Objectives: We evaluated limbal stem cell transplant success in limbal stem cell deficiency due to chemical injury at a tertiary eye care center in Turkey with a novel system for describing limbal stem cell deficiency, as developed by the Limbal Stem Cell Working Group.
Materials and Methods: Medical records of 80 eyes of 80 patients after limbal stem cell transplant for limbal stem cell deficiency secondary to chemical injury were included, with patients grouped according to surgical procedure, ie, limbal autograft, limbal allograft, and cultivated limbal epithelial cell transplant. Surgical success was defined as improvement in postoperative year 1 of limbal stem cell deficiency stage.
Results: Patients’ mean age was 37.9 ± 15.7 years (range, 4-71 years). Male/female ratio was 2.4. Forty-five patients (56.3%) were injured with alkaline substance, and 16 (20%) with acid substance. Mean follow-up time was 60.3 ± 30.6 months (range, 6-118.6 months). Limbal autograft, allograft, and cultivated limbal epithelial cell transplants were performed in 58 (72.5%), 12 (15%), and 10 (12.5%) eyes, respectively. Intervals between injury and surgery in limbal autograft, limbal allograft, and cultivated limbal epithelial cell transplants were 43.3 ± 94.1 months (range, 0.5-592 months), 14.5 ± 10.6 months (range, 2.4-32.5 months), and 122.8 ± 158.9 months (range, 21.1-504 months), respectively (P = .02); and surgical success rates in each group were 65.5%, 41.7%, and 90%, respectively (P = .03). Overall surgical success rate was 65%.
Conclusions: Accurate determination of the limbal stem cell deficiency stage is crucial for proper evaluation of surgical success. Surgery type and interval between injury and surgery were the most important factors associated with higher surgical success rates. Despite the limited number of patients in the subgroups, the results were remarkable to emphasize the significance of a novel limbal stem cell deficiency scoring system.
Key words : Limbal stem cell deficiency, Ocular surface chemical injury
Chemical injury of the ocular surface is a critical ocular emergency that requires urgent and intensive management.1 The Dua classification system for grading the severity of ocular chemical injury may predict the visual prognosis.2,3
Severe ocular chemical injury may cause limbal stem cell deficiency (LSCD) that results from the dysfunction of limbal stem cells and impairment of the limbal barrier. Limbal stem cell deficiency secondary to chemical injury is usually unilateral and may either be total or partial.4-6 The accurate evaluation of LSCD is crucial for the choice of appropriate management of the disease. Although medical treatment may be useful in mild LSCD, replacement of the limbal stem cells via graft or stem cell transplant is essential for treatment of severe LSCD.7-11 There was no consensus regarding a descriptive scale for LSCD until 2019, when a new system for evaluation of LSCD severity was defined by the Limbal Stem Cell Working Group.9
Overall surgical success rates for limbal stem cell transplant (LSCT) vary from 50% to 80% according to the tissue source. In addition to the tissue source selected for LSCT, there are several other factors, such as surgery type, LSCD stage, and the duration between the chemical injury and surgical intervention, that may affect the success rates.10-12
Objective criteria are vital for the accurate evaluation of the surgical success after LSCT.13,14 The aim of this study was to use the Limbal Stem Cell Working Group LSCD staging system to evaluate LSCT outcomes for treatment of LSCD due to chemical injury in patients at a tertiary eye care center in Turkey.
Materials and Methods
This retrospective study was conducted with the approval of the Ethics Committee of Ege University and in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants.
The study comprised patients admitted to Ege University Department of Ophthalmology from 2007 to 2019 with LSCD secondary to ocular chemical injury who underwent LSCT. Medical records of patients were reviewed, and details were recorded, including age, sex, type of chemical agent, the associated ocular injuries (eg, eyelid malformation, symblepharon), the time interval between injury and LSCT, and transplant methods. Preoperative and postoperative (month 6, year 1, year 3 follow-ups) best-corrected visual acuity (BCVA) measurements (Snellen eye chart) and intraocular pressure measurements were documented. Anterior segment photographs of the patients’ eyes were analyzed by 2 different researchers, and chemical injury grades and LSCD stages were recorded. We included patients with grade 3 and higher chemical injury according to the Dua classification2 system. Preoperative and postoperative LSCD stages (month 6, year 1, and year 3) were categorized according to the system established by the Limbal Stem Cell Working Group,9 for which the baseline (stage 1) is defined as normal corneal epithelium within the central 5-mm zone of the cornea. Stage 2 is defined to include the involvement of the limbus in addition to the central 5-mm zone of the cornea. Stage 3 comprises the involvement of the entire corneal surface. Additionally, according to the degree of limbal involvement. Stage 1 is divided into subgroups A, B, and C and Stage 2 is divided into subgroups A and B. In the present study, we included medical records of patients with stage 1B and/or higher LSCD stages at preoperative examination.
