Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications.

: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate-coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization-Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme-substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

Nanofiber-integrated miniaturized systems: an intelligent platform for cancer diagnosis.

Cancer diagnostic tools enabling screening, diagnosis, and effective disease management are essential elements to increase the survival rate of diagnosed patients. Low abundance of cancer markers present in large amounts of interferences remains the major issue. Moreover, current diagnostic technologies are restricted to high-resourced settings only. Integrating nanofibers into miniaturized analytical systems holds a significant promise to address these challenges as demonstrated by recent publications. A large surface area, three-dimensional porous network, and diverse range of functional chemistries make nanofibers an excellent candidate as immobilization support and/or transduction elements, enabling high capture yield and ultrasensitive detection in miniaturized devices. Functional nanofibers have thus been used to isolate and detect various cancer-related biomarkers with a high degree of success in both on-chip and off-chip platforms. In fact, the chemical and functional adaptability of nanofibers has been exploited to address the technical challenges unique to each of the cancer markers in body fluids, where circulating tumor cells are prominently investigated among others (proteins, nucleic acids, and exosomes). So far, none of the work has exploited the nanofibers for cancer-derived exosomes, opening an avenue for further research effort. The trend and future prospects signal possibilities to strengthen the implementation of nanofiber-miniaturized system hybrid for a next generation of cancer diagnostic platforms both in clinical and point-of-care testing.

Practical considerations on redesigning internal quality control process to verify the traceability of clinical results for decision-making.

Traceability is an important tool in the harmonization and standardization of reporting laboratory results, making them comparable across measurement systems. Driven by International Standardization Organization (ISO) 15189 accreditation requirements, medical laboratories have entered the era of metrological traceability. Although calibrators are a key component in the entire metrological traceability system, there is controversy over internal quality control (IQC) materials. It has been proposed that trueness materials supplied by the system's manufacturer with metrological traceability can be used to confirm that the performance of the measuring system is properly unbiased. This article focuses on the implementation challenges and operational hurdles of applying traceability concepts to IQC materials for trueness verification in medical laboratories regarding the most recent 2022 edition of ISO 15189 standard requirements for IQC and metrological traceability. There are practical considerations concerning the acquiring of IQC materials. We must acknowledge the limitations and restrictions that manufacturers and laboratories face before the recommendations can be applied in routine practices.

Suggested guide to using lactate gap as a surrogate marker in the diagnosis of ethylene glycol overdose.

BBACKGROUND: Ethylene glycol (EG) poisoning, if not diagnosed rapidly, can lead to poor patient outcomes. Gas Chromatography (GC) is primarily used for EG quantitation which is rarely available, and the turn-around time may be prolonged. Most lactate results from point-of care (POCT) methods are falsely elevated in EG poisoning compared with automated chemistry analyser results. In combination the lactate gap (POCT-Automated chemistry) can be used as surrogate marker in just about all laboratories to indicate likely EG toxicity and guide treatment. CASE REPORT: A male presented by ambulance to hospital with severe agitation requiring mechanical ventilation to facilitate ongoing management. Venous blood gas analysis confirmed a high anion gap metabolic acidosis (HAGMA) with an elevated lactate. The lactate and osmolarity measured in the laboratory showed a normal lactate and high osmolarity, giving a large osmolar gap. The patient was immediately commenced on renal replacement therapy for presumed EG poisoning to minimize kidney injury, and the treatment continued for 19 hours. A very high EG concentration was confirmed by GC the next day. CONCLUSION: An elevated lactate gap along with a HAGMA and osmolar gap can provide rapid surrogate laboratory data indicating EG poisoning enabling timely treatment and better patient outcomes.

Sample comparison of BÜHLMANN fCAL Turbo and OC-FCa faecal calprotectin methods.

