Ординатура / Офтальмология / Английские материалы / Eye Banking_Bredehorn-Mayr, Duncker, Armitage_2009

Bredehorn-Mayr T, Duncker GIW, Armitage WJ (eds): Eye Banking.

Dev Ophthalmol. Basel, Karger, 2009, vol 43, pp 1–14

Corneal Grafting and Banking

Niels Ehlers Jesper Hjortdal Kim Nielsen

Danish Eye Bank, Department of Ophthalmology, Århus University Hospital, Århus, Denmark

Abstract

Corneal transplantation was conceptualized at the end of the 18th century, but it took more than 100 years before human corneal grafting was introduced. The greatest step forward was the demonstration by Filatov that corneal tissue can be collected and used post mortem. The history of eye banking includes the development of preservation techniques. Storage in cold to minimize microbial growth and tissue disintegration was first choice but during the last 30 years this has been taken over by warm storage (organ culture) where the donor cornea proves its sterility and vitality before being transferred to the recipient. The long-term organ culture storage makes exchange between centres possible and allows for histocompatibility matching. The internationalization led to the establishing of the European Eye Bank Association but also to an increasing number of governmental regulations. Developments in years to come may lead to control of graft biomechanics and optics. This technical development tends to favour a centralization.

The Beginning of Corneal Transplantation

The replacement of the opaque cornea by an artificial transparent structure was mentioned in 1789 by de Quengsy [1] of Montpellier, France. Later (in 1824) Reisinger [2] suggested the use of an animal cornea, a procedure he named keratoplasty. During the 19th century, many animal experiments were done, and it was gradually realized that homologous tissue was necessary to avoid opacification of the graft. Human transplantations were attempted, and Zirm [3] (1906) is usually credited as the first to have performed and reported a successful grafting. It was originally believed that very fresh tissue, used in a kind of status nascendi, was important. It was therefore a most important step forward when in 1935 and 1937 Filatov [4, 5] of Odessa, Ukraine, documented the applicability of post-mortem tissue (fig. 1). This opened up to a supply of donor material, the limitations now being ethics, legislation and practical organization.

This work was presented at the Polish-Ukrainian Ophthalmological Conference in Lublin, Poland, June 29 to July 1, 2006.

Fig. 1. F.P. Filatov.

Refinement of Surgical Technique

Over some decades in the middle of the 20th century, the corneal handling and the techniques of grafting were developed. Milestones were the routine use of a surgical microscope and the manufacturing of sufficiently delicate instruments. Every step could now be performed precisely and under direct visual control.

Early Eye Banking

Corneal preservation of the donor cornea before grafting was reported by Magitot [6] in 1912. He kept a human cornea in haemolysed blood for 8 days at 5°C before successful use as a lamellar graft. With the later observation by Filatov of the use of post-mortem tissue, the road to proper eye banking was found.

On the European continent, donor eyes were routinely obtained from hospital morgues, and the tissue was used immediately after retrieval. To overcome the practical problems in retrieving, donor tissue banks were organized, e.g. in London and New York, where donor eyes were collected and quickly redistributed.

The banking technique was originally very simple with the use of small glass bottles, in which the eyebulbs were kept under moist and cool conditions (fig. 2). Enucleation was performed as shortly after death as possible. The post-mortem time

Fig. 2. Donor eye in moist chamber.

and the immediate direct appearance of the cornea were the only quality controls. The importance of a viable endothelium for a successful outcome was soon realized by Stocker [7]. Consequently corneal banking research was directed at the maintenance of viability and integrity of this layer.

Current Preservation Techniques

Several methods for corneal preservation are currently in use throughout the world. None of these techniques is ideal but each provides benefits and disadvantages. The main differences regard storage temperature, medium composition and presence of an osmotic agent to prevent stromal swelling. In the following paragraphs the moist chamber storage, the cold medium storage and the cryopreservation techniques will be briefly presented before turning to a more detailed discussion of the organ culture methods.

Moist Chamber

The entire globe or the excised cornea may be kept under moist and cool conditions for hours and up to 1–2 days. It is the traditional method when donor corneas are collected at

the hospital morgue and used the same day. The technique is simple and requires almost no equipment. It is therefore still in use in some locations. With access to fresh donor material, the results are excellent. However, aspects such as microbiological contamination, transmission of donor diseases and histocompatibility are neglected. Today, therefore, the method is replaced by preservation of the isolated cornea in various solutions kept at 4°C (cold storage) or at body temperature (30–37°C, organ culture conditions).

Cold Storage

This technique of storage at 4°C was developed by McCarey and Kaufman [8] in 1974 using a standard culture medium (TC-199) supplemented with antibiotics and dextran as antiswelling agent. Due to the low temperature, the metabolism of the cornea is reduced to a minimum but viability is not demonstrated, infection not disclosed and the cornea can only be maintained for days (up to 7–10 days).

Cryopreservation

For years the establishment of a bank of cryopreserved donor corneas was considered a goal. The donor corneas should be waiting for the patients. This idea was based on reports on successful use of frozen and thawed human tissue [9, 10], and for some time it was thought that cryopreservation could provide the solution to the cornea banking problems. In 1965, Capella et al. [11] improved the technique with dimethylsulphoxide as cryoprotectant. This method was clinically useful but its complexity, e.g. the requirement of very fresh tissue to be frozen, prevented a widespread use. In 1981, Sperling [12] developed a technique with cryopreservation of tissue at first kept under organ culture conditions, then frozen. After thawing the tissue was once again organ cultured to prove its vitality. The clinical results with this cryopreservation procedure were reported in 1982 [13]. After 1 year 71% clear grafts were found, and even after 12 years [14] the graft survival was 58%.