Preoperative and postoperative medical treatment regimens were standard for all patients in different transplant groups. In the acute phase of the treatment (0-7 days), topical antibiotics, steroids, and pre-servative-free artificial tears eyedrops were initiated after copious irrigation. In severe cases, systemic doxycycline and vitamin C were also added. Topical antibiotic was ceased when the epithelial defect healed. Topical steroid was ceased at week 1 of the incident, restarted at week 3, and was reduced according to ocular surface inflammation.
The LSCT procedures comprised limbal autograft, living relative limbal allograft, and cultivated limbal epithelial transplant (CLET). The CLET procedure was performed as an explant culture that was expanded on human amniotic membrane with autologous serum. Limbal autograft was generally preferred for unilateral LSCD, but limbal allograft was performed in bilateral cases. Cultivated limbal epithelial transplant was performed according to the availability allowed by the laboratory infrastructure in eligible patients with unilateral chemical burns.
After LSCT, the recipient eyes were treated daily with a combination of 0.5% moxifloxacin (Vigamox, Alcon), 0.5% loteprednol etabonate (Lotemax, Bausch and Lomb), 0.05% cyclosporine (Restasis, Allergan), and preservative-free artificial tears. Topical antibiotic was discontinued after 1 month, and topical steroid was tapered over the course of 3 months. In limbal autograft and CLET groups, donor eyes were treated solely with 0.5% moxifloxacin for 10 days. The patients were examined daily for the first postoperative week, weekly for the first month, monthly for the first 3 months, and once in the 3-month period thereafter.
Successful surgical outcome was defined as improvement of the LSCD stage at 1 year after transplant, regardless of visual acuity improvement. Increase in the LSCD score means worsening of the LSCD stage. While evaluating the change in LSCD scores, subgroups of A-B-C in each stage were also evaluated separately. For example, changing from Stage 2A to Stage 2B was a 1-unit worsening of the LSCD stage. The patients were divided into 3 groups according to the LSCT type: group 1, limbal autograft; group 2, limbal allograft; and group 3, CLET.
Exclusion criteria were (1) limbal stem cell deficiency secondary to causes other than ocular chemical injury, (2) follow-up less than 6 months, (3) previous ocular surgery or other trauma history, and (4) absence of anterior segment photographs or standard medical records.
Descriptive statistics are presented as mean, standard deviation, median, minimum, maximum, frequency, and percentage. The Shapiro-Wilk test was used to test the normality assumption of quantitative variables. In univariate analyses, 1-way analysis of variance was used for normally distributed variables, and the Kruskal-Wallis test (Dunn test for paired comparisons) was used for nonnormally distributed variables. Categorical variables were compared by the Pearson chi-square test and the Fisher exact test. To examine relationships between quantitative variables, the Spearman correlation analysis was performed. Univariate logistic regression analysis was performed to determine the factors that affected the rates of surgical success. Parameters with P < .10 in univariate tests were included in the multiple logistic regression analyses. SPSS software (version 25.0, IBM Corporation) was used for statistical analyses. P < .05 was considered significant.
The mean age of the patients was 37.9 ± 15.7 years (range, 4-71 years) with a male-to-female ratio of 2.4. Forty-five patients (56.3%) had been injured with an alkaline substance, and 16 patients (20%) with an acid substance. Nineteen patients (23.7%) had been exposed to unknown or mixed substances. The mean follow-up time after the surgical intervention was 60.3 ± 30.6 months (range, 6-118.6 months). The chemical injury severity in 48 of 80 patients (60%) was grade 5 or higher at presentation. Most of these injuries were grade 6 (n = 27; 33.8%), followed by grade 5 (n = 21; 26.2%), grade 4 (n = 18; 22.5%), and grade 3 (n = 14; 17.5%). At the preoperative examination, 58 patients (72.5%) were assessed as LSCD stage 2B or higher. For the entire group of 80 patients, most were stage 3 n = 30; 37.5%), followed by stage 2B (n = 28; 35%), stage 2A (n = 10; 12.5%), and stage 1B (n = 12; 15.0%).
