A Specular Microscopic Viewing System for Donor Corneas

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A Specular Microscopic

Viewing System for Donor Corneas

ANTHONY B. NESBURN, MD, SIDNEY MANDELBAUM, MD,* DRU E. WILLEY, PhD, MELVIN D. TROUSDALE, PhD, EZRA MAGUEN, MD, DONALD E. WARD, BA

Abstract: A system for routine specular microscopic assessment of donor corneal endothelium including a chamber that allows both storage and viewing of the donor tissue was evaluated. A specular microscope modified to enable noncontact scanning of donor cornea from the endothelial side was used. Forty-five human corneas were examined upon arrival at the eyebank. Storage in the new chamber was compared with that in standard vials for 13 paired corneas. This study demonstrates the feasibility and simplicity of routine specular microscopy on donor corneal tissue, and discusses its advantages. [Key words: corneal endothelium, cornea storage, cornea viewing, 'donor, donor election, eye bank procedures, human cornea, specular microscopy, video viewing.] Ophthalmology 90:686-691, 1983

Despite stringent donor criteria and slit-lamp biomicroscopy of harvested corneas, inadequate endothelial function or frank donor failure subsequent to penetrating keratoplasty is occasionally encountered. To minimize this possibility, ophthalmologists as well as eyebank personnel have sought a convenient, noninvasive method to assess morphology, cell count, and function of donor corneal endothelium prior to surgery. As a first step toward this goal, we evaluated a prototype donor corneal endothelial viewing system, composed of a specular microscope and corneal viewing chamber. This system was particularly helpful in evaluating the endothelial morphology of donor corneal tissue when it arrived at the eyebank.

From the Estelle Doheny Eye Foundation and the Department of Ophthalmology, University of Southern California School of Medicine, Los Angeles, California. Supported in part by Fight for Sight, Inc. New York City (Dr. Mandelbaum) and Discovery Fund for Eye Research, Los Angeles, California. * Dr. Mandelbaum is now with the Bascom Palmer Eye Institute.

Reprint requests to Anthony B. Nesburn, MD, Estelle Doheny Eye Foundation, 1355 San Pablo Street, Los Angeles, CA 90033.

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MATERIALS AND METHODS CORNEAL VIEWING AND STORAGE (CVS) CHAMBER

The CVS chamber is a modification of a device first presented by Bourne in 1976. 1 The prototype is constructed of polypropylene and high temperature acrylic, allowing ethylene oxide gas sterilization. The top has a silicon rubber gasket that forms a water and airtight seal. The assembled unit holds 20 ml McCarey-Kaufman (MK) medium. The cornea rests on a pedestal designed to support the tissue safely and bring it within focusing range of most laboratory specular microscopes equipped with long working distance objectives (Fig 1). EYE BANK VIDEO MICROSCOPE

We used the PRO EB-l (Professional Research Organization, Irvine, CA) specular microscope developed in conjunction with the chamber. The EB-l is an eyebank version of a specular microscope (Syber and HeyerSchulte, Irvine, CA) in wide clinical use. The lamp housing is modified to permit vertical mounting of the mi-

0161-6420/83/0600/0686/$1.10 © American Academy of Ophthalmology

NESBURN, et al •

DONOR CORNEA VIEWING AND STORAGE CHAMBER

Fig I. The corneal viewing and storage (CVS) chamber used in this study consisting of the outer storage container, the pedestal supporting the donor cornea, and the screw-on viewing cap. The support pedestal is within the storage container in this photograph.

croscope assembly on a table top stand with room for a video camera. The optics increase the size of the viewing field and provide a longer working distance that requires no contact with the chamber. Using a modified low light level video camera, excellent high magnification (final magnification 500X) specular reflection images of the endothelium can be obtained. The addition of an X -Y mechanical stage aids in the systematic scanning of the tissue (Fig 2). An alphanumeric typing terminal allows relevant information to be entered on the lower portion of the video screen. The specular microscopic examination may be photographed at the time of examination or recorded on videotape.

counts were obtained from the Polaroid photographs by averaging the number of cells counted in six separate fields, each 100 microns by 100 microns. Group I: Endothelial screening only. Of the 45 corneas, 19 were suitable for corneal transplantation. After the endothelium was screened as described, the corneas were transferred to a standard M-K storage vial and stored at 4 C until assigned for clinical use. They were not examined further. Group II: Comparison ofM-K vial and CVS chamberstored corneas. The remaining 26 corneas were obtained from 13 consecutive donors whose medical condition or age (over 67 years) precluded use of the cornea for penetrating keratoplasty. Of each pair, one was randomly stored in a CVS chamber while the other was stored in a M-K vial. All were stored at 4 C for a period of two, three, or four days. At the end of the storage period, each pair was warmed to room temperature in the containers in which they had been stored. The cornea in the M-K vial was transferred to a CVS chamber. Both corneas were reexamined immediately and photographed with the eyebank specular microscope. After storage, four pairs of these corneas were mounted on an artificial anterior chamber, 2 and applanation spec-

