Surgical Maneuvers

Several surgical approaches available for patients with limbal stem cell deficiency

A systematic approach is needed for ocular surface reconstruction.

Global bilateral blindness is estimated by the World Health Organization to be around 45 million people, of which more than 20% are secondary to corneal diseases. Of all the corneal disorders, limbal stem cell deficiency may be considered as one of the most severe and difficult-to-treat clinical entities.

Limbal stem cells are of significant importance with regard to corneal homeostasis. They form a somewhat circular garland on the ocular surface, sandwiched between the corneal and conjunctival epithelia. Human limbal stem cells can be detected both in vivo and in vitro by their expression of p63 transcription factor. The protein p63 retains the proliferative potential of limbal stem cells. These stem cells maintain corneal stromal avascularity and corneal epithelial integrity and thus play a vital role in the sustenance of a clear cornea, which is essential to vision. In the absence of a viable, healthy stem cell population, conjunctival progression to the corneal territory and vascular invasion of the cornea can result in a cloudy or opaque cornea and can potentially lead to corneal blindness. In cases of such corneal blindness, an allogenic corneal transplantation such as a penetrating keratoplasty can provide a clear cornea and restore vision only temporarily because conjunctival cell invasion onto the grafted cornea will lead to possible blindness. Hence, any attempt at vision restoration must first address the restoration of the limbal stem cells.

Limbal stem cell restoration may be approached from various routes. These include autologous culture of limbal cells as a source of transplant tissue; conjunctival limbal autograft; living-related conjunctival allograft; and keratolimbal allograft using a donor cornea from the eye bank. Once limbal stem cells have been established, then corneal opacity may be addressed in terms of partial-thickness anterior lamellar keratoplasty, full-thickness penetrating keratoplasty, or an artificial cornea such as the Boston keratoprosthesis, depending on the extent of the corneal damage.

Under optimal culture conditions, human keratinocytes generate holoclones, meroclones and paraclones. Holoclone-forming cells are the stem cells of almost all human squamous epithelia and are located in the limbus. The discovery that human limbal cell cultures contain holoclones resulted in the first therapeutic application of such cultures for the purpose of corneal epithelial regeneration. Although this technique is utilized in other parts of the globe, it is currently not available in the United States as a technique approved by the U.S. Food and Drug Administration. Hence, the other three surgical options may be considered, depending on the extent of limbal stem cell deficiency and whether the involvement is unilateral or bilateral.

In this column, Drs. Chan and Holland describe their surgical approach to limbal stem cell deficiency.

Thomas “TJ” John, MD
OSN Surgical Maneuvers Editor

Ocular surface reconstruction requires a systematic approach. The ophthalmologist should first reliably make the diagnosis of limbal stem cell deficiency (LSCD). Staging of disease severity needs to be considered. Control of glaucoma and optimizing the lids, tear film and conjunctival inflammation must occur before any limbal stem cell transplantation (LSCT) surgery.

Patients with LSCD may present with decreased vision, foreign body sensation or photophobia. Clinical findings include various combinations of conjunctivalization of the cornea with vascularization and pannus (Figures 1 and 2); corneal scarring or haze (Figure 2); a wave-like late fluorescein staining pattern (Figure 3); persistent or recurrent epithelial defects; and keratoplasty failure. Patients may have associated conjunctival disease: symblepharon, loss of the fornices, surface keratinization and mucin deficiency.

Figure 1.

Figure 1. Stage 2C ocular surface disease. Severe conjunctivalization of the cornea and symblephara in a patient 3 months after a base chemical ocular injury.

Images: Chan CC, Holland EJ

Figure 2.

Figure 2. Stage 2B ocular surface disease. Conjunctivalization, corneal scarring and stromal haze in a patient with congenital aniridia.

Figure 3.

Figure 3. Stage 2B ocular surface disease. Wave-like late fluorescein staining pattern in a patient with contact lens wear-related limbal stem cell deficiency.

