Lecture: Introduction to SLET

[Samara] Good morning and good evening. My name is Samara Andrade and I’m the Senior Director for Clinical Training and Prevention at SightLife. We’re thrilled that you’ve joined this webinar today to learn more about simple limbal epithelial techniques. Before introducing our incredible faculty that’s here to share with you today, I wanted to take a little bit of time to let you know about SightLife, what we do, and how we work to achieve our mission to eliminate corneal blindness by 2040.

Next slide. So to start, I really want to thank Orbis for our long-standing partnership of our education events for corneal care providers in low and middle-income countries. On the international side of SightLife, we focus on five main program areas. The first is advocacy and policy, which works to ensure that barriers to successful health systems are removed by working with key opinion leaders, and advocating to governments and working with them to build policies that work to address care, and to increase the number of donated corneas, and ensure that the health system overall is working for their communities.

The second area that we’re dedicated to working on is prevention and awareness, which works to prevent basic eye injuries, like abrasions, from progressing into corneal blindness, by working with community health workers and eye hospitals in rural communities. The third area that we work on is clinical training, of which today’s program is part of. Our focus in this is to ensure that there are qualified eye health care providers who can treat patients who need sight-saving surgery and to better manage corneal disease.

The fourth area that we work is eye banking. We support the eye banking ecosystem to ensure that eye banks are implementing best practices and functioning at high-efficiency rates to provide tissue for transplantation. The last area we work on is access and innovation. Bringing innovative solutions to improve access to high-quality, affordable care, empowering local healthcare providers to treat their communities more quickly and more efficiently. Through these five programs, we ensure that the health system is supported to be self-sufficient and meet the needs of the countries that we support work in.

Next slide. Most of you here today are clinical care providers. And you probably have a particular interest in learning more about what we do on clinical training. Since our inception in 2013, we’ve directly changed more than 1,400 ophthalmologists directly in-person in low and middle income countries, and nearly 2,000 online. The way we provide in-person learning is through development of peer-reviewed curriculum. This curriculum is for different programs such as short-term corneal fellowships in PKP, where we take high-volume cataract surgeons and support them in transitioning into cornea. We also provide training in various surgical techniques including DMEK, DSEK, SLET, as well as different educational programs for ophthalmic personnel like nurses and transplant coordinators.

The faculty of SightLife are also serving as coaches and mentors for hundreds of corneal surgeons around the world. So today we’re very lucky to have Dr. Virender Sangwan here to give this webinar on Simple Limbal Epithelial Transplantation. It’s absolutely my pleasure to welcome him. His passion for teaching comes through everything he does. Not only was he the first to describe this specific surgical technique in 2012, but he also worked closely with SightLife as a faculty for training courses, and to develop a peer-reviewed curriculum on SLET, which is the first in the world.

He also teaches many courses globally each year on SLET as well as many other techniques, and you may have even recently seen him a couple of weeks ago right here on Cybersight. His generosity of time and commitment to education and training ophthalmologists is inspiring. So without further ado, I’d like to hand over to Dr. Virender.

[Virender] Good evening and good morning to everyone. Thank you, Samara, for the kind introduction. And I would like to thank Cybersight and SightLife for providing this opportunity to share the ideas about simple limbal epithelial transplantation. I will talk about simple limbal epithelial transplantation for the beginner and give you insight how we’d have loved it.

I have no financial disclosures to make in the context of this presentation.

For understanding the SLET, let me make a brief comment about the normal ocular surface. Here in this short video, what you are seeing is the front part of the eye and it’s called the ocular surface. There are three critical element which are essential for normal ocular surface. These are: preocular tear film with all its components, surface epithelia, which are shown in this cartoon here. Which essentially talks about the limbal epithelium going onto the cornea and then exfoliating that classical XYZ hypothesis. And then there has to be an adequate amount of ocular surface sensitivity. Once you have all these three components, which is important for you to check before you do any surgery. Obviously, in patients with stem cell deficiency, some of their epithelium is abnormal.

