Use of fundus imaging in quantification of age-related macular change

This review will discuss the use of manual grading scales, digital photography, and automated image analysis in the quantification of fundus changes caused by age-related macular disease. Digital imaging permits processing of images for enhancement, comparison, and feature quantification, and these techniques have been investigated for automated drusen analysis. The accuracy of automated analysis systems has been enhanced by the incorporation of interactive elements, such that the user is able to adjust the sensitivity of the system, or manually add and remove pixels. These methods capitalize on both computer and human image feature recognition and the advantage of computer-based methodologies for quantification. The histogram-based adaptive local thresholding system is able to extract useful information from the image without being affected by the presence of other structures. More recent developments involve compensation for fundus background reflectance, which has most recently been combined with the Otsu method of global thresholding. This method is reported to provide results comparable with manual stereo viewing. Developments in this area are likely to encourage wider use of automated techniques. This will make the grading of photographs easier and cheaper for clinicians and researchers. © 2007 Elsevier Inc. All rights reserved.

Development of a prescribing indicator for objective quantification of antibiotic usage in secondary care

Objectives: To compare the recognized defined daily dose per 100 bed-days (DDD/100 bed-days) measure with the defined daily dose per finished consultant episode (DDD/FCE) in a group of hospitals with a variety of medicines management strategies. To compare antibiotic usage using the above indicators in hospitals with and without electronic prescribing systems. Methods: Twelve hospitals were used in the study. Nine hospitals were selected and split into three cohorts (three high-scoring, three medium-scoring and three low-scoring) by their 2001 medicines management self-assessment scores (MMAS). An additional cohort of three electronic prescribing hospitals was included for comparison. MMAS were compared to antibiotic management scores (AMS) developed from a questionnaire relating specifically to control of antibiotics. FCEs and occupied bed-days were obtained from published statistics and statistical analyses of the DDD/100 bed-days and DDD/FCE were carried out using SPSS. Results: The DDD/100 bed-days varied from 81.33 to 189.37 whilst the DDD/FCE varied from 2.88 to 7.43. The two indicators showed a high degree of correlation with r = 0.74. MMAS were from 9 to 22 (possible range 0-23) and the AMS from 2 to 13 (possible range 0-22). The two scores showed a high degree of correlation with r = 0.74. No correlation was established between either indicator and either score. Conclusions: The WHO indicator for medicines utilization, DDD/100 bed-days, exhibited the same level of conformity as that exhibited from the use of the DDD/FCE indicating that the DDD/FCE is a useful additional indicator for identifying hospitals which require further study. The MMAS can be assumed to be an accurate guide to antibiotic medicines management controls. No relationship has been found between a high degree of medicines management control and the quantity of antibiotic prescribed. © The British Society for Antimicrobial Chemotherapy; 2004 all rights reserved.

Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.

Quantitative methods in neuropathology

The last decade has seen a considerable increase in the application of quantitative methods in the study of histological sections of brain tissue and especially in the study of neurodegenerative disease. These disorders are characterised by the deposition and aggregation of abnormal or misfolded proteins in the form of extracellular protein deposits such as senile plaques (SP) and intracellular inclusions such as neurofibrillary tangles (NFT). Quantification of brain lesions and studying the relationships between lesions and normal anatomical features of the brain, including neurons, glial cells, and blood vessels, has become an important method of elucidating disease pathogenesis. This review describes methods for quantifying the abundance of a histological feature such as density, frequency, and 'load' and the sampling methods by which quantitative measures can be obtained including plot/quadrat sampling, transect sampling, and the point-quarter method. In addition, methods for determining the spatial pattern of a histological feature, i.e., whether the feature is distributed at random, regularly, or is aggregated into clusters, are described. These methods include the use of the Poisson and binomial distributions, pattern analysis by regression, Fourier analysis, and methods based on mapped point patterns. Finally, the statistical methods available for studying the degree of spatial correlation between pathological lesions and neurons, glial cells, and blood vessels are described.

