Adriano Fasolo

Analysis of limbal stem cell deficiency by corneal impression cytology

Purpose. The impaired function of corneal epithelial stem cells, located in the limbus, is responsible for corneal surface damage and is clinically characterized by recurrent epithelial defects, conjunctivalization, neovascularization, and corneal opacity. The aim of this study was to investigate corneal limbal stem cell deficiency (LSCD) by means of the impression cytology (IC) technique, using antibodies against cytokeratin 19 (CK19) and cytokeratin 3 (CK3), and to evaluate the diagnostic potential of this approach. Methods. Over a 3-year period (October 1998–June 2001), we collected 113 pairs of IC samples from the eyes of 85 patients with a range of ocular surface diseases and performed an immunocytochemical analysis of CK19 and CK3. Samples with more than 50% cellularity were considered suitable for diagnostic purposes, while samples with less than 50% cellularity were considered with caution. CK19-positive cells in corneal IC were considered an expression of LSCD. We arbitrarily scored LSCD as mild (<25% of CK19-positive cells), moderate (25–50%), and severe (>50%). Results. One hundred thirteen pairs of IC specimens were obtained from 85 patients; 32 patients (37.6%) had alkaline burns, 18 (21.2%) had other chemical or physical corneal injuries, 13 (15.3%) had complications from wearing contact lenses, 8 (9.4%) had severe microbial keratitis, and 14 (16.5%) had suspicious limbal deficit due to other causes. Nine patients underwent bilateral sampling and 12 had to be resampled. Thirteen pairs of IC specimens were obtained during the follow-up of 8 patients who had undergone limbal stem cell transplantation. In 3 of these patients, IC confirmed reversion to corneal immunophenotype (CK3+/CK19−), whereas in 4, residual limbal damage was still evident; 1 patient relapsed. In the remaining 100 pairs of impressions, we found 77 cases of LSCD, whereas in 16 pairs, we did not find LSCD. Seven pairs were defined as “not valuable” because of the poor quality of both CK samples. Diffuse LSCD, moderate or severe in degree, was found in 26 of 32 patients (81.2%) with alkali burns, whereas mild diffuse LSCD or sectoral LSCD was found in 13 of 18 patients (72.2%) with other chemical-physical injuries, in 10 of 13 patients (76.9%) wearing contact lenses, in 7 of 8 patients (87.5%) with severe microbial keratitis, and in 12 of 14 patients (85.7%) with other corneal pathologies. The quality of impressions was assessed in 77 cases and found to be good or discrete for both CKs in 32 cases (41.5%) and poor in 45 (58.5%): in 46.7% of these cases, the IC was poor only for CK19 and in 45.4% only for CK3. Conclusions. Immunocytochemistry for seeking out CK19- and CK3-positive cells on corneal IC is a simple and practical method to investigate LSCD. We believe that this technique could have an important role in evaluating patients undergoing therapeutic penetrating keratoplasty to select those who would benefit from limbal stem cell transplantation. Since sampling has been shown to be a critical point, we believe that any improvement in this area will also help to improve the methodology and will contribute to its wider utilization.

The CORTES study: Corneal transplant indications and graft survival in an italian cohort of patients

