Novel Colorimetric and Light Scatter Methods to Identify and Manage Peritoneal Dialysis-Associated Peritonitis at the Point-of-Care.

Introduction: Peritoneal dialysis (PD)-related peritonitis (PDRP) is a common cause of transfer to hemodialysis, patient morbidity, and is a risk factor for mortality. Associated patient anxiety can deter selection of PD for renal replacement therapy. Diagnosis relies on hospital laboratory tests; however, this might be achieved earlier if such information was available at the point-of-care (POC), thereby significantly improving outcomes. The presence of culturable microbes and the concentration of leukocytes in effluent both aid peritonitis diagnosis, as specified in the International Society for Peritoneal Dialysis (ISPD) diagnostic guidelines. Here, we report the development of 2 new methods providing such information in simple POC tests. Methods: One approach uses a tetrazolium-based chemical reporting system, primarily focused on detecting bacterial contamination and associated vancomycin-sensitivity. The second approach uses a novel forward light-scatter device (QuickCheck) to provide an instant quantitative cell count directly from PD patient effluent. Results: The tetrazolium approach detected and correctly distinguished laboratory isolates, taking 10 hours to provide non-quantitative results. We compared the technical performance of the light scatter leukocyte counting approach with spectrophotometry, hemocytometer counting and flow cytometry (Sysmex) using patient effluent samples. QuickCheck had high accuracy (94%) and was the most precise (coefficient of variation <4%), showing minimal bias, overall performing similarly to flow cytometry. Conclusion: These complementary new approaches provide a simple means to obtain information to assist diagnosis at the POC. The first provides antibiotic sensitivity following 10 hours incubation, whereas the second optical approach (QuickCheck), provides instant accurate total leukocyte count.

An investigative study into an oscillatory reaction in acoustically levitated droplets.

For the first time we have studied an oscillatory chemical reaction (the well-known Belousov-Zhabotinsky (BZ) reaction) in acoustically levitated droplets. Acoustically levitated droplets allow wall-less reaction studies, reduce consumption of sample/reagents, offer high throughput measurements, and enable environmentally friendly chemistry by significantly reducing plastic waste. In this work, microdroplets of the BZ reactants were mixed at the central axis of a low-cost acoustic levitator. The chemical reaction observed in acoustically levitated droplets proceeded in the same way as that in both stirred and unstirred vials where the volume of droplets was 750-fold lower than the solutions in vials. The observed oscillation frequency in droplets was lower than that observed in vials, possibly as a result of evaporative cooling of the droplets. This work has shown that oscillatory reactions can be successfully carried out in acoustically levitated droplets, which allows the application of this technique to areas such as analysis, synthesis and actuation of smart materials and studies of the origins of life.

A study of diffraction-based chitosan leaky waveguide (LW) biosensors.

The waveguide layer of diffraction-based leaky waveguides (LWs) must be made of materials that have low refractive index, are permeable to analytes, can be deposited by spin coating, and can be functionalised and crosslinked. These requirements are fulfilled by thin films of chitosan hydrogels. In this work, we studied the reproducibility of diffraction-based LWs with chitosan waveguides. The average refractive index sensitivity (RIS) and RI limit of detection (LOD) of the eight devices investigated herein were 125.5 ± 3.8 deg RIU-1 and 1.9 × 10-6 ± 1.3 × 10-6 RIU, respectively. While several challenges associated with the realisation of reproducible chitosan LWs have been addressed, reducing the variations in RI LOD requires improving the adhesion of chitosan films to glass substrates, minimising bubbles trapped in microfluidic channels, and using pumps with minimal pulsations. We showed that the buffer baseline of LWs with unmodified chitosan before and after introducing 750 µM bovine serum albumin (BSA), which is equal to the physiological levels of serum albumin, was different by 3.6%. Nevertheless, using biotin, anti-biotin antibody and BSA as exemplar recognition element, analyte and interferent, respectively, we demonstrated that diffraction-based chitosan LWs were suitable for monitoring analyte-RE binding in the presence of 750 µM BSA.

Musculoskeletal ultrasound in hemophilia: Results and recommendations from a global survey and consensus meeting.

