Evaluation of a Portable Blood Gas Analyzer for Prehospital Triage in Carbon Monoxide Poisoning: Instrument Validation Study.

BACKGROUND: Carbon monoxide (CO) poisoning is an important cause of morbidity and mortality worldwide. Symptoms are mostly aspecific, making it hard to identify, and its diagnosis is usually made through blood gas analysis. However, the bulkiness of gas analyzers prevents them from being used at the scene of the incident, thereby leading to the unnecessary transport and admission of many patients. While multiple-wavelength pulse oximeters have been developed to discriminate carboxyhemoglobin (COHb) from oxyhemoglobin, their reliability is debatable, particularly in the hostile prehospital environment. OBJECTIVE: The main objective of this pilot study was to assess whether the Avoximeter 4000, a transportable blood gas analyzer, could be considered for prehospital triage. METHODS: This was a monocentric, prospective, pilot evaluation study. Blood samples were analyzed sequentially with 2 devices: the Avoximeter 4000 (experimental), which performs direct measurements on blood samples of about 50 µL by analyzing light absorption at 5 different wavelengths; and the ABL827 FLEX (control), which measures COHb levels through an optical system composed of a 128-wavelength spectrophotometer. The blood samples belonged to 2 different cohorts: the first (clinical cohort) was obtained in an emergency department and consisted of 68 samples drawn from patients admitted for reasons other than CO poisoning. These samples were used to determine whether the Avoximeter 4000 could properly exclude the diagnosis. The second (forensic) cohort was derived from the regional forensic center, which provided 12 samples from documented CO poisoning. RESULTS: The mean COHb level in the clinical cohort was 1.7% (SD 1.8%; median 1.2%, IQR 0.7%-1.9%) with the ABL827 FLEX versus 3.5% (SD 2.3%; median 3.1%, IQR 2.2%-4.1%) with the Avoximeter 4000. Therefore, the Avoximeter 4000 overestimated COHb levels by a mean difference of 1.8% (95% CI 1.5%-2.1%). The consistency of COHb readings by the Avoximeter 4000 was excellent, with an intraclass correlation coefficient of 0.97 (95% CI 0.93-0.99) when the same blood sample was analyzed repeatedly. Using prespecified cutoffs (5% in nonsmokers and 10% in smokers), 3 patients (4%) had high COHb levels according to the Avoximeter 4000, while their values were within the normal range according to the ABL827 FLEX. Therefore, the specificity of the Avoximeter 4000 in this cohort was 95.6% (95% CI 87%-98.6%), and the overtriage rate would have been 4.4% (95% CI 1.4%-13%). Regarding the forensic samples, 10 of 12 (83%) samples were positive with both devices, while the 2 remaining samples were negative with both devices. CONCLUSIONS: The limited difference in COHb level measurements between the Avoximeter 4000 and the control device, which erred on the side of safety, and the relatively low overtriage rate warrant further exploration of this device as a prehospital triage tool.

Association between prehospital arterial hypercapnia and mortality in acute heart failure: a retrospective cohort study.

BACKGROUND: Acute Heart Failure (AHF) is a potentially lethal pathology and is often encountered in the prehospital setting. Although an association between prehospital arterial hypercapnia in AHF patients and admission in high-dependency and intensive care units has been previously described, there is little data to support an association between prehospital arterial hypercapnia and mortality in this population. METHODS: This was a retrospective study based on electronically recorded prehospital medical files. All adult patients with AHF were included. Records lacking arterial blood gas data were excluded. Other exclusion criteria included the presence of a potentially confounding diagnosis, prehospital cardiac arrest, and inter-hospital transfers. Hypercapnia was defined as a PaCO2 higher than 6.0 kPa. The primary outcome was in-hospital mortality, and secondary outcomes were 7-day mortality and emergency room length of stay (ER LOS). Univariable and multivariable logistic regression models were used. RESULTS: We included 225 patients in the analysis. Prehospital hypercapnia was found in 132 (58.7%) patients. In-hospital mortality was higher in patients with hypercapnia (17.4% [23/132] versus 6.5% [6/93], p = 0.016), with a crude odds-ratio of 3.06 (95%CI 1.19-7.85). After adjustment for pre-specified covariates, the adjusted OR was 3.18 (95%CI 1.22-8.26). The overall 7-day mortality was also higher in hypercapnic patients (13.6% versus 5.5%, p = 0.044), and ER LOS was shorter in this population (5.6 h versus 7.1 h, p = 0.018). CONCLUSION: Prehospital hypercapnia is associated with an increase in in-hospital and 7-day mortality in patient with AHF.

