Year : 1966 | Volume
: 14 | Issue : 2 | Page : 75--82
Preservation and suitability of donor human corneas for keratoplasty
Corneal Surgery Unit & Regional Eye-Bank, M.G.M. Medical College & M.Y. Hospital, Indore, India
Corneal Surgery Unit & Regional Eye-Bank, M.G.M. Medical College & M.Y. Hospital, Indore
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Kalevar V. Preservation and suitability of donor human corneas for keratoplasty.Indian J Ophthalmol 1966;14:75-82
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Kalevar V. Preservation and suitability of donor human corneas for keratoplasty. Indian J Ophthalmol [serial online] 1966 [cited 2020 May 29 ];14:75-82
Available from: http://www.ijo.in/text.asp?1966/14/2/75/38567
A very important factor in the evolution of keratoplasty from a rare operation in the recent past to an accepted surgical procedure today, is the development of precise knowledge about usability of donor material. The important aspect is the problem of preservation of donor human eye balls for periods longer than what is possible now so that the corneal surgeons can be busy doing their job with a regular supply of good donor material available whenever required. This is of particular significance in our country where active corneal surgery units are needed so urgently.
As in actual surgery, in preservation of donor human eyes also, our problems are quite different from those of the Western countries, because of the extreme climatic variations, unsatisfactory handling of the dead bodies, chronic ailments associated with dehydration being a common cause of death and the unhygienic circumstances in which the bodies of the destitute and unclaimed are found. Our main source of donor material is from such destitute bodies and the eyeballs from such bodies are materially affected by the factors mentioned above.
Review of Literature
Donor material used to be available only from eyes enucleated for some ocular pathology till as late as 1935. It was Filatov (1937) who first reported use of eyes from cadavers after preservation for periods varying from 10-56 hours at 4-6°C temperature and reported 18.9% surgical successes. Weiss & Taylor (1944) first tried freezing and drying method on rat corneas, which was later reported by Leopold et al (1947) and Katzin (1947) on rabbit corneas. This method of rapid freezing at temperature of liquid nitrogen and dehydrating in vacuum at -40°C failed to produce a transparent transplant. The grafts took alright but did not remain transparent. Smelser and Ozanics (1946) tried freezing the whole eyeball in isopentane chilled with liquid nitrogen but the result was the same. The graft remained transparent for some few days and then became opaque. These workers concluded that although the method preserved the corneal tissue in as nearly as normal physical and structural conditions as possible, the tissue was presumably no longer living.
Eastcott and his co-workers (1954) later reported success in transplantation of frozen human corneal tissue. They used a mixture of carbon dioxide and alcohol at -79°C temperature. The storage time for lamellar grafts was three days to nine months and for penetrating 5 days to 7 months.
Billingham Rycroft (1955) pretreated the donor eyeball with antibiotic solution and 15% glycerol in Ringer's solution at room temperature for 1 hour, then placed the eyeball in a glass bottle which was stored in contact with solid carbon dioxide at -79°C in deep freeze. These were thawed in warm saline before use and gave good results in lamellar keratoplasty but not in penetrating surgery.
Stocker and his colleagues (1952) developed a method of glycerol freezing at -45°C by which viability of the endothelium was reported for as long as 45 weeks of storage. If it could be demonstrated that these preserved viable donor corneas can be successfully used for perforating grafts, great progress in the technique of corneal transplantation will have been made.
McNair and King (1955) modified the method of drying in vacuum and kept the tissue in 100% glycerine. They kept these at room temperature and maximum period of storage was four months. They concluded that corneas stored in glycerine without dehydration and at 4°C were not sufficiently protected from autolysis while grafts dehydrated in glycerine but stored in vacuum without refrigeration were suitable for lamellar keratoplasty. King (1957) from his further experiments concluded that glycerine protected the cellular structure and maintained viability with cellular metablism at a stand still. He used a modified manifold vacuum system which enabled him to dehydrate excised corneas of cats and dogs in glycerine and preserved in vacuum.
Storage under liquid paraffin is another tissue storage method first introduced by Carrel in 1908. Forty years later it was used by Burki (1948) for preservation of donor eyes. Rycroft (1958) adopted the method and found it very satisfactory. He reported successful results by using donor material preserved in liquid paraffin at 4°C for as long as 3 weeks.