INTRODUCTION: For persons with hemophilia, optimization of joint outcomes is an important unmet need. The aim of this initiative was to determine use of ultrasound in evaluating arthropathy in persons with hemophilia, and to move toward consensus among hemophilia care providers regarding the preferred ultrasound protocols for global adaptation. METHODS: A global survey of hemophilia treatment centers was conducted that focused on understanding how and why ultrasound was being used and endeavored to move toward consensus definitions of both point-of-care musculoskeletal ultrasound (POC-MSKUS) and full diagnostic ultrasound, terminology to describe structures being assessed by ultrasound, and how these assessments should be interpreted. Next, an in-person meeting of an international group of hemophilia health care professionals and patient representatives was held, with the objective of achieving consensus regarding the acquisition and interpretation of POC-MSKUS and full diagnostic ultrasound for use in the assessment of musculoskeletal (MSK) pathologies in persons with hemophilia. RESULTS: The recommendations were that clear definitions of the types of ultrasound examinations should be adopted and that a standardized ultrasound scoring/measurement system should be developed, tested, and implemented. The scoring/measurement system should be tiered to allow for a range of complexity yet maintain the ability for comparison across levels. CONCLUSION: Ultrasound is an evolving technology increasingly used for the assessment of MSK outcomes in persons with hemophilia. As adoption increases globally for clinical care and research, it will become increasingly important to establish clear guidelines for image acquisition, interpretation, and reporting to ensure accuracy, consistency, and comparability across groups.

Hydrogel gratings with patterned analyte responsive dyes for spectroscopic sensing.

This is an unprecedented report of hydrogel gratings with an analyte responsive dye immobilised in alternating strips where the patterned dye is its own dispersive element to perform spectroscopy. At each wavelength, the diffraction efficiency of hydrogel gratings is a function of dye absorbance, which in turn is dependent on the concentration of analytes in samples. Thus, changes in intensity of diffracted light of hydrogel gratings were measured for sensing of analytes. Equally, the ratio of diffracted intensities at two wavelengths was used for quantification of analytes to reduce errors caused by variations in intensity of light sources and photobleaching of dyes. 15.27 µm pitch gratings were fabricated by exposing 175 µm thick films of photofunctionalisable poly(acrylamide) hydrogel in a laser interferometric lithography setup, generating an array of alternating lines with and without free functional groups. The freed functional groups were reacted with pH sensitive fluorescein isothiocyanate to create gratings for measurement of pH. The ratio of intensity of diffracted light of hydrogel gratings at 430 and 475 nm was shown to be linear over 4 pH units, which compares favourably with ∼2 pH units for conventional absorption spectroscopy. This increased dynamic range was a result of cancellation of the opposite non-linearities in the pH response of the analyte responsive dye and the diffraction efficiency as a function of dye absorbance.

A Self-Referenced Diffraction-Based Optical Leaky Waveguide Biosensor Using Photofunctionalised Hydrogels.

We report a novel self-referenced diffraction-based leaky waveguide (LW) comprising a thin (~2 µm) film of a photofunctionalisable hydrogel created by covalent attachment of a biotinylated photocleavable linker to chitosan. Streptavidin attached to the chitosan via the photocleavable linker was selectively removed by shining 365 nm light through a photomask to create an array of strips with high and low loading of the protein, which served as sensor and reference regions respectively. The differential measurements between sensor and reference regions were used for measuring analytes (i.e., biotin protein A and IgG) while reducing environmental and non-specific effects. These include changes in temperature and sample composition caused by non-adsorbing and adsorbing species, leading to reduction in effects by ~98%, ~99%, and ~97% respectively compared to the absolute measurements. The novelty of this work lies in combining photofunctionalisable hydrogels with diffraction-based LWs for referencing. This is needed to realise the full potential of label-free optical biosensors to measure analyte concentrations in real samples that are complex mixtures, and to allow for sample analysis outside of laboratories where drifts and fluctuations in temperature are observed.

Optimization Synthesis and Biosensing Performance of an Acrylate-Based Hydrogel as an Optical Waveguiding Sensing Film.

