Comparison Of Conjunctival Autograft And Combined Amniotic Membrane Mini-Simple Limbal Epithelial Transplantion After Primary Pterygium Excision

Abstract

Background: To compare conjunctival autograft and combined amniotic membrane mini-simple limbal epithelial transplant after primary pterygium excision

Methods: A prospective randomized interventional study was conducted on 264 eyes with Primary Pterygium.The patients were divided into Group I (conjunctival autograft)  and Group II (mini-simple limbal epithelial transplant). 133 eyes in Group I underwent pterygium excision with a conjunctival autograft using fibrin glue. 131 eyes in Group II underwent mini Simple Limbal Epithelial Transplant with amniotic membrane using fibrin glue. Post-operatively, the patients were reviewed on day 1,3,7,14 & 30 and then at three,six and nine months. Primary outcome measure was the recurrence rate whereas the secondary outcome measures were the intraoperative time and other complications.Recurrence rate was calculated using Fisher’s exact test. Variables like age , preoperative BCVA , operative time and the dimensions of graft were compared using unpaired t test . Other baseline characteristics like gender, Laterality, grades of pterygium(I-III),Occupation and indication of surgery were expressed between the two groups using Pearson’s Chi-Square test.

Results: Two hundred and thirty three eyes(118 in group I and 115 in group II) could complete nine months follow-up period. Recurrence was seen in 2(1.6%) cases in group I whereas 3 cases (2.6%) had recurrence in group II(p=0.681).Operative time for group (II) (20.33±1.28 min) was significantly higher (p<0.001) than group I (12.01±1.26). Graft displacement occurred in one case in group II (p=0.999).

Conclusions: Despite a longer time,(p<0.001)  mini-SLET seems to be a viable and equally effective alternative to CAG in the management of primary pterygium ,especially in cases where conjunctiva needs to be spared.

Ethical Clearance Certificate Number :     29/MH/2015 dated 11 Aug 2015

BACKGROUND

Pterygium is a  benign degenerative ocular surface disorder with wing-shaped fibrovascular growth of the subconjunctival tissue onto the cornea¹. It usually invades the nasal limbus and spreads along the interpalpebral fissure and may necessitate surgical removal.  postoperative recurrence is not uncommon. Various adjunctive measures like beta irradiation²and medications such as Mitomycin C^3,4have been used in the past. However, these methods are associated with side effects, such as punctate epitheliopathy, bacterial “superinfection”, delayed-onset scleral melting, and elevation of intraocular pressure (IOP). To circumvent these complications and to achieve better results, human amniotic membrane grafting⁵ and autologous conjunctival grafting⁶ came into vogue. However, ipsilateral autologous conjunctival grafting has become the most common and effective surgical procedure of choice.The efficacy and low recurrence rate of this method has been substantiated by many studies.^{6 7 8 9}Tissue adhesive like fibrin glue¹⁰ offers many advantages, primarily  shorter operating time, increased postoperative comfort and freedom from  suture-related complications^11,12.CAG using autologous in situ blood coagulum is an emerging concept in pterygium surgery in recent years which has been corroborated by many reports.^13,14,15However, lately the induction of autologous simple epithelial limbal transplant (mini-SLET)¹⁶in the armamentarium has offered one more option of  rehabilitating bare sclera . The purpose of the current study is to compare the efficacy of pterygium excision with CAG and pterygium excision with mini-SLET.

Methods

This was a prospective, randomised ,interventional clinical study , duly approved by the Institutional Ethics Committee (Ethical clearance certificate no. 29/MH/2015 dated 11 Aug 2015 of Military Hospital Jammu Cantt) and adheres to the tenets of the declaration of Helsinki.Two hundred and sixty four consecutive patients (264 eyes) with primary pterygium were enrolled in this study which was conducted between between Aug 2015 and Jan 2019. All consecutive cases of Primary nasal Pterygium were enrolled in the study. Exclusion criteria included ocular surface disorders , hypersensitivity to blood components and seropositivity to Hepatitis B, Hepatitis C and HIV (These patients were excluded as part of the norm followed by the Ethical committee of the institute). 