Purpose: To describe the corneal transplantation activity in Italy, to assess the long-term graft survival, and to begin to outline the potential risk factors for graft outcome. Methods: We followed a consecutive series of penetrating (PK) and lamellar (LK) keratoplasties performed with corneas procured and distributed by the Veneto Eye Bank Foundation, which provides about one third of the corneas grafted in Italy each year. Results: Data on 4415 PKs and 489 LKs performed in 174 clinical centers are reported. Keratoconus was the major transplant indication (47% and 66%, respectively, for the 2 groups), followed by regraft (14%) and bullous keratopathy (14%) in the PK group and keratitis (8%) and refractive reasons (4%) for the LKs. In the 2 groups, graft survival, after 1 year, was estimated to be 95% and 93%, respectively, showing a decrease of the survival rate during the second and third years of the study. Graft survival in patients with keratoconus indication was 98% in the PK group and 95% in the LK group for the whole period of observation, whereas the patients with other indications reported a survival rate ranging from 92% after 1 year to 52% after 3 years (PK) and from 89% to 85% (LK). Conclusions: CORTES is the most extensive survey on corneal transplantation in Italy that involves a large cohort of patients and a significant number of surgeons with corneal tissues processed and distributed by a single eye bank. In the first 3 years, a picture of the epidemiology of the corneal transplant has been defined. The graft survival rates were comparable to those reported by other studies for the same follow-up period. However, the follow-up of a sample of this cohort for a further 3 years will allow us to precisely estimate the long-term graft survival and to better evaluate the risk factors related to graft failure.

Evaluation of ocular surface disorders: a new diagnostic tool based on impression cytology and confocal laser scanning microscopy

Aim To provide a new tool for the evaluation of altered ocular surfaces by using a combination of impression cytology, laser scanning confocal microscopy and advanced image analysis. Methods The expression of keratin 3 (K3), keratin 12 (K12), keratin 19 (K19) and mucin 1 (MUC1) was analysed by immunofluorescence on both histological sections of nine corneoscleral buttons from normal donors comprising conjunctiva, limbus and cornea and impression cytology specimens from six healthy normal subjects (12 eyes) and 12 patients with chronic ocular surface disorders. Levels of fluorescence expression of the different markers were quantified through quantitative fluorescence immunohistochemistry (Q-FIHC). Results Impression cytology specimens from normal and diseased ocular surfaces showed distinct expression patterns for K12 and MUC1. Healthy corneas expressed only K12 (but not MUC1), while conjunctivalised corneas from patients with limbal stem cell deficiency (LSCD) were characterised by the presence of MUC1 and the disappearance of K12. Similar clear-cut results were not seen with the K3/K19 markers, which showed lack of specificity and overlapping signals in cornea and conjunctiva impression cytology specimens. Conclusions The ability of K12 and of the antibody against MUC1 to discriminate clearly between limbus/cornea and conjunctiva in impression cytology specimens could become a valuable diagnostic tool for ophthalmologists in order to evaluate alterations of the ocular surface and the grading of LSCD.

Long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns.

Background:  Chemical burns cause depletion of limbal stem cells and eventually lead to corneal opacity and visual loss. We investigated the long‐term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency.

Localization and expression of CHST6 and keratan sulfate proteoglycans in the human cornea

Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder caused by mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence of unsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visual impairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressed and exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of the KSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in cultured primary keratinocytes (n = 3) and keratocytes (n = 4). Immunohistochemistry, semiquantitative RT-PCR, in situ RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneas KS was predominantly found in the stroma, but absent, or barely detectable, in the corneal epithelium. A similar pattern of distribution was found in the epidermis, with KS mainly localised in the derma. As expected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues onto KSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and in the endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytes express and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarly expression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromal keratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and express very little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare genetic disorder must therefore involve and target stromal keratocytes. If coupled to the accuracy of HPLC-based assay that we developed to determine the amount of KS in serum, our findings could lead to more targeted therapeutic treatments of the ocular features in MCD patients.

Sixty-kilohertz femtosecond laser-assisted endothelial keratoplasty: clinical results and stromal bed quality evaluation.

