Corneal Endothelium (corneal + endothelium)

Distribution by Scientific Domains


Selected Abstracts


Function of indoleamine 2,3-dioxygenase in corneal allograft rejection and prolongation of allograft survival by over-expression

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2006

Abstract Indoleamine 2,3-dioxygenase (IDO) suppresses T cell responses by its action in catabolising tryptophan. It is important in maintenance of immune privilege in the placenta. We investigated the activity of IDO in the cornea, following corneal transplantation and the effect of IDO over-expression in donor corneal endothelium on the survival of corneal allografts. IDO expression was analysed and functional activity was quantified in normal murine cornea and in corneas following transplantation as allografts. Low levels of IDO, at both mRNA and protein levels, was detected in the normal cornea, up-regulated by IFN-, and TNF. Expression of IDO in cornea was significantly increased following corneal transplantation. However, inhibition of IDO activity in vivo had no effect on graft survival. Following IDO cDNA transfer, murine corneal endothelial cells expressed functional IDO, which was effective at inhibiting allogeneic T cell proliferation. Over-expression of IDO in donor corneal allografts resulted in prolonged graft survival. While, on one hand, our data indicate that IDO may augment corneal immune privilege, up-regulated IDO activity following cytokine stimulation may serve to inhibit inflammatory cellular responses. While increasing IDO mRNA expression was found in allogeneic corneas at rejection, over-expression in donor cornea was found to significantly extend survival of allografts. [source]


Feline immunodeficiency virus vectors.

THE JOURNAL OF GENE MEDICINE, Issue 5 2002
Gene transfer to mouse retina following intravitreal injection
Abstract Background Transduction of the murine retinal pigmented epithelium (RPE) with adenovirus vectors requires technically difficult and invasive subretinal injections. This study tested the hypothesis that recombinant vectors based on feline immunodeficiency virus (FIV) could access the retina following intravitreal injection. Methods FIV vectors expressing E. coli ,-galactosidase (FIV,gal) were injected alone, or in combination with adenovirus vectors expressing eGFP, into the vitreous of normal mice and eyes evaluated for transgene expression. In further studies, the utility of FIV-mediated gene transfer to correct lysosomal storage defects in the anterior and posterior chambers of eyes was tested using recombinant FIV vectors expressing ,-glucuronidase. FIV,gluc vectors were injected into ,-glucuronidase-deficient mice, an animal model of mucopolysacharridoses type VII. Results The results of this study show that similar to adenovirus, both corneal endothelium and cells of the iris could be transduced following intravitreal injection of FIV,gal. However, in contrast to adenovirus, intravitreal injection of FIV,gal also resulted in transduction of the RPE. Immunohistochemistry following an intravitreal injection of an AdeGFP (adenovirus expressing green fluorescent protein) and FIV,gal mixture confirmed that both viruses mediated transduction of corneal endothelium and cells of the iris, while only FIV,gal transduced cells in the retina. Using the ,-glucuronidase-deficient mouse, the therapeutic efficacy of intravitreal injection of FIV,gluc (FIV expressing ,-glucuronidase) was tested. Intravitreal injection of FIV,gluc to the eyes of ,-glucuronidase-deficient mice resulted in rapid reduction (within 2,weeks) of the lysosomal storage defect within the RPE, corneal endothelium, and the non-pigmented epithelium of the ciliary process. Transgene expression and correction of the lysosomal storage defect remained for at least 12,weeks, the latest time point tested. Conclusion These studies demonstrate that intravitreal injection of FIV-based vectors can mediate efficient and lasting transduction of cells in the cornea, iris, and retina. Copyright © 2002 John Wiley & Sons, Ltd. [source]


