Quality Assessment of Retinal Fundus Images using Elliptical Local Vessel Density

Diabetic retinopathy is the leading cause of blindness in the Western world. The World Health Organisation estimates that 135 million people have diabetes mellitus worldwide and that the number of people with diabetes will increase to 300 million by the year 2025 (Amos et al., 1997). Timely detection and treatment for DR prevents severe visual loss in more than 50% of the patients (ETDRS, 1991). Through computer simulations is possible to demonstrate that prevention and treatment are relatively inexpensive if compared to the health care and rehabilitation costs incurred by visual loss or blindness (Javitt et al., 1994). The shortage of ophthalmologists and the continuous increase of the diabetic population limits the screening capability for effective timing of sight-saving treatment of typical manual methods. Therefore, an automatic or semi-automatic system able to detect various type of retinopathy is a vital necessity to save many sight-years in the population. According to Luzio et al. (2004) the preferred way to detect diseases such as diabetic retinopathy is digital fundus camera imaging. This allows the image to be enhanced, stored and retrieved more easily than film. In addition, images may be transferred electronically to other sites where a retinal specialist or an automated system can detect or diagnose disease while the patient remains at a remote location. Various systems for automatic or semi-automatic detection of retinopathy with fundus images have been developed. The results obtained are promising but the initial image quality is a limiting factor (Patton et al., 2006); this is especially true if the machine operator is not a trained photographer. Algorithms to correct the illumination or increase the vessel contrast exist (Chen & Tian, 2008; Foracchia et al., 2005; Grisan et al., 2006;Wang et al., 2001), however they cannot restore an image beyond a certain level of quality degradation. On the other hand, an accurate quality assessment algorithm can allow operators to avoid poor images by simply re-taking the fundus image, eliminating the need for correction algorithms. In addition, a quality metric would permit the automatic submission of only the best images if many are available. The measurement of a precise image quality index is not a straightforward task, mainly because quality is a subjective concept which varies even between experts, especially for images that are in the middle of the quality scale. In addition, image quality is dependent upon the type of diagnosis being made. For example, an image with dark regions might be considered of good quality for detecting glaucoma but of bad quality for detecting diabetic retinopathy. For this reason, we decided to define quality as the 'characteristics of an image that allow the retinopathy diagnosis by a human or software expert'. Fig. 1 shows some examples of macula centred fundus images whose quality is very likely to be judged as poor by many ophthalmologists. The reasons for this vary. They can be related to the camera settings like exposure or focal plane error (Fig. 1.(a,e,f)), the camera condition like a dirty or shuttered lens (Fig. 1.(d,h)), the movements of the patient which might blur the image (Fig. 1.(c)) or if the patient is not in the field of view of the camera (Fig. 1.(g)). We define an outlier as any image that is not a retina image which could be submitted to the screening system by mistake. Existing algorithms to estimate the image quality are based on the length of visible vessels in the macula region (Fleming et al., 2006), or edges and luminosity with respect to a reference image (Lalonde et al., 2001; Lee & Wang, 1999). Another method uses an unsupervised classifier that employs multi-scale filterbanks responses (Niemeijer et al., 2006). The shortcomings of these methods are either the fact that they do not take into account the natural variance encountered in retinal images or that they require a considerable time to produce a result. Additionally, none of the algorithms in the literature that we surveyed generate a 'quality measure'. Authors tend to split the quality levels into distinct classes and to classify images in particular ones. This approach is not really flexible and is error prone. In fact human experts are likely to disagree if many categories of image quality are used. Therefore, we think that a normalized 'quality measure' from 0 to 1 is the ideal way to approach the classification problem. Processing speed is another aspect to be taken into consideration. While algorithms to assess the disease state of the retina do not need to be particularly fast (within reason), the time response of the quality evaluation method is key towards the development of an automatic retinopathy screening system

A Health Insurance Portability and Accountability Act–Compliant Ocular Telehealth Network for the Remote Diagnosis and Management of Diabetic Retinopathy

In this article, we present the design and implementation of a regional ocular telehealth network for remote assessment and management of diabetic retinopathy (DR), including the design requirements, network topology, protocol design, system work flow, graphics user interfaces, and performance evaluation. The Telemedical Retinal Image Analysis and Diagnosis Network is a computer-aided, image analysis telehealth paradigm for the diagnosis of DR and other retinal diseases using fundus images acquired from primary care end users delivering care to underserved patient populations in the mid-South and southeastern United States

Highly Sensitive In Vitro Methods for Detection of Residual Undifferentiated Cells in Retinal Pigment Epithelial Cells Derived from Human iPS Cells

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research

Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina

The retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment.We isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals.This comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly

Lipid Composition of the Human Eye: Are Red Blood Cells a Good Mirror of Retinal and Optic Nerve Fatty Acids?