Preoperative, postoperative month 6, and posto-perative year 1 LSCD stages showed positive correlation with chemical injury grade, and these results were statistically significant (P < .05; r = 0.73, r = 0.61, and r = 0.44, respectively).
Overall, we observed surgical success in 52 of 80 patients (65%) according to the novel LSCD staging system described by the Limbal Stem Cell Working Group. The mean improvement in LSCD stage in patients with successful surgical outcomes was 1.9 ± 0.9 (range, 1-4). The LSCD stages were stable in 22 patients (27.5%) and increased in 6 patients (7.5%). In these 6 patients with worsening LSCD scores, the mean increase in the LSCD stage was 1.3 ± 0.8 (range, 1-3) unit. For the variables of sex, age, type of chemical agent, presence of symblepharon, and chemical injury grade, there were no significant associations with surgical success (P > .05 for all categories). The preoperative LSCD stage did not have a statistically significant effect on surgical success rates (P = .1). In a multivariate logis-tic regression analysis controlling for the variables of age, type of chemical agent, presence of symblepharon, and chemical injury grade, we observed that surgical success was predicted by the time interval from injury to transplant. Longer time intervals between injury and transplant were asso-ciated with higher rates of surgical success (P = .05).
The patients were divided into 3 groups according to the type of eye surgery. Limbal autograft was performed in 58 eyes (72.5%), limbal allograft was performed in 12 eyes (15%), and CLET was performed in 10 eyes (12.5%) (Figure 1). There were no significant differences among these 3 groups with regard to mean age, sex, type of chemical agent, chemical injury grade, and preoperative LSCD stage (P > .05 for all categories). The mean time interval between injury and surgery in limbal autograft, limbal allograft, and CLET was 43.3 ± 94.1 months (range, 0.5-592 months), 14.5 ± 10.6 months (range, 2.4-32.5 months), and 122.8 ± 158.9 months (range, 21.1-504 months), respectively, and was significantly higher in the CLET group compared with the other 2 groups (P = .02) (Table 1).
At postoperative year 1, we observed surgical success for 38 of 58 patients (65.5%) in the limbal autograft group. The rate of surgical success was 41.7% in the limbal allograft group and 90% in the CLET group. The mean improvement in LSCD stage in the limbal autograft, limbal allograft, and CLET group was 1.7 ± 0.8 (range, 1-3), 2.1 ± 1.1 (range, 1-4), and 2.3 ± 1.2 (range, 1-4), respectively. Surgical success at postoperative year 1 was significantly higher in the CLET group (P = .03). Although we observed a better rate of surgical success in the limbal autograft group compared with the limbal allograft group, the difference was not statistically significant (P > .05) (Table 1).
Additional penetrating keratoplasty (PK) after LSCT was required in 11 of 80 patients (13.7%). Seven of these 11 patients were in the limbal autograft group, and 4 were in the limbal allograft group. The mean time interval from LSCT to PK was 22.4 ± 17.7 months (range, 4.8-67.7 months). After PK, at postoperative month 6, the mean BCVA improved in 10 of 11 patients (90.9%) from 20/5000 ± 20/2222 (range, 20/20000 to 20/666) to 20/137 ± 20/143 (range, 20/20000 to 20/50). In 1 patient (9.1%), the BCVA and the LSCD stage remained unchanged. No increase in LSCD stage and no decrease in BCVA were observed after PK in any patients. The increase in BCVA after PK was significantly higher in patients with a higher initial LSCD stage (P = .04).
Chemical injury of the ocular surface is a significant public health problem with a serious economic effects caused by prolonged hospitalization, long-term medical treatment, multiple surgeries, lost workdays, and, in extreme cases, blindness.15 In severe ocular chemical injury, the most common cause of blindness is LSCD. The current and definitive treatment for LSCD is the replacement of healthy limbal stem cells.7,16,17 Young male individuals are at a higher risk of ocular chemical injuries as well as other traumas, especially trauma due to work accidents, versus other population groups.18,19 Similar to the other reports in the literature, most of the patients in our present study were male (n = 57; 71.2%), and the mean age of our patients was 37.9 ± 15.7 years.20
In the setting of chemical ocular surface injury, exposure to alkaline agents is more frequently the cause of the injury versus exposure to acid agents.6,21 In the present study, 56.3% of the injuries, overall, were caused by alkaline substances, which is consistent with previously published studies.