Then came in vogue the method of lyophilisation or what is also called freeze-drying reported by Payrau et al (1957). The method consists in taking out all the contents of an enucleated eye-ball by a posterior route and separating the cornea and sclera. Placing this outer coat of the eye-ball on a special tray which maintains the original shape of the corneo-scleral sac, the eye-ball is quickly immersed in a mixture of 90°; alcohol, and dry ice and left for 5 minutes. This is then placed in a jar reduced previously to the same temperature and has an opening protected by a filter. Such prepared jars are put in a desiccation tank where the temperature is maintained for several hours at -50°C, in vacuum jars. The jars containing the eyes are then sealed hermetically in nitrogen atmosphere and stored at room temperature without any special precautions. At the time of use, the jars are filled with physiological saline at 35° C for rehydration. Later this method was modified by Payrau and Pauliquen in 1959 by making use of quick freezing and rapid drying and storing with a small quantity of microporous silca jel. Later still they even omitted the freezing process from the procedure. They wrapped the cornea in cellophane and placed in a flask containing silica jel and then sealed in vacuum. Tissue culture studies and oxygen consumption studies of such preserved corneas showed that lyophilised corneas were not viable.
King (1961) modified his glycerine dehydration method so that the cumbersome vacuum manifold system could be obviated and evolved the method of dehydration with a molecular sieve. These are a new series of adsorbents composed of sodium and calcium alumino-silicates. They are chemically inert, insoluble in water and glycerine and are capable of removing water to an extremely low vapour pressure. The size of the pores in these synthetic crystals is comparable to that of a molecule of water and it is thought that the physical adsorptive power of the crystals is due to a trapping of the water molecules in these pores. The excised donor corneas with a seleral rim are placed in a 15 c.c. bottle which contains 10 gms. of molecular sieve, filled with 95% glycerine and sealed with wax. These are stored as such without any refrigeration. King by this modification of the original glycerine method, made an important advance in the field of preservation of donor material for corneal grafting. He showed that a cornea preserved for as long as 9 months can be as successfully used for a lamellar keratoplasty.
Inspite of all the researches the position at present continues to be that fresh donor cornea is the only suitable donor material for penetrating keratoplasty because maintenance of endothelial viability has not become possible by any method of preservation to date. Problem of donor material for lamellar keratoplasty is not so acute because the endothelium does not form a part of the lamellar graft and therefore not only fresh corneas can be used for longer intervals after enucleation but it is practicable to preserve the corneas from 9-10 months or more for use in lamellar keratoplasty.
Suitabilities of Donor Material
The cadaver tissue constitutes the major source of donor material. Autoplastic cornea is an excellent donor material but it is only rarely available. Homoplastic material is obtained from the cadavers of adults, children, infants or premature and still-born children as well as eyes enucleated for some ocular pathology. Use of heteroplastic material is however still in experimental stages.
It is generally accepted that 15-45 years is the most suitable age of the donor source. Eyes from very young donors and infants are not suitable because of the extreme plasticity and friability of cornea which presents operative difficulties. Similarly eyes from very old persons also make an inferior donor material because senile changes in the endothelial layer of cornea have probably started and naturally these are detrimental to a penetrating graft. An arcus senilis can of course be avoided by keeping the size of the graft well within the arcus.
Cornea from eyes with intraocular malignancy limited to the posterior segment is a safe donor material. Similarly a malignancy in the donor body even if generalised does not metastatise in the cornea and therefore corneas from such donors when transplanted cannot transmit it to the recipient. However eyes from a source where the cause of death is due to a neurotropic organism or toxin, such as rabies or tetanus should better be rejected. Similarly corneas with any evidence of intraocular malignancy extending into the anterior chamber are better not accepted. Glaucomatous eyes are usually unsuitable because of the endothelial changes and the poor state of nutrition of cornea in such cases.
The perishable nature of corneal tissue imposes an urgent time factor. It is obvious therefore, that sooner the eye ball or the graft tissue is removed from the donor after death, the better. There have been different opinions as to the maximum permissible time limit for enucleation after death. McLean (1948) Paufique (1948) and others advocated removal of corneal graft within an hour of the donor's death. Bock (1950) although recommended the in-situ removal of partial lamellar graft within 2 hours of death, the enucleation of whole eye, he said can be done upto 12 hours after death provided the cadaver is refrigerated. Paton (1955) permitted an interval of 5 hours only after death and that too provided the eye lids are carefully closed after death, antibiotic drops instilled in the conjunctival sac and the body refrigerated. Paufique permitted an interval of 24 hours but stressed the importance of enucleation within 6 hours after death if the above condition cannot be fulfilled.