The metal-clad leaky waveguide (MCLW) is an optical biosensor consisting of a metal layer and a low index waveguide layer on a glass substrate. This label-free sensor measures refractive index (RI) changes within the waveguide layer. This work shows the development and optimization of acrylate based-hydrogel as the waveguide layer formed from PEG diacrylate (PEGDA, Mn 700), PEG methyl ether acrylate (PEGMEA, Mn 480), and acrylate-PEG2000-NHS fabricated on a substrate coated with 9.5 nm of titanium. The acrylate-based hydrogel is a synthetic polymer, so properties such as optical transparency, porosity, and hydrogel functionalization by a well-controlled reactive group can be tailored for immobilization of the bioreceptor within the hydrogel matrix. The waveguide sensor demonstrated an equal response to solutions of identical RI containing small (glycerol) and large (bovine serum albumin; BSA) analyte molecules, indicating that the hydrogel waveguide film is highly porous to both sizes of molecule, thus potentially allowing penetration of a range of analytes within the porous matrix. The final optimized MCLW chip was formed from a total hydrogel concentration of 40% v/v of PEGMEA-PEGDA (Mn 700), functionalized with 2.5% v/v of acrylate-PEG2000-NHS. The sensor generated a single-moded waveguide signal with a RI sensitivity of 128.61 ± 0.15° RIU-1 and limit of detection obtained at 2.2 × 10-6 RIU with excellent signal-to-noise ratio for the glycerol detection. The sensor demonstrated RI detection by monitoring changes in the out-coupled angle resulting from successful binding of d-biotin to streptavidin immobilized on functionalized acrylate hydrogel, generating a binding signal of (12.379 ± 0.452) × 10-3°.

Effectiveness, cost-effectiveness and safety of gabapentin versus placebo as an adjunct to multimodal pain regimens in surgical patients: protocol of a placebo controlled randomised controlled trial with blinding (GAP study).

INTRODUCTION: Gabapentin is an antiepileptic drug currently licensed to treat epilepsy and neuropathic pain but has been used off-label to treat acute postoperative pain. The GAP study will compare the effectiveness, cost-effectiveness and safety of gabapentin as an adjunct to standard multimodal analgesia versus placebo for the management of pain after major surgery. METHODS AND ANALYSIS: The GAP study is a multicentre, double-blind, randomised controlled trial in patients aged 18 years and over, undergoing different types of major surgery (cardiac, thoracic or abdominal). Patients will be randomised in a 1:1 ratio to receive either gabapentin (600 mg just before surgery and 600 mg/day for 2 days after surgery) or placebo in addition to usual pain management for each type of surgery. Patients will be followed up daily until hospital discharge and then at 4 weeks and 4 months after surgery. The primary outcome is length of hospital stay following surgery. Secondary outcomes include pain, total opioid use, adverse health events, health related quality of life and costs. ETHICS AND DISSEMINATION: This study has been approved by the Research Ethics Committee . Findings will be shared with participating hospitals and disseminated to the academic community through peer-reviewed publications and presentation at national and international meetings. Patients will be informed of the results through patient organisations and participant newsletters. TRIAL REGISTRATION NUMBER: ISRCTN63614165.

Disposable Injection Molded Conductive Electrodes Modified with Antimony Film for the Electrochemical Determination of Trace Pb(II) and Cd(II).

This work describes a novel electrochemical sensor fabricated by an injection molding process. This device features a conductive polymer electrode encased in a plastic holder and electroplated in situ with a thin antimony film. The antimony film sensor was applied to the determination of Pb(II) and Cd(II) by anodic stripping voltammetry (ASV). The deposition of Sb on the sensor was studied by cyclic voltammetry (CV) and microscopy. The experimental variables (concentration of the antimony plating solution, deposition potential and time, stripping waveform) were investigated, and the potential interferences were studied and addressed. The limits of detection were 0.95 µg L-1 for Pb(II) and 1.3 for Cd(II) (at 240 s of preconcentration) and the within-sensor percentage relative standard deviations were 4.2% and 4.9%, respectively, at the 25 µg L-1 level (n = 8). Finally, the sensor was applied to the determination of Pb(II) and Cd(II) in a phosphorite sample and a lake water sample.

A feasibility study of a leaky waveguide aptasensor for thrombin.

This proof-of-principle study demonstrates the feasibility of a leaky waveguide (LW) aptasensor, where aptamers were immobilised in a mesoporous chitosan waveguiding film for the detection of thrombin. This work has demonstrated that aptamers immobilised in hydrogels retain their affinity and selectivity towards their target and thus can be used as bioreceptors. The use of antibodies as bioreceptors for sensing thrombin is not viable because it is a serine protease, which will cleave the antibodies. Currently used assays based on clotting time and chromogenic/fluorogenic substrates have limited potential for thrombin measurement in whole blood. Using the initial binding rate over the first 5 min, the limit of detection of our LW aptasensor for thrombin was ∼22 nM. The sensor was tested with spiked serum samples, giving a reading of 46.1 ± 4.6 nM for a sample containing 50 nM thrombin. Our proposed sensor combines the robustness and low cost of aptamers as molecular recognition elements with the simple fabrication process of the chitosan-based leaky waveguide, making LW aptasensors highly attractive for applications in point-of-care diagnostics and healthcare monitoring.

