Comprehensive mass spectrometry lipidomics of human biofluids and ocular tissues.

Evaluating lipid profiles in human tissues and biofluids is critical in identifying lipid metabolites in dysregulated metabolic pathways. Due to various chemical characteristics, single-run lipid analysis has not yet been documented. Such approach is essential for analyzing pathology-related lipid metabolites. Age-related macular degeneration, the leading cause of vision loss in western countries, is emblematic of this limitation. Several studies have identified alterations in individual lipids but the majority are based on targeted approaches. In this study, we analyzed and identified approximately 500 lipid species in human biofluids (plasma and erythrocytes) and ocular tissues (retina and retinal pigment epithelium) using the complementarity of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RPC), coupled to high-resolution mass spectrometry. For that, lipids were extracted from human eye globes and blood from 10 subjects and lipidomic analysis was carried out through analysis in HILIC and RPC, alternately. Furthermore, we illustrate the advantages and disadvantages of both techniques for lipid characterization. RPC showed greater sensitivity in hydrophobicity-based lipid separation, detecting diglycerides, triglycerides, cholesterol, and cholesteryl esters, whereas no signal of these molecules was obtained in HILIC. However, due to coelution, RPC was less effective in separating polar lipids like phospholipids, which were separated effectively in HILIC in both ionization modes. The complementary nature of these analytical approaches was essential for the detection and identification of lipid classes/subclasses, which can then provide distinct insights into lipid metabolism, a determinant of the pathophysiology of several diseases involving lipids, notably age-related macular degeneration.

Treatment of corticosteroid-resistant Graves' orbitopathy with tocilizumab: a single-centre prospective study.

PURPOSE: To assess tocilizumab (TCZ) as an emergent treatment for corticosteroid-resistant active Graves' orbitopathy (GO). METHODS: We conducted a single-centre prospective study. We assessed TCZ in patients with active corticosteroid-resistant GO. Each patient received intravenous TCZ every four weeks until symptom stabilization. Our primary outcome was GO activity evaluated by the clinical activity score (CAS). The secondary outcomes included variation in thyroid-stimulating immunoglobulin (TSI). RESULTS: We included ten patients. Three patients had compressive neuropathy with visual field impairment and vision loss. CAS improved significantly in 100% of the patients included in the analysis, with a decrease in the mean CAS of 4.5 ± 1.2 points (p = .003). There was a significant decrease in the TSI after therapy, from 21.7 ± 22.9 at baseline to 4.0 ± 3.3 (p = .006). A mean of three infusions was necessary to drastically decrease the TSI amount. The baseline mean before TCZ was 4.7 ± 1.2 and the final mean after TCZ IV infusion was 0.2 ± 0.4. CONCLUSIONS: Our study showed the efficiency of TCZ in patients with GO resistant to corticosteroid therapy, as shown in previous studies. Our present work adds two important pieces of information: TCZ might be particularly useful for GO with compressive neuropathy and it is efficient regardless of initial TSI level. Considering the numerous advantages over steroids (high response rate and lower rate of adverse events), further randomized controlled trials should be conducted to assess the possible place of TCZ as a first-line treatment.

Exploration of the ocular surface infection by SARS-CoV-2 and implications for corneal donation: An ex vivo study.

BACKGROUND: The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. METHODS AND FINDINGS: To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. CONCLUSIONS: In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. TRIAL REGISTRATION: Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

A retrospective study of the efficacy of intense pulsed light delivered by the Lacrystim® for meibomian gland dysfunction therapy.