STUDY CORNEAS

A total of 45 human corneas divided into two groups were examined using this system. Donor age ranged from 18 to 78 years (mean, 45 years). As soon postmortem as possible, specimens consisting of the cornea and an adjacent 2 to 3 mm of scleral rim were harvested. All tissue was transported to the eyebank and processed according to routine procedures, including slit-lamp examination of donor tissue. EXAMINATION TECHNIQUES

Each cornea was placed epithelial side down in a CVS chamber filled with M-K medium prepared in our eyebank. The chamber was then placed on the stage of the PRO EB-l specular microscope and the central corneal endothelium was scanned systematically. The video monitor was used because it provides magnification and better contrast than direct viewing through the microscope. The quality of the images obtained was noted, and representative photographs were taken directly from the video screen with a Polaroid camera. Central cell

Fig 2. Photograph of the specular microscope; video camera is mounted above the specular microscope. The CVS chamber is in position on the microscope stage.

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OPHTHALMOLOGY • JUNE 1983 • VOLUME 90 • NUMBER 6

ular microscopy was performed from the epithelial side, using the Syber clinical specular microscope. The central corneal endothelium was photographed, and the central cell count was determined by averaging the number of cells counted in six separate fields, each 40 microns by 200 microns. An additional three pairs of these corneas were randomly chosen for electron microscopic (EM) study after storage. Corneas (including scleral rims) were bisected from the endothelial side by using a razor blade knife. Hemicorneas were fixed in half strength Karnovsky's solution for 6 to 12 hours and then transferred to phosphate buffered saline. One-half of each cornea was submitted for scanning EM, while the other half was processed for transmission EM using standard procedures. Each pair of corneas was processed simultaneously and identically. CVS CHAMBER TESTING

To test whether cytotoxic substances were introduced by the chamber, cell cultures were inoculated with medium incubated in the CVS chamber or medium not incubated in the chamber. Minimal essential medium (GIBCO) supplemented with 10% NCTC 135, 2 mM Lglutamine, 10% fetal bovine serum, 50 mg per ml gentamycin, and 2 mg per ml fungizone was employed. Aliquots of this medium were incubated in four CVS chambers for 0, 2, 24, 72, or 168 hours. Rabbit kidney and human embryonic lung cell cultures were trypsinized, dispensed in tissue culture dishes, and inoculated when monolayers were 50 to 75% confluent. At each time interval, 4 ml of chamber-incubated medium or the unincubated medium were added to replicate dishes. Cultures were incubated and examined microscopically every other day for one week in a "masked" fashion.

Fig 3. Representative specular micrograph obtained from a donor cornea on arrival at the eye bank, using the CVS chamber and specular microscope. Cell count = 2200 cells/mm2,

cell count and large cells (pair # I, 2770 and 1500; pair #7, 2480 and 680) (Fig 4). An additional pair of specimens was noted to have significant endothelial guttata (Fig 5) not recognized on initial slit-lamp evaluation of the cornea. POSTSTORAGE SPECULAR MICROSCOPIC EXAMINATION

Endothelial examination was generally more difficult after two to four days of storage, owing to a considerable increase in the folding of Descemet's membrane. This led to shadowing and difficulty in focusing on broad areas of endothelium at one time. Figure 6 shows a specular micrograph obtained with this system on a cornea when it arrived in the eyebank and after three days of

RESULTS INITIAL SPECULAR MICROSCOPIC EXAMINATION

The endothelium of all 45 corneas could be imaged at evaluation upon arrival in the eyebank. In 40 of these, examination was adequate to determine the morphologic character and cell count of the central endothelium in at least six separate fields. Figure 3 is a representative specular micrograph obtained from one donor cornea upon initial evaluation. Although endothelium could be imaged in the remaining five corneas, too much folding of Descemet's membrane was already present at the initial examination to obtain full fields of endothelial cells in at least six separate areas. Initial specular microscopic examination showed normal appearing endothelial morphology in most cases. Of the corneas examined, 14 were from donors 68 to 78 years old. Ten of the 14 corneas had normal endothelial appearance and cell count. In two instances, one member of a donor pair exhibited an unexpectedly low 688