The elements of ocular surface defense include the stable preocular tear film, which consists of compositional factors and hydrodynamic factors. And all of you are familiar with these components, how they help in stabilizing the normal ocular surface.

This, at the brain level or the midbrain level is controlled by these two reflex arcs. Which is the lid blinking and tear clearance. And both these motors control the tear clearance. A stable tear film is essential for the health of the ocular surface. A healthy ocular surface is essential for the tear film. So they are interdependent and they are helpful to each other.

When you have a injury, or an insult, or an autoimmune problem, or a developmental problem, we get a condition called limbal stem cell deficiency. This consists of ingrowth conjunctiva to the corneal surface, chronic inflammation, recurrent epithelial breakdown. And this happens because the limbal epithelial barrier function is gone and they are no longer able to resurface. And that’s why the conjunctiva come onto the corneal surface. Obviously you lose vision, there’s pain, redness and the eye may look very unsightly.

The clinical spectrum of this condition can be total stem cell deficiency, partial, there can be classification-based, from congenital causes, idiopathic, or secondary to trauma. Or primary immunity diseases like Stevens-Johnson or ocular cicatricial pemphigoid. In this video you are seeing some of the pre-op videos of my patient.

Here at the bottom, I would like to draw your attention. This is a patient with VKC, and you see the dysfunction. This is not still a total deficiency because the cornea had to be healed. And this is also destruction, due to Mooren’s ulcer. This is total stem cell deficiency due to chemical burn. So it’s very important to make a distinction between dysfunction and destruction. In both of these cases, you may not need a limbal transplantation but here in this patient, you would need a limbal transplantation.

Before we go further, let us just understand the terminology related to the limbal transplantation. This is based on the source of the tissue, which can be allogenic, when the tissue is taken from another human being, which can be a donor, a live-related or a cadaveric. If it is autologous, meaning from the same patient’s other eye or the same eye. So this is an autologous procedure. Classification can also be based on the surgical technique. This is like direct limbal transplantation, where there’s a risk of donor site limbal decompensation because the amount of tissue we had risk is much higher. If we grow cells in the lab, it’s called ex-vivo cultivation. And the procedure does not have any risk for decompensation or deficiency at the donor site. And the more newer technique is simple limbal epithelial transplantation, which we will talk about today.

So the rationale or the scientific basis of doing a limbal stem cell transplantation, or limbal transplantation for LSCD, or limbal stem cell deficiency, is following. That you take a small amount of the donor limbus and transplant on to the affected cornea after cleaning up the pannus and then these cells grow and resurface the corneal epithelium.

In direct, or what we call conjunctival limbal autografting, the amount of tissue as you see here, it needs six to eight clock hours. And that is why there is risk of deficiency of the donor site. In cultivation, when you have a lab-based cultification, the amount of tissue is about one clock hours and there’s no chance of deficiency. And now SLET, what we are talking about, it’s the best part of the both CLAU and CLET. And the side effect, or the downside, so the negatives of both the techniques are eliminated with this SLET.

The treatment for limbal stem cell deficiency essentially is surgical. And you will be hearing again and again, CLAU, CLET and SLET. Conjunctival limbal autograft, cultivated limbal epithelial transplantation, and simple limbal epithelial transplantation. Live or cadaver, it depends on the limbal tissue source and it’s useful in allograph and therefore systemic immunosuppressant is acquired. I’m not going to talk about the allografting today. We are talking about autologous simple limbal epithelial transplantation. I’m not going to talk about the conjunctival limbal autograft or cultivated limbal epithelial transplants. However, I’ll give a brief introduction about CLET.

The first question. For normal functioning of the ocular surface, which statement is not correct? Surface sensitivity, pre ocular tear film, surface epithelium, the elements which are critical for normal functioning, and keratin expression by the epithelium? Practically 80% of you got that answer correct, that is keratin expression by the epithelium is incorrect answer.