International audienceBACKGROUND: The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics. METHODOLOGY AND PRINCIPAL FINDINGS: Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens. CONCLUSIONS AND SIGNIFICANCE: LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration

A Smart Biosensor-Enabled Intravascular Catheter and Platform for Dynamic Delivery of Propofol to Close the Loop for Total Intravenous Anesthesia

Background: Target-controlled infusion anesthesia is used worldwide to provide user-defined, stable, blood concentrations of propofol for sedation and anesthesia. The drug infusion is controlled by a microprocessor that uses population-based pharmacokinetic data and patient biometrics to estimate the required infusion rate to replace losses from the blood compartment due to drug distribution and metabolism. The objective of the research was to develop and validate a method to detect and quantify propofol levels in the blood, to improve the safety of propofol use, and to demonstrate a pathway for regulatory approval for its use in the USA. Methods: We conceptualized and prototyped a novel smart biosensor-enabled intravenous catheter capable of quantifying propofol at physiologic levels in the blood, in real time. The clinical embodiment of the platform is comprised of a smart biosensor-enabled catheter prototype, a signal generation/detection readout display, and a driving electronics software. The biosensor was validated in vitro using a variety of electrochemical methods in both static and flow systems with biofluids, including blood. Results: We present data demonstrating the experimental detection and quantification of propofol at sub-micromolar concentrations using this biosensor and method. Detection of the drug is rapid and stable with negligible biofouling due to the sensor coating. It shows a linear correlation with mass spectroscopy methods. An intuitive graphical user interface was developed to: (1) detect and quantify the propofol sensor signal, (2) determine the difference between targeted and actual propofol concentration, (3) communicate the variance in real time, and (4) use the output of the controller to drive drug delivery from an in-line syringe pump. The automated delivery and maintenance of propofol levels was demonstrated in a modeled benchtop patient applying the known pharmacokinetics of the drug using published algorithms. Conclusions: We present a proof-of-concept and in vitro validation of accurate electrochemical quantification of propofol directly from the blood and the design and prototyping of a smart, indwelling, biosensor-enabled catheter and demonstrate feedback hardware and software architecture permitting accurate measurement of propofol in blood in real time. The controller platform is shown to permit autonomous, closed-loop delivery of the drug and maintenance of user-defined propofol levels in a dynamic flow model

Propofol detection and quantification in human blood: the promise of feedback controlled, closed-loop anesthesia

The performance of a membrane-coated voltammetric sensor for propofol (2,6-diisopropylphenol) has been characterized in long term monitoring experiments using an automated flow analytical system (AFAS) and by analyzing human serum and whole blood samples by standard addition. It is shown that the signal of the membrane-coated electrochemical sensor for propofol is not influenced by the components of the pharmaceutical formulation of propofol (propofol injectable emulsion). The current values recorded with the electrochemical propofol sensor in buffer solutions and human serum samples spiked with propofol injectable emulsion showed excellent correlation with the peak heights recorded with an UV-Vis detector during the HPLC analysis of these samples (R(2) = 0.997 in PBS and R(2) = 0.975 in human serum). However, the determination of propofol using the electrochemical method is simpler, faster and has a better detection limit (0.08 ± 0.05 μM) than the HPLC method (0.4 ± 0.2 μM). As a first step towards feedback controlled closed-loop anesthesia, the membrane-coated electrochemical sensor has been implemented onto surface of an intravenous catheter. The response characteristics of the membrane-coated carbon fiber electrode on the catheter surface were very similar to those seen using a macroelectrode

Propofol detection and quantification in human blood: the promise of feedback controlled, closed-loop anesthesia

The performance of a membrane-coated voltammetric sensor for propofol (2,6-diisopropylphenol) has been characterized in long term monitoring experiments using an automated flow analytical system (AFAS) and by analyzing human serum and whole blood samples by standard addition. It is shown that the signal of the membrane-coated electrochemical sensor for propofol is not influenced by the components of the pharmaceutical formulation of propofol (propofol injectable emulsion). The current values recorded with the electrochemical propofol sensor in buffer solutions and human serum samples spiked with propofol injectable emulsion showed excellent correlation with the peak heights recorded with an UV-Vis detector during the HPLC analysis of these samples (R(2) = 0.997 in PBS and R(2) = 0.975 in human serum). However, the determination of propofol using the electrochemical method is simpler, faster and has a better detection limit (0.08 ± 0.05 μM) than the HPLC method (0.4 ± 0.2 μM). As a first step towards feedback controlled closed-loop anesthesia, the membrane-coated electrochemical sensor has been implemented onto surface of an intravenous catheter. The response characteristics of the membrane-coated carbon fiber electrode on the catheter surface were very similar to those seen using a macroelectrode

Polyplex-mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age-related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome-mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex-mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex-mediated gene transfer, but stable integration does occur at low frequencies with and without selection