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.

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.

Evaluation of corneal epithelial wound healing after penetrating keratoplasty in patients receiving a new matrix therapy agent (regenerating agent).

OBJECTIVES: Complete epithelial wound healing is a milestone in early postoperative care after penetrating keratoplasty. The re-epithelialization rate after penetrating keratoplasty was measured in patients receiving a new matrix therapy agent (regenerating agent, Cacicol®) that mimics heparan sulphates. METHODS: This was a prospective, open-label, uncontrolled, single-centre observational study. A total of 33 consecutive patients (33 eyes) who underwent an 8.25-mm diameter penetrating keratoplasty were treated with regenerating agent eye drops: one drop in the operating theatre immediately after graft, then on alternate days. Patients were divided into those at low risk (13 patients) and high risk (20 patients) of delayed wound healing, and follow-up was performed by digital slit lamp with fluorescein-dye testing repeated daily at a fixed time. Dye area was measured using ImageJ freeware. The main endpoint was epithelial healing after regenerating agent therapy. RESULTS: The mean ± standard deviation time to complete healing for all patients was 2.7 ± 1.1 (median: 3, range: 1-6) days. This was obtained on Day 1 for 15% of patients, Day 2 for 33%, Day 3 for 88%, Day 4 for 94% and Day 6 for 100%. There was no significant difference between low- and high-risk patients. The area of epithelial defect decreased by a mean ± standard deviation of 75% ± 22% between Day 1 and Day 2, corresponding to a mean ± standard deviation wound-healing rate of 11.5 ± 6.5 mm2/D. There were no systemic or local side effects related to regenerating agent. CONCLUSION: These preliminary data suggest that regenerating agent could be a useful, non-invasive therapeutic approach in postoperative management of penetrating keratoplasty with the potential to accelerate re-epithelialization.

Transplantation Blues: Inadvertent Staining of Amyloid Deposits With Trypan Blue.

PURPOSE: To describe inadvertent persistent staining of stromal amyloid deposits by trypan blue (TB) after penetrating keratoplasty (PK) and Descemet membrane endothelial keratoplasty (DMEK) performed in patients with corneal amyloidosis. METHODS: Case series of patients with corneal amyloidosis in whom intraoperative TB was used. RESULTS: One patient, hospitalized for acute rejection 6 weeks after DMEK, presented with an intense blue staining of small, spindle-shaped structures in the anterior half of the cornea. DMEK had been performed for endothelial failure of a previous PK procedure done 13 years earlier for advanced lattice corneal dystrophy (LCD). After 6 months, the stromal blue tattoo persisted with impaired visual acuity, and PK was performed. Blue-stained structures were amyloid deposits characteristic of LCD recurrence. In parallel, among 85 consecutive triple procedures (PK + cataract + intraocular lens [IOL]) performed over 7 years, in which TB was used, only patients with LCD (n = 18 eyes in 17 patients) or presumed secondary amyloidosis due to chronic inflammation (n = 1), presented an isolated intense blue ring of the graft-host interface. This persisted up to 7 years with no clinical consequence. CONCLUSIONS: TB can stain corneal amyloid deposits. After PK, staining is limited to the recipient peripheral cornea and has no apparent clinical consequence. However, during DMEK performed after a failed PK, TB stains fibrils accumulated during slow LCD recurrence and scattered on the whole graft. The long-term staining duration indicates strong interactions between TB and amyloid.

Very early endothelial cell loss after penetrating keratoplasty with organ-cultured corneas.

AIMS: After keratoplasty, postoperative endothelial cell loss is calculated between the eye bank endothelial cell density (ebECD) and the postoperative specular microscopy (SM). To elucidate the very early cell loss, always described after penetrating keratoplasty (PK), we designed two complementary studies. METHODS: (1) Clinical prospective study of 90 consecutive PKs (keratoconus, Fuchs' corneal dystrophy, lattice dystrophy, bullous keratopathy) with organ-cultured corneas and postoperative follow-up by SM at day 5 (D5), D15, month 1 (M1) and M3. This series provided a quantification of the difference between ebECD performed 2 days before graft and very early postoperative ECD. (2) Ten pairs of corneas with comparable ebECD in both corneas and same organ-culture (OC) duration were randomised: one cornea was grafted, and, at the same time, the viable ECD (vECD) of the other was measured after labelling with Hoechst/ethidium/calcein-AM. The relationship between vECD and very early postoperative ECD was studied. RESULTS: vECD at the time of graft did not differ from ECD 5 days after PK, with a difference of 39 (-356; 355) cells/mm2 (median (10°; 90° percentile, p=0.799)), whereas a significant difference of 755 (359; 1146) cells/mm2, corresponding to 28% (95% CI 26 to 30) of cells, was measured between ebECD and ECD 5 days after PK (p<0.001). CONCLUSIONS: In OC, ebECD provided to surgeons significantly overestimate the number of viable ECs grafted to patients. The absence of difference between the vECD at D0 and ECD at D5 indicates that the very early endothelial cell loss is almost negligible in recipients.