The preoperative staging of ocular surface disease is important when deciding upon the type and timing of LSCT. Ocular factors include laterality, extent of LSCD and extent of conjunctival disease. Additional factors include extent of stromal scarring, mechanical eyelid problems, glaucoma and retinal disease. Non-ocular factors include age, systemic health and compliance ability. In cases of simple 
focal LSCD, only limbal reconstruction is required. In complicated cases, a step-by-step approach is taken.

Conjunctival limbal autograft surgery

Conjunctival limbal autograft (CLAU) surgery (Figure 4) is performed in partial or total unilateral LSCD. The fellow eye must be normal with no limbal stem cell problems. A 360° conjunctival peritomy is performed in the recipient eye, then superficial keratectomy of any corneal pannus. In the donor eye, 2 to 3 clock hours of limbus from superior and inferior locations and 2 mm to 3 mm of conjunctiva are marked. The conjunctiva is dissected toward the adherent portion at the limbus. Reflect the conjunctiva over the cornea and dissect with a crescent blade, a shallow depth of 1 mm onto the cornea, before amputation of the graft to ensure the stem cells are harvested. The grafts are stored in a sterile Petri dish with balanced salt solution. The donor sites are sutured with 8-0 Vicryl or tissue glue. When placing the donor tissue, interrupted 10-0 nylon sutures ensure that each CLAU graft is secure at the limbus. Tissue glue is used to secure the base of the graft. A bandage contact lens is placed. Topical steroids and antibiotics are used four times daily and tapered as the cornea re-epithelializes. Non-preserved artificial tears are used liberally.

Figure 4.

Figure 4. Schematic of CLAU or LR-CLAL surgery. Marking and harvesting of the conjunctival and limbal grafts of the donor tissue at 12 and 6 o’clock (a). Recipient site is prepared using a 360° peritomy and resection of the conjunctiva at 12 and 6 o’clock for placement of the donor tissue (b). Superficial keratectomy of the recipient corneal fibrovascular pannus (c). Securing of the grafts using tissue glue and 10-0 nylon sutures. A bandage contact lens is then placed on the eye (d).

Living-related conjunctival limbal allograft surgery

Living-related conjunctival limbal allograft (LR-CLAL) surgery (Figures 4 and 5) can be performed in cases of bilateral, partial or total LSCD. The donor tissue is usually from a sibling or parent. Donor and recipient immunologic matching is possible based on ABO blood type and human leukocyte antigens. Recipient and donor preparation, along with graft placement, is similar to that for CLAU.

Figure 5a.

Figure 5b.

Figure 5c.

Figure 5. Patient with contact lens wear-related limbal stem cell deficiency who underwent LR-CLAL surgery. Persistent epithelial defect and whorl-like irregular epithelium demonstrating a late staining pattern with fluorescein dye (a). Superior LR-CLAL tissue secured at the limbus with 10-0 nylon 1 month postoperatively (b). Smooth normal corneal epithelium and best corrected visual acuity of 20/30, 3 months after LR-CLAL surgery (c).

Keratolimbal allograft surgery

Keratolimbal allograft (KLAL) surgery (Figures 6 and 7) is for unilateral severe LSCD in which the fellow eye has risk for limbal disease or bilateral severe LSCD and there is no available living-related donor. The corneoscleral rims from both eyes of one donor are used for one recipient eye. The recipient eye is prepared in the same fashion as in CLAU or LR-CLAL surgery. The KLAL donor corneoscleral rim is trephined with a 7.5-mm blade as in routine penetrating keratoplasty. The rim is cut in half and trimmed, leaving 2 mm to 3 mm of sclera. The posterior sclera and corneal stroma of each segment are removed using lamellar dissection techniques with a crescent blade and curved Vannas scissors. When each graft is secured at the limbus with two 10-0 nylon sutures, the corneal edge then sits smoothly on the plane of the cornea, avoiding a step. The segment length can be trimmed to avoid overlap. Tissue glue secures the base of the grafts to recipient sclera and the posterior edge to the conjunctiva. Avoid gaps between the KLAL lenticules, where conjunctival invasion could occur. After subconjunctival antibiotics and steroid, a large bandage contact lens with a diameter of 18 mm to 20 mm is placed.