The conclusion to be drawn is that cryopreservation can be done and a bank of frozen tissue could be established, but the technique would be very demanding. Still, however, research is going on and the possibility of undercooling, i.e. maintenance of the tissue at a temperature below zero but without crystallization in the tissue, is another not yet fully explored possibility [15].

Organ Culture

The organ culture technique is based on the idea of a long-term preservation of the isolated human cornea under simulated physiological conditions. The main advantage

of such an approach is that the donor cornea can prove its viability and sterility in the culture flask rather than failing in the patient’s eye. So-called primary graft failures do not occur or are extremely rare.

The organ culture technique was introduced in the 1970s [16, 17] and brought to Europe by Sperling, who in the following years modified and developed the technique into clinical routine (fig. 3). The technique was later reintroduced in the USA as the ‘Minnesota system’ [18, 19]. Extensive research and long-term clinical use in Europe document the safety of the organ culture method. The organ culture method is today the method of choice in Western Europe.

Eye Banking Procedures in Denmark

The number of corneas required for grafting in Denmark is less than 500/year. All corneas in the country are supplied by one eye bank, the Danish Eye Bank, at Århus University Hospital. Since 1979 exclusively organ-cultured corneas have been used.

After laboratory development of the technique [20] (fig. 4–7) and quality documentation by comparison to fresh material [21] (fig. 8), the routine procedure described below was established.

Retrieval and Primary Evaluation

Retrieval of donor tissue is done by specially trained technicians removing the whole globe. Simultaneously a blood sample is taken. The post-mortem time is not critical. The eyebulbs and the blood samples are transported to the eye bank and stored in a refrigerator.

Primary evaluation of the donor cornea is done by visual inspection followed by 30 s of rinsing in tap water. The cornea is excised with a scleral rim and the endothelium inspected under an ordinary light microsope after trypan blue staining. The cells are outlined by the temporary swelling of the intercellular spaces [22]. The cornea is then suspended by a suture in a culture bottle with minimal essential medium containing 10% fetal calf serum.

Organ Culture and Sterility Checks

The cornea is kept in the closed bottle at 31°C until 1 day before use.

Under sterile conditions, a sample of the medium is withdrawn after 1–2 weeks and examined at the microbiological laboratory for bacterial and fungal contamination. The tissue is in quarantine until negative test results are received. The ‘banking time’ is usually 2–4 weeks but may, after exchange of medium, be extended to 7 weeks [23] and probably even longer.

Fig. 3. S. Sperling developed the organ culture technique for clinical use.

Fig. 4. Preparation of a cornea with a scleral rim.

Preparation for Clinical Use

On the day prior to use, the medium is inspected for turbidity. If the medium is clear, the bottle is opened in a laminar flow bench. The endothelium is inspected after staining and cell border swelling. Cell density is estimated but not routinely quantitated. The cornea is then transferred to new medium with addition of 5% dextran to deswell the stroma.

Fig. 5. Staining endothelium with trypan blue.

Fig. 6. Closed-system preparation.

Fig. 7. Donor corneas packed for shipping.

Fig. 8. No difference in immediate and late deswelling of fresh and organ-cultured corneas. CCT = Central corneal thickness.

For transportation, the bottle is packed in a shock-absorbing box and sent by ordinary surface mail to the receiver.

Documentation

The cornea is labelled for identification.

Modern-Day Results

Today results from keratoplasty are generally satisfactory. The success rate depends upon the quality of the donor tissue and the condition of the recipient corneal bed.

Vascularization and earlier graft rejection have a negative influence. In non-risk cases, the success rate is between 90 and 100%. In risk cases, the success rate falls to 50% or less [21] (fig. 9).

These percentages refer to a limited observation time. Even if the risk of rejection decreases with time, endothelial rejection can still occur after many years. A recently observed rejection in a keratoconus patient occurred after 18 years and was reversed by steroid treatment.

In biology the concept of compatibility is recognized. Organs are successfully exchanged between identical twins. The compatibility is described by the complex HLA system comprising at least 2 classes (I and II) that are of importance. Most clinical studies find histocompatibility to be of importance for the outcome.

What is however still an unsettled question is the logistics of handling compatibility and corneal grafting.

With a given HLA genotype and knowledge of the prevalence of the different HLA genes, an expected waiting time for a compatible donor can be calculated [24]. This must be compared to the patient’s age and life expectancy. Evidently, the more donors, the shorter the waiting time. Therefore exchange of tissue between banks (and countries) becomes an interesting issue. This situation was in fact recognized 30 years ago. The need for a collaboration between eye banks has been a stimulus for creation of an eye bank association.

The European Eye Bank Association

The first meeting, in what turned out to be the European Eye Bank Association (EEBA), was held in Århus, Denmark, in 1989. From that meeting, it was clear that there was a need to coordinate our efforts to organize and develop the eye bank concept, mainly following the procedures for organ culture. Since then annual meetings have been held, and the name EEBA was suggested by Andrew Tullo, Manchester,