Now, let me share a story of a girl who I treated in 2001. She had a lime burn in one of her eye and came with a total stem cell deficiency. Underwent a cultivated limbal epithelial transplantation in one eye, that is the left eye, followed by penetrating keratoplasty. And she underwent squint surgery, she underwent ptosis surgery and now she is a grown up woman. She is from Delhi, so I follow her up here. And almost 20 years now, and her eyes are still doing very well. She maintains 20/25 vision. The eye, last year I had to repeat her corneal graft, but other than that, she’s doing absolutely fine.

So what is this cultivated limbal epithelial transplant technique I’m talking about? This is the technique of growing the stem cells in the lab, which we developed when I was in LVP. We started in ‘99-2000 and the first cultivated limbal epithelial transplant I did in 2001. In this technique, you have submerged explant culture on de-epithelialized amniotic membrane, which you saw just here. And these are the plan for growing the cells. Each of these fed all the explants, which you can see here under microscope. And this is the green fluorescence. In this technique, there’s nothing off an animal region, we use autologous serum and we use to obtain it, single monolayer. And our protocol has been published in nature protocols and being followed all over.

Using the technique we have published extensively in peer review literature and shown that the outcomes of this technique are excellent and have been doing very well.

Except that this technique has not worked very well in pediatric limbal stem cell deficiency. Why it has not worked, really did not understand well. But in this paper in JAMA, published in 2013, we showed that the success rate of CLET in pediatric LSCD is only 40%. And then a large percentage of them develop what we call amblyopia or lazy eye.

So we started feeling that there are problems with the CLET. Cost is one of the major considerations and we’ll talk towards the end of the talk what I’m talking about, how and why this is expensive. We need a culture media, non-human products, xenogenic material, the laboratory needs very high maintenance cost. You need for CLET, two surgeries. First, therefore, taking the biopsy and then you grow the cells for two weeks, then after two weeks you go back and then transplant is done. And that is one patient.

Now this patient, a nine-year-old boy presented to our clinic with his father. And father said, can you remove this eye? Because the boy refused to go to school because the eye looks very bad and he’s being bullied by his friends. So what we did was, for us, and by that time we had started developing the simple limbal epithelial transplantation. So in this boy we did this technique. And as you see on the left panel, pre op and after about six weeks, post-operatively, the cornea has cleared. There is residual symblepharons superiorly, which was treated with the top of SLET. About three months, six months later you can see there’s 20/40 vision. And this is the histology of that size pannus, which looks very intense inflammation and granuloma formation.

This is how we do this technique. This is a very brief introduction taking the biopsy. And I’ll be showing you next, a lot of videos about surgical steps, and I’ll take you through each step.

So you take the biopsy, deposit the cones back, if you don’t need as a conjunctival autograft. Then you clean up. This was very mild, superficial limbal stem cell deficiency. After cleaning up you do a little bit of cautery, and put fibrin glue, bring the amniotic membrane, spread it all out nicely. And the biopsy which was taken, you keep in a BS solution. Then you cut, either with a 15 number blade, later you will see, I started using scissors. Anything is fine, whichever works.

And this is one of my very earlier videos when I was developing. That I did the fibrin glue first and then put the explants. Which is quite messy. But later on, I revised and I would put the explant first and then put the fibrin glue, you will see in subsequent videos. And in the bottom, you see the different time point and about six months later, the eye becomes almost normal.

So what is a good case for doing SLET for a beginner. In my view, an uncomplicated chemical burn induced unilateral limbal stem cell deficiency, total or partial, without much symblepharon. Which is typified here on the top right corner. Any adult or children with lime injury, lime injury is preferable because the pannus comes off easily, you can find the plane, as compared to acid injuries. No underlying systemic autoimmune conditions. The other eye should be normal and you should be able to see a good, what we call the limbal palisades. Examine the other eye and these are the pictures I showed you earlier of this dysfunction and destruction. These don’t need surgery, therefore do not operate them.

