Evaluation of hemifield sector analysis protocol in multifocal visual evoked potential (MFVEP) objective perimetry for the diagnosis and early detection of glaucomatous field defects

Visual field assessment is a core component of glaucoma diagnosis and monitoring, and the Standard Automated Perimetry (SAP) test is considered up until this moment, the gold standard of visual field assessment. Although SAP is a subjective assessment and has many pitfalls, it is being constantly used in the diagnosis of visual field loss in glaucoma. Multifocal visual evoked potential (mfVEP) is a newly introduced method used for visual field assessment objectively. Several analysis protocols have been tested to identify early visual field losses in glaucoma patients using the mfVEP technique, some were successful in detection of field defects, which were comparable to the standard SAP visual field assessment, and others were not very informative and needed more adjustment and research work. In this study, we implemented a novel analysis approach and evaluated its validity and whether it could be used effectively for early detection of visual field defects in glaucoma. OBJECTIVES: The purpose of this study is to examine the effectiveness of a new analysis method in the Multi-Focal Visual Evoked Potential (mfVEP) when it is used for the objective assessment of the visual field in glaucoma patients, compared to the gold standard technique. METHODS: 3 groups were tested in this study; normal controls (38 eyes), glaucoma patients (36 eyes) and glaucoma suspect patients (38 eyes). All subjects had a two standard Humphrey visual field HFA test 24-2 and a single mfVEP test undertaken in one session. Analysis of the mfVEP results was done using the new analysis protocol; the Hemifield Sector Analysis HSA protocol. Analysis of the HFA was done using the standard grading system. RESULTS: Analysis of mfVEP results showed that there was a statistically significant difference between the 3 groups in the mean signal to noise ratio SNR (ANOVA p<0.001 with a 95% CI). The difference between superior and inferior hemispheres in all subjects were all statistically significant in the glaucoma patient group 11/11 sectors (t-test p<0.001), partially significant 5/11 (t-test p<0.01) and no statistical difference between most sectors in normal group (only 1/11 was significant) (t-test p<0.9). sensitivity and specificity of the HAS protocol in detecting glaucoma was 97% and 86% respectively, while for glaucoma suspect were 89% and 79%. DISCUSSION: The results showed that the new analysis protocol was able to confirm already existing field defects detected by standard HFA, was able to differentiate between the 3 study groups with a clear distinction between normal and patients with suspected glaucoma; however the distinction between normal and glaucoma patients was especially clear and significant. CONCLUSION: The new HSA protocol used in the mfVEP testing can be used to detect glaucomatous visual field defects in both glaucoma and glaucoma suspect patient. Using this protocol can provide information about focal visual field differences across the horizontal midline, which can be utilized to differentiate between glaucoma and normal subjects. Sensitivity and specificity of the mfVEP test showed very promising results and correlated with other anatomical changes in glaucoma field loss.

The role of hemifield sector analysis in multifocal visual evoked potential objective perimetry in the early detection of glaucomatous visual field defects

Objective: The purpose of this study was to examine the effectiveness of a new analysis method of mfVEP objective perimetry in the early detection of glaucomatous visual field defects compared to the gold standard technique. Methods and patients: Three groups were tested in this study; normal controls (38 eyes), glaucoma patients (36 eyes), and glaucoma suspect patients (38 eyes). All subjects underwent two standard 24-2 visual field tests: one with the Humphrey Field Analyzer and a single mfVEP test in one session. Analysis of the mfVEP results was carried out using the new analysis protocol: the hemifield sector analysis protocol. Results: Analysis of the mfVEP showed that the signal to noise ratio (SNR) difference between superior and inferior hemifields was statistically significant between the three groups (analysis of variance, P<0.001 with a 95% confidence interval, 2.82, 2.89 for normal group; 2.25, 2.29 for glaucoma suspect group; 1.67, 1.73 for glaucoma group). The difference between superior and inferior hemifield sectors and hemi-rings was statistically significant in 11/11 pair of sectors and hemi-rings in the glaucoma patients group (t-test P<0.001), statistically significant in 5/11 pairs of sectors and hemi-rings in the glaucoma suspect group (t-test P<0.01), and only 1/11 pair was statistically significant (t-test P<0.9). The sensitivity and specificity of the hemifield sector analysis protocol in detecting glaucoma was 97% and 86% respectively and 89% and 79% in glaucoma suspects. These results showed that the new analysis protocol was able to confirm existing visual field defects detected by standard perimetry, was able to differentiate between the three study groups with a clear distinction between normal patients and those with suspected glaucoma, and was able to detect early visual field changes not detected by standard perimetry. In addition, the distinction between normal and glaucoma patients was especially clear and significant using this analysis. Conclusion: The new hemifield sector analysis protocol used in mfVEP testing can be used to detect glaucomatous visual field defects in both glaucoma and glaucoma suspect patients. Using this protocol, it can provide information about focal visual field differences across the horizontal midline, which can be utilized to differentiate between glaucoma and normal subjects. The sensitivity and specificity of the mfVEP test showed very promising results and correlated with other anatomical changes in glaucomatous visual field loss. The intersector analysis protocol can detect early field changes not detected by the standard Humphrey Field Analyzer test. © 2013 Mousa et al, publisher and licensee Dove Medical Press Ltd.