BACKGROUND: Meibomian gland dysfunction is the most common etiology of dry eye disease worldwide and intense pulsed light appears to be a promising treatment with encouraging results. Lacrystim® is a new IPL device (CE marking in 2019) and no studies have yet been published on it. We propose the first study on this device with an objective assessment of its efficacy and an extended follow-up over 6 months. METHODS: Patients presenting with a dry eye disease (DED) with stable mild to moderate MGD and having received Lacrystim® treatment between june 2019 and june 2020 were included. 3 IPL sessions were performed at D0, D15 and D45 with 4 shots per side at a fluence of 8 mJ/cm2. DED clinical evaluation was performed at D0, D15, D45, 3rd month and 6th month: Oxford scale and break up time, Schirmer test and Ocular Surface Disease Index (OSDI) questionnaire. Lacrydiag® imaging device carried out an objective examination of tear film: interferometry, meibography, tear meniscus height and non-invasive break up time (NIBUT). The primary endpoint was the evolution in NIBUT between the first visit D0 and 3rd month. Data collection was done retrospectively. Statistical analysis was done using a linear mixed-effects model and a non-parametric linear mixed-effects model (R software). RESULTS: Forthy five consecutive patients were included. NIBUT significantly increased between D0 and 3rd month: mean difference of 1.63 seconds, IC95% [0.51; 2.62], (p = 0.002) with a prolonged effect at 6th month. OSDI and OXFORD scores and interferometry were also significantly improved at 3rd month and 6th month. There was no significant change in BUT, Schirmer test and tear meniscus height. No adverse event was noted. CONCLUSIONS: IPL delivered by Lacrystim® appears effective and safe to treat MGD although a randomized controlled trial is needed to validate its results. TRIAL REGISTRATION: This work was approved by a local ethics committee "Terre d'éthique" (institutional review board number: IRBN672019/CHUSTE) and registered on the clinicaltrial.gov website ( NCT04147962 , 01/11/2019).

Corneal endothelial cell therapy: feasibility of cell culture from corneas stored in organ culture.

In 2013, a clinical trial was initiated to investigate cell therapy for the treatment of corneal endothelial decompensation. Cultivating human corneal endothelial cells (CECs) while maintaining their functional phenotype is challenging; therefore, establishment of a confirmed protocol is pivotal for obtaining approval from regulatory authorities for use of cellular therapy products. In this study, we evaluated organ culture (OC) as a storage method for donor corneas used as a raw material for establishing CEC cultures. OC allows storage of corneal tissue for conventional corneal transplantation at 31-37 °C for up to 5 weeks, whereas storage at 4 °C is limited to 2 weeks. We investigated 20 pairs of corneas: one cornea of each pair was stored in OC and the other in cold storage for one week before CEC culture. In 15/20 cases, the CECs assumed a hexagonal sheet-like monolayer structure and expressed endothelial function-related markers. CECs were also obtained from OC corneas that had been stored for 1 (n = 19) and 2 (n = 7) months. As a further test, CECs were cultivated from 5 OC corneas that had been transported from France to Japan. In all cases, these corneas, even after international transport, generated CECs that formed hexagonal monolayers with clinically applicable and sufficiently high cell densities. In conclusion, the CEC cultures required for endothelial cell therapy can be obtained from OC corneas without changing the standard storage operating procedures of the eye banks.

Detection of refractive photokeratectomy traces during eye banking: impossible with organ culture but possible with an active storage machine: case report.

The detection of corneas operated on for refractive surgery [LASIK or photorefractive keratectomy (PRK)] will become a major concern for eye banks in the coming years because this surgery is often forgotten during the interview with the deceased's relatives. We present here 2 corneas operated on with PKR and stored successively in organ culture (OC) and in the active storage machine (ASM) that restores intraocular pressure, restores the cornea to its original shape, respects transparency and incorporates non-invasive controls. The 2 corneas of a 49-year-old donor operated 17 years earlier by PRK for -2 and -3 diopters myopia were stored in OC for 14 days and then placed in ASM for 48 h. Thickness map and OCT topography were performed under the 2 storage conditions, histology and electron microscopy were then performed. Traces of PRK remained unnoticed in OC while they were evident in ASM with central epithelial anomaly, central thinning and flattening of central keratometry shown by OCT. Histology and ultrastructure confirmed the absence of Bowman's membrane in the center. By placing the cornea under physiological conditions, and in particular by triggering its deswelling and by restoring its natural curvature, the ASM allows effective detection of subtle refractive surgery traces like those present after PRK.

Prospective study of serum and aqueous humour anti-Hsp70.1 IgG antibody levels in ocular toxoplasmosis.