The severity of a chemical ocular surface injury is generally dependent on the duration of exposure and the type of chemical agent.1 Potent corrosive agents (which are most often alkaline substances) may penetrate deep into the eye and cause prolonged inflammation not only on the ocular surface but also in deep structures of the eye, with greater severity of LSCD and higher risk for additional complications such as glaucoma and cataracts.22,23 In the present study, the eyes with higher Dua grades of chemical injury were associated with higher stages of LSCD.
To evaluate the rates of surgical success for LSCT, various sets of criteria have been published. Some authors have described surgical success on the basis of anatomic improvements in the ocular surface; in other studies, surgical success has been defined on the basis of functional improvement and patient-reported symptoms.13 In a recent meta-analysis of 40 clinical studies (2202 eyes), the overall success rate of all LSCT procedures was 67.4%, and the rate of improvement of the ocular surface was 74.5%.14 Furthermore, the comparison of outcomes of different treatment modalities remains difficult because of the lack of standardized criteria for evaluation of LSCD. Although the increase in visual acuity was considered as a success criterion in most previous studies, the goal of LSCT is mitigation of the LSCD severity rather than direct improvement of vision acuity. Stromal opacities are the main cause of poor vision in patients with severe ocular chemical injury, so the absence of visual improvement does not necessarily indicate failure of treatment.14,24,25 In 2019, the Limbal Stem Cell Working Group established an objective scale to evaluate LSCD.9 This staging system facilitates accurate evaluation of surgical success, diagnosis and definition of LSCD stages, and the determination of appropriate treatment.9
For LSCD, there are several options for surgical intervention. Limbal autograft is the most commonly performed surgical option in unilateral cases.25-27 Consistent with previously published studies, we observed that limbal autograft was the most common surgical intervention.
Surgery type is a crucial factor for successful treatment of ocular injury. Many different techniques have been described for the various sources of the cells and the carrier tissue.7 Limbal autograft is generally preferred in unilateral LSCD; however, in bilateral cases, limbal allograft is the preferred choice.28 The main disadvantages of limbal autograft are lack of repeatability and the risk of LSCD at the contralateral healthy eye.10 In limbal allografts, there is the risk of rejection and the requirement for systemic immunosuppressive therapy.29 On the other hand, CLET is a relatively new and promising method with a success rate of approximately 80%.4 Repeatability is the most important advantage of CLET, and the main disadvantage is the high financial cost of this procedure.30 In a meta-analysis of 40 clinical studies (2202 eyes), improvement of the ocular surface was observed in 85.7% (range, 33%-100%) of limbal autografts, 57.8% (range, 0%-89%) of limbal allografts, and 84.7% (range, 44%-91%) of CLET.14 Herein, surgical success rates at postoperative year 1 were 65.5% for limbal autograft, 41.7% for limbal allograft, and 90% for CLET. The improvement in postoperative LSCD stage was statistically higher in the CLET group. There were no differences among the 3 groups with respect to the type of chemical agent, grade of chemical injury, and stage of preoperative LSCD; however, the time interval between injury and limbal transplant was significantly longer in the CLET group. These results suggest that the surgical success rate of CLET may be associated with the longer time interval between chemical injury and LSCT surgery, ie, inflammation tends to regress over time and thereby promotes limbal graft survival.
Factors other than the surgery type may affect LSCT outcomes.31 Cheng and colleagues32 reported that preoperative symblepharon severity and presence of inflammation may have significant effects on CLET outcomes. In a series of 80 eyes with symblepharon secondary to chemical or thermal injury, Cheng and colleagues observed that the success rate of CLET was higher in eyes with grade 1 or grade 2 symblepharon versus eyes with grade 3 or grade 4 symblepharon.32 El-Hofi and Helaly33 reported better final BCVA results after limbal allograft in eyes with Dua grade 4 chemical injury versus eyes with Dua grade 5 chemical injury. In their study of 20 patients with chemical eye injury who underwent limbal allograft, all patients selected for regraft were Dua grade 5, and all patients who received delayed re-epithelization after limbal transplant had eye injuries caused by alkali insult.33 However, in the present study, for the variables of sex, age, type of chemical agent (alkaline or acid or other), presence of symblepharon, and chemical injury grade, there were no significant relationships with postoperative year 1 surgical success rates.
