Living-Related Conjunctival Limbal Allograft

Indications

The value of a normal limbus in maintaining a normal ocular surface has been well documented on the basis of anatomical and experimental evidence.1–7 The objectives of epithelial stem cell transplantation are to restore phenotypic corneal epithelium to the corneal surface, to promote the barrier function of the limbus, and to improve surface lubrication, thus providing an improved milieu for maintenance of corneal clarity. Depending on the cause, as outlined in earlier chapters, stem cell deficiency may be bilateral and may also involve the conjunctiva with varying severity ranging from subconjunctival scarring, to keratinization, cicatrization, symblepharon formation, and secondary lid abnormalities. In bilateral disease, the only option for stem cell restoration is the use of allogeneic sources.8–10 Procedures available include a cadaveric keratolimbal allograft (KLAL), living-related conjunctival limbal allograft (lrCLAL), and more recently, ex vivo stem cell allograft expansion.

Keratolimbal allograft transplantation has the advantage of providing a complete limbus for transplantation and thus a larger load of stem cells with restoration of a barrier to the whole limbus. The practical disadvantage is that it is almost impossible to obtain immune histocompatibility and, with such highly antigenic tissue transplanted into a highly vascularized recipient bed, there is the potential increase risk of graft rejection.11,12 This risk of rejection may account for the reduced long-term survival of nonmatched keratolimbal allografts. Tsubota reported long-term survival of only 51%,13 and Ilari reported 50%.14 Furthermore, depending on cadaver and storage time, there may be a degree of stem cell death, thus reducing the volume of cells transplanted. While there may be limitations on the amount of tissue that can be transplanted in a lrCLAL, there is the advantage of providing some degree of immune histocompatibility,15,16 thereby reducing the de-

pendency on and dosage of systemic immunosuppressive agents. Additionally, cell death is theoretically limited because of the rapidity of transplantation from donor to recipient and the rapid revascularization of grafted material.

Ocular surface reconstruction can be divided into two components, structural reconstruction and surface restoration. Structural reconstruction involves reduction of ankyloblepharon and symblepharon and treatment of lid abnormalities, whereas surface restoration includes establishment of the barrier function at the limbus and epithelialization of the cornea with phenotypic corneal epithelium. Cadaveric KLAL transplantation alone or in combination with amniotic transplantation may accomplish both successful structural reconstruction as well as surface restoration. On a long-term basis, KLAL may fail to maintain phenotypic corneal epithelium even if structural reconstruction is usually maintained without recurrence of sym-

blepharon.10,13,17,18

Since lrCLAL requires a procedure on an individual other than the patient, it is best reserved for eyes in which there is a minimal risk of failure. Eyes that require surface restoration alone are best suited for lrCLAL. Eyes requiring both reconstruction as well as restoration are unsuitable for lrCLAL and are best initially treated by KLAL with amniotic membrane transplant as a first stage, followed by lrCLAL in the event of epithelial failure.

Contraindications and

Preoperative Considerations

Contraindications to lrCLAL include:

1.Any ocular surface requiring structural reconstruction.

2.Severe keratinization of the palpebral conjunctiva.

3.Inadequate lid closure.

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4.Dry eye.

5.Severe inflammation.

In severe cases of bilateral disease, such as those following chemical injuries and Stevens–Johnson syndrome, severe cicatrization may be associated with lid abnormalities including keratinization, trichiasis, and entropion. Lid reconstructive and corrective procedures are best performed well in advance of epithelial transplantation. The goal of these procedures is to ensure adequate and atraumatic coverage of the ocular surface. Inadequate lid closure and severe keratinization of palpebral conjunctiva are incompatible with successful epithelial transplantation and are an absolute contraindication to lrCLAL. An ocular surface requiring reconstruction has been discussed above and is also a relative contraindication to lrCLAL.

Surface lubrication is crucial for survival of epithelium. Bilateral stem cell deficiency can be associated with severe dryness as a result of cicatrization of the main and accessory lacrimal ducts. The tear function index (TFI)19 is a method of evaluating status of lubrication, and methods to improve the TFI such as punctal occlusion are best carried out prior to surgery. Epithelial transplant procedures are contraindicated in instances in which there is absolute dryness with no tear function and where improvement is unlikely in the future.19

Severe inflammation is a poor prognostic factor, and its presence is also a contraindication to lrCLAL.16 Treatment during the acute phase of an illness, for instance, soon after a chemical injury, is contraindicated. The goal in this instance would be to stabilize the eye and reduce inflammation. Other procedures, such as amniotic membrane transplantation with or without KLAL, are best utilized to promote epithelialization and downregulation of inflammation.20–23 Underlying causes of stem cell deficiency, such as ocular cicatricial pemphigoid, must also be adequately controlled prior to surgery. Inflammation, where present, can be controlled by topical and systemic steroids. Furthermore, it is best to have a period of 3 to 6 months free of inflammation prior to surgery.