Purpose: To evaluate preliminary clinical results and lamellar and side cut obtained with a 60-kHz femtosecond laser (IntraLase) using a new software designed to create penetrating cuts for keratoplasty to perform the donor and recipient dissection in femtosecond laser-assisted endothelial keratoplasty. Design: Prospective interventional case series. Methods: A femtosecond laser set in a raster spot pattern was used to create the posterior lamellar dissection and posterior side cut in 4 donor corneas and in 4 recipient human eyes of 4 patients with endothelial decompensation. The laser cut was performed to obtain a 150-μm thick and 9-mm wide posterior lamellar disk from the donor and the recipient eyes. Postoperatively, patients underwent slit-lamp examination and Snellen visual acuity evaluation, Visante optical coherence tomography, optical pachymetry, and endothelial microscopy analysis. Three residual donor corneas were analyzed by scanning electron microscopy and transmission electron microscopy to observe the stromal bed surface, the side cut, and the effects of the laser dissection on the cornea ultrastructure. Main Outcome Measures: Thickness of the implanted donor lamella, adhesion and clarity of the transplanted cornea in the postoperative follow-up, and smoothness of the interface. Results: All the patients showed good adhesion and fit of the donor disk to the recipient and a circle side cut observed at the slit-lamp examination. Visante optical coherence tomography evaluation confirmed the adhesion and the precalibrated thickness of the implanted lamella. A good corneal transparency was achieved within the first 2-3 weeks. The scanning electron microscopy analysis showed a smooth stromal residual surface and a precise side cut. The transmission electron microscopy images showed that the laser randomly modified the structural design of the collagen fibers for approximately 10-μm depth, although below a regular stromal structure was maintained. Conclusions: The 60-kHz IntraLase femtosecond laser creates a smooth stromal bed and precise side cut for endothelial keratoplasty. The posterior lamellar cut performed at the same depth in both the donor and the recipient makes it possible to create a corneal disk from the donor that fits exactly with the cut in the recipient.

Improved Preparation and Preservation of Human Keratoplasty Lenticules

Purpose: To improve the preparation of lenticules from human cornea and to obtain their preservation without loss of viable keratocytes. Methods: The epithelium was manually removed after bathing the surface of the cornea with a solution of trypsin and EDTA. Lenticules were prepared by microkeratome resection and viable keratocytes were visualized by staining with thiazolyl blue (MTT). Results: The pretreatment with trypsin-EDTA allowed the removal of the epithelium without damage to the keratocytes and the stroma. When these lenticules were incubated in Optisol-GS for 7 days at 4°C, they showed a limited thickness increase and a preservation of keratocyte viability. Conclusion: This procedure allows the preparation of lenticules with viable keratocytes that can be preserved in the cold for at least 1 week.

In Vivo Confocal Microscopy 1 Year after Autologous Cultured Limbal Stem Cell Grafts

PURPOSE To correlate clinical, impression cytologic, and in vivo confocal microscopy findings on the corneal surface after cultured limbal stem cell transplantation. DESIGN Prospective, interventional, noncomparative, masked case series. PARTICIPANTS Thirteen patients with limbal stem cell deficiency after unilateral (9 eyes) or bilateral (2 eyes) chemical burn, liquid nitrogen injury (1 eye), or herpes simplex virus infection (1 eye). METHODS Limbal cells were harvested from healthy or less affected eyes, cultured on 3T3 cells and fibrin glue, and transplanted to the patients injured eye. Patients underwent clinical examination and impression cytologic examination of the central cornea before and 1 year after intervention. In vivo confocal microscopy scans were obtained in all corneal quadrants after 1 year. The interexamination agreement was established by calculation of the Cohens κ coefficient. MAIN OUTCOME MEASURES Results of surgery were assessed considering clinical signs (successful: restoration of transparent, avascular, and stable corneal epithelium without neovascularization in central corneal surface; partially successful: recurrence of superficial neovascularization; failed: recurrent epithelial defects, pannus, and inflammation), phenotype of cells covering the corneal surface (conjunctivalized corneal surface: cytokeratin 12 [cK12]-negative and mucin 1 [MUC1]-positive cells; mixed epithelium: cK12-positive and MUC1-positive cells; corneal epithelium: cK12-positive and MUC1-negative cells), and cell morphologic features (corneal epithelium: multilayered polygonal and flat cells with hyperreflective nuclei; conjunctival epithelium: stratified cuboidal or polygonal cells, hyperreflective cytoplasm, and barely defined borders; epithelial transition: transition of epithelial cells from the cornea to the conjunctiva over the corneal surface). RESULTS We found a moderate to substantial degree of concordance between confocal microscopy and clinical evaluation (κ = 0.768) and between confocal microscopy and impression cytologic analysis (κ = 0.629). Confocal microscopy showed that 46.2% of patients exhibited corneal epithelium in the central and peripheral cornea, 30.8% showed an irregular mixed corneal and conjunctival epithelium, and 23.0% showed conjunctival epithelium. Palisades of Vogt were absent in all (100.0%) patients, and the cornea-conjunctiva epithelial transition localized approximately 1 mm internally on the cornea. CONCLUSIONS Confocal microscopy provides objective measures of the corneal epithelium and may significantly improve the evaluation of outcomes after cultured limbal stem cell graft.

