Storage Period

As part of the globe, the corneal endothelium is in contact with the stagnant aqueous humour. Accordingly, necrotising tissue, metabolic waste products and hydrolytic enzymes accumulate with time. Since these are toxic for the endothelium, the storage time is limited to 48–72 h.

The storage time of the whole eye generally does not exceed 24 h prior to corneoscleral button excision and storage.

Microbiological Safety

The effectiveness of antibiotic solutions in reducing microbial flora and contamination is debatable since their effectiveness at low temperatures cannot be verified. However, antibiotics may accumulate in the tissue and may be an effective prophylaxis following transplant surgery.

Tissue Evaluation

The slit lamp is the usual method of evaluating the cornea. This can be supplemented with examination of the endothelium by specular microscopy provided the cornea is not too swollen and therefore it needs to be performed as soon as possible after retrieval surgery.

Hypothermic Storage of the Corneoscleral Button

Technical Aspects

The corneoscleral buttons are placed in a modified tissue culture solution and stored at 2–6°C. Handling of the cornea is minimised as the evaluation of the endothelium by slit lamp and specular microscopy is usually performed with the cornea remaining in its container. The formula for some older storage solutions, such as M-K [2] are in the published literature while the more modern storage solutions are commercially available. Storage parameters such as temperature and maximum time of storage vary between solutions and need to be followed as prescribed. The corneoscleral buttons are directly available for surgery provided that the donor’s medical history and tissue serology permits release.

The fundamental composition of hypothermic storage solutions is tissue culture medium supplemented with antibiotics and an osmotic agent such as dextran to compensate for the inactivity of the cornea’s normal water removal mechanism at 4°C, thus preventing cell swelling and maintaining corneal deturgescence. More modern variants have increased storage times through the addition of energy sources, antioxidants, membrane stabilising factors and growth factors. Most importantly, the addition of chondroitin sulphate has provided better endothelial cell viability during storage while also acting as an additional osmotic agent, extending acceptable storage times [2]. However, degenerative changes in the endothelium as a result of post-mortem time and other variables such as cause and circumstances of death will progress during hypothermic storage and thus limit storage times. In addition, epithelial healing at hypothermic temperatures is not possible and indeed

the epithelium can be compromised when the hypothermic storage is prolonged [10]. To reduce these risks, the recommended storage periods are kept far below the original reported maxima and the benchmark time interval from death to storage of the corneoscleral button is often kept well within 12 h.

Storage Period

The original M-K medium was reported to provide a safe storage period up to 10 days, although the generally accepted period in practice is limited to 3–4 days. Some of the later introduced storage solutions such as K-sol, Dexsol, Likorol, Optisol (GS) report longer periods and storage for 7–10 days is generally accepted.

Microbiological Safety

The reported overall incidence of postoperative keratitis and ophthalmitis caused by microbes transferred with the cornea varies from 0.1% to 2% [11]. This is despite reported figures of positive donor rims of up to 19% [12], indicating that the antibiotics in storage solutions cannot be very effective at these low temperatures of storage. Instead it can be assumed that, upon return to physiological temperature, the residual antibiotic effect in the donor cornea coupled with the recipient’s ocular immune defense normally proves to be sufficient to control any organisms.

Tissue Evaluation

Non-invasive specular microscopy of the endothelium (coupled with slit lamp examination) is the favoured method for evaluating hypothermically stored corneas. Indeed, since 2001, determination of the endothelial cell density is required by the Medical Standards of the EEBA. Most specular microscopes are today equipped with software programmes to determine parameters such as cell count, variation in cell size, cell shape although careful calibration needs to be performed to obtain reliable morphometric results [13].

A limitation with specular microscopy is that the area of the central corneal endothelium is much larger than can be sampled by the specular microscope even with multiple field examinations. Therefore, the information gained from specular microscopy must be interpreted within the context of the slit lamp evaluation. In addition, the best observation of the corneal endothelium is at room temperature after the placement of the cornea in storage media, yet allowing sufficient time for adequate deturgescence of the cornea necessary to view the endothelial cells [9]. Therefore, degenerative changes and possible endothelial cell loss during storage, which may result in inferior quality and/or primary graft failure, cannot always be readily evaluated, but it must be taken into account [14, 15].

The incidence of primary graft failure has been reported to be between 0% and 10% and is currently believed to be about 1% [15]. Evaluation of the endothelium may reduce the risk of primary graft failure and increase long-term graft survival, but studies supporting this premise are lacking. However, examination of the endothelium certainly plays a role in setting higher and more uniform standards and helps to increase the donor supply by assessing corneas that may otherwise be arbitrarily excluded for transplantation on basis of donor age or previous ocular surgery.