Decay Accelerating Factor is Essential for Successful Corneal Engraftment

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2010
A. Esposito
In contrast to immune restrictions that pertain for solid organ transplants, the tolerogenic milieu of the eye permits successful corneal transplantation without systemic immunosuppression, even across a fully MHC disparate barrier. Here we show that recipient and donor expression of decay accelerating factor (DAF or CD55), a cell surface C3/C5 convertase regulator recently shown to modulate T-cell responses, is essential to sustain successful corneal engraftment. Whereas wild-type (WT) corneas transplanted into multiple minor histocompatibility antigen (mH), or HY disparate WT recipients were accepted, DAF's absence on either the donor cornea or in the recipient bed induced rapid rejection. Donor or recipient DAF deficiency led to expansion of donor-reactive IFN-, producing CD4+ and CD8+ T cells, as well as inhibited antigen-induced IL-10 and TGF-,, together demonstrating that DAF deficiency precludes immune tolerance. In addition to demonstrating a requisite role for DAF in conferring ocular immune privilege, these results raise the possibility that augmenting DAF levels on donor corneal endothelium and/or the recipient bed could have therapeutic value for transplants that clinically are at high risk for rejection. [source]


Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl-xL

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 9 2007
R. N Barcia
Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immune-privileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium. [source]


Clinical phenotype of posterior polymorphous corneal dystrophy in a family with a novel ZEB1 mutation

ACTA OPHTHALMOLOGICA, Issue 6 2010
Dan Q. Nguyen
Acta Ophthalmol. 2010: 88: 695,699 Abstract. Purpose:, To describe the clinical phenotype in a family with posterior polymorphous corneal dystrophy (PPCD) and a novel mutation in the ZEB1 gene. Methods:, Clinical examination, anterior segment photography, specular microscopy and electrophysiological investigations were performed and quantified. Genomic DNA extracted from peripheral blood was sequenced for ZEB1 exons. Cosegregation of identified mutation with the disease status in the family was confirmed using polymerase chain reaction and restriction fragment length polymorphism. Results:, Ocular examination was performed on five family members from two generations. Three had anomalies of the corneal endothelium that were consistent with PPCD. Endothelial cell counts ranged from 2306 to 2987 mm2 (ref. 2000,4000 cells/mm2). No evidence of glaucoma or retinal abnormalities was observed. Extraocular abnormalities such as inguinal herniation, hydrocoele and possible bony or connective tissue anomalies were part of the disease spectrum in this family. Mutation analysis revealed a novel change in exon 5 of ZEB1 (c.672delA) that cosegregated with the affected disease status. Conclusion:, The detailed clinical features of PPCD associated with a novel ZEB1 mutation are supportive of the previously proposed range of phenotype parameters. Further phenotype,genotype correlations may provide insights into the clinical variability and pathological processes affecting the corneal endothelium, Descemet's membrane, retinal photoreceptor function and extraocular tissues of some patients. [source]


Serious complications of cosmetic NewColorIris implantation

ACTA OPHTHALMOLOGICA, Issue 6 2010
Justin E. Anderson
Acta Ophthalmol. 2010: 88: 700,704 Abstract. Purpose:, This case report describes serious postoperative complications and markedly elevated intraocular pressure (IOP) associated with the NewColorIris cosmetic implant. Methods:, We report an interventional case series of two patients who suffered multiple complications after NewColorIris implantation carried out in Panama. Assessment included visual acuity, photography, endothelial cell count and anterior segment optical coherence tomography (OCT) when possible. Results:, Both patients presented with endothelial cell loss, uveitis, pigment dispersion and elevated IOP. Anterior segment OCT demonstrated irregularities in the position and configuration of the implants within the anterior chamber with resultant areas of implant,iris and implant,endothelial contact. One patient had acute postoperative hyphaema that resolved with anterior chamber tissue plasminogen activator injection. Both patients required explantation OU, one eye has required trabeculectomy, and one eye with bullous keratopathy is being evaluated for Descemet's stripping endothelial keratoplasty. Conclusions:, Implantation of the NewColorIris cosmetic implant can lead to serious complications including hyphaema, uncontrolled IOP, severe endothelial cell loss, bullous keratopathy and anterior uveitis. Explantation may lead to improvement, but permanent damage to the trabecular meshwork and corneal endothelium persists. [source]