Which are the cases that you should avoid in the beginning? Bilateral cases. Unless you have done, even in the wet eyes, because Graves’ need allo-SLET and needs systemic immunosuppressant. So I wouldn’t do those cases in the first 10 or 15 unless you are very familiar. Any patient with severe dry eyes. Like SJS, OCP, Sjogren’s syndrome, GVHD, unknown etiological LSCD. Especially in India and the frequent subcontinent, you’ll have patients who have measles or viral fevers and they develop total bone dry eye. Don’t touch those eyes for doing these surgery, it won’t work.

Poor surface with decreased ocular or corneal sensitivity, post radiation, post OSSN excision. Local or systemic immune-mediated diseases which cause a limbal stem cell deficiency. So in the beginning, avoid those cases. Select simple, straight-forward wet eyes.

What do you need to do a SLET? You should be familiar with the surface surgeries, meaning you should be doing region surgery, OSSN or dermoid removal. You should be familiar with doing corneal surgeries. You should be able to assess the visual potential in the eye to be operated. Sometimes you may consider doing surgery even when they appear negative eye, for cosmetic enhancement. But those should not be your first few cases. You should be able to do a proper work up and informed consent. You should be able to understand post operative requirement for the course of action should be. And BCL should be put and kept for two weeks, at least. And when in doubt, do a tarsorrhaphy at the time of surgery.

I’m showing these two videos. The video which says bone dry, which is essentially a different stages of different types of Stevens-Johnson, please do not do any kind of limbal transplantation in these eyes, they don’t work. There can be modified protocol but I can talk about that later, or in question/answer, or some other form.

The video on the wet surface, these are chemical burns, they are good enough, but they’re different stages of disease. Select simple, effective, or simple uncomplicated cases in the beginning.

What are the indications of SLET? Unilateral or bilateral limbal stem cell deficiency. Which is total or partial, with decrease in vision or symptomatic patients. LSCD is a surgical disease and medical treatment is not indicated, not required. Cosmetic enhancement if visual prognosis is poor. But that is only after you have done five or ten cases and you’re familiar. SLET is now being done as a preventive, or a primary surgery, along with excision of OSSN or recurrent ptergium to prevent limbal stem cell deficiency.

Time for the second question. What is the best treatment for unilateral total limbal stem cell deficiency caused by a chemical burn? Only about 4-5% of people did not get it correct. And you should be able to get it by the end of this. Simple limbal epithelial transplantation is the correct answer.

Now let’s talk about surgical technique, post operative management in detail. This video is the original video of Kent Kenyon and Scheffer Tseng, who described the limbal stem cell transplantation for limbal stem cell deficiency, way back in 1988-1989, when both of them were in Mass Eye and Ear Infirmary. As you would notice, they’re starting to harvest the limbus from the corneal site. And if you see my video, how I start, I start exactly opposite. I start from the conjunctival side. And you will see this way of harvesting biopsy again and again. But the downside of harvesting from the corneal side is you don’t know what the plane, how much deep you have to go. If you start from the conjunctiva side you know where the conjunctiva is, where the tenon’s capsule is, and where the sclera is. So you want to go, there’s sclera, then dissect the blunt and shove the six in towards limbus, and then you keep harvesting until you reach limbus. And we talk about this in further videos.

Before that, I’ll show you how we do the pannus dissection. All of you will agree that it’s a total stem cell deficiency. And barely you can make out if there’s a cornea or no cornea. As we start this video, you start dissecting the pannus from periphery of what you think or where the cornea might be. And it’s always good idea to put your scissors closed prongs of the scissor, and go underneath, undermine and then remove the abrasions and cut it. Make a space, and then bit by bit, use the 15 number blade, sharp instrument, as much avoid as possible. And certainly I don’t recommend using supersaw blades or diamond knives or crystal knives. They will create multiple planes and you’ll go in unwanted areas. So my best instrument for this dissection is a 15 number blade on BP handle.