Evaluation of hemifield sector analysis protocol in multifocal visual evoked potential objective perimetry for the diagnosis and early detection of glaucomatous field defects

CONCLUSIONS: The new HSA protocol used in the mfVEP testing can be applied to detect glaucomatous visual field defects in both glaucoma and glaucoma suspect patients. Using this protocol can provide information about focal visual field differences across the horizontal midline, which can be utilized to differentiate between glaucoma and normal subjects. Sensitivity and specificity of the mfVEP test showed very promising results and correlated with other anatomical changes in glaucoma field loss. PURPOSE: Multifocal visual evoked potential (mfVEP) is a newly introduced method used for objective visual field assessment. Several analysis protocols have been tested to identify early visual field losses in glaucoma patients using the mfVEP technique, some were successful in detection of field defects, which were comparable to the standard automated perimetry (SAP) visual field assessment, and others were not very informative and needed more adjustment and research work. In this study we implemented a novel analysis approach and evaluated its validity and whether it could be used effectively for early detection of visual field defects in glaucoma. METHODS: Three groups were tested in this study; normal controls (38 eyes), glaucoma patients (36 eyes) and glaucoma suspect patients (38 eyes). All subjects had a two standard Humphrey field analyzer (HFA) test 24-2 and a single mfVEP test undertaken in one session. Analysis of the mfVEP results was done using the new analysis protocol; the hemifield sector analysis (HSA) protocol. Analysis of the HFA was done using the standard grading system. RESULTS: Analysis of mfVEP results showed that there was a statistically significant difference between the three groups in the mean signal to noise ratio (ANOVA test, p < 0.001 with a 95% confidence interval). The difference between superior and inferior hemispheres in all subjects were statistically significant in the glaucoma patient group in all 11 sectors (t-test, p < 0.001), partially significant in 5 / 11 (t-test, p < 0.01), and no statistical difference in most sectors of the normal group (1 / 11 sectors was significant, t-test, p < 0.9). Sensitivity and specificity of the HSA protocol in detecting glaucoma was 97% and 86%, respectively, and for glaucoma suspect patients the values were 89% and 79%, respectively.

Immunochemical detection of glyoxal DNA damage

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.

Simultaneous estimation of global background synaptic inhibition and excitation from membrane potential fluctuations in layer III neurons of the rat entorhinal cortex in vitro

It is becoming clear that the detection and integration of synaptic input and its conversion into an output signal in cortical neurons are strongly influenced by background synaptic activity or "noise." The majority of this noise results from the spontaneous release of synaptic transmitters, interacting with ligand-gated ion channels in the postsynaptic neuron [Berretta N, Jones RSG (1996); A comparison of spontaneous synaptic EPSCs in layer V and layer II neurones in the rat entorhinal cortex in vitro. J Neurophysiol 76:1089-1110; Jones RSG, Woodhall GL (2005) Background synaptic activity in rat entorhinal cortical neurons: differential control of transmitter release by presynaptic receptors. J Physiol 562:107-120; LoTurco JJ, Mody I, Kriegstein AR (1990) Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex. Neurosci Lett 114:265-271; Otis TS, Staley KJ, Mody I (1991) Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res 545:142-150; Ropert N, Miles R, Korn H (1990) Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus. J Physiol 428:707-722; Salin PA, Prince DA (1996) Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75:1573-1588; Staley KJ (1999) Quantal GABA release: noise or not? Nat Neurosci 2:494-495; Woodhall GL, Bailey SJ, Thompson SE, Evans DIP, Stacey AE, Jones RSG (2005) Fundamental differences in spontaneous synaptic inhibition between deep and superficial layers of the rat entorhinal cortex. Hippocampus 15:232-245]. The function of synaptic noise has been the subject of debate for some years, but there is increasing evidence that it modifies or controls neuronal excitability and, thus, the integrative properties of cortical neurons. In the present study we have investigated a novel approach [Rudolph M, Piwkowska Z, Badoual M, Bal T, Destexhe A (2004) A method to estimate synaptic conductances from membrane potential fluctuations. J Neurophysiol 91:2884-2896] to simultaneously quantify synaptic inhibitory and excitatory synaptic noise, together with postsynaptic excitability, in rat entorhinal cortical neurons in vitro. The results suggest that this is a viable and useful approach to the study of the function of synaptic noise in cortical networks. © 2007 IBRO.