BACKGROUND: Faecal calprotectin is an inflammatory marker used to triage patients for further investigation with suspected inflammatory bowel disease (IBD). Our current method requires faecal samples be sent to the laboratory, where calprotectin is extracted before analysis. This is a time-consuming, potential bottleneck in the pathway. We have recently evaluated the OC-SENSOR PLEDIA fCAL method that uses the same sampling device as used in some bowel cancer screening and symptomatic colorectal cancer programmes that detect faecal haemoglobin. The below study is a comparison of the OC-FCa method with the BÜHLMANN fCAL Turbo which is used routinely within BSPS. METHOD: 150 homogenised and 110 non-homogenised faecal samples were loaded into OC-Sampling Bottle 3 and BÜHLMANN CALEX cap sampling devices. The samples were then analysed on their respective systems according to manufacturer's instructions. RESULTS: The OC-FCa assay had a mean positive bias of 67.3% (homogenised) and 88.4% (non-homogenised). Homogenised samples showed substantial agreement between the methods for normal (<50 µg/g) and elevated (150+µg/g) risk categories (k = 0.794, k = 0.788, respectively) and moderate agreement for borderline (51-150 µg/g) (k = 0.25) according to the current Berkshire and Surrey Pathology Service (BSPS) guidelines. Non-homogenised samples had none to slight agreement for normal and borderline values (k = 0.02 for both) and moderate agreement for elevated (k = 0.596). CONCLUSION: The OC-FCa method is a viable alternative for faecal calprotectin testing, but requires an adjustment to clinical cut-off values due to the lack of standardisation and strong positive bias. A clinical comparative study is required to assess the impact of patients collecting their own samples into the devices, as this may negate any potential degradation samples may exhibit during transit to the laboratory.

Natural Indigenous Paper Substrates for Colorimetric Bioassays in Portable Analytical Systems: Sustainable Solutions from the Rain Forests to the Great Plains.

Point-of-care (POC) devices can provide inexpensive, practical, and expedited solutions for applications ranging from biomedicine to environmental monitoring. This work reports on the development of low-cost microfluidic substrates for POC systems suitable for analytical assays, while also satisfying the need for social and environmentally conscious practices regarding circular economy, waste reduction, and the use of local resources. Thus, an innovative greener process to extract cellulose from plants including abaca, cotton, kozo, linen, and sisal, originating from different places around the world, is developed, and then the corresponding paper substrates are obtained to serve as platforms for POC assays. Hydrophobic wax is used to delineate channels that are able to guide solutions into chambers where the colorimetric assay for total cholesterol quantification is carried out as a proof of concept. Morphological and physicochemical analyses are performed, including the evaluation of fiber diameter, shape and density, and mechanical and thermal properties, together with peel adhesion of the printed wax channels. Contact angle and capillary flow tests ascertain the suitability of the substrates for liquid assays and overall viability as low-cost, sustainable microfluidic substrates for POC applications.

UK National audit of the measurement of ammonia.

BACKGROUND: Severe hyperammonaemia is associated with significant morbidity and mortality. Rapid analysis and reporting of ammonia results is essential to prevent patient harm. The aim was to investigate the laboratory sample acceptance criteria, ammonia analysis and the reporting of ammonia results. METHODS: A questionnaire was distributed to clinical biochemistry laboratories in the United Kingdom. The results were collated and compared to updated best practice guidelines on hyperammonaemia issued by the Metabolic Biochemistry Network (MetBio.net). RESULTS: Seventy-six laboratories responded to the audit questionnaire. Although 83% laboratories are aware of the updated MetBio.net hyperammonaemia guidelines, most laboratories continue to reject samples for ammonia that are 'too old' for analysis (64%), haemolysed (72%) or not sent on ice (24%). Rapid ammonia analysis is available in 96% laboratories and all laboratories offer ammonia analysis on a 24/7 basis. Nearly all laboratories had implemented critical phoning limits for ammonia. CONCLUSIONS: Laboratories are rejecting samples for ammonia analysis that have not been collected/transported in an optimal manner. Laboratories should review their sample acceptance criteria for ammonia and accept all samples in order to avoid delaying the diagnosis and management of hyperammonaemia.

Closed-loop control systems for pumps used in portable analytical systems.

The demand for accurate control of the flowrate/pressure in chemical analytical systems has given rise to the adoption of mechatronic approaches in analytical instruments. A mechatronic device is a synergistic system which combines mechanical, electronic, computer and control components. In the development of portable analytical devices, considering the instrument as a mechatronic system can be useful to mitigate compromises made to decrease space, weight, or power consumption. Fluid handling is important for reliability, however, commonly utilized platforms such as syringe and peristaltic pumps are typically characterized by flow/pressure fluctuations and slow responses. Closed loop control systems have been used effectively to decrease the difference between desired and realized fluidic output. This review discusses the way control systems have been implemented for enhanced fluidic control, categorized by pump type. Advanced control strategies used to enhance the transient and the steady state responses are discussed, along with examples of their implementation in portable analytical systems. The review is concluded with the outlook that the challenge in adequately expressing the complexity and dynamics of the fluidic network as a mathematical model has yielded a trend towards the adoption of experimentally informed models and machine learning approaches.