Global Survey of Corneal Transplantation and Eye Banking.

IMPORTANCE: Corneal transplantation restores visual function when visual impairment caused by a corneal disease becomes too severe. It is considered the world's most frequent type of transplantation, but, to our knowledge, there are no exhaustive data allowing measurement of supply and demand, although such data are essential in defining local, national, and global strategies to fight corneal blindness. OBJECTIVE: To describe the worldwide situation of corneal transplantation supply and demand. DESIGN, SETTING, AND PARTICIPANTS: Data were collected between August 2012 and August 2013 from a systematic review of published literature in parallel with national and international reports on corneal transplantation and eye banking. In a second step, eye bank staff and/or corneal surgeons were interviewed on their local activities. Interviews were performed during international ophthalmology or eye-banking congresses or by telephone or email. Countries' national supply/demand status was classified using a 7-grade system. Data were collected from 148 countries. MAIN OUTCOMES AND MEASURES: Corneal transplantation and corneal procurements per capita in each country. RESULTS: In 2012, we identified 184,576 corneal transplants performed in 116 countries. These were procured from 283,530 corneas and stored in 742 eye banks. The top indications were Fuchs dystrophy (39% of all corneal transplants performed), a primary corneal edema mostly affecting elderly individuals; keratoconus (27%), a corneal disease that slowly deforms the cornea in young people; and sequellae of infectious keratitis (20%). The United States, with 199.10-6 corneal transplants per capita, had the highest transplantation rate, followed by Lebanon (122.10-6) and Canada (117.10-6), while the median of the 116 transplanting countries was 19.10-6. Corneas were procured in only 82 countries. Only the United States and Sri Lanka exported large numbers of donor corneas. About 53% of the world's population had no access to corneal transplantation. CONCLUSIONS AND RELEVANCE: Our survey globally quantified the considerable shortage of corneal graft tissue, with only 1 cornea available for 70 needed. Efforts to encourage cornea donation must continue in all countries, but it is also essential to develop alternative and/or complementary solutions, such as corneal bioengineering.

Corneal endothelium self-healing mathematical model after inadvertent descemetorhexis.

UNLABELLED: An 85-year-old man presented with an unusual complication of cataract surgery. During injection of an ophthalmic viscosurgical device, Descemet membrane was accidentally dissected and removed during the capsulorhexis. Early postoperative visual acuity was counting fingers as a result of massive corneal edema. Using a mathematical model of the endothelium, we evaluated the corneal endothelial cell self-healing capacity from the periphery by calculating the theoretical endothelial cell density (ECD) after corneal endothelial cell redistribution. The calculated theoretical ECD was 1592 cells/mm(2). At 22 months, the corrected distance visual acuity improved to 20/25. The central ECD was 549 cells/mm(2), 516 cells/mm(2), 987 cells/mm(2), and 1344 cells/mm(2) at 5, 8, 16, and 22 months, respectively. The originality of this case is based on the mathematical simulation of the corneal endothelial cell's spontaneous self-healing process, deferring keratoplasty. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

In vivo confocal microscopy of pine processionary caterpillar hair-induced keratitis.

PURPOSE: Multimodal imaging of processionary caterpillar hair-induced keratitis with anterior segment optical coherence tomography and in vivo confocal microscopy. METHODS: Case report. RESULTS: A 25-year-old woman presented with acute keratitis induced by multiple tiny processionary caterpillar hairs. She initially experienced severe pain and moderate vision loss, which gradually improved within a few weeks. Diagnosis was confirmed by in vivo confocal microscopy showing a pathognomonic image strictly comparable with ex vivo microscopy photography. CONCLUSIONS: To the best of our knowledge, this is the first case of corneal in vivo confocal imaging of a caterpillar hair with confirmation by ex vivo microscopy.