Method for Determining Average Iron Content of Ferritin by Measuring its Optical Dispersion.

We report a method where the refractive index increments of an iron storage protein, ferritin, and apoferritin (ferritin minus iron) were measured over the wavelength range of 450-678 nm to determine the average iron content of the protein. The protein used in this study had ∼3375 iron atoms per molecule. The measurement of optical dispersion over the broad wavelength range was enabled by the use of mesoporous leaky waveguides (LWs) made of chitosan. We present a facile approach for fabricating mesoporous chitosan waveguides for improving the measurement sensitivity of macromolecules such as ferritin. Mesoporous materials allow macromolecules to diffuse into the waveguide, maximizing their interaction with the optical mode and thus increasing sensitivity by a factor of ∼9 in comparison to nonporous waveguides. The sensitivity was further improved and selectivity toward ferritin was achieved by the incorporation of antibodies in the waveguide. The method presented in this work is a significant advance over the state of the art method, the enzyme linked immunosorbent assay (ELISA) used in clinics, because it allows determining the average content of ferritin in a single step. The average iron content of ferritin is an important marker for conditions such as injury, inflammation, and infection. Thus, the approach presented here of measuring optical dispersion to determine the average iron content of ferritin has a significant potential to improve the point of care analysis of the protein for disease diagnosis and screening.

Over two decades of orthopaedic surgery in patients with inhibitors-Quantifying the complication of bleeding.

Patients with haemophilia who have developed inhibitors against factor VIII (FVIII) or factor IX present a significant concern to those surgeons who operate on them. The evidence base for bypassing agents such as recombinant factor VIIa and activated prothrombin complex concentrate has amassed over several decades. The literature is open to positive interpretation on the successful use of these agents in the treatment of inhibitor-positive patients. However, there are equally persistent concerns amongst surgeons, in particular orthopaedic surgeons, regarding the high complication rate of bleeding. To explore and quantify this concern, we present a literature review spanning two decades of publications on haemophilia patients with inhibitors undergoing orthopaedic surgery. Irrespective of the progress made with haemostatic protocols, trepidation on embarking on surgery is valid. The high risk of bleeding is a function of the inherent complexity of the disease and rightfully translates into difficulties in its management. Combined with the prospect of orthopaedic surgery, those involved in the care of such patients are justified in their continued anxiety and diligence when considering the benefits in quality of life against the prevalent complications.

Determination of Pb(II) by sequential injection/stripping analysis at all-plastic electrochemical fluidic cells with integrated composite electrodes.

This work reports the development of a sequential injection/stripping analysis method for the determination of trace Pb(II) at injection-moulded electrochemical fluidic cells. Conducting carbon fibre-loaded polystyrene electrodes were integrated within the plastic cells. The flow-through cells were incorporated into a home-made sequential injection analysis (SIA) manifold. Different experimental parameters for the detection of Pb(II) were investigated including the type and concentration of the supporting electrolyte, the conditions of the stripping step, the volume of the sample, the flow rate and the accumulation potential. The LOD for Pb(II) was 0.5µgL(-1), the within-cell % relative standard deviation (n=8) was 3.1% and the between-cell % relative standard deviation (n=5) was 8.9% for 25µgL(-1) Pb(II). The cells were applied to the determination of Pb(II) in tapwater and a phosphate fertilizer sample.

Accurate lung volume measurements in vitro using a novel inert gas washout method suitable for infants.

BACKGROUND: Multiple breath washout (MBW) in infants presents a number of technical challenges. Conventional MBW is based on simultaneous measurement of flow and gas concentrations. These two signals are aligned and combined to derive expired gas volumes from which lung volumes and measures of ventilation inhomogeneity are calculated. Accuracy of measurement becomes increasingly vulnerable to errors in gas signal alignment at fast respiratory rates. In this paper we describe an alternative method of performing MBW in infants. Expired gas is collected and analyzed to derive functional residual capacity (FRC) and lung clearance index (LCI). This eliminates the need for simultaneous measurement of flow, and integration of flow and gas signals, and significantly reduces deadspace. METHODS: A highly accurate lung model incorporating BTPS conditions was used to generate realistic infant breathing parameters: FRC of 100-250 mls with respiratory rate of 20-60 min(-1) . In vitro accuracy of FRC measurement using the novel MBW method was assessed using the model. RESULTS: Overall mean error (standard deviation) of FRC measurement was -1.0 (3.3)% with 90% of tests falling within ±5%. DISCUSSION: FRC measurement using the novel method has superior accuracy in vitro than previously described systems. By uncoupling the measurement of gas volumes from real-time flow and gas measurement, this system offers an alternative method of MBW which is well suited to infants.