Safety outcomes and long-term effectiveness of ex vivo autologous cultured limbal epithelial transplantation for limbal stem cell deficiency

Purpose To evaluate the safety and effectiveness of ex vivo autologous cultured limbal stem cell transplantation (CLET). Methods We reviewed the clinical records of 59 consecutive patients treated with 65 CLETs. Efficacy was graded 1 year after surgery as successful, partially successful or failed. A safety analysis was performed considering side effects and complications that were recorded during the first year after CLET and those reported later than 1 year, including the events related to subsequent treatments. Results The mean post-CLET follow-up was 6.0±4.1 years. 69% of CLETs had either one or more adverse events (AEs), or adverse drug reactions (ADRs), within 1 year of surgery, with inflammation being the most common (42%), followed by corneal epithelium defects/disepithelialisation (31%), and blood coagula under the fibrin (24%). One year after surgery, 41% of the 59 primary CLET procedures were successful, 39% partially successful and 20% failed. The most common ADRs recorded for the primary unsuccessful CLETs were ulcerative keratitis, melting/perforation, and epithelial defects/disepithelialisation. Six failed CLETs required reconstructive penetrating keratoplasty (PK). Among CLETs with a favourable outcome, 13 underwent corrective PK (mean 4.8±3.4 years), and thereafter seven eyes maintained integrity of the corneal epithelium, five showed corneal surface failure, and one had recurrent epithelial defects. Corneal graft rejection episodes were reported in 71% and 58% of patients following corrective or reconstructive PK, respectively. Seven primary CLETs with a favourable outcome worsened thereafter, and the overall 3-year long-term effectiveness was 68%. Conclusions This study addresses important issues regarding possible risks associated with disarray of the ocular surface homeostasis following autologous CLET in patients with limbal stem cell deficiency, despite the fact that the majority of patients experienced a favourable long-term benefit.

Advances in corneal surgery and cell therapy: challenges and perspectives for eye banks

Recent advances in the fields of eye banking, ophthalmology and regenerative medicine are challenging the traditional activities of eye banks. Serum-free media are being evaluated for corneal storage at 30.5–37°C (organ culture) or 2–6°C (cold storage). Autologous serum eye drops and amniotic membrane transplantation are being offered as complementary or alternative remedies for ocular surface disorders. While penetrating keratoplasty remains the most common procedure for cornea transplantation, advances in corneal surgery have led to the development of lamellar keratoplasty as a way to replace only the anterior stroma or the posterior stromal and endothelial layers. Progress in femtosecond lasers and microkeratome-based techniques in creating pre-cut endothelial donor tissues are at the basis of the success of lamellar procedures. Autologous-cultured limbal stem cells have been used to restore damaged corneal surfaces in hundreds of patients with limbal stem cell deficiency. Strategies aimed at the regeneration of conjunctival epithelium and corneal endothelium through cell therapy and genetic engineering of corneal stem cells are also under evaluation. Many of these developments might eventually lead to new therapeutic services and treatments offered by eye banks to patients with ocular surface diseases.