23 Eye Bank Preparation of Endothelial Keratoplasty Grafts The current era of corneal transplantation is marked by tremendous surgical innovation, technological advancement, and expansion in the role of eye banks.1 The 20th century witnessed the inception of full-thickness penetrating keratoplasty (PK) by Dr. Edward Zirm in 19052 and the establishment of the first eye bank by Dr. R. Townley Paton in 1944.3 Arguably, the greatest step forward in eye banking was the demonstration by Filatov that corneal tissue can be collected and used postmortem in 1937.4 The Eye Bank Association of America (EBAA) was then established in 1961 to promote medical standards.5 McCarey-Kaufman (MK) medium was used for corneal tissue storage in the 1970s and was later transitioned to Optisol-GS medium (Bausch & Lomb) in the 1980s, after demonstration of its superiority in maintaining corneal deturgescence, the appearance of the endothelial cells, and overall tissue preservation.6 The 21st century then witnessed the subsequent transition to partial-thickness endothelial keratoplasty (EK) with Gerrit Melles, who laid the foundation of modern EK in 1998 with posterior lamellar keratoplasty (PLK),7 followed by Mark Terry in 2003 with deep lamellar endothelial keratoplasty (DLEK),8 and Francis Price Jr. in 2005 with Descemet stripping endothelial keratoplasty (DSEK).9 Mark Gorovoy thereafter, in 2006, introduced the technique known as Descemet stripping automated endothelial keratoplasty (DSAEK), with the use of an artificial anterior chamber and blade microkeratome for cutting donor grafts.10,11 Eye banks swiftly excelled in the midst of the multitude of surgical advancements. Though faced with initial surgeon hesitation to allow nonsurgeons to prepare their donor corneal grafts, in 2006, eye banks began supplying precut tissue for DSEK. Early studies further substantiated eye bank efforts with the demonstration of minimal difference in tissue quality and postoperative outcomes with the use eye bank-prepared precut tissue as compared with surgeon-prepared grafts.12,13,14,15,16 This shift in paradigm led to the rapid adoption of DSEK. From 2005 to 2014, the EBAA reported an increase in the number of endothelial transplants in the United States from 1429 to 28,961, with a simultaneous decrease in the number of full-thickness PK transplants from 45,821 to 38,919.17,18 The reduction in surgical time and the increase in graft preparation precision yielded faster visual recovery and lower complication rates than ever before.16,19 Today, eye banks are taking their repertoire to the next level with the preparation of Descemet membrane endothelial keratoplasty (DMEK) grafts,20,21,22 and more recently, in pioneering graft preparation for Amar Agarwal’s pre-Descemet endothelial keratoplasty (PDEK) technique.23,24,25 Thanks to the collaborative efforts of expert eye surgeons and eye banks globally, the world of EK is in the midst of history in the making, with the unified goal to achieve safe and effective tissue for sight restoration and lessen the global burden of corneal blindness.1,26 With the concurrent evolutionary advancements in eye banking and in corneal transplantation surgery tissue recovery, corneal quality evaluation, precutting, and, at times, even preloading into a delivery system are seamlessly being taken care of for the surgeon. The advantages are numerous. Eye banks can perform postdissection assessments and use specular microscopy and slit-lamp biomicroscopy to evaluate endothelial health, which cannot be readily performed when a surgeon dissects donor tissue in the operating room.14 Additionally, in electing to have the eye bank prepare the EK graft, the surgeon transfers the risk of tissue perforation during preparation to the eye bank, thus minimizing the inherent risks of tissue wastage and potentially needing to cancel surgery. The resulting improvement in operating room efficiency has demonstrated improved postoperative outcomes and has shown financial benefit, eliminating the need for expensive equipment.27 Notably, the use of eye bank-prepared EK tissue decreases the number of new skills a surgeon needs to learn when adopting the procedure. The experience some eye bank technicians have with EK graft preparation today far surpasses that of many ophthalmic surgeons.16 In an effort to optimize graft quality, many eye banks aim to remove corneoscleral tissue from the donor and place it into a storage medium within 18 to 24 hours from the time of death. Indeed, a longer death-to-preservation (DTP) time, time from corneal preservation to subsequent transplantation, as well as the donor’s cause of death have all