AIMS: We evaluate whether the serum and aqueous humour (AH) level of IgG anti-Hsp70.1 antibodies improved the biological diagnosis of ocular toxoplasmosis. METHODS AND RESULTS: In this prospective cross-sectional and multicentre study, serum and AH were collected at the time of active uveitis. Anti-Hsp70.1-antibody levels were determined by ELISA. Patients with confirmed (Group A1, n = 21) or suspected ocular toxoplasmosis (group A2, n = 30) were enrolled, as well as a control group of patients with cataract (group B, n = 42). Serum IgG anti-Hsp70.1 antibody levels were not significantly different within the group of uveitis patients (A1, n = 21 vs A2, n = 30, P = .8) and were significantly associated with the affected retinal zone (P = .006) and with the size of the retinal lesion (P = .03). Serum anti-Hsp70.1 antibody level was positive in 10 out of the 18 patients of group A2. Significant anti-Hsp-70.1 antibody level in AH was reported in only three patients (3 eyes) with confirmed ocular toxoplasmosis. CONCLUSION: While the level of IgG anti-Hsp-70.1 antibody in AH did not improve the laboratory diagnosis of ocular toxoplasmosis, its level in serum was of major significance for retinal damage diagnosis.

Regional trends in corneal transplantation from 2004 to 2015 in France: a 12-year review on indications, technique and waiting period.

The aim of this study was to report the 12-year longitudinal trends in regional disparity in terms of indications, techniques and waiting period for corneal transplantation in France from 2004 to 2015. The records of all corneal transplantations performed from 2004 to 2015 in France were retrospectively reviewed by analasing the registry and the territorial organization [divided France into 7 interregional areas of collection and distribution of grafts (IACD)] of the French Biomedicine Agency. A total of 46,658 corneal transplantations were performed between 2004 and 2015. In 2014, there was 65.8 keratoplasties per million inhabitants (10-6 per capita in France, but there were some regional disparities, from 44.9 × 10-6 per capita in IACD 2 to 87.2 × 10-6 per capita in IACD 5. In 2014, IACD 7 performed the highest number of transplantations for keratoconus with 15.7 × 10 - 6 per capita; IACD 5 ranked first for Fuchs endothelial disease and secondary endothelial failure with, respectively, 20.5 × 10-6 per capita and 21.2 × 10-6 per capita, and IACD 4 ranked first for graft failure with 17.4 × 10-6 per capita. All regions over the years began to perform more lamellar keratoplasties (4.3% in 2004 vs 45.2% in 2015) and fewer penetrating keratoplasties (85.8% in 2004 vs 48.2% in 2015). The mean waiting time was 3.4 ± 5.2 months in France over 12 years, with minimal disparities between regions: all of them under 4 months waiting time in 2015, from 1.4 months for IACD 1-3.8 months for IACD 5. Regional disparities have changed over the years, with a modification of indications, and upgrading surgical techniques for some indications. Some disparities remain, mainly because of the variability in the number and activity of transplantation centres and eye banks. Measures could be taken to minimize these disparities, such as increasing communication between eye banks. The waiting time for keratoplasty decreasing below the 4-month mark is a good indicator of the progress made. Regional disparities have decreased over the years, but some regions remain disadvantaged in terms of needs and access to transplants. Tomorrow's challenge is to identify solutions and adapt the offer to the needs.

Innovative corneal active storage machine for long-term eye banking.

Optimal ex vivo corneal storage in eye banks is crucial to increase both the number of corneas suitable for graft and their intrinsic quality, mainly the number of viable endothelial cells, which dictates graft survival in recipients. With both passive storage methods used worldwide (short-term cold storage in the United States, long-term organ culture in Europe), significant endothelial cell loss is inevitable. Here we show that, with an active storage machine, also called a bioreactor, which restores 2 fundamental physiological parameters, intraocular pressure and medium renewal, endothelial cell survival is improved by 23% compared with organ culture after 4 weeks' storage. Also observed in the bioreactor is a 4-fold higher expression of Na+ /K+ ATPase, which supports one of the major endothelial cell pumping functions. In addition, corneas remain thin and transparent, so they are suitable for surgery at any time. This new active eye banking method may help to reduce the severe global scarcity of donor corneas.

How transparent film applied on dermatologic imaging devices in order to prevent infections affects image quality?

BACKGROUND: In the clinical practice, transparent films are used as sterile interfaces in in vivo dermatologic imaging in order to prevent the transmissions of infections. However, in our experience, the use of a transparent film can alter skin images. Our study aimed to compare the optical quality of a series of different plastic films used as interfaces in order to understand if some might be more suitable for imaging. MATERIALS AND METHODS: We tested the optical properties of 11 different protective transparent films that are marketed in France with a transparency meter and a spectrophotometer. RESULTS: Transmission, minimal diffusion, amount of gray, and contrast were obtained for each transparent film. Transmission ranged from 93.24% to 96.88% (mean 95.36; standard deviation SD 1.02), minimal diffusion from 88.28% to 123.87% (mean 101.04; standard deviation SD 10.02) and contrast from 11.01 to 15.88 (mean 13.93 and SD 1.3). For some films, the transmission was lower at lower wavelengths. CONCLUSION: All tested films had excellent optical properties. However, some of them had better optical qualities and seemed more suitable for their use in dermatologic imaging.