Since many affected corneas are not transparent, some surgeons might consider performing a simultaneous visually rehabilitative procedure such as a penetrating keratoplasty. One report16 demonstrated a reduction in corneal opacification with improvement in visual outcome in 70% of eyes following lrCLAL alone, obviating the need for a penetrating or lamellar keratoplasty. In most cases, therefore, it is best to perform a lrCLAL alone and wait for at least 6 to 12 months before considering corneal graft.24 This allows the cornea to clear and also provides an opportunity for the ocular surface to improve, thereby optimizing graft survival, should it be required.

S.M. Daya, E.J. Holland, and M.J. Mannis

Surgical Techniques

Donor Selection

Since immune histocompatibility is one of the main reasons for embarking on this course of management, the best HLA-matched relative is the ideal donor. Human leukocyte antigen (HLA) typing is performed on all possible donors. If only parents are available, then HLA matching is not necessary, since the recipient will be a haplotype. Since expression of blood-group antigens has been demonstrated on epithelial cells,25 ABO matching is also advisable when there are several potential donors available.

Both eyes of the donor are evaluated to ensure there is no underlying ocular history that may preclude donation. A history of glaucoma, for example, would be a relative contraindication because of the possible need for a trabeculectomy in the future. Additionally, chronic use of topical medications and prior contact lens wear with Thimerosal-containing preservatives suggest potential stem cell depletion that could be exacerbated by removal of limbal conjunctiva. A prior surgical history would also be a relative contraindication because of the possibility of iatrogenic stem cell deficiency.26 One should measure tear breakup time, Schirmer test, and employ rose bengal staining to ensure there is no underlying abnormality.

Following identification of the best-matched living relative, screening for the possibility of blood-borne diseases is necessary. The standard serologic tests required by the Eye Bank Association of America are advised; these include screening for human immunodeficiency virus 1 and 2 and hepatitis B and C.

Anesthesia

Topical retroor peribulbar anaesthesia can be used for harvesting donor tissue. However, based on cultural and practical considerations, general anesthesia can be employed. The recipient’s eye can be similarly anesthetized using retroor peribulbar anesthesia or, if necessary, general anesthesia.

Harvesting Tissue From the Relative

The nondominant eye of the donor is selected for the conjunctival limbal allograft, if it has not been used for tissue donation before. Since it is customary for surgeons to operate out of one operating room, removal of the donor tissue needs to be accomplished first. The procedure includes standard sterile preparation and draping of the eye. The sites for donation are marked on the donor eye (Figure 17.1A). Methods include a marking pen on the conjunctival surface or a 12-blade radial ker- atot-omy marker with a gentian violet marking pad. The

cornea and conjunctiva are marked. It is best to incorporate the gentian violet markings within the donor tissue. These marks safeguard against inverting the donor tissue on the recipient eye.

Two pieces of tissue, each between 2 and 3 clock hours at the limbus, are resected from the 12 o’clock and 6 o’- clock areas, and 5 mm posterior to the limbus (Figure 17.1B). Some surgeons elevate the site with a subconjunctival injection of balanced salt solution. Radial incisions are made at the lateral borders and dissection is performed, undermining the tissue as much as possible. Blunt forceps are used in order to prevent tearing the tis-

sue. The posterior edge is then cut 5 mm posterior to the limbus, and the tissue is reflected anteriorly on to the corneal surface (Figure 17.2). Dissection is continued into the peripheral cornea 1 mm anterior to the vascular arcade. A blunt scarifier (e.g., Tooke knife) is useful in accomplishing this dissection. This does not involve a lamellar dissection into the corneal stroma. Since stem cells are in the basal layer of the epithelium, only a superficial keratectomy is necessary. The author finds Vannas scissors useful in cutting the anterior portion of the tissue once adequate blunt dissection has been performed. The lower blade is placed under the tissue and

Figure 17.2. Donor conjunctival limbal allograft tissue reflected onto corneal surface and dissection carried anteriorly without involving corneal stroma.

directed anteriorly and flat on the ocular surface. The scissors are advanced after small cuts are made with the mid-shaft of the blade until the tissue is completely resected. The tissue is allowed to float on balanced salt solution or is immersed into corneal storage media if available. Some surgeons leave the donor site alone and proceed to harvest tissue from the second site. Others prefer to advance the posterior conjunctiva to 2 mm posterior to the limbus and to suture the conjunctiva to the episclera using 8-0 polyglactin (Vicryl™, Ethicon) suture. Advancement of conjunctiva minimizes postoperative discomfort and inflammation on the donor eye. Tissue is harvested from the second site in a similar fashion. An antibiotic steroid ointment is placed in the eye, and the eye is patched until the patient is seen the following day.