Analysing olive ripening with digital image RGB histograms.

Digital images are commonly used to monitor processes that are based on colour changes due to their simplicity and easy capture. Colour information in these images can be analysed objectively and accurately using colour histograms. One such process is olive ripening, which is characterized by changes in chemical composition, sensory properties and can be followed by changes in physical appearance, mainly colour. The reference method to quantify the ripeness of olives is the Maturity Index (MI), which is determined by trained experts assigning individual olives into a colour scale through visual inspection. Instead, this study proposes a methodology based on Chemometrics Assisted Colour Histogram-based Analytical Systems (CACHAS) to automatically assess the MI of olives based on R, G, and B colour histograms derived from digital images. The methodology was shown to be easily transferable for routine analysis and capable of controlling the ripening of olives. The study also confirms the high potential of digital images to understand the ripening process of olives (and potentially other fruits) and to predict the MI with satisfactory accuracy, providing an objective and reproducible alternative to visual inspection of trained experts.

Validation of the Martin method to estimate low-density lipoprotein cholesterol concentrations in Japanese populations and a modified method for laboratory information system application.

OBJECTIVES: High concentrations of low-density lipoprotein cholesterol (LDL-C) are a risk factor for cardiovascular disease. We validated the efficacy of the Martin method is useful in the estimation of LDL-C concentrations was validated in Japanese populations and derived a modified Martin method for easy laboratory information system applications. METHODS: We created 3 subject groups, including 2664 health check-up participants registered with the Resource Center for Health Science, 29,806 clinical patients (A) in the Gifu University Hospital, and 113,716 clinical patients (B) in the Fujita Health University Hospital. Each method to estimate serum LDL-C concentrations (Friedewald formula, Martin method and modified Martin method) was validated by correlation analysis with serum LDL-C concentrations measured using a direct method. RESULTS: The correlation coefficients with the direct method in terms of the Friedewald formula, Martin method, and modified Martin method were 0.963, 0.972 and 0.970 in the health check-up participants; 0.946, 0.962 and 0.961 in clinical patients A; and 0.961, 0.979 and 0.978 in clinical patients B, respectively. Concordance ratios with using the direct method in the Friedewald formula, Martin method and modified Martin method were 82.8%, 85.5% and 85.3% in the health check-up participants; 76.4%, 80.5% and 80.2% in clinical patients A; and 76.1%, 82.6% and 82.6% in clinical patients B, respectively. CONCLUSION: Our results show that the Martin and modified Martin methods display good performance in terms of the estimation of LDL-C concentrations among triglyceride concentrations of a wide range, and they may thus be useful for estimating LDL-C concentrations.

Factoring ethics in management algorithms for municipal information-analytical systems.

The discourse on the ethics of artificial intelligence (AI) has generated a plethora of different conventions, principles and guidelines outlining an ethical perspective on the use and research of AI. However, when it comes to breaking down general implications to specific use cases, existent frameworks have been remaining vague. The following paper aims to fill this gap by examining the ethical implications of the use of information analytical systems through a management approach for filtering the content in social media and preventing information thrusts with negative consequences for human beings and public administration. The ethical dimensions of AI technologies are revealed through deduction of general challenges of digital governance to applied level management technics.

Binding the gap between experiments, statistics, and method comparison: A tutorial for computing limits of detection and quantification in univariate calibration for complex samples.

The present tutorial aims to review the most frequently reported criteria for the calculation of the limits of detection (LOD) and quantification (LOQ) in univariate calibration, summarizing their fundamentals, advantages, and limitations. The current criteria for estimating LOD and LOQ are based on diverse theoretical and/or empirical assumptions and require different amounts of experimental data, making the calculation rather complex in some cases. Moreover, alternative forms for calculating LOD/LOQ frequently lead to dissimilar results. This scenario might worsen in the case of complex analytical systems. Throughout this tutorial, different forms of calculating LOD/LOQ are illustrated using previously reported experimental datasets in the environmental chemistry field as examples. The influence of the sample matrix during the estimation of LOD/LOQ parameters is investigated through one calibration approache. The discrepancies in the obtained results with different criteria for the calculation of LOD/LOQ are highlighted. Finally, general guidelines and recommendations regarding experimental and data processing issues are proposed, aiming to promote fair criteria for the comparison of different analytical methodologies in terms of prediction ability and detection capability.

Potentiometric pH Sensor Based on Flexible Screen-Printable Polyaniline Composite for Textile-Based Microfluidic Applications.

