold induction of the alpha class Yc GST subunit and a 2 fold elevation in the Ya subunit. In mouse bone marrow cells following the administration in vivo of a low 'primary dose' of cyclophosphamide, transient increase in alpha Ya and particularly mu Yb GST subunits were found. These increases have been associated with a subsequent protection against a higher lethal dose of the same agent. The changes observed in GST isozyme composition were confined to the granulocyte population. Differences in selenium-dependent GPX, GSH and fGT were also found in cells resistant to high oxygen, with only marginal changes in GST sub-unit profiles. In preneoplastic foci, significant elevations in pi class Yf GST and also alpha Ya and mu Yb were detected. Selenium- dependent GPX was decreased and TfGCS and GRD elevated in this model. These studies indicate therefore: 1) GST levels in certain cells appear to be directly related to resistance to cytotoxic chemicals; 2) Changes in GST expression associated with preneoplasia and in acquired drug resistance are not confined to one sub group of GST enzymes; 3) Changes in GST expression are often paralleled by changes in GSH and other glutathione- dependent enzymes; 4) The most consistent phenotypic changes observed in the drug resistant models studied were in GSH and fGT levels; 5) There seemed to be an inverse relationship in the regulation of selenium and non-selenium-dependent GPX activity in different drug resistant models. GSH and glutathione-dependent enzyme changes in acquired drug resistance, appear to be due to an adaptive response which may be of central importance in the resistance of tumours to chemotherapeutic agents.

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To evaluate and identify new candidate cancer drug targets, there is an ongoing need for a reliable, sensitive and quantitative assay that enables the analysis of larger numbers of compounds in preclinical research. This thesis has developed, and optimized a sensitive enzyme-release assay for monitoring natural cytotoxicity. It measures the release of the intracellular enzyme adenylate kinase into the culture supernatant after membrane rupture and is evaluated as an indicator of cell death. This assay was proven to correlate and compete with currently used methodologies for the assessment of cytotoxicity and with its superior sensitivity; convenience and in expense, it should be applicable to the study of other cytotoxicity reactions. The resulting ToxiLight® kit is now being sold world-wide and rapidly became the top selling product for Lonza Bio Science with many references to its use in publications. It was proven from this investigation that to truly comprehend the effect a cytotoxic drug has on cells, two assays are required in combination; one to measure cytotoxicity and a second to measure viability. The two most sensitive kits tested in this study, the ViaLight® Plus assay and the newly designed ToxiLight® assay were used in combination to monitor the effect of commonly used cytotoxicity drugs on melanoma cells. It was hoped to find both a sensitive and resistant cell line for further analysis by MALDI-MS. The study revealed how cells of the same histological and tissue type can respond differently to the same anticancer drug with one cell line revealing cell death after treatment and another remaining unaffected. This is representative of how individual patients may respond differently to the treatments given in vitro and explains the vast biochemical heterogeneity of tumour cells and the complexity involved in developing anticancer drugs that will specifically kill tumours arising from a given cell. The primary melanoma cells used for the research were representative of the clinical situation and were a kind gift from the OYSTER (Outcome and Impact of Specific Treatment in European Research in melanoma) tissue bank; with the established cell lines obtained from ESTDAB. A selection of three of these cell lines (Ma Mel 28, Ma Mel 26a and MEWO) were chosen after investigating their sensitive/resistant nature to certain chemotherapeutic drugs and were further investigated with a novel agent currently in its early stages of drug trials, the histone deacetylase inhibitor, trichostatin A. To investigate this resistance further, MALDI-MS was performed on the chosen melanoma cell lysates. The results demonstrated that good quality proteomic data could be achieved from cell lines and that it is possible to generate discriminatory protein profiles that correlate with the cytotoxicity assays when analysed using artificial neural networks (ANNs). Through the analysis of the proteome the ANNs was able to train itself using the raw dataset from the MALDI-MS analysis and distinguish differences between those samples that were drug-treated and those that were left untreated. The differences between the two classes of treated and untreated cells revealed biomarkers that may correlate to cell death and thus the effect of the drug trichostatin A. These findings could lead to the discovery of proteins that are up regulated when a patient is responding to therapy. This could be of prognostic and therapeutic benefit to patients enabling them to find out in the early stages of treatment if they are responding to a given treatment; the long term outcome leading to personalised treatments for individuals in which a decision can be made on the best suited treatment.

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