Maintaining the Link Between Patients and Their Families During the COVID Pandemic Using Visio: Iterative Development and Evaluation.

Access to hospitals has been dramatically restricted during the COVID 19 pandemic. As a result, the patients were unable to communicate with their families other than through virtual communication channels. A still significant number of patients, which do not have access to modern videoconference tools, were completely isolated. As a result, the University Hospitals of Geneva decided to implement a Visio conference system inside their patient app ecosystem to allow every patient to remain connected. This article presents the iterative development of the solution in order to respond to the specific timely constraint of the situations as well as its evaluation by the patients and caregivers. Two iterations of the tool have been developed. The first relied on an existing commercial platform whereas the second is a fully integrated solution in our patient app ecosystem. The very positive evaluation at the first stage by more than 300 patients, relatives and caregivers convinced us to invest additional effort to provide a fully integrated solution. The second version, evaluated by 16 patients, confirmed that the Visio reaches its objective of reducing isolation during hospitalization. This initiative is completely in line with the objective of the hospital of providing human centered care.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes.

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

Imaging mass spectrometry using peptide isoelectric focusing.

Imaging Mass Spectrometry (IMS) has emerged as a powerful technique in the field of proteomics. The use of Immobilized pH Gradient-IsoElectric Focusing (IPG-IEF) is also a new trend, as the first dimension of separation, in shotgun proteomics. We report a combination of these two outstanding technologies. This approach is based on the separation of shotgun-produced peptides by IPG-IEF. The peptides are then transferred by capillarity to a capture membrane, which is then scanned by the mass spectrometer to generate MS images. This high-throughput methodology allows a preview of the sample to be obtained in a single day. We report the application of this new pipeline for differential comparison of the membrane proteome of two different strains of Staphylococcus aureus bacteria in a proof-of-principle experiment.

Proteomic analysis of a podocyte vesicle-enriched fraction from human normal and pathological urine samples.

Podocytes (glomerular visceral epithelial cells) release vesicles into urine. Podocyte vesicle-enriched fractions from normal and pathological human urine samples were prepared for proteomic analysis. An immunoadsorption method was applied and enrichment of podocyte vesicles was assessed. We identified 76 unique proteins. One protein, serum paraoxonase/arylesterase 1 (PON-1), was newly identified in normal human urine sample. We confirmed this result and showed PON-1 expression in normal human kidney. These results demonstrated the potential for using the urine samples enriched in podocyte vesicles as a starting material in studies aimed at discovery of biomarkers for diseases.

Exploring glycopeptide-resistance in Staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers.

BACKGROUND: To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. RESULTS: In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. CONCLUSION: Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

The molecular scanner in microscope mode.

The combination of microscope mode matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) with protein identification methodology: the molecular scanner, was explored. The molecular scanner approach provides improvement of sensitivity of detection and identification of high-mass proteins in microscope mode IMS. The methodology was tested on protein distributions obtained after separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). High-quality, high-spatial-resolution ion images were recorded on a TRIFT-II ion microscope after gold coating of the MALDI sample preparation on the poly(vinylidenedifluoride) capture membranes. The sensitivity of the combined method is estimated to be 5 pmol. The minimum amount of sample consumed, needed for identification, was estimated to be better than 100 fmol. Software tools were developed to analyze the spectral data and to generate broad mass range and single molecular component microscope mode ion images and single mass-to-charge ratio microprobe mode images.

MSight: an image analysis software for liquid chromatography-mass spectrometry.

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.

Gold coating of non-conductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis prevents charging effect.

