Excised donor cornea in the slotted lucite dish. (from Max Fine, 1908-1989, Mannis, History of Keatoplasty) (From Max Fine, 1908-1989, Lanier, J.D., Webster, R.G., in Mannis, M.J. and Mannis, A.A., eds. Corneal Transplantation: A History in Profiles, J.P. Wayenborgh, Belgium, 1999)

After the globe is received in the eye bank, the cornea  with its scleral rim is removed and preserved.  The sclera may also be preserved at this time.  In-situ removal is very similar.  Each eye bank, depending on their retrieval routines and locations will have an individualized protocol for in-situ removal.  The principles are the same and combine the preparation phase of the enucleation procedure with the corneo-scleral rim excision procedure.  The excision begins with preparation of the sterile field, hood, media and transport containers (eye jars or similar). The globes are irrigated while in their transport containers.   The technician then scrubs and gloves in a standard operating room fashion.  An impermeable sterile wrap (such as sterile aluminum foil) is used to hold the container while the globe and cage are removed from the jar. The globe is then wrapped with a sterile 4x4, and the gauze secured with a hemostat. A scalpel blade is then used to scrape any conjunctiva from the limbus and subsequently discarded as contaminated.  A trephine may be used to mark a cutting line for the scleral rim.  A new sharp-pointed scalpel blade is used to incise the sclera 2-3mm posterior to the limbus without penetrating the choroid (Figure 11a).  With a curve tipped scissors the sclera can then be cut around the circumference of the scleral rim (Figure 11b).  Care must be taken not to penetrate and cause a vitreous leak or physically damage the cornea while completing the incision.   Using a toothed forceps to hold the corneo-scleral rim, the ciliary body, choroid, and other posterior structures are very carefully pushed away and separated from the corneo-scleral rim at the scleral spur (Figure 11c).  Extreme care must be taken not to bend, fold or stretch (stress) the cornea.  The corneo-scleral tissue may then be placed in a corneal storage and viewing chamber (CSVC) filled with media or placed in a sterile container filled with preservation media (Figure 12). This is repeated for the second globe using new instruments that have not touched the conjunctiva.  The tissue must be appropriately labeled including which eye, right or left, is in the container.  The posterior globe should be examined for whether a lens or a lens implant is present, or whether a lens is absent (aphakia).  The sclera can be preserved in alcohol or other media by removing remnants of the conjunctiva, Tenon's, the nerve and any of the internal structures.  Separate protocols are available to explain this procedure in depth. Corneal Cap Removal The sclera is incised tangentially with a scalpel blade (a).  Scissors are then used to cut the sclera (b).  The cap is lifted with care, and the ciliary body teased posteriorly, avoiding any buckling of the tissue (c). (Figure 11a-c) (Figure 11a) (Figure 11b) (Figure 11c) Media vial The sterile media and its vial can used to store the corneal cap, or to fill the corneal storage and viewing chamber.  (Figure 12) EBAA Laboratory Corneal Excision Protocol E1.220  Laboratory Corneal Excision Purpose: To provide a standardized method for the aseptic preservation of corneal tissue in the laboratory that will minimize endothelial cell loss and contamination and maximize the number and quality of cells that are ultimately grafted. Materials needed: Sterile Supplies Sterile gown or sleeves Sterile gloves Sterile scrub brush for scrubbing hands 1 sterile towel Sterile ophthalmic irrigating solution Sterile ophthalmic broad spectrum antibiotic 2 vials corneal storage medium 2 mini tipped culturettes (if cultures are performed  by the eye bank) Sterile cotton-tipped applicators Sterile gauze Jars containing whole eyes Class 100 Hood or Class II or Class III Biosafety     Cabinet or an operating room Appropriately wrapped sterilized instrument tray containing the following: 2 Small toothed forceps 2 Scalpel handles 2 #11 or #15 blades 1 Corneal section scissors, or Castroviejo or Aebli scissors 2 Tenotomy or iris scissors 1 Hemostat 1 Forceps to handle cages and/or solution bottles 2 Medicine cups or other small 30 cc glass/steel container Non-Sterile Supplies Moisture impermeable protective clothing Mask Cap to cover hair Protective eyewear (goggles or face shield) Slit Lamp Evaluation Form Procedure with Rationale in Italics 1.  