Endothelial quality of eye bank-prestripped DMEK prepared form organ-cultured corneas with the Muraine technique.

Our aim was to measure the endothelial quality of prestripped Descemet membrane endothelial keratoplasty (DMEK) 48 h after preparation in an eye bank with the Muraine technique and shipping in a distant center. Ten pairs of human corneas with similar eye bank endothelial cell density (ebECD) were stored in organ-culture (OC) for 25 days (20, 28) [median (10-90 percentiles)]. One cornea was then randomized to DMEK preparation using the Moria Muraine trephine, the other served as control. The grafts were left attached to the center of the cornea, immersed in the OC medium (without Dextran) and shipped to a distant center. After 48 h, the viable ECD (vECD) was assessed by image analysis after staining with Hoechst/Ethidium/Calcein-AM. In addition, immunostaining was performed on flat mounted tissues for structural (ZO-1, NCAM, CD166) and functional (Na+/K+ ATPase) proteins of ECs, and for collagen I. Just before stripping, ebECD was 2428 (2268-2669) cells/mm2 for DMEK and 2471 (2135-2714) for controls (P = 1). Forty-eight hours after stripping, vECD was 2057 (1829-2463) cells/mm2 for DMEK and 2119 (1496-2525) for controls (P = 0.508). The expression patterns of the 5 proteins were similar in ECs of both groups. Notably, the deep posterior folds observed in OC controls almost disappeared in prestripped DMEK due to the lack of a link between Descemet membrane and stroma. As a consequence of the elimination of mechanical stress in these zones, EC evenly covered the whole graft. In conclusion, DMEK prestripping with the Muraine technique and shipping away can be used safely by eye banks.

'En face' ex vivo reflectance confocal microscopy to help the surgery of basal cell carcinoma of the eyelid.

BACKGROUND: Ex vivo confocal microscopy is a recent imaging technique for the perioperative control of skin tumour margins. Up to date, it has been used in the fluorescence mode and with vertical sections of the specimen margins. The aim of this study was to evaluate its use in the reflectance mode and with a horizontal ('en face') scanning of the surgical specimen in a series of basal cell carcinoma of the eyelid. DESIGN: Prospective consecutive cohort study was performed at the University Hospital of Saint-Etienne, France. PARTICIPANTS: Forty-one patients with 42 basal cell carcinoma of the eyelid participated in this study. METHODS: Basal cell carcinomas were excised with a 2-mm-wide clinically safe margin. The surgical specimens were analysed under ex vivo confocal microscopy in the reflectance mode and with an en face scanning in order to control at a microscopic level if the margins were free from tumour invasion. Histopathogical examination was later performed in order to compare the results. MAIN OUTCOME MEASURES: Sensitivity and specificity of ex vivo confocal microscopy for the presence of tumour-free margins. RESULTS: Ex vivo confocal microscopy results were consistent with histopathology in all cases (tumour-free margins in 40 out of 42 samples; sensitivity and specificity of 100%). CONCLUSIONS: Ex vivo confocal microscopy in the reflectance mode with an 'en face' scanning can control tumour margins of eyelid basal cell carcinomas and optimize their surgical management. This procedure has the advantage on the fluorescent mode of not needing any contrast agent to examine the samples.

Corneal transplantation: study of the data of a regional eye bank for the year 2013 and analysis of the evolution of the adverse events reported in France since 2010.