1361: Main anterior entities 2: granulomatous

ACTA OPHTHALMOLOGICA, Issue 2010
F WILLERMAIN
Purpose During uveitis, inflammatory cells and epitheloid cells can aggregates on the corneal endothelium forming keratic precipitates (KPs). Methods On slit lamp examination, KP's can have different forms and locations. Once they are large with a mutton fat appearance, uveitis will be classified as granulomatous. Results Granulomatous uveitis is a heterogeneous group of disease with anterior, intermediate and panuveitis. Granulomatous uveitis can be due to several infectious and non infectious causes, but masquerade syndromes and idiopathic cases must also been ruled out. Conclusion This course will first describe the main clinical characteristics of those different entities. A standard work-up procedure will then be proposed. [source]


3132: The presence and suggested role of mesothelial proteins in the human corneal endothelium

ACTA OPHTHALMOLOGICA, Issue 2010
K JIRSOVA
Purpose To determine whether proteins that are characteristic of the human mesothelial cell phenotype are expressed in human corneal endothelium. Methods Cadaverous human corneo-scleral discs and pathological corneal specimens (melted and rejected corneas) were used. The detection of mesothelin, proteinase inhibitor-9 (PI-9), calretinin and HBME-1 protein (a membrane protein of mesothelial cells) was performed on cryosections and corneal endothelial imprints using indirect immunofluorescent or enzymatic immuncytochemistry. The staining intensity was assessed using fluorescent or light microscopy, and the percentage of positive cells was calculated. Semi-quantitative RT PCR of PI-9, mesothelin and calretinin was performed using mRNA isolated from endothelial imprints. Results A strong signal for mesothelin was present in the corneal epithelium, while less intensive staining was visible in the endothelium. These results were confirmed using qRT-PCR. Immunostaining for PI-9 was observed in almost all corneal epithelial cells and in approximately 50% of the endothelial cells. An increased number of PI-9-positive cells was detected in stromal infiltrates of most melted corneal explants, while almost no positivity was observed in rejected explants. Calretinin was detected in the corneal epithelium and less intensively in the corneal endothelium, where both cytoplasmic and nuclear localisation were demonstrated. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Conclusion The proteins expressed constitutively in mesothelial cells (PI-9, mesothelin, calretinin and HBME-1 protein) are present in adult human corneal endothelium, confirming that this layer expresses markers typical of mesothelial cells. [source]


3134: Identification of potential human corneal endothelial stem-like cell niches

ACTA OPHTHALMOLOGICA, Issue 2010
G THURET
Purpose to study the localization of potential stem-(like) cells in human adult corneal endothelium Methods Fresh (6-12h post mortem) and organ cultured (OC) corneas were studied after flat mount. The whole endothelium and posterior limbus (PL) was observed after triple staining with Trypan blue, Alizarin red and Hoechst 33342, in order to determine cells shape, localization and viability. The level of endothelial cell (EC) differenciation was determined after immunostaining (fluorescence) for ZO-1, Na+/K+ ATPase and COX IV; the cell proliferation status was assessed using Ki67; four markers for stem cells were used: Oct-4, BCRP, Nestin and Telomerase; ability for cell migration was evaluated from Myosin IIA expression Results In several corneas, the nuclei of peripheral EC were centripetally aligned suggesting continuous slow central migration. Numerous small cells with a reduced expression of differenciation markers were accumulated near peripheral Hassall Henle bodies. In these potential niches, cells were distributed in 3-5 layers. A high expression of Myosin II was found in peripheral cells. Ki67+ cells were found in PL and peripheral EC only after OC. None of the 4 stem cell markers was found in EC, and their expression in PL was poorly reliable because of high background noise. Numerous trypan blue positive cells were located at the PL and in the extreme periphery of endothelium Conclusion several strong arguments suggest the location of corneal endothelial stem-like cell niches in endothelial periphery or in the PL, and the capacity of EC to migrate from these niches toward the centre. Trypan blue staining pattern suggests that they could rapidly die in ex vivo corneas, and be therefore hard to indentify [source]


3136: Donor and recipient endothelial cell populations in transplanted corneas: new insights from endothelial imaging

