The existence of dead cells in donor corneal endothelium preserved with storage media.

AIM: To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination. METHODS: Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33 342, annexin V, anti-vimentin antibody and toluidine blue. RESULTS: Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%±3.3% (mean ±SD) (range: 0.6%-10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells. CONCLUSION: Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells.

A case of fungal keratitis and endophthalmitis post penetrating keratoplasty resulting from fungal contamination of the donor cornea.

PURPOSE: Fungal infections post keratoplasty due to contamination of the donor corneal graft have become important issues that need to be addressed. Here we report a case of fungal keratitis and endophthalmitis post penetrating keratoplasty (PKP) due to fungal contamination of the donor corneal graft. OBSERVATIONS: We present a 52-year-old male who underwent PKP with a donor corneal graft that was later found to be contaminated with fungus. At 4-weeks postoperative, infectious infiltrates suddenly appeared at the border between the host and donor corneal graft, and endophthalmitis concomitantly occurred. A culture of the remnant donor corneoscleral rims and the vitreous fluid obtained during vitreous surgery was found to be positive for Candida albicans. At 6-months post vitreous surgery and intensive anti-fungal medical treatment, both corneal infiltrates and vitreous opacity completely disappeared, and the patient's best-corrected visual acuity recovered to 20/40, with a transparent cornea. CONCLUSIONS AND IMPORTANCE: The findings of this case show that prompt intensive medical treatment and surgical intervention effectively saved the vision in a patient with fungal keratitis and endophthalmitis due to contamination of the donor corneal graft.

Femtosecond laser-assisted keratoplasty: full and partial-thickness cut wound strength and endothelial cell loss across a variety of wound patterns.

PURPOSE: To evaluate wound strength for patient safety during transport and endothelial viability when partial and complete femtosecond laser-assisted keratoplasty (FLAK) incisions are made in cadaveric corneas. METHODS: 19 human corneoscleral rims were divided into six groups, mounted on an anterior chamber maintainer and cut with a femtosecond laser programmed to the following patterns: 'zigzag' (A), 'mushroom' (B) and 'top hat' (C) in both full (1) and partial (2) thicknesses. The pressure required to produce leakage from the corneal incision was then measured. Eight additional corneas were cut with the 'zigzag' pattern: four full and four partial thickness, prepared and transported per standard eye bank protocol, and analysed for endothelial cell loss with trypan blue staining and digital image analysis. RESULTS: Mean leakage pressure in mm Hg for group A1 was 110 (SD 94); group A2, 1180 (SD 468); group B1, 978 (SD 445); group B2, 987 (SD 576); group C1, 710 (SD 474); group C2, 1290 (SD 231). There was a significant difference in leakage pressure between groups A1 and A2 (p=0.05), groups A1 and B1 (p=0.05), and groups A1 and C1 (p=0.05). Mean percentage endothelial damage after full-thickness cuts was 8.40 (SD 2.34) and 5.30 (SD 1.33) in partial-thickness cuts (p=0.11). CONCLUSIONS: Partial thickness zigzag, top hat and mushroom-style partial FLAK incisions left an intact tissue wall with high resistance to rupture, whereas full-thickness cuts were more variable. Laser trephination and eye bank handling protocol for donor FLAK buttons leads to moderate peripheral endothelial cell loss in tissue with both complete and partial cuts.

In situ ultrahigh-resolution optical coherence tomography characterization of eye bank corneal tissue processed for lamellar keratoplasty.

PURPOSE: To use optical coherence tomography (OCT) as a noninvasive tool to perform in situ characterization of eye bank corneal tissue processed for lamellar keratoplasty. METHODS: A custom-built ultrahigh-resolution OCT (UHR-OCT) was used to characterize donor corneal tissue that had been processed for lamellar keratoplasty. Twenty-seven donor corneas were analyzed. Four donor corneas were used as controls, whereas the rest were processed into donor corneal buttons for lamellar transplantation by using hand dissection, a microkeratome, or a femtosecond laser. UHR-OCT was also used to noninvasively characterize and monitor the viable corneal tissue immersed in storage medium over 3 weeks. RESULTS: The UHR-OCT captured high-resolution images of the donor corneal tissue in situ. This noninvasive technique showed the changes in donor corneal tissue morphology with time while in storage medium. The characteristics of the lamellar corneal tissue with each processing modality were clearly visible by UHR-OCT. The in situ characterization of the femtosecond laser-cut corneal tissue was noted to have more interface debris than shown by routine histology. The effects of the femtosecond laser microcavitation bubbles on the corneal tissue were well visualized at the edges of the lamellar flap while in storage medium. CONCLUSIONS: The results of our feasibility study show that UHR-OCT can provide superb, in situ microstructural characterization of eye bank corneal tissue noninvasively. The UHR-OCT interface findings and corneal endothelial disc thickness uniformity analysis are valuable information that may be used to optimize the modalities and parameters for lamellar tissue processing. The UHR-OCT is a powerful approach that will allow us to further evaluate the tissue response to different processing techniques for posterior lamellar keratoplasty. It may also provide information that can be used to correlate with postoperative clinical outcomes. UHR-OCT has the potential to become a routine part of tissue analysis for any eye bank or centers creating customized lamellar corneal tissue for transplantation.