Towards a high-throughput microfluidic single-cell proteomic platform for analysing patient blood samples

A critical driver in the development of single-cell analysis platforms has been the recognition that cellular heterogeneity is crucial to understanding disease. Single cell proteomics offer significant insights of cellular function; however, currently suffers from low-throughput. The work outlined here presents the development and application of several single-cell protein analysis systems. Each aim to address technological gaps regarding throughput, cell selectivity and amenability to processing samples directly from patients. To achieve higher-throughput, we have developed the CellWell platform, a high-density microwell array which can capture thousands of cells within minutes; however, posed challenges relating to the simultaneous lysis of these cells. We developed a facile method to produce surface microelectrodes to achieve single-cell lysis on-chip, but the demanding surface chemistry requirements imposed by the necessity to support simultaneously both the microelectrodes and single-molecule antibody microarrays proved difficult to overcome. Instead, we investigated how implementing semi-permeable hydrogel-based microwells could overcome these issues. To assay cells in patient blood samples with the CellWell, pre-processing is necessary. With a clinical setting in mind, it would be advantageous to process raw samples directly from patients with little or no off-chip pre-processing. To address this, we develop our methodology into the Hydrodynamic Trapping Centrifugal Release (HTCR) chip which is specifically designed to isolate cancer cells from patient liquid biopsies. The HTCR implements a method by which cells can be easily released from hydrodynamic traps and subsequently moved to isolated compartments. We conclude in validating the single-molecule single-cell method using fluorescence and immunofluorescence microscopy. While necessary to validate our single-molecule approach, we also show that the equivalence of these measurements of the steady-state distribution of protein abundance can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy.Open Acces

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

Background: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. Methods: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. Findings: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96–1·28). Interpretation: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. Funding: National Institute for Health Research Health Services and Delivery Research Programme

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

BACKGROUND: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. METHODS: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. FINDINGS: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96-1·28). INTERPRETATION: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. FUNDING: National Institute for Health Research Health Services and Delivery Research Programme

Hand-portable HPLC with broadband spectral detection enables analysis of complex polycyclic aromatic hydrocarbon mixtures

Portable liquid chromatography instruments enable a myriad of applications in field research. Here a handportable system incorporating a broadband absorption detector is used to separate and classify polycyclic aromatic hydrocarbons from environmental water samples based solely on spectral fingerprinting