Written informed consent was obtained from all the patients recruited in the study. This included consent for the images to be published. Comprehensive medical and ocular histories besides age, sex of the subjects was obtained. Thereafter, a detailed ocular examination was performed. Pterygia were graded into three grades based on classification given by Tan and coworkers¹⁷: T1 (episcleral vessels underlying the body of the pterygium seen unobscured and distinguished), T2 (episcleral vessels indistinctly seen or partially obscured) and T3 (epislceral vessels totally obscured by fibrovascular tissue). The Patients were divided into two groups by simple random sampling using lottery method: group I (133 eyes)underwent CAG with fibrin glue and group II (131 eyes) underwent mini-SLET. An allocation concealment and masking of the patients was done. Formula used for calculation of sample size is n= (Zα+z(1-β)2×(p1q1+p2q2)/ d2,n= sample size,Zα = Standard normal variate for α=0.05(95%CI),Z 1-β = Standard normal variate for 1-β=0.80(80%),P1 = proportion (%) in one gp; qi =100-p1 &p2…,d = p1-p2

Surgical Technique

All surgeries were performed under peribulbar anaesthesia (2 % lidocaine) by the same surgeon (AJ).Using the Westcott scissors horizontal incisions along the superior and inferior borders of the body of pterygium were made.Thereafter, an incision parallel to the limbus at the periphery of the pterygium 2mm nasal to the limbus was made. The pterygium was subsequently reflected centrally i.e. towards the limbus using Moore field’s conjunctival forceps. The remaining underlying fibrovascular tissues in the bulbar conjunctiva were dissected.

 The area of the bare sclera was measured using a caliper. For group I a tenon-free supero-temporal conjunctival autograft one mm larger than the bare sclera in horizontal and vertical dimensions was obtained.Fibrin glue (Tisseel,Baxter) was applied to the bare and dried up sclera and the graft was transferred onto the bare sclera with correct orientation. The angled flat end of two iris repositors were utilized to iron out the graft. This was done superior- inferior axis(parallel to limbus) and nasal-temporal axis ,if required. This manoeuvre with iris spatulas has been termed as ‘cheese-spreading technique’ (video 1) not only leads to expansion of the graft to its maximum possible size but also ensures removal of excess glue on the sclera bed. 

In group II, after performing 3mm or 1 clock hour of (10-11 o’clock for Right Eye and 1-2 o’clock for left Eye) peritomy, 2x 2 mm size limbal tissue was harvested with the help of a crescent blade commencing from the conjunctival side. This strip was then cut into 6 to 8 pieces, using Vannas scissors under higher magnification, i.e.X16. These pieces were placed and fixed with fibrin glue on the inlay AM closer to the limbus.Subsequently, the overlay AM was used to sandwich the limbal tissues.Freeze dried Amniotic membrane (Amnio-care, Biocover Labs), which is available in (3X3) cm size was used. A bandage contact lens was left in place.

The fibrin glue (Tisseel VH, Baxter AG) was prepared by reconstituting freeze-dried protein concentrate and thrombin in fibrinolysis inhibitor solution and calcium chloride solution, respectively, and were warmed for 20 min in a patented fibrinotherm device. Both fibrin and the thrombin were loaded in the separate syringes mounted with 26G needle.This was mainly done to be able to use one fibrin glue kit of 2ml for at least 10-15 cases(for both groups) to avoid any logistic wastage. To use the glue, equal number of drops of (fibrin) and (thrombin) were used to stick conjuctival autograft, Limbal tissues and Amniotic membrane. The operating time was noted for both the groups.

Both the groups were patched for 24 hours. Post-operatively all the patients were treated with1 % prednisolone acetate eye drops 4 hourly ,0.5 % Moxifloxacin eye drops 6 hourly in the first week, followed by application of only a topical steroid in tapering dosage over a period of 1 month.

The patients were reviewed on next day after surgery and then on day 3,7,14 and 30 and subsequently at third , sixth and ninth month. Each visit initially included a slit-lamp examination to check autograft/AM status, and complications like graft retraction, graft/AM displacement, corneal thinning and later on for recurrence.