To analyze the data of the adverse events collected in a single major eye bank (EFS Bourgogne Franche Comté, Besançon, France) for the year 2013 and to report the French data of biovigilance provided by the French National Agency for Medicines and Health Products Safety (ANSM) between 2010 and 2013. we have set up a study of adverse events in 2013, in collaboration with a single eye bank (EFS Bourgogne Franche Comté, Besançon, France). A survey was sent to the surgeon for each delivered corneal button by the eye bank in 2013. They were asked for each grafted patient performed in their center, the type of graft (penetrating keratoplasty, anterior keratoplasty or endothelial keratoplasty), the occurrence of adverse events (primary failure, infectious keratis, endophthalmitis, immune rejection, and other events) and the time interval between surgery and events (Less than 1 postoperative month, between 1 month and 1 year postoperatively, >1 year postoperatively). In 2013, 407 corneal buttons were delivered by the eye bank of Besançon in 21 medical centers which performed corneal grafts and we sent 407 surveys. We received 243 completed questionnaires (59.75%) from 11 centers (52.38%). The global reported rate of adverse events was 27.54% of the graft (n = 65/236 corneal grafts performed in 11 centers in 2013; 20% of Primary graft failure, 11% of infectious keratitis, 1% of endophthalmitis, 34% of rejection, 34% of other incidents). 30.16% of complications were noticed before the first month after surgery versus 52.38% of complications noticed between the first month and the first year after surgery and 17.46% of complications noticed after the post-operative first year The most common causes of adverse events after PK were Immune rejection (13.17%), surgical causes (5.98%) and infection (4.79%) and after EK were Primary graft failure (8.2%) and surgical causes (19.67%). In 2013, in France 0.83% of adverse events were notified in ANSM. For the 236 performed graft issued from a major eye bank (EFS Besançon) in 2013 the global reported rate of post-graft adverse events was 27.54% of the grafts (20% of Primary graft failure, 11% of infectious keratitis, 1% of endophthalmitis, 34% of rejection and 34% of other incidents). Compared to the ANSM data (0.83% of adverse events reported in 2013) this rate is high. This difference can be explained by the low rate of annual notification to the ANSM and shows that biovigilance in France must be more developed. Since biovigilance needs constant improvement for the safety of the graft system, training, information for practitioners, simplifications of procedures and international standardization of the definition are the main points that could be improved.

Considering 3D topography of endothelial folds to improve cell count of organ cultured corneas.

The posterior side of the cornea is covered by the endothelial monolayer, which governs corneal transparency but cannot proliferate. Determination of endothelial cell density (ECD) is therefore the minimal and mandatory quality control in all eye banks. It avoids primary graft failures caused by endothelial insufficiency, and allows allocation of corneas to surgical techniques requiring different numbers of endothelial cells (ECs). Corneas stored in organ culture (17% of grafts worldwide), are characterized by heavy stromal swelling and numerous deep endothelial folds, up to 200 µm high. During microscopic en face observation, flat surfaces are thus exceptional and EC counting is biased by parallax errors, resulting in overestimated eye bank ECD (ebECD). We used a motorized transmitted light microscope to acquire Z-stacks of images every 10 µm, and processed them to reconstruct the 3D surface of the folded endothelium. This method (3D-ECD) takes into account the local point-by-point slope in order to correct ECD. On a set of 30 corneas, we compared 3D-ECD and ebECD determined on five identical zones at the center of the cornea. 3D reconstruction allowed us to visualize twice as many cells, and ebECD was 8.1 ± 4.5% (95%CI 6.4-9.7) higher than 3D-ECD, with 1744 ± 488 versus 1606 ± 473 cells/mm2. 3D counting makes it possible to increase cell sampling and to correct overestimation by the conventional en face counting still routinely performed in eye banks.

Optimization of immunostaining on flat-mounted human corneas.