been shown to affect graft quality.28 Thus eye banks quickly need to obtain tissue recovery authorization, conduct a detailed medical and social interview with the surviving family, review the donor’s medical record, perform a thorough physical assessment of the body, obtain a qualified blood sample, and recover the donated ocular tissue.13,18,29 Recovery of the tissue can be performed via whole eye enucleation or, more commonly, via an in situ excision of the corneoscleral rim, and is generally done in the morgue, at the bedside, in the funeral home, or in an operating room. The diameter of the donated corneoscleral button should be > 16 mm, with the scleral rim being 2 to 4 mm wide and uniform. During graft preparation, these margins help to minimize the risk of the tissue being cut too small or asymmetrically with the microkeratome.13 Gentle manipulation of the tissue is critical, as excessive mechanical trauma of the tissue during recovery and preparation may decrease the viability of the grafts.30 The history of eye banking includes the development of preservation techniques. Once eye banks were established in 1944, the original method used for preservation was the moist chamber for storing entire globes or excised corneas for up to 1 to 2 days. The later development of MK medium in 1974, consisting of a standard culture medium tissue culture (TC) 199, antibiotics, and dextran as an antiswelling agent, provided viable cold corneal storage at 4°C for 3 to 4 days and was the preferred choice31 for a number of years, until it was taken over by the organ culture method, which not only allowed for long-term preservation (∼ 48 days), but also simulated physiological conditions, at storage temperatures of 31 to 37°C.32 At the same time, short-term storage media alternatives, such as K-Sol, CSM, and Dexsol were being formulated, which allowed for 7 to 10 days of storage, adding chondroitin sulfate to the MK medium to help minimize donor swelling during storage.33,34 Optisol (Bausch & Lomb, Irvine, CA), a hybrid of K-Sol and Dexsol,6 was later introduced in the 1980s to further prolong endothelial survival during storage at 4°C for up to 2 weeks.34 Optisol GS (Bausch & Lomb, Irvine, CA),35,36,37 with the addition of antibiotics gentamicin and streptomycin to Optisol, is currently the most widely used cold storage medium (► Fig. 23.1) in the United States,34 though newer products, such as Eusol-C (Al.Chi.mia., Padova, Italy),38 LIFE4°C (Numendis, Istanti, MN), and Chen Medium (Chen Laboratories, Baltimore, MD)39,40,41 are proving to be effective alternatives.13,34 Researchers have evaluated the impact of culture conditions on visual outcomes in EK. Laaser et al demonstrated that tissue storage in Optisol-GS short-term culture at 4°C and organ culture at 34°C for DMEK tissue storage demonstrated comparable best-corrected visual acuity (BCVA), postoperative endothelial cell density (ECD), and central corneal thickness.42,43 Further evidence suggests that longer storage times may allow better outcomes in high-risk grafts because of the depletion of donor T cells from the donor cornea into the storage media. Thus storage media will need to be optimized for preserving the endothelium for longer periods of time.34 The addition of fibroblast growth factor-2 and nitric oxide synthase inhibitors is being investigated to further enhance the viability.34,44,45,46 Fig. 23.1 Optisol-GS (Optisol with the addition of antibiotics gentamicin and streptomycin) is currently the most widely used cold storage medium in the United States. Areas of ongoing research include epithelial and limbal stem cell preservation, endothelial preservation, and antimicrobial prophylaxis.47,48,49 EBAA has laid out extensive criteria that each donor and associated tissue must meet in order to allow safe and effective transplantation. Endothelial grafts should meet the same criteria as those for PK, except that tissue with noninfectious pathology limited to the anterior stroma is also eligible for use in EK with notification of the surgeon. Although these criteria are in place to prevent potential harm to the recipient, the ultimate responsibility and decision to transplant a particular tissue lie with the surgeon. The lower limit of endothelial cell count for PK is 2000 cells/mm2 and surgeons often request a density of at least 2500 cells/mm2, but the optimal density for EK has not been well established. However, preoperative endothelial cell count does not appear to be correlated with graft dislocation rates or cell counts at 1 year postoperatively.50 Storage time also does not seem to affect the average percentage of cell loss at time