Quantification of visual field loss in age-related macular degeneration

Background - An evaluation of standard automated perimetry (SAP) and short wavelength automated perimetry (SWAP) for the central 10–2 visual field test procedure in patients with age-related macular degeneration (AMD) is presented in order to determine methods of quantifying the central sensitivity loss in patients at various stages of AMD. Methods - 10–2 SAP and SWAP Humphrey visual fields and stereoscopic fundus photographs were collected in 27 eyes of 27 patients with AMD and 22 eyes of 22 normal subjects. Results - Mean Deviation and Pattern Standard Deviation (PSD) varied significantly with stage of disease in SAP (both p<0.001) and SWAP (both p<0.001), but post hoc analysis revealed overlap of functional values among stages. In SWAP, indices of focal loss were more sensitive to detecting differences in AMD from normal. SWAP defects were greater in depth and area than those in SAP. Central sensitivity (within 1°) changed by -3.9 and -4.9 dB per stage in SAP and SWAP, respectively. Based on defect maps, an AMD Severity Index was derived. Conclusions - Global indices of focal loss were more sensitive to detecting early stage AMD from normal. The SWAP sensitivity decline with advancing stage of AMD was greater than in SAP. A new AMD Severity Index quantifies visual field defects on a continuous scale. Although not all patients are suitable for SWAP examinations, it is of value as a tool in research studies of visual loss in AMD.

Quantification of visual field loss in age-related macular degeneration

Presentation Purpose:To determine methods of quantifying the sensitivity loss in the central 10o visual field in a cross section of patients at various stages of age-related macular degeneration (AMD). Methods:Standard and short-wavelength automated perimetry (SAP and SWAP) visual fields were collected using program 10-2 of the Humphrey Field Analyzer, in 44 eyes of 27 patients with AMD and 41 eyes of 22 normal subjects. Stereoscopic fundus photographs were graded by two independent observers and the stage of disease determined. Global indices were compared for their ability to delineate the normal visual field from early stages of AMD and to differentiate between stages. Results:Mean Deviation (MD) and Pattern Standard Deviation (PSD) varied significantly with stage of disease in SAP (both p<0.001) and SWAP (both p<0.001), but post-hoc analysis revealed overlap of functional values between stages. Global indices of focal loss, PSD and local spatial variability (LSV) were the most sensitive to detecting differences between normal subjects and early stage AMD patients, in SAP and SWAP, respectively. Overall, defects were confined to the central 5°. SWAP defects were consistently greater in depth and area than those in SAP. The most vulnerable region of the 10° field to sensitivity loss with increasing stage of AMD was the central 1°, in which the sensitivity decline was -4.8dB per stage in SAP and -4.9dB per stage in SWAP. Based on the pattern deviation defect maps, a severity index of AMD visual field loss was derived. Threshold variability was considerably increased in late stage AMD eyes. Conclusions:Global indices of focal loss were more sensitive to the detection of early stage AMD from normal. The sensitivity decline with advancing stage of AMD was greater in SWAP compared to SAP, however the trend was not strong across all stages of disease. The less commonly used index LSV represents relatively statistically unmanipulated summary measure of focal loss. A new severity index is described which is sensitive to visual field change in AMD, measures visual field defects on a continuous scale and may serve as a useful measure of functional change in AMD in longitudinal studies. Keywords: visual fields • age-related macular degeneration • perimetry

Relationships between fundus image quantification and visual field status in age-related macular degeneration

