Virender S. Sangwan

Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study

Purpose Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency. Methods This retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10–14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patients affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed. Results A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3±1.6 (range: 1–7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy. Conclusions Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.

Simple limbal epithelial transplantation (SLET): a novel surgical technique for the treatment of unilateral limbal stem cell deficiency

This study describes a novel surgical technique of limbal transplantation, which combines the benefits of existing techniques while avoiding their difficulties. Six patients with unilateral and total limbal stem cell deficiency following ocular surface burns underwent a single-stage procedure. A 2×2 mm strip of donor limbal tissue was obtained from the healthy eye and divided into eight to ten small pieces. After surgical preparation of the recipient ocular surface, these tiny limbal transplants were distributed evenly over an amniotic membrane placed on the cornea. After surgery, a completely epithelialised, avascular and stable corneal surface was seen in all recipient eyes by 6 weeks, and this was maintained at a mean±SD follow-up of 9.2±1.9 months. Visual acuity improved from worse than 20/200 in all recipient eyes before surgery to 20/60 or better in four (66.6%) eyes, while none of the donor eyes developed any complications. This technique requires less donor tissue than previously used for conventional autografting and does not need a specialist laboratory for cell expansion. Although long-term results are awaited, this simple limbal epithelial transplantation promises to be an easy and effective technique for treating unilateral limbal stem cell deficiency following ocular burns.

International Results with the Boston Type I Keratoprosthesis

PURPOSE To determine the indications and outcomes of Boston type 1 keratoprosthesis (Massachusetts Eye and Ear Infirmary, Boston, MA) surgery performed outside of North America and to compare them with those obtained in the United States by the surgeon who trained the international surgeons. DESIGN Retrospective review of consecutive clinical case series. PARTICIPANTS One hundred ninety-four patients (223 keratoprosthesis procedures performed in 205 eyes) who received Boston type 1 keratoprosthesis at 11 ophthalmology centers in Armenia, India, Indonesia, Nepal, Philippines, Russia, and Saudi Arabia between May 1, 2006, and July 1, 2011 (international series), and at the Jules Stein Eye Institute between May 1, 2004, and July 1, 2011 (University of California, Los Angeles [UCLA] series). METHODS Data were collected for each procedure regarding the preoperative characteristics of each eye, the surgical procedure(s) performed, and the postoperative outcomes. Statistical analysis was performed to identify significant differences between the international and UCLA series in terms of retention and complications. MAIN OUTCOME MEASURES Interval visual acuities, keratoprosthesis retention, and significant postoperative complications. RESULTS In the international series, 113 Boston type I keratoprostheses were implanted in 107 eyes of 100 patients. The most common indication for surgery was corneal graft failure (n = 50; 44%) followed by chemical injury (n = 30; 27%). Although only 2% of eyes had a preoperative corrected distance visual acuity (CDVA) of 20/20 to 20/200, 70%, 68%, and 59% of eyes had a postoperative CDVA of 20/20 to 20/200 at 6 months, 1 year, and 2 years after surgery, respectively. Ninety-one of the 113 keratoprostheses implanted (80.5%) were retained at a mean follow-up of 14.2 months, for a retention failure rate of 22 per 134.6 eye-years (0.163/eye-year). The most common postoperative complications were retroprosthetic membrane formation (27%) and sterile corneal necrosis (18%). The only postoperative complication that was more common in the international series than in the UCLA series was infectious endophthalmitis, which developed in 9% of eyes. CONCLUSIONS Boston keratoprosthesis is a viable means of managing repeat graft failure and ocular chemical injury outside of North America, with similar visual acuity outcomes, retention rates, and incidence rates of postoperative complications to those obtained by North American surgeons.

Amniotic membrane transplantation for ocular surface reconstruction

The amniotic membrane, composed of 3 layers, the epithelium, basement membrane, and the stroma, was first used along with the chorion as a biologic membrane to promote healing of skin burns in 1910. In ophthalmology, it was used in 1940 in the management of conjunctival defects. Its revival in the 1990s was due to its ability to reduce ocular surface inflammation and scarring, promote rapid epithelialization due to the presence of growth factors, and antimicrobial properties. This has resulted in its application in several ocular disorders. A review of the literature shows that amniotic membrane is definitely beneficial in some but not all pathology. The future of amniotic membrane transplantation is very exciting, especially in the field of limbal stem cell research. However, further work is needed to elucidate whether it functions merely as a biologic contact lens or whether it has additional benefits.

Amniotic membrane transplantation for ocular surface reconstruction in stevens-johnson syndrome

PURPOSE To evaluate amniotic membrane transplantation (AMT) for ocular surface reconstruction in Stevens-Johnson syndrome (SJS). DESIGN Prospective interventional case series. PARTICIPANTS Ten consecutive patients (10 eyes) with SJS that underwent AMT as the first step in staged ocular surface reconstruction were included. METHODS Amniotic membrane was processed under sterile conditions from a fresh placenta obtained from cesarean section in a seronegative pregnant woman and stored at -700 degrees C. Symblepharon release, excision of epibulbar fibrous tissue, and clearing of the fibrovascular membrane over the cornea was performed in all cases. Amniotic membrane covered the entire bulbar surface up to the fornices in five eyes; cornea and the perilimbal area in two eyes; cornea, the inferior bulbar surface, and the lower fornix in two eyes; and cornea and the superior bulbar surface in one eye. Obliterated fornices were deepened by use of fornix-formation sutures in all eyes. Symblepharon ring was placed postoperatively for 3 weeks to 2 months. Mean postoperative follow-up was 13.5 months (SD, +3.8 months; range, 9-30 months). MAIN OUTCOME MEASURES Restoration of adequate bulbar surface free of symblepharon and good fornix depth were the main outcome measures. RESULTS Complete corneal reepithelization occurred in all eyes between 1 and 6 weeks. Adequate bulbar surface and fornix depth were achieved in nine eyes, all of which were free of symblepharon at the final follow-up visit. Cicatricial entropion resolved in four of five lower eyelids and one of two upper eyelids after AMT. One patient had a central corneal melt that required or necessitated a penetrating keratoplasty. CONCLUSIONS AMT restores adequate bulbar surface and fornix depth and prevents recurrence of symblepharon in severe cases of SJS.