Inflammation is the main cause of graft failure in LSCT,34 and in the subacute phase of chemical injury, inflammation may persist at a low level despite anti-inflammatory therapy.23 Therefore, most studies have shown that LSCT in the chronic period of chemical injury is much better for graft survival compared to LSCT in the acute phase.23,33 However, there are few published reports of the direct effect of LSCT timing on LSCT outcomes, and the ideal postinjury time interval preceding LSCT has not been determined.26 Rao and colleagues35 observed that surgery at the acute phase of injury (<4 months) is associated with delayed corneal re-epithelialization and worse visual outcomes. Sejpal and colleagues23 reported a high rate of surgical failure in patients who underwent CLET within 4 months after chemical injury to their eyes. On the contrary, Ozdemir and colleagues36 reported that early LSCT may prevent corneal neovascularization in cases of chemical injury with greater epithelial defects and less limbal ischemia. Herein, with 80 eyes, longer intervals between injury and transplant were associated with higher rates of surgical success.
The preoperative LSCD stage may also be associated with the prognosis after LSCT. In the past, the lack of consensus with regard to a severity scale for LSCD has prevented any substantive evaluation of the preoperative and postoperative associations of LSCD severity. In the present study, a lower LSCD stage at the initial examination was associated with a better surgical outcome at 1 year after transplant, but there was no statistically significant difference. To the best of our knowledge, this is the first study to use the Limbal Stem Cell Working Group’s staging system to demonstrate the relationship between preoperative LSCD stages and rates of LSCT success.
The LSCT procedure is an effective treatment for cases of ocular chemical injury limited to the corneal epithelium and limbus,37 and for these cases LSCT promotes ocular integrity and improves visual acuity. However, if the corneal stroma is affected, then PK is required for visual improvement.24 PK simultaneously with LSCT is should only be preferred for cases with additional corneal perforation, since it has a high graft failure rate.38,39 The presence of inflammation and vascularization in the recipient bed increases the risk of rejection of the corneal graft.40 Therefore, PK is recommended once ocular surface stability is achieved with LSCT.38 Although there is no consensus with regard to the timing of PK after LSCT, Ozer and colleagues have recommended that PK should be delayed until 12 months after LSCT.25 In addition, there are few reports on the potential effect of PK on limbal graft survival. Figueiredo and colleagues37 have recommended PK to be delayed at least 12 months after CLET, and that PK at least 12 months after CLET did not negatively affect CLET survival and provided significant improvement in visual acuity.37 In the present study, 11 of 80 patients underwent PK after LSCT, and the mean time interval between LSCT and PK was 22.4 months. None of the patients had a worse LSCD stage and a worse BCVA after PK. Although the number of patients who underwent PK was limited, there were sufficient data to show that PK did not affect limbal graft survival. In 10 of 11 patients, a significant increase in BCVA was achieved after PK. The increase in BCVA after PK was higher in patients with a higher initial LSCD stage. In the LSCD scale, involvement of the central 5-mm zone of the cornea is defined as the most important criteria. The LSCD stage is likely to be high in patients with central corneal involvement. Therefore, in patients with involvement of the central cornea, better visual acuity may be achieved after PK.
The limitations of this study are its retrospective nature and the limited number of patients in the CLET and limbal allograft groups; however, the results are remarkable to highlight the benefits of the novel LSCD scoring system introduced by the Limbal Stem Cell Working Group.
Limbal stem cell transplant is the preferred surgical treatment for severe ocular chemical injuries that cause LSCD. The accurate determination of LSCD is vital for proper assessment of the surgical success. The most important factors that affect LSCT outcomes are (1) the method of transplant and (2) the time interval between surgery and injury. For this reason, to achieve higher rates of success with LSCT, a hasty rush to surgery after a chemical injury should be avoided.
DOI : 10.6002/ect.2021.0393
From the 1Department of Ophthalmology and the 2Department of Medical Biochemistry, Ege University, Izmir, Turkey
Acknowledgements: The authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no declarations of potential conflicts of interest. This study was presented at Turkish Ophthalmological Association 54th Virtual National Congress, November 4-8, 2020, Antalya, Turkey.
Corresponding author: Ozlem Barut Selver, Department of Ophthalmology, Ege University Medical Faculty Hospital, 35100 Bornova-Izmir, Turkey
Phone: +90 505 648 7268
Figure 1. Preoperative and Postoperative Year 1 Anterior Segment Image Samples From Patients of Limbal Autograft, Limbal Allograft, and Cultivated Limbal Epithelial Cell Transplant
Table 1. Variables for Different Methods of Limbal Stem Cell Transplant