Enea Poletti

Standardized Baseline Human Corneal Subbasal Nerve Density for Clinical Investigations With Laser-Scanning in Vivo Confocal Microscopy

PURPOSE We established a baseline value for central corneal subbasal nerve density in a large, healthy cohort. METHODS A total of 106 healthy volunteers (207 eyes) underwent full ophthalmic examination, including laser-scanning in vivo confocal microscopy (IVCM) of the central cornea. Images of the corneal subbasal nerve plexus were acquired and analyzed based on defined criteria. Nerve tracing was performed by two human observers and by a fully automated method. Subbasal nerve density was stratified by eye, observer, tracing method, calculation method, and age group. Association of nerve density with age was examined by linear regression and population distribution was examined by nonlinear regression. RESULTS We analyzed 892 distinct, high quality images of the subbasal nerve plexus (mean, 4.3 images/eye) from 207 eyes. An overall mean central subbasal nerve density of 19 mm/mm(2) was found in 106 subjects aged 15 to 88 years, independent of eye, sex, or nerve tracing method, while the SD was a consistent 4 to 5 mm/mm(2). Subbasal nerve density followed a normal Gaussian distribution, and correlated negatively with age, with a mean decline of 0.25% to 0.30% per year, independent of eye, observer, or nerve tracing method. Moreover, the use of automated tracing techniques and randomized sampling may improve the speed and reproducibility of subbasal nerve density assessment for clinical applications. CONCLUSIONS A baseline human corneal subbasal nerve density has been determined by laser-scanning IVCM using rigorous methods. The methods and results could aid in the future assessment of corneal nerves in various patient populations.

Automatic Segmentation and Disentangling of Chromosomes in Q-Band Prometaphase Images

Karyotype analysis is a widespread procedure in cytogenetics to assess the possible presence of genetics defects. The procedure is lengthy and repetitive, so that an automatic analysis would greatly help the cytogeneticist routine work. Still, automatic segmentation and full disentangling of chromosomes are open issues. We propose an automatic procedure to obtain the separated chromosomes, which are then ready for a subsequent classification step. The segmentation is carried out by means of a space-variant thresholding scheme, which proved to be successful even in presence of hyper- or hypofluorescent regions in the image. Then, the tree of choices to resolve touching and overlapping chromosomes is recursively explored, choosing the best combination of cuts and overlaps based on geometric evidence and image information. We show the effectiveness of the proposed method on routine data acquired with different microscope-camera setup at different laboratories: from 162 images of 117 cells totaling 6683 chromosomes, 94% of the chromosomes were correctly segmented, solving 90% of the overlaps and 90% of the touchings. In order to provide the scientific community with a public dataset, the data used in this paper are available for public download.

A review of thresholding strategies applied to human chromosome segmentation

Karyotype analysis is a widespread procedure in cytogenetics to assess the presence of genetic defects by the visualization of the structure of chromosomes. The procedure is lengthy and repetitive and an effective automatic analysis would greatly help the cytogeneticist routine work. Still, automatic segmentation and the full disentangling of chromosomes are open issues. The first step in every automatic procedure is the thresholding step, which detect blobs that represent either single chromosomes or clusters of chromosomes. The better the thresholding step, the easier is the subsequent disentanglement of chromosome clusters into single entities. We implemented eleven thresholding methods, i.e. the ones that appear in the literature as the best performers, and compared their performance in segmenting chromosomes and chromosome clusters in cytogenetic Q-band images. The images are affected by the presence of hyper- or hypo-fluorescent regions and by a contrast variability between the stained chromosomes and the background. A thorough analysis of the results highlights that, although every single algorithm shows peculiar strong/weak points, Adaptive Threshold and Region Based Level Set have the overall best performance. In order to provide the scientific community with a public dataset, the data and manual segmentation used in this paper are available for public download at http://bioimlab.dei.unipd.it.

Corneal Nerve Regeneration After Collagen Cross-Linking Treatment of Keratoconus: A 5-Year Longitudinal Study.

IMPORTANCE It is unknown whether a neurotrophic deficit or pathologic nerve morphology persists in keratoconus in the long term after corneal collagen cross-linking (CXL) treatment. Nerve pathology could impact long-term corneal status in patients with keratoconus. OBJECTIVE To determine whether CXL treatment of keratoconus results in normalization of subbasal nerve density and architecture up to 5 years after treatment. DESIGN, SETTING, AND PARTICIPANTS Observational study of 19 patients with early-stage keratoconus indicated for a first CXL treatment with longitudinal follow-up to 5 years postoperatively (examinations were performed from 2009 to 2015; analysis was performed from February to May 2015) and 19 age-matched healthy volunteers at a primary care center and a university hospital ophthalmology department. EXPOSURE The patients with keratoconus underwent standard epithelial-off UV-A/riboflavin CXL treatment with 30-minute UV-A exposure at 3 mW/cm2 irradiance. MAIN OUTCOMES AND MEASURES Central corneal subbasal nerve density and subbasal nerve architecture by use of laser-scanning in vivo confocal microscopy; subbasal nerve analysis by 2 masked observers and by use of a fully automated method; wide-field mosaics of subbasal nerve architecture by use of an automated method; and ocular surface touch sensitivity by use of contact esthesiometry. RESULTS Mean (SD) age of the 19 patients with keratoconus was 27.5 (7.1) years (range, 19-44 years), and minimal corneal thickness was 428 (36) μm (range, 372-497 μm). Compared with the mean (SD) preoperative subbasal nerve density of 21.0 (4.2) mm/mm2 in healthy corneas, the mean (SD) preoperative subbasal nerve density of 10.3 (5.6) mm/mm2 in the corneas of patients with stage 1 or 2 keratoconus was reduced 51% (mean difference, 10.7 mm/mm2 [95% CI, 6.8-14.6 mm/mm2]; P < .001). After CXL, nerves continued to regenerate for up to 5 years, but nerve density remained reduced relative to healthy corneas at final follow-up (mean reduction, 8.5 mm/mm2 [95% CI, 4.7-12.4 mm/mm2]; P < .001) despite recovery of touch sensitivity to normal levels by 6 months. Preoperatively, more frequent nerve loops, crossings, and greater crossing angles were observed in the corneas of patients with keratoconus compared with healthy corneas. Postoperatively, the frequency of nerve looping increased, crossings were more frequent, and nerve tortuosity increased. Wide-field mosaics indicated persistent disrupted orientation of the regenerating subbasal nerves 5 years after CXL. CONCLUSIONS AND RELEVANCE Keratoconus is characterized by a neurotrophic deficit and altered nerve morphology that CXL treatment does not address, despite providing a positive biomechanical effect in the stroma. Given the widespread use of CXL in the management of patients with keratoconus, the progression of abnormal innervation after CXL should be recognized.