When you reach this, now you are convinced that this is a cornea. Looks nice and you do a cautery a little bit and then put the amniotic membrane. And we’ll see in the further videos like here. So first you harvest biopsy. Watch it carefully how I am doing it. 15 number blade, tenons are cutting. And then my assistant start pouring continuously the water. Because at this stage of cut, you should not do cautery. And you should learn to harvest or operate under the water. Here the very reason I’m showing this. I did a snip biopsy from the area and did at the donor site. So that preventing the limbal stem cell deficiency at that site. Now this is the affected eye. So I did the limbal biopsy first, put it in the BSS, on the surgical trolley, and then I started dissecting. And make sure that you tell your nurse do not throw that limbal biopsy, don’t send it to pathology, don’t put it in the formalin, that you need it.

Look at that, I’m doing dissections in different ways. Trying to find the plane of insertion of the pannus onto the cornea. And how do you know that? Once you start from the periphery, away from the limbus, the reasons are most severe at the limbus. Once you dissect them, come towards the cornea, you will be able to see the plane. And you should try to find the plane and do not remove the corneal fibers at all. Once you clean up a hemostate is achieved, fibrin glue, both component, pick one first. That is the thrombin and then fibrin glue and then you bring the amniotic membrane. Or you can put the amniotic membrane and lift up one half, put the glue, iron it out.

Some other people do, that you grab the membrane first and then under the membrane, you go and inject the glue. Then I’m now using the scissors and forceps. These are both micro scissors designed by Georgia surgicals for me. But they are available from him. You can also use this intraocular retinal forceps and scissors, maybe 18-20 gauge or 23 gauge. Which may be a bit fine, but they are very, very good. Then use the fibrin glue, both the component, wait for a minute and then put the bandaged contact lens. You can make a choice if you want to do a tarsorrhaphy or not. If you do, then do for two weeks temporary tarsorrhaphy to prevent the loss of contact lens.

Here I went a little too deep in the cornea. If you see here, this is corneal fibers. So you have to be careful that you don’t perforate.

The rest of the procedure is the same. You put the fibrin glue, excess membrane and a BCL. Here, if you see there’s limbus is superiorly not very healthy. This area’s not very healthy. So I am taking a slightly temporal area. You can take the limbal biopsy from most preferred areas is the superior limbus. Inferior limbus, then temporal, or nasal. Harvesting inferior, any other site other than superior is a little more difficult. You need to be ambidextrous. And you should be able to manage. This is the affected eye. And you start dissecting pannus, you will clean away from the limbus, because you have a clear definition of the cones, subtenon and the cones in this area. And then you start going towards into the clear cornea.

Remember not to remove the corneal fibers. Even if there is scars, sometimes you may need to do pachymetry, if you are not sure, take measurements there, good enough or not. I would like at least 200 to 250 micron, this is good or bad. What you’re seeing, this is a BLV membrane which I’m developing as a substitute for the human amniotic membrane. This is still under development and one day I hope that we can do SLET without amniotic membrane, without fibrin glue. Because in most of the world, these two things are not that easily available. And I’m actively working and trying to find a solution to these problems. And this is an example of a temporary tarsorrhaphy that I did in this patient for two weeks until the BCL is in place.

Now this is a video which has its own audio. And you should be able to enjoy all the steps which we have talked about so far.

[Narrator] Healthy cornea is lined by nonkeratinized stratified squamous epithelium, demarcated by the limbus from the conjunctival epithelial phenotype. The limbus contains radially-oriented fibrovascular ridge called the palisades of Vogt. Immunohistopathological studies involviing the limbus and ultrastructural imagining involving the palisades of Vogt, using in vivo confocal microscopy, have revealed that these limbal stem cells are located in a protected micro environment known as the limbal niche. It comprises of a limbal epithelial crypt, harboring the stem cells, which proliferate and migrate centrifaly to form the corneal epithelial cells.

(gentle piano music)

Chemical injuries to the eye, caused by acids or alkalis, destroy the limbal stem cells. Severe surface insults eventually lead to propagation of conjunctival epithelial cells. Limbal stem cell deficiency, either partial or total, characterized by vascularization, conjunctivalization, corneal scarring, symblepharon, and in extreme cases, inability to open the eye.

Pannus is a well-documented clinical feature. It has to be dissected and replaced with a scaffold of healthy, live limbal stem cells, which in unilateral cases can be harvested from contralateral healthy limbus. Simple limbal epithelial transplantation, commonly called SLET, is a technique first described by Dr. Sangwan, et al, for unilateral cases.