Combination surgery

In cases of severe bilateral conjunctival and limbal disease with significant symblepharon formation, LR-CLAL has been used in combination with KLAL at the 3 and 9 o’clock meridians to prevent conjunctival invasion. In unilateral cases, CLAU can be used in combination with KLAL lenticules. In these procedures, only one corneoscleral rim is needed (Figure 8).

Figure 6.

Figure 6. Schematic of KLAL surgery. Donor keratolimbal allograft lenticules are fashioned from two cadaver corneoscleral rims with the central 7.5 mm of cornea removed by trephination (a). A 360° conjunctival peritomy and tenectomy is performed, allowing the conjunctiva to retract (b). Abnormal corneal epithelium and fibrovascular pannus are removed by superficial dissection using blunt and sharp techniques, such as with a 64 Beaver blade (c). KLAL lenticules are secured to the recipient limbus using 10-0 nylon sutures and tissue glue (d).

Figure 7a.
Figure 7b.
Figure 7c.
Figure 7d.

Figure 7. Severe ocular surface disease with both limbal stem cell deficiency and conjunctival disease due to chemical and thermal injury (a). Visual acuity was hand motions. Glaucoma drainage device implantation was performed 3 months before ocular surface stem cell transplantation. Immediately after KLAL surgery (b). Stable ocular surface, diminished conjunctival inflammation and residual corneal stromal scarring 3 months after KLAL surgery. The glaucoma drainage device was positioned at 2 o’clock with a suture in situ (c). PK was performed 3 months after KLAL. Best corrected visual acuity measured 20/40 at 3 months after PK (d).

 

Figure 8.

Figure 8. Schematic of the Cincinnati or modified Cincinnati procedures in which both conjunctival-limbal grafts (autografts and living-related allografts, respectively) and keratolimbal allografts are used in order to provide enough limbal stem cells and the necessary conjunctiva for ocular surface rehabilitation in eyes with severe ocular surface disease. The conjunctival limbal autografts or living-related conjunctival limbal allografts are secured at 12 and 6 o’clock and KLAL tissue is secured at 3 and 9 o’clock.

Topical steroids and cyclosporine, and oral prednisone, tacrolimus and mycophenolate mofetil are necessary in all patients who receive any form of allografts to prevent rejection. For successful LSCT, multiple factors need to be optimized preoperatively. As ex vivo techniques are refined, they will be useful, especially for patients with bilateral disease. Until then, CLAU, LR-CLAL, KLAL and combination surgeries are viable options to rehabilitate patients with LSCD.

References:
Biber JM, et al. Cornea. 2011;doi:10.1097/ICO.0b013e318201467c.
Chan CC, et al. Cornea. 2012;doi:10.1097/ICO.0b013e31823f8e95.
Daya SM, et al. Cornea. 2011;doi:10.1097/ICO.0b013e318207f135.
Holland EJ, et al. Cornea. 1996;15(6):549-556.
Holland EJ, et al. Cornea. 2012;doi:10.1097/ICO.0b013e31823f8b0c.
Skeens HM, et al. Cornea. 2010;doi:10.1097/ICO.0b013e3181b6489e.
For more information:
Clara C. Chan, MD, FRCSC, FACS, can be reached at University of Toronto, Department of Ophthalmology and Vision Sciences, 340 College St., Suite 400, Toronto, ON M5T 3A9; 416-603-5401; fax: 416-603-1993; email: clarachanmd@gmail.com.
Edward J. Holland, MD, can be reached at Cincinnati Eye Institute, 580 South Loop Road, Edgewood, KY 41017; 859-331-9000; email: eholland@holprovision.com.
Thomas “TJ” John, MD, is a clinical associate professor at Loyola University at Chicago and is in private practice in Oak Brook, Tinley Park and Oak Lawn, Ill. He can be reached at 708-429-2223; fax: 708-429-2226; email: tjcornea@gmail.com.
Disclosure: Chan, Holland and John have no relevant financial disclosures.

