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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

Date of Web Publication12-Jan-2008

Correspondence Address:
V Kalevar
Corneal Surgery Unit & Regional Eye-Bank, M.G.M. Medical College & M.Y. Hospital, Indore
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Source of Support: None, Conflict of Interest: None

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How to cite this article:
Kalevar V. Preservation and suitability of donor human corneas for keratoplasty. Indian J Ophthalmol 1966;14:75-82

How to cite this URL:
Kalevar V. Preservation and suitability of donor human corneas for keratoplasty. Indian J Ophthalmol [serial online] 1966 [cited 2020 Dec 2];14:75-82. Available from: https://www.ijo.in/text.asp?1966/14/2/75/38567

Table 1

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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 Top

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 Top

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.

  Time Factors Top

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 Top

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

a) Transparent.

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.

  Conclusions Top

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.

  Summary Top

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.

  Acknowledgement Top

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.[20]

  References Top

Burki, E., (1948): Ophthalmologica, 116: 221.  Back to cited text no. 1
Rock. R. H., (1950): Arch. Ophth. 44: 293-299.  Back to cited text no. 2
Billingham, R. E. and Rycroft, B. W., (1955): Corneal Grafts. Butterworth & Co., Ltd., London.  Back to cited text no. 3
Eastcott, H. H. et al. (1954): Lancet 1: 237.  Back to cited text no. 4
Filatov, V. P. (1937): Lancet 1: 1395.   Back to cited text no. 5
Katzin, H. M., (1947): Am. J. Ophth. 30: 1128.  Back to cited text no. 6
King, J. H., (1957): Am. J. Ophth. 43: 353.  Back to cited text no. 7
King. J. H., et al (1961): Tr. Am. Ophth. Soc. 59.  Back to cited text no. 8
Kalevar. V., (1965): Brit. J. Ophthal. 49.   Back to cited text no. 9
Leopold. I. H. et al. (1947): Arch. Ophth. 37: 168.  Back to cited text no. 10
McLean, J. M., (1948): Symposium: Corneal Transplantation. If Technique. Am. J. Ophth. 31: 1370.  Back to cited text no. 11
McNair, J. N. and King, J. H. (1955): Arch. Ophth. 53: 519­  Back to cited text no. 12
Paufique, L., et al. (1948): "Les Grelles de la cornee". Masson & Cie, Paris.  Back to cited text no. 13
Paton, R. T., (1955): "Keratoplasty". The Blakiston Division McGraw-Hill Book Co., Inc., New York.  Back to cited text no. 14
Payrau, P. et al. (1959): Ann. d' Ocul. 191: 636.  Back to cited text no. 15
Payrau, P. and Pauliquen. Y. (1959): Bull. Soc. Ophth. France, 3.  Back to cited text no. 16
Rycroft, B. W., (1958): Excerpta Med. XII 12-c98 Sept.  Back to cited text no. 17
Smelser, G. K. & Ozanics. V., (1946): Proc. Soc. Exper. Biol. and Med. 62: 274.  Back to cited text no. 18
Stocker, F. W., et al. (1962): Am. J. Ophth. 53: 279.  Back to cited text no. 19
Weiss, P. and Taylor, A. C. (1944): Anat. Rec. 88: 464  Back to cited text no. 20


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