Considering these factors in a tropical country like ours and the existing circumstances of a general hospital mortuary it would appear that the enucleation of eye balls should be carried out soonest possible after death of the donor. This however is not always possible and at times one has to wait for quite a few hours before the eyes can be removed. With this in view, the effect of seasonal variations on the viability of corneal tissue in the Indian tropical climate formed an important aspect of studies on preservation of donor human eyes, during the last 5 years and reported Kalevar (1965). It has been observed that in the hot summer months, the corneas become unsuitable much earlier and therefore eyes should be removed within the accepted time limit of 6-12 hours after death. In the colder winter months however this time interval can be extended even upto 18-24 hours as the corneal condition remains satisfactory for a longer time.
The important criterion of suitability of a donor cornea therefore is its condition at the time of enucleation and still more important is the condition at the time of use.
Processing of Donor Material
The routine adopted in the corneal surgery unit, Indore is simple and easily practicable. Whenever eyes are available but immediate enucleation is not possible due to some factors, the eyes are examined and a drop of sterile liquid paraffin is instilled in the conjunctival sac and lids closed properly. Enucleation is carried out as far as possible under as strict aseptic circumstances as in the living. The eyes are transferred on to sterile cotton pads in a sterile bowl and brought to the eye-bank along with the available particulars of the donor body. They are washed thoroughly with sterile normal saline and examined once more under better illumination and magnification. The observations on the state of transparency and tension of eye balls are recorded. The eye balls are next immersed in a 1:5000 solution of merthiolate for 5 minutes and then transferred to small autoclaved glass bottles with a moist cotton pad at the bottom. Antibiotic drops, (Ne-ba-Sulf) are instilled on the cornea, the caps screwed on and the bottle put in refrigerator out side the ice-chamber at a temperature of 4°-6°C. The relevant information about the age and sex of donor, cause of death, date and time of death and enucleation are recorded. At the end of every 24 hours till 96 hours the eyes are daily examined under identical circumstances of illumination and magnification and the following points noted :
1) Transparency of cornea : - We grade it in either of the following categories
b) Folds + : Fine folds in one quadrant visible under bright illumination only.
c) Folds ++ : Folds affecting central part of cornea also.
d) Folds +++ : Folds affecting central 2/3 of cornea.
e) Folds + + + + : Folds all over the cornea.
Superficial epithelial haze is not taken into account because it is of no consequence as it is due to epithelial oedema the deeper cornea being transparent.
2) Tension of the eye ball is graded as follows :
a) T. Good: Normal Tension.
b) T. 1 - Slightly low tension.
c) T. 2 - Moderately low tension.
d) T. 3 - Very low tension with concave cornea.
The viability of corneal tissue is based on different criteria in penetrating and lamellar surgery because in the former a vital layer like the endothelium forms an integral part of the donor graft while in lamellar surgery only the anterior layers of cornea are involved in the donor button and viability of endothelium is not essential. For purposes of judging the viability clinically, the following criteria have been adopted.
1) Cornea perfectly transparent or showing only a few fine folds (F +) and with undisturbed curvature is considered suitable donor material for penetrating keratoplasty.
2) Cornea with transparent lamellar layers but with deep folds not exceeding ++ is considered still suitable but for lamellar keratoplasty only.
We are not very sceptical about the tension of the eye ball if the cornea is good because the tension can always be built up either by injection of normal saline in the posterior segment or simply by tight rolling the eye ball in packing gauze before application of trephine.
Adoption of a method of preservation practicable in Indian tropical circumstances is as important as any another aspect in corneal surgery. In the Regional Eye-Bank at Indore studies on preservation of donor human eyes have been carried out. This study comprises observations on 890 donor eyes which became available in the last five years to the Regional Eye-Bank.