A simple, sensitive and selective quantum-dot-based western blot method for the simultaneous detection of multiple targets from cell lysates

Quantum dots (Qdots) are fluorescent nanoparticles that have great potential as detection agents in biological applications. Their optical properties, including photostability and narrow, symmetrical emission bands with large Stokes shifts, and the potential for multiplexing of many different colours, give them significant advantages over traditionally used fluorescent dyes. Here, we report the straightforward generation of stable, covalent quantum dot-protein A/G bioconjugates that will be able to bind to almost any IgG antibody, and therefore can be used in many applications. An additional advantage is that the requirement for a secondary antibody is removed, simplifying experimental design. To demonstrate their use, we show their application in multiplexed western blotting. The sensitivity of Qdot conjugates is found to be superior to fluorescent dyes, and comparable to, or potentially better than, enhanced chemiluminescence. We show a true biological validation using a four-colour multiplexed western blot against a complex cell lysate background, and have significantly improved previously reported non-specific binding of the Qdots to cellular proteins.

The combination of smart hydrogels and fibre optic sensor technology for analyte detection

The subject of investigation of the present research is the use of smart hydrogels with fibre optic sensor technology. The aim was to develop a costeffective sensor platform for the detection of water in hydrocarbon media, and of dissolved inorganic analytes, namely potassium, calcium and aluminium. The fibre optic sensors in this work depend upon the use of hydrogels to either entrap chemotropic agents or to respond to external environmental changes, by changing their inherent properties, such as refractive index (RI). A review of current fibre optic technology for sensing outlined that the main principles utilised are either the measurement of signal loss or a change in wavelength of the light transmitted through the system. The signal loss principle relies on changing the conditions required for total internal reflection to occur. Hydrogels are cross-linked polymer networks that swell but do not dissolve in aqueous environments. Smart hydrogels are synthetic materials that exhibit additional properties to those inherent in their structure. In order to control the non-inherent properties, the hydrogels were fabricated with the addition of chemotropic agents. For the detection of water, hydrogels of low refractive index were synthesized using fluorinated monomers. Sulfonated monomers were used for their extreme hydrophilicity as a means of water sensing through an RI change. To enhance the sensing capability of the hydrogel, chemotropic agents, such as pH indicators and cobalt salts, were used. The system comprises of the smart hydrogel coated onto an exposed section of the fibre optic core, connected to the interrogation system measuring the difference in the signal. Information obtained was analysed using a purpose designed software. The developed sensor platform showed that an increase in the target species caused an increase in the signal lost from the sensor system, allowing for a detection of the target species. The system has potential applications in areas such as clinical point of care, water detection in fuels and the detection of dissolved ions in the water industry.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Optical fibre-based detection of DNA hybridization

A dual-peak LPFG (long-period fibre grating), inscribed in an optical fibre, has been employed to sense DNA hybridization in real time, over a 1 h period. One strand of the DNA was immobilized on the fibre, while the other was free in solution. After hybridization, the fibre was stripped and repeated detection of hybridization was achieved, so demonstrating reusability of the device. Neither strand of DNA was fluorescently or otherwise labelled. The present paper will provide an overview of our early-stage experimental data and methodology, examine the potential of fibre gratings for use as biosensors to monitor both nucleic acid and other biomolecular interactions and then give a summary of the theory and fabrication of fibre gratings from a biological standpoint. Finally, the potential of improving signal strength and possible future directions of fibre grating biosensors will be addressed.