Skin pH can be used for monitoring infections in a healing wound, the onset of dermatitis, and hydration in sports medicine, but many challenges exist in integrating conventional sensing materials into wearable platforms. We present the development of a flexible, textile-based, screen-printed electrode system for biosensing applications, and demonstrate flexible polyaniline (PANI) composite-based potentiometric sensors on a textile substrate for real-time pH measurement. The pH response of the optimized PANI/dodecylbenzene sulfonic acid/screen-printing ink composite is compared to electropolymerized and drop-cast PANI sensors via open circuit potential measurements. High sensitivity was observed for all sensors between pH 3-10, with a composite based on PANI emeraldine base, demonstrating sufficient response time and a linear sensitivity of -27.9 mV/pH. This exceeded prior flexible screen-printed pH sensors in which all parts of the sensor, including the pH sensing material, are screen-printed. Even better sensitivity was observed for a PANI emeraldine salt composite (-42.6 mV/pH), although the response was less linear. Furthermore, the sensor was integrated into a screen-printed microfluidic channel demonstrating sample isolation during measurement for wearable, micro cloth-based analytical devices. This is the first fully screen-printed flexible PANI composite pH sensor demonstrated on a textile substrate that can additionally be integrated with textile-based microfluidic channels.

Uninterpretable cerebrospinal fluid absorbance scans caused by antibiotic therapy.

BACKGROUND: The revised national guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage (UK) provide an objective means of assessing cerebrospinal fluid samples to determine the risk of subarachnoid haemorrhage. The guidelines are intended for general use, but samples rendered uninterpretable due to the presence of the antibiotic doxycycline have been described. Here, further cases of antibiotic-based interference, and their implications, are presented. METHODS: An archival search of cerebrospinal fluid spectra performed at Hallands County Hospital Halmstad was performed for the years 2011 and 2016-2019 in an attempt to locate instances of interference. Each case of suspected interference was further investigated with in vitro reproduction experiments as a means of confirmation and assessment of potential clinical impact. RESULTS: A total of 10 cases of cerebrospinal fluid curve interference were discovered: six due to doxycycline, three due to metronidazole and one due to tetracycline. Interference caused by the tetracycline class was revealed through in vitro experimentation to cause an apparent decrease in the sample's net bilirubin absorbance; the presence of xanthochromia on visual inspection was, however, conserved. CONCLUSIONS: The problem of cerebrospinal fluid absorbance curve interference might be more common than previously suspected. Due to the potential net bilirubin absorbance-lowering effect of tetracyclines, the author recommends visual examination of cerebrospinal fluid samples in every case.

EXPRESS: Uninterpretable cerebrospinal fluid absorbance scans caused by antibiotic therapy.

Background The revised national guidelines for analysis of cerebrospinal fluid for bilirubin in suspected subarachnoid haemorrhage (UK) provide an objective means of assessing cerebrospinal fluid (CSF) samples to determine risk of subarachnoid haemorrhage (SAH). The guidelines are intended for general use, but samples rendered uninterpretable due to the presence of the antibiotic doxycycline have been described. Here, further cases of antibiotic- based interference, and their implications, are presented.Methods An archival search of CSF spectra performed at Hallands County Hospital Halmstad was performed for the years 2011 and 2016- 2019 in an attempt to locate instances of interference. Each case of suspected interference was further investigated with in vitro reproduction experiments as a means of confirmation and assessment of potential clinical impact.Results A total of 10 cases of CSF curve interference were discovered: 6 due to doxycycline, 3 due to metronidazole and 1 due to tetracycline. Interference due to the tetracycline class was revealed through in vitro experimentation to cause an apparent decrease in the sampleâs net bilirubin absorbance(NBA); the presence of xanthochromia on visual inspection was, however, conserved.Conclusions The problem of CSF absorbance curve interference might be more common than previously suspected. Due to the potential NBA- lowering effect of tetracyclines, the author recommends visual examination of CSF samples in every case.

Clinical Chemistry for Developing Countries: Mass Spectrometry.