PURPOSE: In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts frequently encountered at tissue borders. We previously proposed several protocols, using fixation in 0.5% paraformaldehyde (PFA) or in methanol, allowing immunostaining on flatmounted corneas for proteins of different cell compartments. In the present study, we further refined the technique by systematically assessing the effect of fixative temperature. Last, we used optimized protocols to further demonstrate the considerable advantages of immunostaining on flatmounted intact corneas: detection of rare cells in large fields of thousands of ECs and epithelial cells, and accurate subcellular localization of given proteins. METHODS: The staining of four ubiquitous proteins, ZO-1, hnRNP L, actin, and histone H3, with clearly different subcellular localizations, was analyzed in ECs of organ-cultured corneas. Whole intact human corneas were fixed for 30 min in 0.5% paraformaldehyde or pure methanol at four temperatures (4 °C for PFA, -20 °C for methanol, and 23, 37, and 50 °C for both). Experiments were performed in duplicate and repeated on three corneas. Standardized pictures were analyzed independently by two experts. Second, optimized immunostaining protocols were applied to fresh corneas for three applications: identification of rare cells that express KI67 in the endothelium of specimens with Fuch's endothelial corneal dystrophy (FECD), the precise localization of neural cell adhesion molecules (NCAMs) in normal ECs and of the cytokeratin pair K3/12 and CD44 in normal epithelial cells, and the identification of cells that express S100b in the normal epithelium. RESULTS: Temperature strongly influenced immunostaining quality. There was no ubiquitous protocol, but nevertheless, room temperature may be recommended as first-line temperature during fixation, instead of the conventional -20 °C for methanol and 4 °C for PFA. Further optimization may be required for certain target proteins. Optimized protocols allowed description of two previously unknown findings: the presence of a few proliferating ECs in FECD specimens, suggesting ineffective compensatory mechanisms against premature EC death, and the localization of NCAMs exclusively in the lateral membranes of ECs, showing hexagonal organization at the apical pole and an irregular shape with increasing complexity toward the basal pole. Optimized protocols were also effective for the epithelium, allowing clear localization of cytokeratin 3/12 and CD44 in superficial and basal epithelial cells, respectively. Finally, S100b allowed identification of clusters of epithelial Langerhans cells near the limbus and more centrally. CONCLUSIONS: Fixative temperature is a crucial parameter in optimizing immunostaining on flatmounted intact corneas. Whole-tissue overview and precise subcellular staining are significant advantages over conventional immunohistochemistry (IHC) on cross sections. This technique, initially developed for the corneal endothelium, proved equally suitable for the corneal epithelium and could be used for other superficial mono- and multilayered epithelia.

Assessing microstructures of the cornea with Gabor-domain optical coherence microscopy: pathway for corneal physiology and diseases.

Gabor-domain optical coherence microscopy (GD-OCM) was applied ex vivo in the investigation of corneal cells and their surrounding microstructures with particular attention to the corneal endothelium. Experiments using fresh pig eyeballs, excised human corneal buttons from patients with Fuchs' endothelial dystrophy (FED), and healthy donor corneas were conducted. Results show in a large field of view (1 mm×1 mm) high definition images of the different cell types and their surrounding microstructures through the full corneal thickness at both the central and peripheral locations of porcine corneas. Particularly, an image of the endothelial cells lining the bottom of the cornea is highlighted. As compared to healthy human corneas, the corneas of individuals with FED show characteristic microstructural alterations of the Descemet's membrane and increased size and number of keratocytes. The GD-OCM-based imaging system developed may constitute a novel tool for corneal imaging and disease diagnosis. Also, importantly, it may provide insights into the mechanism of corneal physiology and pathology, particularly in diseases of the corneal endothelium.

Anterior segment biometry using spectral-domain optical coherence tomography.

PURPOSE: To compare the anterior chamber and anterior chamber angle measurements obtained with spectral-domain anterior segment optical coherence tomography (AS-OCT) and time-domain AS-OCT. METHODS: The anterior chamber of healthy participants was imaged with spectral-domain AS-OCT (Casia SS-1000; Tomey, Nagoya, Japan) and time-domain AS-OCT (Visante; Carl Zeiss Meditec, Dublin, CA). Central corneal thickness (CCT), anterior chamber depth (ACD), angle opening distance at 500 and 750 µm (AOD 500 and AOD 750), trabecular iris space area at 500 and 750 µm (TISA 500 and TISA 750), and scleral spur angle were measured. The intraclass correlation coefficients (ICCs) were calculated. The Pearson correlation test was used to evaluate the correlation between the measurements and Bland-Altman plots to evaluate the agreement. RESULTS: One hundred one eyes of 101 healthy participants were analyzed. Excellent repeatability was found with both devices for CCT, AOD 500, AOD 750, TISA 750, and scleral spur angle (ICC = 0.90 to 0.98 and 0.89 to 0.97, respectively) and excellent to moderate repeatability was found for TISA 500 (ICC = 0.68 to 0.97 and 0.70 to 0.93, respectively). For all parameters, Casia and Visante measurements were significantly correlated (r = 0.76 to 0.98; P < .02). ACD measured with the Casia was significantly larger (mean difference = 0.12 ± 0.08 mm; P < .0001), and the scleral spur angle measured with the Casia was significantly lower (mean difference = 4.85° ± 5.30°; P < .01). There were nonsignificant differences between the devices for the other parameters (P > .06). CONCLUSIONS: Both Casia and Visante AS-OCT demonstrate high repeatability. Except for the ACD and scleral spur angles, the two devices show good agreement.