A comparative study between preservation under liquid paraffin and moisture, was made on 25 pairs of eyes, one of each pair preserved under liquid paraffin and the other in moisture both kept at 4-6°C. At the end of 24 hours the eye in liquid paraffin was washed in normal saline to remove the oil and examined to note changes in the corneal transparency and tension of the eye ball, and compared with the one preserved in moisture. From such detailed records it was found that for penetrating grafts at the end of 24 hours 84% of eyes in moisture and 76% of those in liquid paraffin were useful. At the end of 48 hours 68% of the eyes in moisture and 48% of those in liquid paraffin were viable for penetrating surgery. At the end of 72 hours the percentage of viable eyes was again better in moisture preservation than paraffin. At the end of 96 hours no eye from liquid paraffin group was suitable for penetrating surgery while two were still viable from the moisture group. The tables summerise these observations and give the corresponding figures of viable eyes for lamellar grafting:
From this figurative data, it may be noted that as far as the clinical viability from the point of view of criteria detailed earlier was concerned, the liquid paraffin method was relatively slightly less satisfactory. Moreover the difficulty encountered with the liquid paraffin procedure is that at the end of every 24 hours, the oil could not be washed off completely which prevented effective action of antibiotic solution used. Thus the sterilisation of the eye ball was not always adequate. In comparison, it is felt that preservation in moisture is a simpler procedure and the eyes are easy to handle while liquid paraffin preservation is relatively cumbersome and offers no special advantage.
For preservation of corneal buttons for lamellar surgery, we were using an apparatus to carry out dehydration of corneas in glycerine under vacuum.
More recently, a Grant-in-Aid from the National Council to Combat Blindness Inc., New York has helped procurement of molecular sieve granules. These are the newest synthetic adsorbents, a combination of calcium and sodium alumino-silicates. The use of these molecular sieve granules avoids the rather elaborate and complicated system of manifold vacuum system for dehydration purposes. The enucleated eyes and the corneo-scleral button excised therefrom are treated in the same manner as before. 5 grammes of the molecular sieve granules enclosed in a small piece of gauze are kept in a dry bottle. The bottle containing the granules with its cap is sterilised in hot air steriliser at 180°C for one hour. After it has cooled, the excised cornea with scleral rim which was pre-treated with Ne-ba-Sulf solution is put in the bottle and the bottle is filled with 95% autoclaved glycerine leaving a small air bubble on top which prevents continuous seepage of glycerine. The cap is screwed on tightly and then sealed with wax. These are also stored at room temperature.
The Molecular sieve preservation in glycerine is a very simple procedure and does not need any special equipment. We have now adopted this method routinely and thus avoid a great wastage of useful donor material which can be supplied to corneal surgeons in different parts of the country for use in lamellar surgery for which it is as good or perhaps better than fresh donor material. These corneas at the time of use have to be rehydrated in sterile normal saline and antibiotic mixture for 15 minutes. The corneal button with scleral rim is fixed in Dastoor's corneal clamp for donor lamellar dissection. This preserved corneal button is firm and needs a special care to maintain the plane during dissection.
From the observations and studies carried out in this Corneal Surgery Unit and Regional Eye-Bank during the last five years, it has been concluded that preservation in moisture of freshly enucleated eyes at 4-6°C. is a very simple and satisfactory procedure as compared to preservation under liquid paraffin. Such moisture preserved eyes remain viable for penetrating transplants for anything from 24 to 72 hours depending of course on many other factors. For lamellar grafting, these may remain viable for 96 hours or more. The glycerine dehydrated corneas either in vacuum or by use of molecular sieve granules provide a very satisfactory donor material for lamellar grafting upto a period of 6 to 8 months or even more.
1. Evolution of different methods of preservation of donor human eyes has been reviewed.
2. Criteria for usefulness of donor cornea have been discussed. It has been suggested that corneas remain usable for longer period in winter than in summer.
3. Methods of processing the donor eyes at the Corneal Surgery Unit and Regional Eye-Bank, Indore, have been discussed.
4. It has been suggested that preservation of eyes in sterile bottles with a moist cotton pad at the bottom is the most satisfactory method for short term preservation and preservation in liquid paraffin does not offer any advantages.
5. It is reported that corneas preserved in glycerine with molecular sieve granules is a very practical and satisfactory method in our circumstances and corneas thus preserved can be used for lamellar keratoplasty even upto 6-9 months after preservation.
Corneal Surgery Unit and Regional Eye-Bank, Indore is highly indebted to the Indian Council of Medical Research for its support in the research activities of this Unit.
We are also grateful to the Fight for Sight organisation of the National Council to Combat Blindness, Inc., New York for the grants-in-Aid with which the vital equipment has been procured through UNICEF.
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