Automatic segmentation of chromosomes in Q-band images

Karyotype analysis is a widespread procedure in cytogenetics to assess the possible presence of genetics defects. The procedure is lengthy and repetitive, so that an automatic analysis would greatly help the cytogeneticist routine work. Still, automatic segmentation and full disentangling of chromosomes are open issues. We propose an automatic procedure to obtain the separated chromosomes, which are then ready for a subsequent classification step. The segmentation is carried out by means of a space variant thresholding scheme, which proved to be successful even in presence of hyper- or hypo-fluorescent regions in the image. Then a greedy approach is used to identify and resolve touching and overlapping chromosomes, based on geometric evidence and image information. We show the effectiveness of the proposed method on routine data: 90% of the overlaps and 92% of the adjacencies are resolved, resulting in a correct segmentation of 96% of the chromosomes.

Automatic classification of chromosomes in Q-band images

The manual analysis of the karyogram is a complex, wearing and time-consuming operation. It requires a very meticulous attention to details and calls for well-trained personnel. Even though existing commercial software packages provide a reasonable support to cytogenetists, they very often require human intervention to correct challenging situations.

A modular framework for the automatic classification of chromosomes in Q-band images

The manual analysis of the karyogram is a complex and time-consuming operation, as it requires meticulous attention to details and well-trained personnel. Routine Q-band laboratory images show chromosomes that are randomly rotated, blurred or corrupted by overlapping and dye stains. We address here the problem of robust automatic classification, which is still an open issue. The proposed method starts with an improved estimation of the chromosome medial axis, along which an established set of features is then extracted. The following novel polarization stage estimates the chromosome orientation and makes this feature set independent on the reading direction along the axis. Feature rescaling and normalizing techniques take full advantage of the results of the polarization step, reducing the intra-class and increasing the inter-class variances. After a standard neural network based classification, a novel class reassignment algorithm is employed to maximize the probability of correct classification, by exploiting the constrained composition of the human karyotype. An average 94% of correct classification was achieved by the proposed method on 5474 chromosomes, whose images were acquired during laboratory routine and comprise karyotypes belonging to slightly different prometaphase stages. In order to provide the scientific community with a public dataset, all the data we used are publicly available for download.

Focused Tortuosity Definitions Based on Expert Clinical Assessment of Corneal Subbasal Nerves.

PURPOSE We examined agreement among experts in the assessment of corneal subbasal nerve tortuosity. METHODS Images of corneal subbasal nerves were obtained from investigators at seven sites (Auckland, Boston, Linköping, Manchester, Oslo, Rostock, and Sydney) using laser-scanning in vivo confocal microscopy. A set of 30 images was assembled and ordered by increasing tortuosity by 10 expert graders from the seven sites. In a first experiment, graders assessed tortuosity without a specific definition and performed grading three times, with at least 1 week between sessions. In a second experiment, graders assessed the same image set using four focused tortuosity definitions. Intersession and intergrader repeatability for the experiments were determined using the Spearman rank correlation. RESULTS Expert graders without a specific tortuosity definition had high intersession (Spearman correlation coefficient 0.80), but poor intergrader (0.62) repeatability. Specific definitions improved intergrader repeatability to 0.79. In particular, tortuosity defined by frequent small-amplitude directional changes (short range tortuosity) or by infrequent large-amplitude directional changes (long range tortuosity), indicated largely independent measures and resulted in improved repeatability across the graders. A further refinement, grading only the most tortuous nerve in a given image, improved the average correlation of a given graders ordering of images with the group average to 0.86 to 0.90. CONCLUSIONS Definitions of tortuosity specifying short or long-range tortuosity and considering only the most tortuous nerve in an image improved the agreement in tortuosity grading among a group of expert observers. These definitions could improve accuracy and consistency in quantifying subbasal nerve tortuosity in clinical studies.

Segmentation of Corneal Endothelial Cells Contour through Classification of Individual Component Signatures

Corneal images acquired by in-vivo specular microscopy provide clinical information on the cornea endothelium health state. At present, the analysis is based on manual or semi-automatic methods and the segmentation of a large number of endothelial cells is required for a meaningful estimation of the clinical parameters (cell density, pleomorphism, polymegethism). For the practical application in clinical settings, a computerized method capable to fully automatize the segmentation procedure would be needed.

Image-level tortuosity estimation in wide-field retinal images from infants with Retinopathy of Prematurity

Tortuosity and dilation of retinal vessels are considered of primary importance for the diagnosis and follow-up of the Retinopathy of Prematurity (ROP) disease. We developed an algorithm to estimate vessel tortuosity in images acquired with a wide-field fundus camera in ROP subjects, offering clinicians a quantitative, objective, and reproducible diagnostic parameter.