Compartment syndrome in patients with haemophilia.

BACKGROUND: Acute compartment syndrome (ACS) is an uncommon but potentially devastating condition. METHODS AND RESULTS: There are scattered case reports and case series in the literature of ACS in persons with haemophilia (PWH), and even fewer in PWH and inhibitors. The management of compartment syndrome in these scenarios is controversial and often anecdotal. In addition haematological outcomes are frequently quoted but functional outcomes are generally overlooked. This article aims to provide an overview of ACS and its contemporary management. We also review the literature and outcomes of patients with haemophilia who develop ACS in an effort to assess the best treatment modality. CONCLUSION: In the majority of cases ACS settles with normalisation of the clotting cascade. Specialist haematological input is mandatory before surgical intervention should be considered, especially in PWH and inhibitors.

Artefacts at the liquid interface and their impact in miniaturized biochemical assay.

Droplet microfluidic technology has the potential to significantly reduce reagent use, and therefore, lower costs of assays employed in drug discovery campaigns. In addition to the reduction in costs, this technology can also reduce evaporation and contamination which are often problems seen in miniaturized microtitre plate formats. Despite these advantages, we currently advise caution in the use of these microfluidic approaches as there remains a lack of understanding of the artefacts of the systems such as reagent partitioning from droplet to carrier oil and interaction of the biological reagents with the water-oil interface. Both types of artefact can lead to inaccurate and misleading data. In this paper, we present a study of the partitioning of a number of drug-like molecules in a range of oils and evidence of protein binding at the water-oil interface which results in reduced activity of a cytochrome P450 enzyme. Data presented show that the drug-like molecules partitioned the least into fluorocarbon oils and the interaction of the 1A2 cytochrome at the water-oil interface resulted in a lower or complete absence of enzyme activity. This loss of activity of cytochrome 1A2 could be restored by the use of secondary blocking proteins although changes in the pharmacology of known 1A2 inhibitors were observed. The artefacts described here due to reagents partitioning into the carrier oil or protein binding at the water-oil interface significantly impact the potential use of these microfluidic systems as a means to carry out miniaturized biological assays, and further work is needed to understand the impact and reduction of these phenomena.

Microfluidic channel-assisted screening of hematopoietic malignancies.

A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro-channel, which resembled a 250-fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50-fold less probe consumption and 10-fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro-channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over-score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH.

A novel method of diagnosing autonomic dysfunction in carpal tunnel syndrome: measuring skin capacitance.

Carpal Tunnel Syndrome (CTS) is normally diagnosed via its sensory and motor manifestations. The associated autonomic dysfunction has not been exploited to its full potential as a diagnostic tool due to the difficulties in quantifying it. We aim to demonstrate that autonomic dysfunction of CTS can be quantified by measuring skin capacitance. Fifty-one patients with clinical signs and electrophysiological evidence of CTS in 89 hands were recruited. Skin capacitance was measured using Corneometer CM825 (C&K Electronic, GmbH) from the palmar aspect of the distal phalanx of the index and little finger of the affected hand. Healthy gender- and age-matched individuals were recruited as controls. The mean ratio of hydration of the index to the little finger was 0.82. The mean difference was 10.98 arbitrary units. The control group consisted of 151 subjects (80 Male & 71 Female) and 302 hands with an average age of 40.1 years (18-81 years). The mean ratio of hydration of the index to the little finger was 0.87. The mean difference was 8.67 arbitrary units. The measurement ratios (index to little finger skin hydration) between the two groups was compared directly and gave a significant mean difference of 0.05 arbitrary units. Statistically significant differences in skin capacitance between CTS patients and controls have been demonstrated and quantified using a rapid and simple method. This can be used in clinic to reduce the reliance on Nerve Conduction Studies for diagnosing CTS.