The role of in vivo confocal microscopy in the diagnosis of eyelid margin tumors: 47 cases.

BACKGROUND: Handheld in vivo reflectance confocal microscopy (IVCM) is a new imaging method that allows noninvasive diagnosis of cutaneous tumors but to date it has not been used in the study of eyelid tumors. OBJECTIVE: We sought to evaluate the suitability of IVCM for eyelid margin tumors. METHODS: We prospectively evaluated the IVCM features of 47 eyelid margin lesions, clinically suspicious of malignancy; 35 of these were excised whereas the other 12, with no IVCM malignant features, were followed up for at least 1 year. Clinical, IVCM, and histologic diagnoses were compared. RESULTS: IVCM showed sensitivity and specificity of 100% and 69.2%, respectively, for malignancy (basal cell carcinoma, squamous cell carcinoma, and melanoma). The follow-up of the 12 nonexcised lesions did not show any clinical progression. LIMITATIONS: The lesions showing neither clinical nor IVCM features for malignancies were not biopsied in view of the potential functional and aesthetic consequences of eyelid margin surgery. CONCLUSION: Used with a handheld dermatology-specific microscope, IVCM can play a role in the noninvasive diagnosis of eyelid margin lesions. Further studies are needed to better define diagnostic criteria of eyelid tumors and improve the specificity of this technique.

Non-invasive measurement of transparency, arcus senilis, and scleral rim diameter of corneas during eye banking.

We developed a non-invasive device to quantify transparency (T), clear corneal diameter (CCD) excluding arcus senilis, and scleral rim diameter (SRD) of stored corneas. The T value (expressed in % on a relative scale), based on the modulation transfer function principle, referred to the ratio of local contrasts of a special LED backlit chart measured with and without cornea. CCD and SRD (in mm) were automatically calculated by morphologic operations. Firstly, we assessed measurement reproducibility. We then determined the agreement of T and CCD values with 3-level scores given independently by three experts on 179 scientific corneas. Thirdly, an eye bank was equipped with the device, and 358 consecutive organ-cultured (OC) corneas were tested for donor- and storage- related factors possibly influencing T and CCD. Reproducibility of T, CCD and SRD measurements was high, with intraclass correlation coefficients of 0.982, 0.886, and 0.999 respectively. Capacity to discriminate the three levels of transparency and arcus senilis was good, with T of 20.0 (10.0-33.6), 38.3 (24.3-75.4) and 57.9 (33.9-90.0) % respectively for T deemed poor, average, and good (P < 0.001), and CCD of 9.8 (7.3-10.6), 10.5 (8.2-11.5), and 11.1 (9.9-12.0) mm respectively for arcus senilis deemed prominent, moderate or absent (P < 0.001). T was correlated with neither donor age nor endothelial cell density nor storage time, but slightly worsened during OC for corneas assessed twice. In conclusion, the device, which can be easily integrated in the facilities of an eye bank, provides reliable objective measurement of T, CCD, and SRD. This could be a useful tool for standardizing quality assessment of stored corneas and consequently optimizing their selection for penetrating, endothelial or anterior lamellar keratoplasty.

Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks.

The aim of this work was to analyze the magnitude of inherent errors associated with the fixed-frame counting method for corneal endothelial cell density (ECD) measurements. This technique is common among most eye banks worldwide. Three types of mosaics were used: regular and irregular tessellated mosaics (eight increasing densities ranging from 800 to 3,600 cells/mm(2) by steps of 400 cells/mm(2)) generated by a computer, and real mosaics (four specimens) obtained from human corneal endothelium flat mounted and stained with Alizarin red. On the three mosaics, the fixed-frame counting method was applied using a computer program. The ECD was calculated for 3,000 successive random positions from calibrated grids which area ranged from 50 × 50 to 300 × 300 µm(2) (incremental steps of 25 µm). For each grid, the ECD was expressed either as a single count, a mean of five or a mean of 10 measures. The fixed-frame count was constantly associated with an inherent variability but repeatability increased with larger grid size and ECD. The mean calculated out of 10 measures was the most reliable, but still, we noted ±5 % of residual variability from the real ECD. The 100 × 100 µm(2) grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 µm(2) grid counts. Digital image analysis with a variable frame counting method would be the best alternative.