Multiplex assays for biomarker research and clinical application: translational science coming of age

Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench-to-bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody-based immunoassays and MS-based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi-staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field.

A 49 year hindcast of surface winds over the Iberian Peninsula

The use of hindcast climatic data is quite extended for multiple applications. However, this approach needs the support of a validation process to allow its drawbacks and, therefore, confidence levels to be assessed. In this work, the strategy relies on an hourly wind database resulting from a dynamical downscaling experiment, with a spatial resolution of 10 km, covering the Iberian Peninsula (IP), driven by the ERA40 reanalysis (1959–2001) extended by European Centre for Medium-Range Weather Forecast (ECMWF) analysis (2002–2007) and comprising two main steps. Initially, the skill of the simulation is evaluated comparing the quality-tested observational database (Lorente-Plazas et al., 2014) at local and regional scales. The results show that the model is able to portray the main features of the wind over the IP: annual cycles, wind roses, spatial and temporal variability, as well as the response to different circulation types. In addition, there is a significant added value of the simulation with respect to driving conditions, especially in regions with a complex orography. However, some problems are evident, the major drawback being the systematic overestimation of the wind speed, which is mainly attributed to a missrepresentation of frictional forces. The model skill is also lower along the Mediterranean coast and for the Pyrenees. In a second phase, the high spatio-temporal resolution of the pseudo-real wind database is used to explore the limitations of the observational database. It is shown that missing values do not affect the characterisation of the wind climate over the IP, while the length of the observational period (6 years) is sufficient for most regions, with only a few exceptions. The spatial distribution of the observational sampling schemes should be enhanced to improve the correct assessment of all IP wind regimes, particularly in some mountainous areas.

Validation of a modified AVHRR aerosol optical depth retrieval algorithm over Central Europe

The Advanced Very High Resolution Radiometer (AVHRR) carried on board the National Oceanic and Atmospheric Administration (NOAA) and the Meteorological Operational Satellite (MetOp) polar orbiting satellites is the only instrument offering more than 25 years of satellite data to analyse aerosols on a daily basis. The present study assessed a modified AVHRR aerosol optical depth τa retrieval over land for Europe. The algorithm might also be applied to other parts of the world with similar surface characteristics like Europe, only the aerosol properties would have to be adapted to a new region. The initial approach used a relationship between Sun photometer measurements from the Aerosol Robotic Network (AERONET) and the satellite data to post-process the retrieved τa. Herein a quasi-stand-alone procedure, which is more suitable for the pre-AERONET era, is presented. In addition, the estimation of surface reflectance, the aerosol model, and other processing steps have been adapted. The method's cross-platform applicability was tested by validating τa from NOAA-17 and NOAA-18 AVHRR at 15 AERONET sites in Central Europe (40.5° N–50° N, 0° E–17° E) from August 2005 to December 2007. Furthermore, the accuracy of the AVHRR retrieval was related to products from two newer instruments, the Medium Resolution Imaging Spectrometer (MERIS) on board the Environmental Satellite (ENVISAT) and the Moderate Resolution Imaging Spectroradiometer (MODIS) on board Aqua/Terra. Considering the linear correlation coefficient R, the AVHRR results were similar to those of MERIS with even lower root mean square error RMSE. Not surprisingly, MODIS, with its high spectral coverage, gave the highest R and lowest RMSE. Regarding monthly averaged τa, the results were ambiguous. Focusing on small-scale structures, R was reduced for all sensors, whereas the RMSE solely for MERIS substantially increased. Regarding larger areas like Central Europe, the error statistics were similar to the individual match-ups. This was mainly explained with sampling issues. With the successful validation of AVHRR we are now able to concentrate on our large data archive dating back to 1985. This is a unique opportunity for both climate and air pollution studies over land surfaces.