Preparation of the Recipient Eye and

Placement of Donor Tissue

The eye is sterilized and draped in a similar fashion to the donor eye. A speculum is inserted to ensure adequate exposure. Symblepharon, if present, can be lysed in the inferior fornix to enable insertion and expansion of the lid speculum. A 360-degree limbal conjunctival peritomy is performed. The conjunctiva is undermined and allowed to recess 2 to 3 mm from the limbus. Areas of conjunctiva failing to recede adequately are excised along with the limbal skirt of tissue. The recession of conjunctiva is enhanced by excision of subconjunctival scar tissue and thickened Tenon’s capsule. This recession is necessary to enable the new source of limbal epithelium to repopulate the corneal surface and to prevent conjunctival tissue from crossing the limbus. Since the area is highly vascularized, there may be excessive

bleeding that can interfere with visualization. Wet-field cautery, topical adrenergics such as viscous phenylephrine 10%, and thrombin can be used to arrest bleeding. Alternatively, simply waiting a short period prior to operating further permits normal clotting mechanisms to achieve hemostasis. The surgeon then prepares the recipient beds for the limbal tissue. This includes removal of recipient conjunctival tissue 3 clock hours at the 6 and 12 o’clock areas and to 5 mm behind the limbus. The abnormal corneal epithelium is then removed. This is usually a vascularized pannus varying in adherence to the underlying stroma. A surgical spear or a lamellar dissector is often most effective in removing the epithelium without removing the stroma. The cornea is frequently thinner as a result of prior inflammation and fibrosis so that the surgeon must avoid removal of deeper layers, which can lead to optical irregularity, further thinning, or even perforation. The harvested tissue is then sutured in the same anatomical orientation with the limbal edge at the recipient limbus. The tissue can be sutured with 10-0 nylon or polyglactin, although some surgeons prefer the former because it is less likely to produce inflammation than absorbable suture. The author’s preference is to use 2 clock hours of donor tissue and to stretch this to occupy 3 clock hours of limbus, suturing the tissue with absorbable material. Sutures are tied but not buried to prevent trauma and possible cheese wiring of donor tissue. During the operation, it is essential to protect the tissue from trauma and desiccation by balanced salt solution and viscoelastic material. Once concluded, a steroid-antibiotic ointment is instilled and the eye is patched until the patient is seen the following day. Figure 17.3 demonstrates a living related conjunctival limbal allograft.

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Postoperative Management

The goal of postoperative management is to minimize inflammation and promote epithelization in both the donor and host. In the donor, a combination of steroid and antibiotic is used in the donor until epithelization is complete, usually within a few days, and steroids can be gradually tapered over a period of 2 to 3 weeks. In the recipient, vascularization of the grafts is important to ensure survival of stem cells. It is, therefore, essential that the grafts do not move as a result of lid action against the graft site. Keeping the eye patched between eyedrops for at least the first week keeps the grafts in contact with the globe and minimizes the risk of dislodgement. A contact lens can also serve as a bandage preventing the grafts from rubbing against the lid. Preservative-free steroids and antibiotics are instilled 4 times daily. Some also advocate the use of autologous plasma or serum16,27,28 to provide growth factors for the epithelium. When there is an underlying reduction in tear production, preservative-free tear supplements should be started early. Evidence of slow epithelial healing is best managed by an early tarsorrhaphy.

Immunosuppression

The role of immunosuppression has been debated. Kwitko15 and Rao29 in their series did not use systemic cyclosporin A. Daya and Ilari used cyclosporin A indefinitely in all recipients and reported 100% survival at a mean of 26.2 months.16 Since rejection is the major cause of allograft failure, systemic immunosuppression is advisable, assuming that there is no contraindication. Systemic immunosuppression is commenced on the day of surgery, using a combination of systemic steroids and cyclosporin A (CsA). Steroid regimens vary. The author commences intravenous methyl prednisolone 2mg/kg on the day of surgery followed by 1 mg/kg intravenously for three additional days. Oral prednisolone is then started, the dose varying depending on the degree of inflammation present. Systemic CsA is started at a dose of between 3 and 5 mg/kg/day as a single dose or in two divided doses. A serum trough level is obtained on the fourth day, and the daily dose is adjusted accordingly. A repeat trough level is obtained again on a weekly basis until stable and then 1 to 2 monthly thereafter. Desired therapeutic levels vary between institutions, although they are typically between 150 and 200 ng/ml. The duration of immunosuppression is also variable with some surgeons discontinuing treatment at 12 to 18 months and others maintaining this indefinitely. While on treatment, periodic evaluation of creatinine and blood pressure is essential. In addition, some authors advocate the use of a third oral immunosuppressive, azathioprine, which allows for reduced doses of CsA.10

S.M. Daya, E.J. Holland, and M.J. Mannis

The conjunctival grafts are revascularized within 4 to 7 days and in the following 2 weeks often become swollen with engorged vessels. This phenomenon has been observed in conjunctival autografts and probably represents reactive hyperemia. In some instances, a subconjunctival hemorrhage occurs in the graft. There is no reason for concern, since the grafts do eventually thin.

Living-related conjunctival limbal allograft provides an opportunity to restore the ocular surface in cases of bilateral ocular surface disease. Although this technique permits a degree of histocompatibility matching, there is still the potential for rejection and ocular morbidity. Hence, systemic immunosuppression is recommended. Careful patient preparation is necessary to optimize outcomes. Corneal grafting for visual rehabilitation may not be necessary and is best performed 6 to 12 months following lrCLAL. As with all allograft procedures, close and regular follow-up care is essential to ensure long-term survival.

References

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