Fig 4. Specular micrograph of a donor cornea showing large endothelial cells with a count of 680 cells/mm2, The specimen appeared normal by slit-lamp examination, The fellow cornea had a count of 2480 cells/mm2,

NESBURN, et al •

DONOR CORNEA VIEWING AND STORAGE CHAMBER

Fig 5. Specular micrograph of a donor cornea showing endothelial guttata.

storage. After storage a smaller area of cells can be imaged because cells at the margin of the field were not in focus. Considerable variability was observed, some corneas having much larger areas that could be seen than others. In most cases, only IO to 20% of the endothelium could be imaged clearly after two to four days of storage, principally those areas that were flat or at the top or bottom of ridges. Generally, the longer the period of storage, the smaller the area of endothelium visible. During the poststorage examination, no differences were noted between corneas stored in CVS chambers and those stored in standard M-K vials. Examination of poststorage corneas on the artificial anterior chamber allows positive pressure to be applied to the posterior surface of the cornea, thus flattening the Descement folds that develop during storage. Morphologically normal endothelium was present when standard epithelial side specular microscopy was performed

on stored corneas mounted on the artificial anterior chamber. This examination verified that endothelial cells were present even in areas where they could not be imaged by specular microscopy from the endothelial side. To determine comparability of the CVS video system with conventional specular microscopy, cell counts obtained on four pairs of corneas examined by using both the CVS chamber and the artificial anterior chamber were performed (Table 1). Less than a 10% variation was found in the counts obtained by the two systems. No ultrastructural differences between those corneas stored in the CVS chamber and those stored in the standard vial were encountered. Alterations observed in the six corneas studied by transmission electron microscopy were those degenerative changes associated with storage in M-K medium. 3,4 Cell cultures grown in medium previously incubated in the CVS chamber were morphologically and quantitatively indistinguishable from those incubated in standard medium. No cytotoxic substances were detected by this method. However, seals on several of these prototype chambers were not tight, allowing slight leakage and abnormally high pH.

DISCUSSION The critical importance of the corneal endothelium has been recognized only in recent years. This is particularly relevant in corneal transplantation inasmuch as the procedure is often performed specifically because of inadequate host endothelial cell function. Selecting healthy donor endothelium for penetrating keratoplasty is a key responsibility of eye banks and corneal surgeons, particularly in view of the multiple potential sources of damage to the endothelium in the harvesting and eyebanking process.

Fig 6. A, specular micrograph of a donor cornea on arrival at the eye bank. B, specular micrograph of the same cornea as in 6A after three days storage in the CVS chamber. Folding of Descemet's membrane has occurred, allowing fewer cells to be imaged.

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OPHTHALMOLOGY •

JUNE 1983 •

Table 1. Comparison of Central Endothelial Cell Counts Obtained Using the CVS Chamber and the Artificial Anterior Chamber

Specimen

Cell Count (CVS Chamber)*

Cell Count (Artificial Anterior Chamber)t

Percentage Differencet

1A 18

1730 2100

1920 2100

-9.9% 0

2A 28

2500 2430

2290 2330

+9.1% +4.3%

3A 38

1950 2030

1870 2150

+4.3% -5.6%

4A 48

2530 2630

2270 2870

-7.5% -8.4%

* Obtained from endothelial side as described in text. t Obtained from epithelial side using standard specular microscope applied to corneal button mounted on an artificial anterior chamber. 2 t Calculated using cell counts obtained with the artificial anterior chamber as the standard. Minus (-) indicates CVS cell count less than, plus (+) greater than count using artificial anterior chamber.

The specular microscope introduced in 1968 by Maurice5 and modified by Laing, 6 as well as Bourne and Kaufman, 7 has been suggested as a useful means of evaluating the quality of donor tissue. Despite the proven utility of this instrument in demonstrating endothelial morphology, only 15% of eyebanks and only 7% of corneal surgeons in a recent survey recommended its use for tissue screening. 8 Instead, most eyebanks rely on history and slit-lamp examination of donor corneas, 8 despite the knowledge that eyes deemed acceptable by these criteria might be considered unacceptable if endothelial specular microscopy was used for evaluation instead. 9 The probable explanation for the infrequent use of specular microscopy in eyebank screening relates to the inconvenience and risk of contamination and endothelial cell damage associated with the additional manipulation required. Most specular microscopes today require viewing of the endothelium from the epithelial side. When the stroma swells in the postmortem state, poor image quality and a narrow field of view make overall endothelial scanning difficult. The system evaluated here allows convenient, rapid, good quality, and apparently safe specular microscopic evaluation of donor corneal endothelium. Using the same chamber for viewing and storage minimizes manipulation of donor tissue with the attendant risks of contamination and endothelial damage. Modifications to the clinical specular microscope in this system allow noncontact examination from the endothelial side, achieving a wider field of view and better image quality, thus facilitating rapid endothelial screening. In its current state of design, this system appears most useful for initial screening when tissue first arrives in the eyebank. Endothelium could be seen in all corneas ex690