Proposal on How To Conduct a Biopharmaceutical Process Failure Mode and Effect Analysis (FMEA) as a Risk Assessment Tool

With the publication of the quality guideline ICH Q9 "Quality Risk Management" by the International Conference on Harmonization, risk management has already become a standard requirement during the life cycle of a pharmaceutical product. Failure mode and effect analysis (FMEA) is a powerful risk analysis tool that has been used for decades in mechanical and electrical industries. However, the adaptation of the FMEA methodology to biopharmaceutical processes brings about some difficulties. The proposal presented here is intended to serve as a brief but nevertheless comprehensive and detailed guideline on how to conduct a biopharmaceutical process FMEA. It includes a detailed 1-to-10-scale FMEA rating table for occurrence, severity, and detectability of failures that has been especially designed for typical biopharmaceutical processes. The application for such a biopharmaceutical process FMEA is widespread. It can be useful whenever a biopharmaceutical manufacturing process is developed or scaled-up, or when it is transferred to a different manufacturing site. It may also be conducted during substantial optimization of an existing process or the development of a second-generation process. According to their resulting risk ratings, process parameters can be ranked for importance and important variables for process development, characterization, or validation can be identified. LAY ABSTRACT: Health authorities around the world ask pharmaceutical companies to manage risk during development and manufacturing of pharmaceuticals. The so-called failure mode and effect analysis (FMEA) is an established risk analysis tool that has been used for decades in mechanical and electrical industries. However, the adaptation of the FMEA methodology to pharmaceutical processes that use modern biotechnology (biopharmaceutical processes) brings about some difficulties, because those biopharmaceutical processes differ from processes in mechanical and electrical industries. The proposal presented here explains how a biopharmaceutical process FMEA can be conducted. It includes a detailed 1-to-10-scale FMEA rating table for occurrence, severity, and detectability of failures that has been especially designed for typical biopharmaceutical processes. With the help of this guideline, different details of the manufacturing process can be ranked according to their potential risks, and this can help pharmaceutical companies to identify aspects with high potential risks and to react accordingly to improve the safety of medicines.

Evidence Based Development of a Novel Lateral Fibula Plate (VariAx Fibula) Using a Real CT Bone Data Based Optimization Process During Device Development

Development of novel implants in orthopaedic trauma surgery is based on limited datasets of cadaver trials or artificial bone models. A method has been developed whereby implants can be constructed in an evidence based method founded on a large anatomic database consisting of more than 2.000 datasets of bones extracted from CT scans. The aim of this study was the development and clinical application of an anatomically pre-contoured plate for the treatment of distal fibular fractures based on the anatomical database. 48 Caucasian and Asian bone models (left and right) from the database were used for the preliminary optimization process and validation of the fibula plate. The implant was constructed to fit bilaterally in a lateral position of the fibula. Then a biomechanical comparison of the designed implant to the current gold standard in the treatment of distal fibular fractures (locking 1/3 tubular plate) was conducted. Finally, a clinical surveillance study to evaluate the grade of implant fit achieved was performed. The results showed that with a virtual anatomic database it was possible to design a fibula plate with an optimized fit for a large proportion of the population. Biomechanical testing showed the novel fibula plate to be superior to 1/3 tubular plates in 4-point bending tests. The clinical application showed a very high degree of primary implant fit. Only in a small minority of cases further intra-operative implant bending was necessary. Therefore, the goal to develop an implant for the treatment of distal fibular fractures based on the evidence of a large anatomical database could be attained. Biomechanical testing showed good results regarding the stability and the clinical application confirmed the high grade of anatomical fit.

Good manufacturing practice-compliant validation and preparation of BM cells for the therapy of acute myocardial infarction

BACKGROUND: Intracoronary application of BM-derived cells for the treatment of acute myocardial infarction (AMI) is currently being studied intensively. Simultaneously, strict legal requirements surround the production of cells for clinical studies. Thus good manufacturing practice (GMP)-compliant collection and preparation of BM for patients with AMI was established by the Cytonet group. METHODS: As well as fulfillment of standard GMP requirements, including a manufacturing license, validation of the preparation process and the final product was performed. Whole blood (n=6) and BM (n=3) validation samples were processed under GMP conditions by gelafundin or hydroxyethylstarch sedimentation in order to reduce erythrocytes/platelets and volume and to achieve specifications defined in advance. Special attention was paid to the free potassium (<6 mmol/L), some rheologically relevant cellular characteristics (hematocrit <0.45, platelets <450 x 10(6)/mL) and the sterility of the final product. RESULTS: The data were reviewed and GMP compliance was confirmed by the German authorities (Paul-Ehrlich Institute). Forty-five BM cell preparations for clinical use were carried out following the validated methodology and standards. Additionally three selections of CD34+ BM cells for infusion were performed. All specification limits were met. Discussion In conclusion, preparation of BM cells for intracoronary application is feasible under GMP conditions. As the results of sterility testing may not be available at the time of intracoronary application, the highest possible standards to avoid bacterial and other contaminations have to be applied. The increased expense of the GMP-compliant process can be justified by higher safety for patients and better control of the final product.