ACTA OPHTHALMOLOGICA, Issue 2010
N LAGALI
Purpose To elucidate the pattern of donor and recipient endothelial cell population in transplanted human corneas and investigate factors impacting this mosaic. Methods 36 corneal grafts were collected from recipients of opposite sex to the donor, at the time of re-transplantation. An endothelial sheet was harvested from each graft, and labeled by fluorescent in situ hybridization of the sex chromosomes, to identify cells as donor or recipient-derived. Images of the graft endothelium were assembled to depict the pattern of cell population of the graft, and the proportion of donor cells present was estimated. Results Endothelial cells of donor origin were found in 26 of 36 grafts, persisting up to 26 years after transplantation. The proportion of donor endothelial cells in the graft was not significantly correlated with postoperative time (P = 0.19). Endothelial images indicated a highly variable pattern of recipient cell repopulation of the graft. A tendency towards donor cell retention in transparent, successful grafts was noted; however, this feature alone was not a reliable indicator of long-term graft transparency. Recent in-vivo optical coherence tomography studies of transplanted corneas indicate a possible mechanism impacting the donor and recipient cell patterns observed on the endothelial surface. Conclusion Two-dimensional imaging of the corneal graft endothelium revealed a variable pattern and extent of donor and recipient cell population, indicating the highly dynamic nature of the corneal endothelium after transplantation. [source]


4332: Determination of corneal endothelial cell density in French eye banks: second look

ACTA OPHTHALMOLOGICA, Issue 2010
N DELESALLE
Purpose Considering the importance of having a precise, robust and especially reproducible ECD counting method, Afssaps organized from April 2008 to June 2009 a second assessment of the reliability of the routine cell count within the 18 french Eye banks. Methods The study design was similar to the first assessment driven by the laboratory ,Biology, engineering and imaging of Corneal Graft' in 2003 (Transplantation 2004; 78: 1299-1302).5 test corneas (1 mm2 of flat mounted, fixed and alizarin stained human corneal endothelium) were selected and sent to the 18 Eye banks. All the usual technicians of each bank had to count the test corneas using the routine method(s) employed to assess grafts. Results 430 counts were carried out by 70 eye banks technicians, by manual and/or image analysis system. 42% (180/430) deviated by more than 10% from the expected ECD. Among them, 128 were over-estimated (max +88%) and 52 were under-estimated (max -31%). 2 banks constantly over-estimated (in the mean +31,7% and +42,7%, no calibration and/or material problem) but the 16 other banks were in average within ±13% from expected ECDs. For manual methods, a statistically significant difference between banks was observed for the 5 test corneas, whereas no difference was observed with image analyzers. ECD obtained with the analysers were closer to expected values than with the manual methods. Compared to the 2003 study, reliability of ECD determination globally improved. Conclusion Image analysis systems prove more reliable (precise and with a lower intra and inter observer variability) than manual counting methods. This ,second look' of Eye banks will allow editing recommendations to improve ECD determination. [source]


4334: Fully automated corneal endothelial morphometry of a large set of images captured by clinical specular microscopy

ACTA OPHTHALMOLOGICA, Issue 2010
C BUCHT
Purpose The endothelial cell density is the most important morphological factor of the corneal endothelium. Morphometry of the corneal endothelium is an important part of several clinical applications. Morphometry of the endothelium is presently carried out by semi automated analysis of pictures captured by Clinical Specular Microscopy (CSM). The need of operator involvement makes this process time consuming. This study presents a method for fully automated analysis of a large range of in vivo images of the corneal endothelium, captured by CSM, using Fourier analysis. Methods Software was developed in the mathematical programming language MATLAB. Pictures of the corneal endothelium, captured by CSM, were read into the analysis software. The software performed automated digital enhancement of the images. The enhanced images were Fourier transformed, using the Fast Fourier Transform. Relevant characteristics of the Fourier transformed images were identified and sampled. The data obtained from each transformed image was used to calculate the mean cell density of the original image, which in turn was compared to a semi automated method cell density estimate. The calculation was based on well known diffraction theory. Results Estimated cell densities of the corneal endothelium were obtained, using fully automated analysis software on 292 images captured by CSM. Using linear regression, a relatively large correlation between the estimates of the fully automated method and the semi automated method was found. Conclusion The results using the considerably faster fully automated method are highly encouraging for further development and implementation of the method. [source]


Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage

ACTA OPHTHALMOLOGICA, Issue 4 2010
Jana Nejepinska
Abstract. Purpose:, To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. Methods:, The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm2 lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. Results:, The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm2. Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm2 and 2478/mm2 centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm2. Conclusion:, The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue. [source]


Monoamine receptors in human corneal epithelium and endothelium

ACTA OPHTHALMOLOGICA, Issue 1 2006
Matthias Grueb
Abstract. Purpose:,Monoamine receptors are found throughout the body. Reports about the presence of monoamine receptors in the human cornea are inconsistent. Methods:,Immunohistochemistry, immunofluorescence and immunoblotting were used to localize monoamine receptor sites on human corneal epithelium and endothelium. Results:,Antibodies to alpha-1, beta-1 and beta-2 adrenergic receptors and to D1-like and 5HT-7 receptors were bound in corneal epithelium. Antibodies to alpha-1, alpha-2A, beta-1 and beta-2 adrenergic receptors and to 5HT-7 receptors were bound in corneal endothelium. Conclusions:,Our data demonstrate the presence of several monoamine receptors in the human cornea. These receptors may play a role in the regulation of fluid transport or corneal homeostasis. [source]


Comparison of the corneal endothelial protective effects of Healon-D and Viscoat

CLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 4 2009
Carolee M Cutler Peck MD MPH
Abstract Background:, The use of dispersive ophthalmic viscosurgical devices (OVDs) has been shown to provide significant protection against air bubble damage to the corneal endothelium when compared with cohesive OVDs. We compared the corneal endothelial protective effects of a new dispersive OVD, Healon-D, with Viscoat. Methods:, Healon-D and Viscoat were used in a randomized and masked fashion in the anterior chamber of 40 rabbit eyes during a procedure where ultrasound at 70% continuous energy was delivered for 2 min. Two millilitres of air bubbles were injected into the anterior chamber during the first minute of the procedure on each eye. Corneas were then stained with trypan blue and alizarin red and evaluated via light microscopy for endothelial injury. Both denuding of the endothelial layer, as well as damage to endothelial cells were quantified by using the Evaluation of Posterior Capsule Opacification digital imaging system. Results:, The denuded area for eyes treated with Healon-D and Viscoat were not significantly different (medians of 0.004167and 0.003333, respectively, P = 0.8908). There was no significant difference in the area of endothelial cell damaged (medians of 0.02183 and 0.01433, respectively, P = 0.4565). When the denuded and damaged areas were calculated together, there was also no difference in the total injured area (medians of 0.05817 and 0.05821, respectively, P = 0.5740). Conclusion:, The new dispersive OVD Healon-D is equally as effective as Viscoat in protecting the corneal endothelial layer from denuding and damage from air bubbles during anterior segment surgery. [source]


Gene transfer to ovine corneal endothelium

CLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 5 2001
Sonja Klebe MBBS
ABSTRACT Purpose: Modification of a donor cornea by gene therapy has potential to modulate irreversible rejection, the major cause of corneal graft failure. The sheep is a useful model for the human in this respect, as ovine endothelial cells are amitotic. The aim of the study was to investigate the ability of various non-viral and viral agents to transfer a reporter gene to ovine corneal endothelium. Methods: The non-viral agents Transfectin-10, Transfectin-20, Transfectin-50, SuperFect, Effectene and CLONfectin were used to deliver the reporter gene, Escherichia coli lacZ, to ovine corneal endothelium in vitro. A Herpes simplex virus-1 and an adenoviral vector each encoding E. coli lacZ were similarly tested. Infected corneas were organ-cultured for up to 7 days in vitro to allow transfection efficiency, duration of gene expression and toxicity attributable to each vector to be compared. Results: Scattered single or clusters of endothelial cells expressing the reporter gene were observed after transfection with CLONfectin, Transfectin-10, Transfectin-20 and Transfectin-50. SuperFect and Effectene were virtually in-effective. At best, the absolute number of infected cells per endothelial monolayer after 3 or 7 days of organ culture was estimated as < 0.01%. The Herpes simplex virus-1 vector also failed to transduce ovine corneal endothelium efficiently. In contrast, transfection rates of up to 70% of endothelial cells were observed with the adenoviral vector. Conclusion: Non-viral vectors and Herpes simplex virus-1 are unlikely to be suitable for gene therapy of corneal endothelium, because the efficiency of transfection is low compared with the rates achieved with adenoviral vectors. [source]


