Developing Raman microspectroscopy as a technique to analyse single Caco-2 Cell

Raman spectroscopy is rapidly advancing as a cell imaging technique due to its advantages over existing techniques: it requires little sample preparation, is label-free and can be carried out in aqueous environments to image both fixed and live cells. However, Raman spectroscopy is not commonly used in clinical practice due to perceived long acquisition times and complex data analysis. The aim of this work is to develop Raman spectroscopy as a technique to image cells, specifically Caco-2 cells, and to use Raman spectroscopy to measure the response of these cells to abiotic perturbations. We have developed methodologies for mapping both fixed and live Caco-2 cells, as well as robust shading parameters to allow for direct comparisons. Using a metal rhenium complex to identify the mitochondria of these cells, we have demonstrated the difficulties in shading Raman maps to specific peaks of interest, and how the shading range needs to be carefully considered to avoid over or under interpretation of the data. Raman spectroscopy was also used to evaluate the effect of the cannabinoids cannabidiol (CBD) and anandamide (AEA) on Caco-2 cells. CBD affects Caco-2 cells differently at different concentrations; at low concentrations it may induce proliferation, but at high concentrations it causes cell death characterised by DNA breakdown. Investigating the mechanism of CBD induced cell death in Caco-2 cells suggested that it is not apoptosis or necrosis, but is mediated by caspases 3/7 with the broken down DNA being exported from the cell, which is more consistent with autophagy-dependent cell death or lysosomal-dependent cell death. At low concentrations, AEA may also induce proliferation, but at high concentrations it affects Caco-2 cells differently and does not cause cell death; instead, we hypothesise that the drug has an anti-proliferative effect on these cells. Overall, we have demonstrated how Raman spectroscopy can be used to gain valuable visual and biochemical information about cells

Mortality in intensive care: The impact of bacteremia and the utility of systemic inflammatory response syndrome

Background: The purpose of this study was to determine the impact of bacteremia on intensive care unit (ICU) mortality and to develop a bacteremia prediction tool using systemic inflammatory response syndrome (SIRS) criteria. Methods: Patients included those aged >18 years who had blood cultures taken in the ICU from January 1, 2011-December 31, 2013. Eligible patients were identified from microbiology records of the Glasgow Royal Infirmary, Scotland. Clinical and outcome data were gathered from ICU records. Patients with clinically significant bacteremia were matched to controls using propensity scores. SIRS criteria were gathered and used to create decision rules to predict the absence of bacteremia. The main outcome was mortality at ICU discharge. The utility of the decision tools was measured using sensitivity and specificity. Results: One hundred patients had a clinically significant positive blood culture and were matched to 100 controls. Patients with bacteremia had higher ICU mortality (odds ratio [OR], 2.35; P = .001) and longer ICU stay (OR, 17.0 vs 7.8 days; P ≤ .001). Of 1,548 blood culture episodes, 1,274 met ≥2 SIRS criteria (106 significant positive cultures and 1,168 negative cultures). There was no association between SIRS criteria and positive blood cultures (P = .11). A decision rule using 3 SIRS criteria had optimal predictive performance (sensitivity, 56%; specificity, 50%) but low accuracy. Conclusions: ICU patients with bacteremia have increased mortality and length of ICU stay. SIRS criteria cannot be used to identify patients at low risk of bacteremia