Successful Reconstruction of Damaged Ocular Outer Surface in Humans Using Limbal and Conjuctival Stem Cell Culture Methods

When the ocular outer surface is badly damaged, subsequent corneal transplantation fails due to the absence of basal cells that are needed to support the graft. With the realization that the limbus and the conjunctiva have adult stem cells that can be cultured, it has been possible for us to explant culture these on de-epithelized human amniotic membrane, and to graft the resulting viable and transparent epithelium to 125 needy human patients with success. Ultrastructural, histological, biochemical and immunological assays establish the identity of the cells and the tissue formed.

Amniotic membrane transplantation in acute chemical and thermal injury

PURPOSE To present a case of chemical injury and a case of thermal injury treated by amniotic membrane transplantation in acute phase. METHODS Case reports. An eye with sodium hydroxide injury, opaque cornea, and limbal ischemia of more than 180 degrees and an eye with hot tea injury, opaque cornea, stromal edema, and scarring were treated by amniotic membrane transplantation within the first few weeks of injury. RESULTS In the eye with sodium hydroxide injury, 4 months after amniotic membrane transplantation, the ocular surface is stable, superficial corneal scarring with vascularization is present, and visual acuity is 20/25. In the eye with thermal injury, 6 months after amniotic membrane transplantation, the ocular surface is stable, but there is superficial scarring and vascularization, and visual acuity is 20/20. CONCLUSIONS Amniotic membrane transplantation can be considered in chemical injury with severe limbal ischemia and in severe thermal injury in acute phase. Long-term studies are warranted to evaluate further the efficacy of amniotic membrane transplantation in these clinical situations.

Use of Autologous Cultured Limbal and Conjunctival Epithelium in a Patient with Severe Bilateral Ocular Surface Disease Induced by Acid Injury: A Case Report of Unique Application

Purpose. Reconstruction of the ocular surface in a case of severe bilateral partial limbal stem cell deficiency (LSCD) with extensive symblephara using autologous cultured conjunctival and limbal epithelium. Case report. A 31-year-old woman presented with severe bilateral ocular surface disease with partial limbal stem cell deficiency, symblephara, lid and facial scarring, with a vision of 20/400 and counting fingers at 1 m in both eyes. Limbal and conjunctival tissue was harvested from the healthy-appearing left eye and used to generate two sheets of composite epithelium consisting of central limbal and peripheral conjunctival cells. The limbal tissues were explanted in the central region while the conjunctival tissues were explanted on the periphery of the deepithelialized human amniotic membrane (HAM) and nurtured using human corneal epithelial cell medium. After successful generation of a monolayer from both tissues had been confirmed, the composite of cultivated limbal and conjunctival epithelium with HAM was transplanted in each eye after excision of fibrous tissue and release of symblephara. One year postoperatively, the patient had a best spectacle-corrected visual acuity of 20/40 in the right eye (preoperative acuity 20/400) and counting fingers at 1 m in the left eye (same as preoperative) with a stable ocular surface. Conclusions. Autologous cultured epithelial transplantation is as an excellent option in selected patients with bilateral partial LSCD with small area(s) of healthy limbus in either eye and avoids the attendant risk of rejection and cost and potential toxicity of immunosuppression in allogeneic tissue transplantation. This case also highlights the feasibility of generating a composite culture of limbal and conjunctival epithelium using a single amniotic membrane.

Role of Confocal Microscopy in the Diagnosis of Fungal and Acanthamoeba Keratitis

PURPOSE To investigate the role of confocal microscopy as a diagnostic modality in microbial keratitis and to determine inter- and intraobserver variation in the analysis and interpretation of confocal microscopy findings. DESIGN Prospective, double masked, nonrandomized, observational clinical trial. PARTICIPANTS We included 146 consecutive patients with clinically suspected microbial keratitis. METHODS Confocal microscopy and microbiology evaluation of study participants. MAIN OUTCOME MEASURES Sensitivity, specificity, and positive and negative predictive values of confocal microscopy in diagnosing fungal and Acanthamoeba keratitis compared with microbiologic evaluation, as well as the intra- and interobserver variation in interpretation of confocal scans. RESULTS We included 148 cases of infiltrative keratitis. Of the 103 microbiologically proven cases of Acanthamoeba or fungal keratitis, the confocal microscope was able to identify fungal filaments or Acanthamoeba cysts in 91 cases with either fungal or Acanthamoeba keratitis with a sensitivity of 88.3% (95% confidence interval [CI], 82.2-94.5) and a specificity of 91.1% (95% CI, 82.8-99.4). The interobserver agreement in interpreting the scans was good (kappa = 0.6; phi = 0.617). The intraobserver agreement was kappa = 0.795 and phi = 0.807. CONCLUSIONS The confocal microscope seems to be an accurate and reliable diagnostic modality in the etiologic diagnosis of fungal and Acanthamoeba keratitis.

In vitro culture and expansion of human limbal epithelial cells

Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes ∼2 weeks to establish a confluent monolayer from which ∼3 × 106 cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.