Intermediate pyrolysis studies of aquatic biomass and potential applications in the BtVB-process

Aquatic biomass is seen as one of the major feedstocks to overcome difficulties associated with 1st generation biofuels, such as competition with food production, change of land use and further environmental issues. Although, this finding is widely accepted only little work has been carried out to investigate thermo-chemical conversion of algal specimen to produce biofuels, power and heat. This work aims at contributing fundamental knowledge for thermo-chemical processing of aquatic biomass via intermediate pyrolysis. Therefore, it was necessary to install and commission an analytical pyrolysis apparatus which facilitates intermediate pyrolysis process conditions as well as subsequent separation and detection of pyrolysates (Py- GC/MS). In addition, a methodology was established to analyse aquatic biomass under intermediate conditions by Thermo-Gravimetric Analysis (TGA). Several microalgae (e.g. Chlamydomonas reinhardtii, Chlorella vulgaris) and macroalgae specimen (e.g. Fucus vesiculosus) from main algal divisions and various natural habitats (fresh and saline water, temperate and polar climates) were chosen and their thermal degradation under intermediate pyrolysis conditions was studied. In addition, it was of interest to examine the contribution of biochemical constituents of algal biomass onto the chemical compounds contained in pyrolysates. Therefore, lipid and protein fractions were extracted from microalgae biomass and analysed separately. Furthermore, investigations of residual algal materials obtained by extraction of high valuable compounds (e.g. lipids, proteins, enzymes) were included to evaluate their potential for intermediate pyrolysis processing. On basis of these thermal degradation studies, possible applications of algal biomass and from there derived materials in the Bio-thermal Valorisation of Biomass-process (BtVB-process) are presented. It was of interest to evaluate the combination of the production of high valuable products and bioenergy generation derived by micro- and macro algal biomass.

A near real-time water surface detection method based on HSV transformation of MODIS multi-Spectral time series data

In the face of global population growth and the uneven distribution of water supply, a better knowledge of the spatial and temporal distribution of surface water resources is critical. Remote sensing provides a synoptic view of ongoing processes, which addresses the intricate nature of water surfaces and allows an assessment of the pressures placed on aquatic ecosystems. However, the main challenge in identifying water surfaces from remotely sensed data is the high variability of spectral signatures, both in space and time. In the last 10 years only a few operational methods have been proposed to map or monitor surface water at continental or global scale, and each of them show limitations. The objective of this study is to develop and demonstrate the adequacy of a generic multi-temporal and multi-spectral image analysis method to detect water surfaces automatically, and to monitor them in near-real-time. The proposed approach, based on a transformation of the RGB color space into HSV, provides dynamic information at the continental scale. The validation of the algorithm showed very few omission errors and no commission errors. It demonstrates the ability of the proposed algorithm to perform as effectively as human interpretation of the images. The validation of the permanent water surface product with an independent dataset derived from high resolution imagery, showed an accuracy of 91.5% and few commission errors. Potential applications of the proposed method have been identified and discussed. The methodology that has been developed 27 is generic: it can be applied to sensors with similar bands with good reliability, and minimal effort. Moreover, this experiment at continental scale showed that the methodology is efficient for a large range of environmental conditions. Additional preliminary tests over other continents indicate that the proposed methodology could also be applied at the global scale without too many difficulties

A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children

Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS). Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 μl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 μm, 2.1x150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization. Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique. Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology. © 2014 Hawwa et al.

The detection of kinin activity in contact lens wear

Many factors can be, and have been, attributed to the appearance of complications in lens wear, but the greatest is associated with deposition. Reduced acuity, irritation and inflammatory responses are often referred to as adverse reactions arising as a result of deposition. In this study, particular attention was paid to the potential role of adsorbed proteins in activating, mediating and/or stimulating a host immune response, i.e., the hypothesis that the adsorption of certain proteins from the tears and ocular surfaces may actively affect successful lens wear. In particular, the purpose of this study was to investigate the presence of a group of proteins previously undiscovered in the ocular environment. The intention was to target a family of proteins/glycoproteins that have become prominent recently in a variety of inflammatory responses and disorders at many other mucosal associated sites around the body, e.g. in nasal rhinitis and in joint inflammation. The protein cascade in question is the kinin family of inflammatory mediators. The aim was to investigate their presence in the ocular environment, specifically in relation to contact lens wear, and consequently assess the implications of their discovery. High molecular weight kininogen (HMWK), with its central role in kinin responses, was investigated initially as the marker of kinin activity, with subsequent members examined thereafter.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

Protein lipoxidation:detection strategies and challenges

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.