Global bilateral blindness is estimated by the World Health Organization to be around 45 million people, of which more than 20% are secondary to corneal diseases. Of all the corneal disorders, limbal stem cell deficiency may be considered as one of the most severe and difficult-to-treat clinical entities.

Limbal stem cells are of significant importance with regard to corneal homeostasis. They form a somewhat circular garland on the ocular surface, sandwiched between the corneal and conjunctival epithelia. Human limbal stem cells can be detected both in vivo and in vitro by their expression of p63 transcription factor. The protein p63 retains the proliferative potential of limbal stem cells. These stem cells maintain corneal stromal avascularity and corneal epithelial integrity and thus play a vital role in the sustenance of a clear cornea, which is essential to vision. In the absence of a viable, healthy stem cell population, conjunctival progression to the corneal territory and vascular invasion of the cornea can result in a cloudy or opaque cornea and can potentially lead to corneal blindness. In cases of such corneal blindness, an allogenic corneal transplantation such as a penetrating keratoplasty can provide a clear cornea and restore vision only temporarily because conjunctival cell invasion onto the grafted cornea will lead to possible blindness. Hence, any attempt at vision restoration must first address the restoration of the limbal stem cells.

Limbal stem cell restoration may be approached from various routes. These include autologous culture of limbal cells as a source of transplant tissue; conjunctival limbal autograft; living-related conjunctival allograft; and keratolimbal allograft using a donor cornea from the eye bank. Once limbal stem cells have been established, then corneal opacity may be addressed in terms of partial-thickness anterior lamellar keratoplasty, full-thickness penetrating keratoplasty, or an artificial cornea such as the Boston keratoprosthesis, depending on the extent of the corneal damage.

Under optimal culture conditions, human keratinocytes generate holoclones, meroclones and paraclones. Holoclone-forming cells are the stem cells of almost all human squamous epithelia and are located in the limbus. The discovery that human limbal cell cultures contain holoclones resulted in the first therapeutic application of such cultures for the purpose of corneal epithelial regeneration. Although this technique is utilized in other parts of the globe, it is currently not available in the United States as a technique approved by the U.S. Food and Drug Administration. Hence, the other three surgical options may be considered, depending on the extent of limbal stem cell deficiency and whether the involvement is unilateral or bilateral.

In this column, Drs. Chan and Holland describe their surgical approach to limbal stem cell deficiency.

Thomas “TJ” John, MD
OSN Surgical Maneuvers Editor

Ocular surface reconstruction requires a systematic approach. The ophthalmologist should first reliably make the diagnosis of limbal stem cell deficiency (LSCD). Staging of disease severity needs to be considered. Control of glaucoma and optimizing the lids, tear film and conjunctival inflammation must occur before any limbal stem cell transplantation (LSCT) surgery.

Patients with LSCD may present with decreased vision, foreign body sensation or photophobia. Clinical findings include various combinations of conjunctivalization of the cornea with vascularization and pannus (Figures 1 and 2); corneal scarring or haze (Figure 2); a wave-like late fluorescein staining pattern (Figure 3); persistent or recurrent epithelial defects; and keratoplasty failure. Patients may have associated conjunctival disease: symblepharon, loss of the fornices, surface keratinization and mucin deficiency.

Figure 1.

Figure 1. Stage 2C ocular surface disease. Severe conjunctivalization of the cornea and symblephara in a patient 3 months after a base chemical ocular injury.

Images: Chan CC, Holland EJ

Figure 2.

Figure 2. Stage 2B ocular surface disease. Conjunctivalization, corneal scarring and stromal haze in a patient with congenital aniridia.

Figure 3.

Figure 3. Stage 2B ocular surface disease. Wave-like late fluorescein staining pattern in a patient with contact lens wear-related limbal stem cell deficiency.