Cyanoacrylate glue for corneal perforations: a description of a surgical technique and a review of the literature

CLINICAL & EXPERIMENTAL OPHTHALMOLOGY, Issue 6 2000
Brendan Jt Vote MBBS
ABSTRACT The effective early application of a cyanoacrylate glue corneal patch can aid in the management of small corneal perforations, corneal melts and wound leaks. Their use gives improved visual outcomes with reduced enucleation rates (6%vs 19%). It may also avoid the need for tectonic penetrating keratoplasty. Cyanoacrylate glue prevents re-epithelialization into the zone of damaged and naked stroma and prevents the development of the critical setting for collagenase production that leads to stromal melting. Cyanoacrylates also have significant bacteriostatic activity against Gram-positive organisms. We describe a simple and easily reproducible method of cyanoacrylate corneal patch application, with neglible risk of inadvertent glue complications. It has the further advantage of a smooth corneal surface rather than an irregular surface as often occurs with direct application methods. With corneal application, the major concern is toxicity of cyanoacrylates through direct contact with the corneal endothelium and lens. Fibrin glues may be less toxic; however, they are not as readily available. The longer alkyl chains of currently available cyanoacrylate glues (e.g. Histoacryl) slows degradation significantly, limiting accumulation of histotoxic by-products to amounts that can be effectively eliminated by tissues. Vigilance in monitoring for infection/corneal infiltrate is necessary at all times, especially when the glue has been present for more than 6 weeks. Corneal patching with cyanoacrylate glue is a temporizing procedure only, buying time to allow healing secondary to medical treatment of the underlying condition, or allowing surgery to be elective and under more optimal conditions once inflammation has been reduced and the integrity of the globe restored. [source]


Could the coefficient of variation (COV) of the corneal endothelium be overestimated when a centre-dot method is used?

CLINICAL AND EXPERIMENTAL OPTOMETRY, Issue 1 2008
Michael J Doughty PhD
Background:, Little has been published on the reliability of estimates of the coefficient of variation (COV) in cell area for human corneal endothelia. The present study compares two methods. Methods:, A non-contact specular micrograph (Topcon SP-2000P) was obtained from the central region of the corneal endothelium of 20 healthy myopic white European subjects, aged from 32 to 53 years, half of whom were successful long-term soft contact lens wearers. The captured image file was either assessed using a machine-based algorithm, in which 25 cells in the middle of the image were marked and their areas reported (designated as ,centre-dot' method) or by a manual method, by which all the cells in the image were outlined on very high magnification prints of the endothelia and the cell areas measured by a manual digitiser in stream mode. The average cell area was used to calculate the endothelial cell density (ECD), while the COV was calculated from the standard deviation (SD) of the cell area measures. Results:, Identical mean cell area values were found (392 µm2) with the two methods, a marginally higher ECD estimate (2,594 versus 2,569) with the centre-dot method (p = NS) but a much higher COV with the centre-dot method (43.8 versus 29.0 per cent). This highly statistically significant difference in COV (p < 0.001) was seen in both contact lens wearers and non-contact lens wearers. A Bland-Altman analysis reveals a bias in the centre-dot method, especially for the COV estimates, that appears to be linked to erroneous definition of a single large cell domain on any individual image. Conclusions:, A centre-dot method can be reliably used to generate useful data on cell area and ECD but it should be used cautiously for estimates of polymegethism (COV). [source]