Acquisition of tandem mass spectra from peptides or other analytes deposited on non-conductive membranes is inhibited on instruments combining matrix-assisted laser desorption/ionization with tandem time-of-flight analyzers (MALDI-TOF/TOF) due to a charging effect. A thin layer of gold renders the membrane conductive. This allows adequate data acquisition on MALDI-TOF/TOF systems. Therefore, this methodology extends the capacity of the molecular scanner concept to tandem mass spectrometry.

Correlation of proteomic and transcriptomic profiles of Staphylococcus aureus during the post-exponential phase of growth.

A combined proteomic and transcriptomic analysis of Staphylococcus aureus strain N315 was performed to study a sequenced strain at the system level. Total protein and membrane protein extracts were prepared and analyzed using various proteomic workflows including: 2-DE, SDS-PAGE combined with microcapillary LC-MALDI-MS/MS, and multidimensional liquid chromatography. The presence of a protein was then correlated with its respective transcript level from S. aureus cells grown under the same conditions. Gene-expression data revealed that 97% of the 2'596 ORFs were detected during the post-exponential phase. At the protein level, 23% of these ORFs (591 proteins) were identified. Correlation of the two datasets revealed that 42% of the identified proteins (248 proteins) were amongst the top 25% of genes with highest mRNA signal intensities, and 69% of the identified proteins (406 proteins) were amongst the top 50% with the highest mRNA signal intensities. The fact that the remaining 31% of proteins were not strongly expressed at the RNA level indicates either that some low-abundance proteins were identified or that some transcripts or proteins showed extended half-lives. The most abundant classes identified with the combined proteomic and transcriptomic approach involved energy production, translational activities and nucleotide transport, reflecting an active metabolism. The simultaneous large-scale analysis of transcriptomes and proteomes enables a global and holistic view of the S. aureus biology, allowing the parallel study of multiple active events in an organism.

Identification of post-mortem cerebrospinal fluid proteins as potential biomarkers of ischemia and neurodegeneration.

Only few biological markers are currently available for the routine diagnosis of brain damage-related disorders including cerebrovascular, dementia, and other neurodegenerative diseases. In this study, post-mortem cerebrospinal fluid samples were used as a model of massive brain insult to identify new markers potentially relevant for neurodegeneration. The protein pattern of this sample was compared to the one of cerebrospinal fluid from healthy subjects by two-dimensional gel electrophoresis. Using gel imaging, N-terminal microsequencing, mass spectrometry, and immunodetection techniques, we identified 13 differentially expressed proteins. Most of these proteins have been previously reported to be somehow associated with brain destruction or with the molecular mechanisms underlying certain neurodegenerative conditions. These data indicate that the identified proteins indeed represent potential biomarkers of brain damage. We recently showed that H-FABP, a protein highly homologous to E-FABP and A-FABP identified in this study, is a potential marker of Creutzfeldt-Jakob disease and stroke.

Fatty acid binding protein as a serum marker for the early diagnosis of stroke: a pilot study.

No biological marker is currently available for the routine diagnosis of stroke. The aim of this pilot study was to determine whether heart-fatty acid binding protein (H-FABP) could be used as a valid diagnostic biomarker for stroke, as compared with neuron-specific enolase (NSE) and S100B proteins. Using two-dimensional gel electrophoresis separation of cerebrospinal fluid proteins and mass spectrometry techniques, FABP was found elevated in the cerebrospinal fluid of deceased patients, used as a model of massive brain damage. Because H-FABP, a FABP form present in many organs, is also localized in the brain, an enzyme-linked immunosorbant assay was developed to detect H-FABP in stroke versus control plasma samples. However, H-FABP being also a marker of acute myocardial infarction (AMI), troponin-I and creatine kinase-MB levels were assayed at the same time in order to exclude any concomitant heart damage. NSE and S100B levels were assayed simultaneously. These assays were assessed in serial plasma samples from 22 control patients with no AMI or stroke, 20 patients with AMI but no stroke, and 22 patients with an acute stroke but no AMI. Twenty-two out of the 22 control patients and 15 out of the 22 stroke patients were correctly classified, figures much better than those obtained with NSE or S100B, in the same study's population. H-FABP appears to be a valid serum biomarker for the early diagnosis of stroke. Further studies on large cohorts of patients are warranted.