Recurrence was recognised as any fibrovascular growth that crossed the limbus. The photographs were taken pre-operatively, intraoperatively and post-operatively on day 1, 30 & at 6 and 9 months for both the groups (Figure 1). 

Statistical Analysis

The statistical software SPSS version 20 was used for the analysis for various variables. A p value of less than 0.05 was considered as significant. Variables like age and preoperative BCVA were compared using unpaired t test . Other baseline characteristics like gender, Laterality, grades of pterygium(I-III),Occupation and indication of surgery were expressed between the two groups using Pearson’s Chi-Square test. Comparison of operative time and the dimensions of graft between CAG and mini-SLET Conjunctivalautograft was done using unpaired t test.

Results

A total of 264 eyes were enrolled in the study during a period of 42 months duration. Thirty one eyes (15 in group I and 16 in group II eyes) were excluded from the data analysis owing to incomplete follow-up during the study interval. Hence, there were 118 eyes in group I and 115 in group II that completed 9 months follow-up.

The baseline characteristics of both CAG and mini-SLET groups are summarized in Table 1. There was no significant difference in the 2 groups for the baseline characteristics. Size of graft and operative time have been compared in Table 2. Operative time for group II (20.33±1.28 min) was significantly higher (p<0.001) than group I (12.01±1.26). Recurrence was assessed at 1, 3 , 6 and 9 months. 2(1.6%) cases in group I exhibited recurrence whereas 3 (2.6%) had recurrence in group II.When these 2 groups were compared using the Fisher exact test, a statistically insignificant difference (p =0.681).Recurrence was noticed within 3 months in two patients in group I and one in group II . Remaining two patients showed recurrence between 3 and 6 months.Revision surgery was performed in two cases in each group whereas on patient in group II declined surgery. None of the patients who underwent revision surgery had recurrence till six months of follow up.One patient with recurrence in group II was advised revision surgery which he declined.

Amniotic membrane Graft displacement occurred in one case (0.87%) in group II on first postoperative day, which was fixed on the very same day by repositioning the graft with fibrin glue. group II (p=0.999).This was the only case in which recurrence was seen.

Discussion

Pterygium occurs primarily due to local deficiency of limbal stem cells(LSCD)secondary to exposure to UV type B(UVB) light¹⁸. The basic difference between the primary and recurrent pterygium is that the body of the former is free from the underlying structures whereas that of the latter is firmly adherent to the underlying tissues namely episclera and sclera.This is primarily the reason one has to be extremely careful and diligent in the surgical treatment of pterygium to avoid complications and financial implications . This forms the basis of local LSC deficiency inclusion of limbal stem cells in the treatment of pterygium. Of all the modalities described in literature ^3,4,5,6, CAG technique is associated with least recurrence ^{7 8 10} . CAG can be fixed to bare sclera either by sutures, fibrin glue or autologous in situ blood coagulum. Amongst the three techniques of fixing CAG, fibrin glue definitely offers the advantage of least operative time ¹⁹. SLET was first used for the treatment of stem cell deficiency by Sangwan and coworkers ²⁰. Subsequently this SLET technique was used in a miniature form as mini-SLET²⁰ for the treatment of primary pterygium in 10 patients. The basic premise of using mini-SLET for the pterygium was local deficiency or dysfunction of limbal stem cells^21,22. AM acts as an ideal substrate for supporting the growth of epithelial progenitor cells by maintaining their clonigenicty and preventing epithelial apoptosis²³. AM also has anti-inflammatory properties by virtue of containing protease inhibitors²⁴. AM has no limbal stem cells so probably better outcomes have been reported with CAG. Since mini-SLET appeared to be an exciting and a viable prospect for the treatment of pterygium we made an endeavour to compare the two different techniques and carried out this prospective study primarily to compare the rate of recurrence. Sati and coworkers²⁵ compared the outcomes between 40 cases of mini-SLET and 42 cases of CAG fixation with fibrin glue following excision of primary pterygium.The study reported insignificant recurrence rate between two groups. Extensive MEDLINE search did not reveal any study of this magnitude in terms of number of subjects and follow up period comparing CAG and mini-SLET. The recurrence rates are almost nil with CAG if a nearly tenon-free graft is taken and spread out properly using spatulas with ‘cheese-spreading’ technique. The key to success of this technique is that moment the CAG is applied to the dry bare sclera, flattened part of two iris repositors are placed vertically in close apposition and are used to spread it along its length and breadth without much time lag. This not only ensures that original size of the graft is maintained but also removes any excess glue component. This technique enables the graft to use its barrier effect to its full potential and prevents dislocation of graft. 