VOLUME 90 •

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amined upon arrival and could be satisfactorily screened in most. Even in this limited series, several corneas were discovered to have specular microscopic characteristics that would have resulted in their exclusion as donors for penetrating keratoplasty (Figs 4, 5). It may be that donors with undetected endothelial abnormalities such as these are responsible for some of the early failures still encountered after keratoplasty. Cell counts are conveniently obtained and correlate well with those calculated by using a standard epithelial applanation specular microscope. Obtaining cell co~nts easily and safely on donor tissue will allow co~pans~)D of pre- and post-surgical cell counts and correlatIOn WIt? the subsequent clinical course on a large scale. T~IS should help to assess relative contributions of the quahty of donor tissue, surgical trauma, and postsurgical care on the ultimate fate of corneal grafts. Some of the corneas studied were not transplanted because of donor age restrictions in our eye bank, ie, older than 67 years. About three quarters of the donor corneas in this study from people over 67 had cell counts greater than 2000 and acceptable morphology. The ability to determine quantitatively which older corneas have morphologically healthy endothelium may allow a safe extension of acceptable donor age, thereby helping to alleviate the current shortage of donor corneas. To obtain a high quality specular image, the angle between the incident light beam and the tissue must be small. lo As storage time increases, the stroma swells, producing Descemet's folds. This extremely irregular surface limits the ability of currently available specular microscopic systems to image the corneal endothelial cells. While the initial evaluation of a cornea is quite feasible with this system, subsequent evaluation becomes increasingly difficult. This technical problem may eventually be overcome with further improvements in microscopic imaging technique and with better storage conditions. Currently available techniques for assessing the status of donor endothelium include histochemical enzyme techniques, II vital dye staining, 12 in vitro perfusion with measurement of temperature reversal,13 electron microscopy,3 and specular microscopy. I Of these, specular microscopy is the least traumatic to tissue; the other modalities may render corneas unsuitable for transplantation. Thus, despite the obvious shortcomings of morphologic rather than functional assessment of donor endothelium, specular microscopy is one of the only currently practical methods for endothelial screening. The general application of a safe, rapid endothelial specular microscopic viewing system such as the one tested here may prove to be of considerable use in donor evaluation prior to keratoplasty. t

t A corneal viewing and storage chamber based on these principles is now commerCially available (Product Research Organization, 14761-F Franklin Avenue, Tustin, CA 92680).

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DONOR CORNEA VIEWING AND STORAGE CHAMBER

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13:859-63. 4. Van Horn DL, Schultz RO, DeBruin J. Endothelial sUNival in corneal tissue stored in M-K medium. Am J Ophthalmol 1975; 80:642-7. 5. Maurice DM. Cellular membrane activity in the corneal endothelium of the intact eye. Experientia 1968; 24:1094-5. 6. Laing RA, Sandstrom MM, Leibowitz HM. In vivo photomicrography of the corneal endothelium. Arch Ophthalmol 1975; 93:143-5. 7. Bourne WM, Kaufman HE. Specular microscopy of the human corneal endothelium in vivo. Am J Ophthalmol 1976; 81 :319-23.

8. Binder PS. Eye banking and corneal preseNation. In: Symposium on Medical and Surgical Diseases of the Cornea; Transactions of the New Orleans Academy of Ophthalmology. St Louis: CV Mosby, 1980;

320-54. 9. Bigar F, Schimmelpfennig B, Gieseler R. Routine evaluation of endothelium in human donor corneas. Albrecht von Graefes Arch Klin Exp Ophthalmol 1976; 200:195-200.

10. Laing RA, Sandstrom MM, Leibowitz HM. Clinical specular microscopy. I. Optical principles. Arch Ophthalmol 1979; 97:1714-9. 11. Robbins JE, Capella JA, Kaufman HE. A study of endothelium in keratoplasty and corneal preservation. Arch Ophthalmol 1965;

73:242-7. 12. Stocker FW, King EH, Lucas DO, Georgiade NA. Clinical test for evaluating donor corneas. Arch Ophthalmol 1970; 84:2-7. 13. Sherrard ES. Method of evaluating donor corneae for transplantation. Br J Ophthalmol 1973; 57:244-52.

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