Introduction CT pulmonary angiography (CTPA) is the recommended imaging modality for suspected pulmonary embolism (PE). Current NICE guidelines recommend using clinical prediction scoring systems to estimate the probability of PE and guide further investigation [i]. A low or intermediate probability score, coupled with a negative D-dimer, reliably excludes PE, thereby avoiding the need for CTPA. Objectives We undertook a retrospective audit to examine adherence to NICE guidelines for diagnosis of suspected PE in patients admitted to a district general hospital, and identify patients who may have undergone unnecessary CTPA. Methods We obtained a list of all CTPAs undertaken in our hospital between December 2012 and February 2013. D-dimer tests are poorly specific within hospitalised patients; therefore, we excluded post-surgical and obstetric patients, and pre-existing inpatients where primary admission was not for suspected PE. We also excluded outpatient CTPAs. We searched the records for contemporaneous PE probability scores and D-dimer results. For patients without a probability score result, we reviewed the clinical notes and calculated a probability score retrospectively using a local scoring system adapted from BTS guidelines. Results There were 115 CTPAs during the study period -36 were excluded and 4 patients' case notes were unavailable. 75 patients fulfilled the inclusion criteria (mean age 68.2 years), and PE was confirmed in 20%. 50 patients (66.7%) had a contemporaneous documented clinical probability score. There were 5 patients (6.7%) with a low/intermediate probability score and negative D-dimer, who underwent unnecessary CTPA (PE excluded in each case). There were 9 patients (12%) with retrospectively calculated low/intermediate clinical probability scores and no D-dimer result, who may have avoided CTPA had Ddimer been undertaken (CTPA excluded PE in each case). Conclusions In our district general hospital, the underuse of clinical probability scoring and D-dimer testing in patients with suspected PE is contributing to unnecessary CTPAs. Introducing mandatory documentation of PE clinical probability score on CTPA request forms may reduce the number of unnecessary CTPAs.
IntroductionCOPD is a progressive neutrophilic lung disease associated with increased risk of cardiovascular complications. Neutrophil elastase (NE) is implicated in COPD pathogenesis but the precise mechanisms of neutrophil-mediated tissue damage are unknown, particularly with respect to systemic manifestations. Inflamed COPD airways are profoundly hypoxic. We therefore hypothesised that hypoxia synergises with inflammatory cytokines to promote a destructive neutrophil phenotype with enhanced capacity for tissue damage, both locally and systemically.MethodsNeutrophils isolated from exacerbating COPD patients and age/sex-matched healthy volunteers were incubated under normoxia (21% O2) or hypoxia (0.8% O2) for 4 hours, before treatment with priming (PAF) and stimulating (fMLP) agents, with/without PI3Kinase inhibitors. NE activity was measured by Enzchek assay. Neutrophil supernatants were incubated with primary human pulmonary artery endothelial cells (HPAEC); cell damage was assessed by confocal microscopy. Normoxic/hypoxic neutrophil supernatants underwent tandem mass tag-labelled mass spectrometry (TMT-MS), and identified protein abundance was quantified. Neutrophil-derived microparticles (NDMPs) were isolated by ultra-centrifugation and quantified by NanoSight nanoparticle tracking technology.ResultsHypoxia increased NE release in a PI3K-dependent manner, with significantly more NE secreted by hypoxic neutrophils from exacerbating COPD patients versus healthy controls (p<0.0001). Supernatants generated from hypoxic, but not normoxic, stimulated neutrophils induced extensive HPAEC damage. Comparing the secretomes of supernatants derived from normoxic/hypoxic stimulated neutrophils, TMT-MS identified several additional proteins with potential to cause tissue damage as upregulated in hypoxia, including resistin and NGAL (neutrophil gelatinase-associated lipocalin). Notably, several of these proteins were not granule-associated, and some granule proteins were downregulated in hypoxia, indicating additional/alternative release mechanisms. Preliminary data show an increase in NDMP release under hypoxia, potentially contributing to the observed differential protein release.ConclusionsHypoxia augments NE release in a PI3K-dependent manner, further increased during COPD exacerbations, and hypoxic neutrophil supernatants injure endothelial cells in vitro. Unbiased characterisation of hypoxic neutrophil secretomes identified several upregulated proteins which may contribute to cellular/tissue damage. In addition to degranulation, NDMP release may underpin differential protein secretion under hypoxia. Hypoxia engenders a neutrophil phenotype with potential to cause local and distant tissue damage in COPD; novel targets in the hypoxic neutrophil secretome may identify new therapeutic opportunities.
IntroductionDamage to host tissue from persistent neutrophilic inflammation is implicated in the pathogenesis of many diseases, including chronic obstructive pulmonary disease (COPD). Infected/inflamed tissues can be profoundly hypoxic; this state may synergise with inflammatory cytokines to promote a destructive neutrophil phenotype with enhanced potential for tissue damage.MethodsNeutrophils isolated from COPD patients or healthy volunteers were incubated under normoxia (21% O2) or hypoxia (0.8% O2) before treatment with priming (GM-CSF/PAF/TNF-α) and stimulating (fMLP) agents, with/without PI3Kinase inhibitors (pan/γ/δ). Neutrophil elastase (NE) activity was measured by Enzchek® assay. Western blotting for total and phosphorylated Akt was performed using cell lysates. Neutrophil extracellular trap (NET) production was assessed by fluorescence absorbance. Neutrophil supernatants were incubated with primary human pulmonary artery endothelial cells (HPAEC); death and detachment were measured by MTT assay and confocal microscopy. Precipitated neutrophil supernatants were separated by SDS polyacrylamide gel electrophoresis (PAGE) and silver stained. S100A8/A9 homo- and heterodimer content of neutrophil supernatants was assessed by ELISA.ResultsHypoxia increased NE release in an agonist- and PI3K-γ-dependent manner, with more pronounced hypoxic degranulation responses seen in exacerbating COPD patients. Hypoxia augmented resting and cytokine-stimulated Akt phosphorylation; PI3K-γ inhibition abrogated Akt phosphorylation and prevented the hypoxic uplift of NE release. Hypoxia did not increase NET production in resting or GM-CSF/fMLP treated cells. Hypoxic neutrophil supernatants induced extensive HPAEC detachment and death, which was prevented by co-incubation with alpha-1 antitrypsin. Silver stained protein bands from precipitated neutrophil supernatants separated by SDS-PAGE were identified by mass spectrometry, suggesting a hypoxic increase in damage associated molecular pattern (DAMP) proteins S100A8 and S100A9. When interrogated by ELISA, there was no difference between the amount of S100A8/A9 hetero- or homodimers in normoxic versus hypoxic supernatants.ConclusionHypoxia augments neutrophil degranulation in an agonist- and PI3K-γ-dependent manner, which may be further increased during COPD exacerbations. Hypoxic neutrophil supernatants have enhanced capacity to damage endothelial cells in vitro, likely due to increased release of NE. The contribution of S100A8/A9 proteins to this damage is currently unclear. Hence, hypoxia promotes a destructive histotoxic neutrophil phenotype with potential relevance to diseases such as COPD.
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