Validation of the Swiss methane emission inventory by atmospheric observations and inverse modelling

Atmospheric inverse modelling has the potential to provide observation-based estimates of greenhouse gas emissions at the country scale, thereby allowing for an independent validation of national emission inventories. Here, we present a regional-scale inverse modelling study to quantify the emissions of methane (CH₄) from Switzerland, making use of the newly established CarboCount-CH measurement network and a high-resolution Lagrangian transport model. In our reference inversion, prior emissions were taken from the "bottom-up" Swiss Greenhouse Gas Inventory (SGHGI) as published by the Swiss Federal Office for the Environment in 2014 for the year 2012. Overall we estimate national CH₄ emissions to be 196 ± 18 Gg yr⁻¹ for the year 2013 (1σ uncertainty). This result is in close agreement with the recently revised SGHGI estimate of 206 ± 33 Gg yr⁻¹ as reported in 2015 for the year 2012. Results from sensitivity inversions using alternative prior emissions, uncertainty covariance settings, large-scale background mole fractions, two different inverse algorithms (Bayesian and extended Kalman filter), and two different transport models confirm the robustness and independent character of our estimate. According to the latest SGHGI estimate the main CH₄ source categories in Switzerland are agriculture (78 %), waste handling (15 %) and natural gas distribution and combustion (6 %). The spatial distribution and seasonal variability of our posterior emissions suggest an overestimation of agricultural CH₄ emissions by 10 to 20 % in the most recent SGHGI, which is likely due to an overestimation of emissions from manure handling. Urban areas do not appear as emission hotspots in our posterior results, suggesting that leakages from natural gas distribution are only a minor source of CH₄ in Switzerland. This is consistent with rather low emissions of 8.4 Gg yr⁻¹ reported by the SGHGI but inconsistent with the much higher value of 32 Gg yr⁻¹ implied by the EDGARv4.2 inventory for this sector. Increased CH₄ emissions (up to 30 % compared to the prior) were deduced for the north-eastern parts of Switzerland. This feature was common to most sensitivity inversions, which is a strong indicator that it is a real feature and not an artefact of the transport model and the inversion system. However, it was not possible to assign an unambiguous source process to the region. The observations of the CarboCount-CH network provided invaluable and independent information for the validation of the national bottom-up inventory. Similar systems need to be sustained to provide independent monitoring of future climate agreements.

Measuring the Social Identity of Entrepreneurs: Scale Development and International Validation

Recent research indicates that social identity theory offers an important lens to improve our understanding of founders as enterprising individuals, the venture creation process, and its outcomes. Yet, further advances are hindered by the lack of a valid scale that could be used to measure founders' social identities - a problem that is particularly severe because social identity is a multidimensional construct that needs to be assessed properly so that organizational phenomena can be understood. Drawing on social identity theory and the systematic classification of founders' social identities (Darwinians, Communitarians, Missionaries) provided in Fauchart and Gruber (2011), this study develops and empirically validates a 12-item scale that allows scholars to capture the multidimensional nature of social identities of entrepreneurs. Our validation tests are unusually comprehensive and solid, as we not only validate the developed scale in the Alpine region (where it was originally conceived), but also in 12 additional countries and the Anglo-American region. Scholars can use the scale to identify founders' social identities and to relate these identities to micro-level processes and outcomes in new firm creation. Scholars may also link founders' social identities to other levels of analysis such as industries (e.g., industry evolution) or whole economies (e.g., economic growth).

Measuring the Social Identity of Entrepreneurs: Scale Development and International Validation

Social identity theory offers an important lens to improve understanding of founders as enterprising individuals, the venture creation process, and its outcomes. Yet, further advances are hindered by the lack of valid scales to measure founders’ social identities. Drawing on social identity theory and a systematic classification of founders’ social identities (Darwinians, Communitarians, and Missionaries), we develop and test a corresponding 15-item scale in the Alpine region and validate it in 13 additional countries and regions. The scale allows identifying founders’ social identities and relating them to processes and outcomes in entrepreneurship. The scale is available online in 15 languages.