Perform the corneal removal (excision) in the laboratory in a Class 100 Hood or Biosafety Cabinet following a whole eye enucleation.  Wipe down and air dry the work surface of the hood or cabinet with a disinfectant solution immediately prior to use. Turn on laminar airflow of hood and allow to run at least fifteen according to manufacturers' instructions prior to use.  2.  Don appropriate protective apparel consistent with the biological safety cabinet being used. Use of a biosafety cabinet with a plexiglass shield protects the technician and tissue. Therefore, protective eye wear and mask in particular may not be necessary. However, if tissue is opened outside of the hood, e.g., while slit lamping the whole globe, full protective  apparel is still required. 3.  Place sterile instrument pack, eye jars, antibiotic solution, and labeled corneal storage medium vials on the prepared surface of the laminar airflow work surface. 4.  Position the eye jars so that they are immediately adjacent to the edge of the sterile field formed when the sterile instrument pack is opened. The eye jar lids are removed and placed with inner side up next to their respective jars. The labeled storage medium vials are positioned so that they also will be adjacent to the sterile field, Remove the caps of the vials.  Position eye jars and medium vials to ensure that left and right specimen bottles are clearly and readily identified. 5.  Uncap a 10 cc bottle of broad range sterile ophthalmic antibiotic solution or a povidone-iodine solution container and place near the eye jars and medium vials, according to your eye bank's policy.  Antibiotic or antiseptic application to the whole eye prior to corneal excision reduces themicrobial population.  (Povidone-Iodine 0.5% solution has demonstrated effectiveness as an anti-fungal agent.  6.  Set up the sterile field by opening the outer and then inner wraps of the sterile instrument tray (if two wrappers are used).  Alternatively, a sterile moisture impermeable barrier drape may be opened and placed on the work surface of the laminar airflow hood or cabinet, followed by opening sterile instruments in peel packs and dropping them on. Avoid contaminating the sterile field created by touching or reaching over the field. Open individually wrapped sterile items, such as gauze or sterile cotton-tipped applicators and flip onto the sterile field with the surgical instruments. 7.  Scrub three to five minutes according to procedure E1.050. Dry hands with a sterile towel. Don sterile gloves and gown or sleeves. Double glove if this is your eye bank's policy. 8.  Fold a sterile 4 x 4 gauze sponge to form a long strip. This is used to hold the eye during the corneal removal. 9.  Lift the eye and the eye cage, if one is used, from the eye jar with sterile forceps (or the cage  with a sterile cotton-tipped applicator.)  Remove the pin if one is in place from the optic nerve with a hemostat.  Remove the eye from the cage using forceps to grasp a rectus muscle. 10. Soak or irrigate the eye using an antibiotic solution for 3 to 5 minutes in a sterile medicine cup according to your eye bank's procedure.  Avoid contaminating the sterile field by wetting of a cloth drape, if one is used.  Irrigation should be performed over a metal instrument pan or a moisture impermeable drape. Studies have shown that whole globe immersion is superior to irrigation for removal of    microbes (see reference list.) 11. Wrap the eye securely with the gauze strip several times around the equator. To prevent strike through contamination of the sterile field. 12. Lift and cut any remaining conjunctiva at the limbus and extending out 5 mm from the limbus using small toothed forceps and iris or tenotomy scissors.  The exposed sclera may be carefully scraped from the limbus outward with a scalpel blade (#11 or #15) to remove all remaining conjunctival tissue.  It is important to remove all conjunctiva flush at the limbus. To remove microbial contaminants that may be present on the conjunctival tissue. 