PAGE BREAK

The preoperative staging of ocular surface disease is important when deciding upon the type and timing of LSCT. Ocular factors include laterality, extent of LSCD and extent of conjunctival disease. Additional factors include extent of stromal scarring, mechanical eyelid problems, glaucoma and retinal disease. Non-ocular factors include age, systemic health and compliance ability. In cases of simple 
focal LSCD, only limbal reconstruction is required. In complicated cases, a step-by-step approach is taken.

Conjunctival limbal autograft surgery

Conjunctival limbal autograft (CLAU) surgery (Figure 4) is performed in partial or total unilateral LSCD. The fellow eye must be normal with no limbal stem cell problems. A 360° conjunctival peritomy is performed in the recipient eye, then superficial keratectomy of any corneal pannus. In the donor eye, 2 to 3 clock hours of limbus from superior and inferior locations and 2 mm to 3 mm of conjunctiva are marked. The conjunctiva is dissected toward the adherent portion at the limbus. Reflect the conjunctiva over the cornea and dissect with a crescent blade, a shallow depth of 1 mm onto the cornea, before amputation of the graft to ensure the stem cells are harvested. The grafts are stored in a sterile Petri dish with balanced salt solution. The donor sites are sutured with 8-0 Vicryl or tissue glue. When placing the donor tissue, interrupted 10-0 nylon sutures ensure that each CLAU graft is secure at the limbus. Tissue glue is used to secure the base of the graft. A bandage contact lens is placed. Topical steroids and antibiotics are used four times daily and tapered as the cornea re-epithelializes. Non-preserved artificial tears are used liberally.

Figure 4.

Figure 4. Schematic of CLAU or LR-CLAL surgery. Marking and harvesting of the conjunctival and limbal grafts of the donor tissue at 12 and 6 o’clock (a). Recipient site is prepared using a 360° peritomy and resection of the conjunctiva at 12 and 6 o’clock for placement of the donor tissue (b). Superficial keratectomy of the recipient corneal fibrovascular pannus (c). Securing of the grafts using tissue glue and 10-0 nylon sutures. A bandage contact lens is then placed on the eye (d).

Living-related conjunctival limbal allograft surgery

Living-related conjunctival limbal allograft (LR-CLAL) surgery (Figures 4 and 5) can be performed in cases of bilateral, partial or total LSCD. The donor tissue is usually from a sibling or parent. Donor and recipient immunologic matching is possible based on ABO blood type and human leukocyte antigens. Recipient and donor preparation, along with graft placement, is similar to that for CLAU.

Figure 5a.

Figure 5b.

Figure 5c.

Figure 5. Patient with contact lens wear-related limbal stem cell deficiency who underwent LR-CLAL surgery. Persistent epithelial defect and whorl-like irregular epithelium demonstrating a late staining pattern with fluorescein dye (a). Superior LR-CLAL tissue secured at the limbus with 10-0 nylon 1 month postoperatively (b). Smooth normal corneal epithelium and best corrected visual acuity of 20/30, 3 months after LR-CLAL surgery (c).

PAGE BREAK

Keratolimbal allograft surgery

Keratolimbal allograft (KLAL) surgery (Figures 6 and 7) is for unilateral severe LSCD in which the fellow eye has risk for limbal disease or bilateral severe LSCD and there is no available living-related donor. The corneoscleral rims from both eyes of one donor are used for one recipient eye. The recipient eye is prepared in the same fashion as in CLAU or LR-CLAL surgery. The KLAL donor corneoscleral rim is trephined with a 7.5-mm blade as in routine penetrating keratoplasty. The rim is cut in half and trimmed, leaving 2 mm to 3 mm of sclera. The posterior sclera and corneal stroma of each segment are removed using lamellar dissection techniques with a crescent blade and curved Vannas scissors. When each graft is secured at the limbus with two 10-0 nylon sutures, the corneal edge then sits smoothly on the plane of the cornea, avoiding a step. The segment length can be trimmed to avoid overlap. Tissue glue secures the base of the grafts to recipient sclera and the posterior edge to the conjunctiva. Avoid gaps between the KLAL lenticules, where conjunctival invasion could occur. After subconjunctival antibiotics and steroid, a large bandage contact lens with a diameter of 18 mm to 20 mm is placed.