In mini-SLET combined use of AM and limbal stem cells could reduce recurrence rates. The same is evident in our study. In our opinion mini-SLET will really be advantageous for patients who are glaucoma suspects or do not have sufficient conjunctiva left owing to previous surgeries. In this category extra expenditure and operating time is fully justified. In institutional practice like ours it is very much justifiable in young patients. There are numerous studies in literature about pretygium excision using CAG however there is paucity of literature regarding mini-SLET.¹³ This newer technique is an offshoot of SLET technique, the procedure developed by Sangwan and coworkers,²⁰for patients with unilateral limbal stem cell deficiency. However, unlike SLET, mini-SLET technique involves placement of ipsilateral stem cells containing limbal pieces over amniotic membrane close to limbus at the site of excision of pterygium. Compared to CAG ,Mini-SLET technique has many advantages. Firstly, this technique has more chances of restoring normal anatomy of limbus,. Secondly, mini-SLET preserves the conjunctiva for future use for instance glaucoma surgeries. Thirdly, this surgery is likely to be the procedure of choice in patients who have undergone multiple ocular surgeries.

In 2015, Hernández-Bogantes E and coworkers,¹⁶ reported this innovative technique for the first time in a small series of 10 patients with primary pterygium and observed that none of the patients had recurrence with 8 months of follow-up. Inspired by the results, we planned the current randomised study i.e. CAG vs mini-SLET following excision of primary pterygium. This study is unique in many aspects. First, the current study of this magnitude is first of its kind(Number of patients and duration of follow up), comparing mini-SLET technique with standard technique. Second, owing to the similar rate of recurrence in both the groups we are hopeful that Mini-SLET will become the procedure of choice as conjunctival sparing surgery (esp in young patients, glaucoma and cicatrizing conjunctivitis). Third, ‘cheese spreading’ technique appears to have a definite role in preventing graft displacement and in maximizing the barrier effect of CAG.

Conclusion

Based on the findings authors recommend that mini-SLET is a viable alternative to CAG  and is equally efficacious for treating primary pterygium. Both the techniques are safe and effective and are associated with similar rates of recurrence. However, more such  studies needed to substantiate the findings

Abbreviations

CAG : Conjunctival autograft

Mini-SLET:  Mini simple limbal epithelial Transplantation

AM : Amniotic membrane

SPSS: Statistical Package for the Social Sciences

IOP : Intraocular pressure

BCVA : Best corrected visual acuity

LSCD : Limbal stem cell deficiency

Declarations

Ethics approval and consent to participte

The current study has been approved by the ethics committee of the institute and consent was taken.

Consent for publication : Taken

Availability of data and material : The raw data has been attached as a supplementary file.

Competing interests : The authors declare that they have no competing interests

Funding: The author(s) received no financial support for the research,authorship, and/or publication of this article.

Authors’ contribution:

AJ and AS planned and designed the study. Data collection and analysis was done by AJ,AS . AJ,and AS prepared the Manuscript. The term ‘cheese spreading’ was coined by AJ. The editing was done by AJ, and AS. AJ is responsible for the overall guarantor. 

Acknowledgement: The authors wish to thank Dr Alok Sati, Associate Professor of Ophthalmology at Armed Forces Medical College for doing critical review of data and providing insights into the possible aspects of improvement and emphasis in the initial drafts of the manuscript.

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