13. The instruments and blade used to remove the conjunctiva should be isolated from the other instruments on the sterile field and should be used only to remove the conjunctiva on the opposite eye. These instruments are contaminated since they have touched the "dirty" conjunctiva. 14. Pick up the gauze-wrapped globe and hold with one hand. 15. Make an incision through the sclera 2 mm - 4 mm from the limbus and parallel to the limbus approximately 5 mm in length using a second scalpel with a #11 or #15 blade and small toothed forceps. Carefully cut all the way through the sclera without perforating the choroid. Perforation of the choroid causes vitreous leakage, which may collapse the globe including the anterior chamber.  This would compromise the corneal endothelium. 16. Extend the scleral incision 360o around the cornea using corneal section scissors (Castroviejo or Aebli). Avoid perforating the choroid, breaking into the anterior chamber, or causing any deformation of the cornea's normal curvature.  The scissors should not be visible in the anterior chamber. Trauma to the cornea during cutting due to bending, loss of the anterior chamber, or collapse of the globe through vitreous loss would severely compromise its suitability for surgical use. Keep the incision parallel to the limbus to produce an even scleral rim between 2 mm and  4 mm in width.  Scleral rim width is important because some surgical corneal holding devices require a minimum 2 mm rim while other devices require a rim no wider than 4 mm.  Also, cutting a rim less than 2 mm wide greatly increases the chance of entering the anterior chamber while performing the peritomy.  17. Inspect to be certain the incision is complete and that the anterior chamber is intact.  If the incision has been made properly, the corneo-scleral button should be attached to the ciliary body-choroid only at the scleral spur.  The risk of endothelial trauma and cell damage is greatest at this stage of the excision process. 18. A culture of the incision site may be performed at this time, per your eye bank's policy. 19. Set the wrapped eye down near the center of the sterile field which may be stabilized by attaching a sterile hemostat.  Complete the corneal removal using one pair of the small forceps to hold the scleral rim stationary and a second set of small forceps, an iris spatula or back of a scalpel blade to push the ciliary body-choroid downward and away from the corneo-scleral button.  Gently pull away remaining adhesions from the corneo-scleral button working side to side.  The corneo-scleral rim must never be pulled in such a way as to cause cross corneal tension.  The corneo-scleral rim should never be allowed to drop back down onto the anterior chamber. To avoid pulling on the cornea and creating folds.  Aqueous fluid should escape from the anterior chamber at this point assuring that the anterior chamber was indeed intact. To avoid stretching or damage to the endothelium. 20. Continue to hold the cornea by the scleral rim with the small toothed forceps and transfer it to  a labeled vial of storage medium from which the caps have already been removed. The vials may remain open under the laminar airflow hood or biosafety cabinet for a period of 1 hour, which is acceptable operating room practice. 21. You may examine the posterior chamber for crystalline lens. Inspect for signs of previous cataract surgery, which would possibly contraindicate use of the corneal tissue for penetrating keratoplasty, depending on your eye bank's policy (See  EBAA Medical Standards (D1.120). 22. Carefully unwrap and return the remaining posterior segment to its respective eye jar. Avoid contaminating the posterior segment, instruments, or surgical gloves. 23. Repeat the procedure on the second eye. 24. After the second cornea is placed in storage medium, replace both vial caps and tighten. Replace the lids on the eye jars. The vials containing the ocular tissue are immediately labeled and sealed and the tissue refrigerated. See procedures I1.000 and J1.000. 25. Instruments and eye jars are immediately cleaned according to your eye bank's policy and procedure. Discard all disposables in a biohazard receptacle. See procedure C3.300. 26. Immediately after use, wipe down the work surface of the hood with a disinfectant and to dry.  Document these cleaning procedures according to your eye bank's policies and procedures. See EBAA Medical Standards C3.300.

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