points of up to 2 years postoperatively, and tissue stored for even 14 days appears to be sufficient. Together, these studies may allow for less stringent donor graft selection practices on the surgeon’s part, increasing the availability of viable grafts. This can have significant ramifications for the international shipment of donor grafts to areas of shortages; precutting and long-distance overseas transportation of DSAEK have been demonstrated to provide an effective source of tissue with preservation of cell counts at an adequate level.51 DSAEK is the most commonly performed type of EK. The DSAEK procedure offers corneal donor graft predictability in the anterior chamber. The transition from manually dissected DSEK grafts to automated dissection with the microkeratome represented a significant advancement for both the surgeon and the eye bank. Today, however, despite the vast improvement in postoperative visual recovery with DSAEK as compared to earlier methods of corneal transplantation, the ophthalmology community continues to look for ways of overcoming what are believed to be the major disadvantages limiting postoperative visual acuity with DSAEK, namely optical degradation associated with the interface between posterior stromal graft tissue and recipient stromal tissue and interface irregularity.51,52,53 A microkeratome system consists of a blade and an artificial anterior chamber (AAC). Commonly used microkeratome systems include those produced by Moria, Horizon, and Amadeus. In the microkeratome-assisted DSAEK technique (► Fig. 23.2), the donor corneoscleral rim is pressurized with balanced salt solution (BSS), Optisol-GS storage medium, or viscoelastic followed by dissection using the appropriately sized microkeratome head, with the goal of leaving approximately 100 to 200 µm of posterior stroma with Descemet membrane and endothelium.9,10,11,54,55,56,57 Fig. 23.2 Reusable artificial anterior chamber (ALTK System, Moria). In 2006, Mark Gorovoy introduced the technique variant known as Descemet stripping automated endothelial keratoplasty (DSAEK), with the use of an artificial anterior chamber and blade microkeratome for cutting donor grafts, a significant advancement for both the surgeon and the eye bank. In compliance with EBAA Medical Standards (► Fig. 23.3), central endothelial density, graft thickness measurements, and specular microscopy are evaluated before and after the corneal graft is cut.58 The corneal stroma may be marked peripherally to outline the area of the trephine cut as well as centrally, to allow proper centration of the tissue. Some surgeons also request that an “S stamp” be placed on the anterior aspect of the graft to assist them intraoperatively with graft orientation in the eye (► Fig. 23.4). These markings can be done with the ink pen or gentian violet.13,14,15,55 The microkeratome blade/head complex is then released and the graft is realigned back with its anterior cap. The proper alignment of the anterior cap with the graft will eliminate gaps in the interface, preventing fluid from leaking in during transit to the surgeon. The anterior cap/graft complex can then be safely transferred to its container with fresh cornea storage media.15,55,59 Fig. 23.3 In compliance with the Eye Bank Association of America (EBAA) Medical Standards, DSAEK tissue is evaluated pre- and post–graft preparation with slit lamp microscopy. Fig. 23.4 “S” stamp on an endothelial keratoplasty graft. The efforts to further minimize any interface irregularity in DSAEK brought forward the utility of femtosecond laser innovation for DSAEK graft preparation.15,60 Compared to microkeratome-prepared tissues, femto-prepared tissues have greater irregularity of the posterior corneal surface, rougher stromal beds, and increased thickness irregularity.61,62 No difference was noted in endothelial cell density and viability between the two techniques. The theory behind the irregular stromal dissections is the morph in donor cornea contour upon application by the femtosecond laser applanation cone.61,62 Some advocate for use of both the microkeratome and the femtosecond laser together in sequential cuts to yield ultrathin DSAEK grafts. Results have shown promise, with no irregular cuts or perforations during tissue preparation and a reduction in stromal roughness and thickness, with improved contour and symmetry, without endothelial cell damage after excimer laser smoothing passes. However, the clinical significance of these findings has not yet been established.59,63,64 Ultrathin DSAEK (UT-DSAEK) corneal donor graft tissue thickness is generally considered to be < 100 µm. Thinner grafts may improve