Presentation Purpose:To relate structural change to functional change in age-related macular degeneration (AMD) in a cross-sectional population using fundus imaging and the visual field status. Methods:10 degree standard and SWAP visual fields and other standard functional clinical measures were acquired in 44 eyes of 27 patients at various stages of AMD, as well as fundus photographs. Retro-mode SLO images were captured in a subset of 29 eyes of 19 of the patients. Drusen area, measured by automated drusen segmentation software (Smith et al. 2005) was correlated with visual field data. Visual field defect position was compared to the position of the imaged drusen and deposits using custom software. Results:The effect of AMD stage on drusen area within the 6000µm was significant (One-way ANOVA: F = 17.231, p < 0.001), however the trend was not strong across all stages. There were significant linear relationships between visual field parameters and drusen area. The mean deviation (MD) declined by 3.00dB and 3.92dB for each log % drusen area for standard perimetry and SWAP, respectively. The visual field parameters of focal loss displayed the strongest correlations with drusen area. The number of pattern deviation (PD) defects increased by 9.30 and 9.68 defects per log % drusen area for standard perimetry and SWAP, respectively. Weaker correlations were found between drusen area and visual acuity, contrast sensitivity, colour vision and reading speed. 72.6% of standard PD defects and 65.2% of SWAP PD defects coincided with retinal signs of AMD on fundus photography. 67.5% of standard PD defects and 69.7% of SWAP PD defects coincided with deposits on retro-mode images. Conclusions:Perimetry exhibited a stronger relationship with drusen area than other measures of visual function. The structure-function relationship between visual field parameters and drusen area was linear. Overall the indices of focal loss had a stronger correlation with drusen area in SWAP than in standard perimetry. Visual field defects had a high coincidence proportion with retinal manifestations of AMD.Smith R.T. et al. (2005) Arch Ophthalmol 123:200-206.

Concepts and methods of quantitative assessment of risk to groundwater resources

This work presents a two-dimensional approach of risk assessment method based on the quantification of the probability of the occurrence of contaminant source terms, as well as the assessment of the resultant impacts. The risk is calculated using Monte Carlo simulation methods whereby synthetic contaminant source terms were generated to the same distribution as historically occurring pollution events or a priori potential probability distribution. The spatial and temporal distributions of the generated contaminant concentrations at pre-defined monitoring points within the aquifer were then simulated from repeated realisations using integrated mathematical models. The number of times when user defined ranges of concentration magnitudes were exceeded is quantified as risk. The utilities of the method were demonstrated using hypothetical scenarios, and the risk of pollution from a number of sources all occurring by chance together was evaluated. The results are presented in the form of charts and spatial maps. The generated risk maps show the risk of pollution at each observation borehole, as well as the trends within the study area. This capability to generate synthetic pollution events from numerous potential sources of pollution based on historical frequency of their occurrence proved to be a great asset to the method, and a large benefit over the contemporary methods.

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.

Development of an enantiomer selective microsampling assay for the quantification of ketorolac suitable for paediatric pharmacokinetic studies

Background: Ketorolac, a potent nonsteroidal anti-inflammatory drug used for pain control in children, exists as a racemate of inactive R (+) and active S (-) enantiomers. Aim: To develop a microsampling assay for the enantioselective analysis of ketorolac in children. Methods: Ketorolac enantiomers were extracted from 50 µl of plasma by liquid–liquid extraction and separated on a ChiralPak AD-RH. Detection was by a TSQ quantum triple quadrupole mass spectrometer with an electrospray ionisation source operating in a positive ion mode. Five children (age 13.8 (1.6) years, weight 52.7 (7.2) kg), were administered intravenous ketorolac 0.5 mg/kg (maximum 10 mg) and blood samples were taken at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h post administration. CL, VD and t1/2 were calculated based on non-compartmental methods. Results: The standard curves for R (+) and S (-) ketorolac were linear in the range 0–2000 ng/ml. The LLOQs of the method were 0.15 ng on column and 0.31 ng on column for R (+) and S (-) ketorolac, respectively. The median (range) VD and CL of R (+) and S (-) ketorolac were 0.12 l/kg (0.07–0.17), 0.017 l/h/kg (0.12–0.29) and 0.17 (0.09–0.31) l/kg, 0.049 (0.02–0.1) l/h/kg, p = 0.043), respectively. The median (range) elimination half-life (t1/2) of the R (+) and S (-) ketorolac was 5.0 h (2.5–5.8) and 3.1 h (1.8–4.4), p = 0.043), respectively. Conclusion: The development of a simple, rapid and reliable ketorolac assay suitable for paediatric PK studies is reported. Copyright © 2013 John Wiley & Sons, Ltd.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.