Early disease diagnosis is necessary to enable timely interventions. Implementation of this vital task in the developing world is challenging owing to limited resources. Diagnostic approaches developed for resource-limited settings have often involved colorimetric tests (based on immunoassays) due to their low cost. Unfortunately, the performance/sensitivity of such simplistic tests are often limited and significantly hinder opportunities for early disease detection. A new criterion for selecting diagnostic tests in low- and middle-income countries is proposed here that is based on performance-to-cost ratio. For example, modern mass spectrometry (MS) now involves analysis of the native sample in the open laboratory environment, enabling applications in many fields, including clinical research, forensic science, environmental analysis, and agriculture. In this critical review, we summarize recent developments in chemistry that enable MS to be applied effectively in developing countries. In particular, we argue that closed automated analytical systems may not offer the analytical flexibility needed in resource-limited settings. Alternative strategies proposed here have potential to be widely accepted in low- and middle-income countries through the utilization of the open-source ambient MS platform that enables microsampling techniques such as dried blood spot to be coupled with miniature mass spectrometers in a centralized analytical platform. Consequently, costs associated with sample handling and maintenance can be reduced by >50% of the total ownership cost, permitting analytical measurements to be operated at high performance-to-cost ratios in the developing world.

Fluorinated Polymer Membranes as Advanced Substrates for Portable Analytical Systems and Their Proof of Concept for Colorimetric Bioassays.

Portable analytical systems are increasingly required for clinical analysis or environmental monitoring, among others, being materials with tailored physicochemical properties among the main needs for successful functional implementation. This article describes the processing of fluorinated poly(vinylidene-co-trifluorethylene), P(VDF-TrFE), membranes with tailored morphological and physicochemical properties to be used as microfluidic substrates for portable analytical systems, commonly called point-of-care systems in the medical field. The morphology of the developed membranes includes spherulitic, porous, randomly oriented, and oriented fibers. Furthermore, the processed hydrophobic P(VDF-TrFE) membranes were post-treated by oxygen plasma to make them superhydrophilic. The influence of morphology and plasma treatment on the physicochemical properties and capillary flow rates was evaluated. Microfluidic systems were then designed and printed by wax printing for the colorimetric quantification of glucose. The systems comprise eight reaction chambers, each glucose concentration (25, 50, 75, and 100 mg dL-1) being measured in two reaction chambers separately and at the same time. The results demonstrate the suitability of the developed microfluidic substrates based on their tailorable morphology, improved capillary flow rate, wax print quality, homogeneous generation of colorimetric reaction, and excellent mechanical properties. Finally, the possibility of being reused, along with their electroactive properties, can lead to a new generation of microfluidic substrates based on fluorinated membranes.

Cardiac well-being indexes: a decision support tool to monitor cardiovascular health.

Despite the increasing awareness about its severity and the importance of adopting preventive habits, cardiovascular disease remains the leading cause of death worldwide. Most people already recognize that a healthy lifestyle, which includes a balanced diet and the practice of physical activity, is essential to prevent this disease. However, since few simple mechanisms allow a self-assessment and a continuous monitoring of the level of cardiac well-being, people are not conscious enough about their own cardiovascular health status. In this context, this paper presents and describes a tool related to the creation of cardiac well-being indexes that allow a quick and intuitive monitoring and visualization of the users' cardiovascular health level over time. For its implementation, data mining techniques were used to calculate the indexes, and a data warehouse was built to archive the data and to support the construction of dashboards for presenting the results.

The impact of the analytical performance specifications of calcium and albumin on adjusted calcium.

AIM: The generation of accurate, comparable results from traceable measurement procedures is a primary goal in harmonization efforts. In this study, the analytical performance of routine methods for calcium and albumin measurement is assessed to define the impact of the analytical bias of calcium and albumin on adjusted calcium equation performance and on reference intervals. METHOD: In collaboration with the Wales External Quality Assurance Scheme, six months' worth of anonymized data that cover a concentration range of clinical interest were collected. The data were grouped by analytical platforms/methods. RESULTS: Albumin BCG methods are positively biased (8%) to BCP methods. The overall bias for BCP methods ranges from 5.1 to -4.3% and the overall bias for BCG methods is from 2 to -6.7%. Bias for both methods is higher than the allowable minimal bias for albumin. Calcium concentrations for Roche Cobas CPC and NM-BAPTA, Beckman Arsenazo III, Abbott Architect Arsenazo III were within bias of 1.5 to -1%. However, Siemens calcium methods CPC and Arsenazo III appear to suffer from concentration-dependent bias ranging from +3 to -6%, which exceeds even the minimal allowable limits for calcium (1.3%). Adjusted calcium shows significant bias of 11%. Even with the exclusion of Siemens Advia, the scatter of adjusted calcium results exceeds that for total calcium. CONCLUSION: This study shows wider than acceptable analytical variation for albumin and calcium. This variation may contribute to overall adjusted calcium equations variation and invalidate the application of a harmonized reference interval for calcium and albumin.