Combination surgery

In cases of severe bilateral conjunctival and limbal disease with significant symblepharon formation, LR-CLAL has been used in combination with KLAL at the 3 and 9 o’clock meridians to prevent conjunctival invasion. In unilateral cases, CLAU can be used in combination with KLAL lenticules. In these procedures, only one corneoscleral rim is needed (Figure 8).

Figure 6.

Figure 6. Schematic of KLAL surgery. Donor keratolimbal allograft lenticules are fashioned from two cadaver corneoscleral rims with the central 7.5 mm of cornea removed by trephination (a). A 360° conjunctival peritomy and tenectomy is performed, allowing the conjunctiva to retract (b). Abnormal corneal epithelium and fibrovascular pannus are removed by superficial dissection using blunt and sharp techniques, such as with a 64 Beaver blade (c). KLAL lenticules are secured to the recipient limbus using 10-0 nylon sutures and tissue glue (d).

Figure 7a.
Figure 7b.
Figure 7c.
Figure 7d.

Figure 7. Severe ocular surface disease with both limbal stem cell deficiency and conjunctival disease due to chemical and thermal injury (a). Visual acuity was hand motions. Glaucoma drainage device implantation was performed 3 months before ocular surface stem cell transplantation. Immediately after KLAL surgery (b). Stable ocular surface, diminished conjunctival inflammation and residual corneal stromal scarring 3 months after KLAL surgery. The glaucoma drainage device was positioned at 2 o’clock with a suture in situ (c). PK was performed 3 months after KLAL. Best corrected visual acuity measured 20/40 at 3 months after PK (d).

 

Figure 8.

Figure 8. Schematic of the Cincinnati or modified Cincinnati procedures in which both conjunctival-limbal grafts (autografts and living-related allografts, respectively) and keratolimbal allografts are used in order to provide enough limbal stem cells and the necessary conjunctiva for ocular surface rehabilitation in eyes with severe ocular surface disease. The conjunctival limbal autografts or living-related conjunctival limbal allografts are secured at 12 and 6 o’clock and KLAL tissue is secured at 3 and 9 o’clock.

Topical steroids and cyclosporine, and oral prednisone, tacrolimus and mycophenolate mofetil are necessary in all patients who receive any form of allografts to prevent rejection. For successful LSCT, multiple factors need to be optimized preoperatively. As ex vivo techniques are refined, they will be useful, especially for patients with bilateral disease. Until then, CLAU, LR-CLAL, KLAL and combination surgeries are viable options to rehabilitate patients with LSCD.

References:
Biber JM, et al. Cornea. 2011;doi:10.1097/ICO.0b013e318201467c.
Chan CC, et al. Cornea. 2012;doi:10.1097/ICO.0b013e31823f8e95.
Daya SM, et al. Cornea. 2011;doi:10.1097/ICO.0b013e318207f135.
Holland EJ, et al. Cornea. 1996;15(6):549-556.
Holland EJ, et al. Cornea. 2012;doi:10.1097/ICO.0b013e31823f8b0c.
Skeens HM, et al. Cornea. 2010;doi:10.1097/ICO.0b013e3181b6489e.
For more information:
Clara C. Chan, MD, FRCSC, FACS, can be reached at University of Toronto, Department of Ophthalmology and Vision Sciences, 340 College St., Suite 400, Toronto, ON M5T 3A9; 416-603-5401; fax: 416-603-1993; email: clarachanmd@gmail.com.
Edward J. Holland, MD, can be reached at Cincinnati Eye Institute, 580 South Loop Road, Edgewood, KY 41017; 859-331-9000; email: eholland@holprovision.com.
Thomas “TJ” John, MD, is a clinical associate professor at Loyola University at Chicago and is in private practice in Oak Brook, Tinley Park and Oak Lawn, Ill. He can be reached at 708-429-2223; fax: 708-429-2226; email: tjcornea@gmail.com.
Disclosure: Chan, Holland and John have no relevant financial disclosures.