visual outcomes65 and possibly reduce the observed postoperative hyperopic shift.66,67,68,69,70 The rationale behind the double-pass microkeratome technique is that it produces more planar and thinner graft lenticules, though some studies report high perforation rates and increased endothelial cell damage in using this method.71,72,73 In an effort to decrease corneal perforation risk, Busin et al hydrated grafts after the initial microkeratome cut by using intrastromal injections of BSS and immersing tissue in hypoosmotic tissue culture medium for 24 hours.72 Both strategies thickened the residual tissue bed and prevented perforations during the second microkeratome pass. However, the hydration technique in particular was found not to be as clinically useful because there were multiple areas of Descemet detachment.13,14,15 Ultrathin tissue can also be prepared using a low-pulse energy, high-frequency femtosecond laser, with no increased endothelial cell damage. The current challenge with this technique is the resulting irregular stromal surface, due to the photodisruptive effects of the femtosecond laser, which is thought to be potentially improved with modified laser settings or an inverse cutting approach.59,74,75 With DMEK, the transplanted lenticule includes only Descemet membrane (DM) and corneal endothelium with no stromal interface, which may explain the potential improvements in visual acuity reported with this technique. Thinner, well-centered, planar tissue may be associated with less induction of higher-order optical aberrations, although this remains an unproven hypothesis.21,76 The challenges associated with DMEK have included both preparing the delicate graft tissue as well as intraoperative handling of the tissue without causing tears or excessive endothelial cell damage. Thus, in current practice, many surgeons delving into DMEK will often have a DSEK corneal graft made available, should the need for conversion to DSEK arise.76,77,78,79 Melles et al described a manual peeling method where a donor corneoscleral rim is initially mounted endothelial side up on a custommade holder with a suction cup. After trephination, a DM was stripped from the posterior stroma with one-point, fine, nontoothed forceps to obtain a 9 mm diameter flap of posterior DM with its endothelial monolayer. Based on the properties of DM, the graft tends to scroll spontaneously with the endothelium on the outer side.20,56,60,77,78 The technique of submerged corneas using backgrounds away (SCUBA) (► Fig. 23.5) was then subsequently described, where a cornea is submerged in corneal storage media or BSS to minimize surface tension and allow for the DM to settle back onto the stroma.20,77,79 Various other important technique modifications have been trialed since, including the use of a second pair of forceps during peel,80,81 use of a curvilinear forceps with a half-moon-shaped, nontoothed anterior segment to equally distribute the force needed for DM separation,82 as well as the use of a microkeratome in combination with a Barraquer sweep to dissect the residual stroma from the DM.83 In DMEK-S, the central graft consists of only DM and endothelium, while an additional layer of posterior stroma is maintained in the periphery.84 The tissue loss rate was noted to be less with this additional rim of tissue in some studies, whereas others demonstrated an even higher rate secondary to bubble rupture and failure of bubble formation.85 The transitions to the DMEK-S and Descemet membrane automated endothelial keratoplasty (DMAEK) techniques parallel the difference between DSEK and DSAEK. With DMAEK, a posterior lamellar dissection is done with a microkeratome84 or femtosecond laser.22 An AAC is used, and mechanical separation of the DM is conducted. This can be done with a 330Æ superficial trephination followed by cannula insertion with BSS injected to detach the DM86 or with the use of an epi-keratome (Senturium; Norwood Abbey EyeCare).87,88
23.1 Background
23.1.1 Evolution of Eye Banking
23.1.2 Advantages of Eye Bank-Prepared Grafts
23.1.3 Donor Graft Recovery
23.1.4 Corneal Storage Media
23.1.5 Graft Eligibility
23.2 Endothelial Keratoplasty Graft Preparation Technique
23.2.1 Descemet Stripping Automated Endothelial Keratoplasty
Microkeratome-Assisted DSAEK
Femtosecond Laser-Assisted DSAEK
23.2.2 Ultrathin Descemet Stripping Automated Endothelial Keratoplasty
Double-Pass Microkeratome Technique
Femtosecond Laser-Assisted UT-DSAEK
23.2.3 Descemet Membrane Endothelial Keratoplasty
Manual Peel
Submerged Corneas Using Backgrounds Away Technique
DMEK-S
DMAEK
Ento Key
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