First, a limbal biopsy is harvested from the healthy eye and preserved, followed by pannus dissection in the affected eye. Amniotic membrane is applied and small bits of limbal biopsy are secured over it with fibrin glue.

We shall now demonstrate the technique of limbal biopsy. The normal eye is draped. The conjunctiva’s cut and the tenon’s dissected. The incision is extended with Vannas scissors to about two to three o’clock hours. The dissection now proceeds with a 15 number blade and the correct plane is reached with the cut limbal blood vessels start to bleed. We proceed further into the cornea, avoiding buttonholing of the tissue.

The limbal tissue is harvested with a pair of scissors at the level of the limbus, leaving a strip of tissue still attached to the cornea. The cut conjunctiva is now deposited back with fibrin glue. The limbal biopsy is placed in normal saline to prevent drying of the tissue. The different techniques of pannus dissection, one of the most crucial steps represented here. The affected eye is draped, the pannus is carefully dissected, starting at the limbus width, and then onto the cornea. 360 degree peritomy is done. The dissected area on the sclera is cauterized. Thrombin powder may be used to achieve hemostasis.

Pannus dissection is done with a 15 number blade until an appropriate plane is found. Dissection is extended with an iris respositer. Every effort must be made to find this plane. Meticulous dissection needs to be done as it has a bearing on the final visual outcome.

Ability to view the iris can be a useful marker. In certain cases, the appropriate plane may be found with simply peeling the pannus from the cornea. In severe cases, the skin too may be adherent, making it difficult to position in the speculum. In such cases, skin tags need to be cut and symblepharon release done in all the quadrants.

The speculum is now easily applied. Further dissection is continued. The bed is dried and the amniotic membrane is placed on the eye. And attached to the ocular surface with fibrin glue.

The excess tissue is neatly trimmed away. The limbal biopsy is then divided into multiple tiny bits against a hard surface like a firm block, with a thin number blade. And placed over the amniotic membrane. It can also be done with micro scissors or more often with Vannus scissors. The limbal tissue bits are arranged equal distantly in a concentric fashion, avoiding the visual axis. Fibrin glue is applied on each of the bits and allowed to polymerize over the tissue. A bandage contact lens is then placed over the eye. The speculum is gently removed and the eye is patched. Post operatively, this technique stabilizes the ocular surface with rapid epithelialization and clearing in the transplanted eye.

The donor site remains healthy, it also heals rapidly in just a few weeks. When done meticulously, this procedure provides gratifying anatomical and visual outcomes. Recent published studies across the globe have shown that SLET results, similar to long-term outcomes from our group, have been replicated by several other groups.

This novel technique is cost-effective, and can be performed as a single stage procedure at any ophthalmic institution without needing a sophisticated laboratory set up. In conclusion, simple limbal epithelial transplant is a novel single stage technique in the management of unilateral limbal stem cell deficiency with gratifying surgical outcomes.

[Virender] Now, what are the intraoperative questions that will come to your mind? Whether you should use a local or a topical or general anesthesia? For pediatric patients, or patients who are not cooperative, use general anesthesia. And the rest of everybody, I would do under peribulbar. I warn you that don’t use topical anesthesia. Even though surgery can be done, but because harvesting of the limbal biopsy is a very delicate procedure and you don’t want to tear and lose the stem cells.

When you’re harvesting the limbal biopsy, some people feel should I do biopsy first, or should I remove the pannus first? What will happen if I remove the pannus and I perforate? My biopsy may go to waste. I have been subconsciously doing the limbal biopsy first and then pannus dissection, and there’s no problem. Some of my fellows taught me that they would like to remove the pannus and are sure that everything is okay and then they harvest the limbal biopsy. And also it may give you an advantage that the tissue is not outside of the eye for a longer time. So whichever way you are comfortable, you can do.

What happens if I have perforation while dissecting the pannus? That will depend on the size of perforation. Say if it is a very tiny perforation which gets sealed with the fibrin glue membrane, it’s okay. If the eye is not soft, you can go ahead. If you detect the perforation while dissecting, you can go back and put the suture and still you can go. If it is a large perforation, which you think it won’t be sealed by usual means, then think of doing a graft. If you have harvested the limbal biopsy, I suggest you put the limbal biopsies, instead of the entire cornea, you can put on the graft host chunks in after putting the amniotic membrane.

While dissecting the pannus or biopsy, how deep you should be? I have shown you in the videos, it’s very difficult to quantify, but visual impression that you want to be is at the level of the depth of the conjunctiva and tenons. Normally, in the cornea, when you come, it should be just where the limbus is inserting into the cornea, follow that plane.

Another question, can we combine PKP or LKP with SLET? As a planned procedure, I don’t recommend. Mainly because first, do the procedure like SLET and once that is successful, then only do a PK or LKP. And now, I’ll show you further that following SLET, the need for PK or LK is very, very low chance. You may not need, in many of the cases.

If you perforate and then you have to do, then it’s okay.

Which one to do first? If you have a patient with a limbal stem cell deficiency, any patient with PK should you do SLET first or PK first, or a combined? Again, depending on the situation, my preference would be to do the SLET first and see if the procedure has worked and then only you do PK. Another rationale for not combining is surface procedure, as far as possible, don’t combine with them intraocular procedures. Because then you are opening the avenue for potential infection. If it had happened during the surgery, then it’s a different situation.

The post operative management of an autologous simple limbal epithelial transplantation would consist of topical steroids. Typically, pred acetate or equellan, every hour for the first week. And then every two hours, then make them six times a day. And the donor eye continues six times and antibiotic four times. Weekly in the donor eye, antibiotics can be stopped after two weeks. And in the recipient the antibiotics should be stopped once you remove the bandage contact lens and when the epithelium is healed. Usually you don’t need systemic medication for the SLET patients because it’s autologous procedure. You don’t need systemic antibiotics, except some patient may need some analgesic orally for a day or two.

What you can expect post operative, what do you look for post operatively? On day one, when you are removing the bandage, be very careful that sometimes the membrane sticking on the eyelid margin, you can pull the BCL or the membrane along with it. So be very vigilant, be careful. You should note the AMG with explants is present, BCL present. Eye is moving properly, there is no infection, and there is no discharge.

Discharge is expected as much as what you do in the operated eye. You may expect some fluid or a hemorrhage under the membrane, very rarely. And you don’t really need to do anything, no intervention required for such. If you find there is no AMG explant or bandaged contact lens, there’s a chance that when you were removing your drape, you have pulled it inadvertently. What you should do in that situation? Don’t panic.

What has to happen has already happened, so continue your treatment and observe the patient for a few days and keep the bandage contact lens. Very high chance that this patient will develop limbal stem cell deficiency, but to reoperate or not, I think you should wait for a few weeks. If the BCL is lost in the first few days, replace it up to two weeks. Or until the epithelium is healed. And the epithelium healing is indicated by negative Fluorescein staining.

Now the time for question. All the statements are true for SLET except? It is equally effective both in adults and children with LSCD. It is a single stage surgery. Fetal calf serum drops are required postoperatively. Expensive stem cell laboratory is not required. The question was all the statements are true except which one? Fetal calf serum, the C is the answer. Because we don’t need any of this fetal calf serum or anything.

This is a patient who had a granuloma-forming chemical burn. And this is the post op appearance and the donor eye. This is a patient preoperative on the left side and post-SLET at one year. This is two year follow up. This is three year follow up. So you can see that as the time goes by, the cornea becomes clearer. And this is demonstrated in this particular patient. So you can see this is PL positive in July 2011, total limbal stem cell deficiency. Undergoes SLET, about a month later you see there’s a lot of scarring, you see the scars there. Then in about six months later, there is some clarity. Further improvement. And at this stage, in March 2012, we offered him doing a DALK or PK, but the patient said they’d rather wait. And he was wise enough. As you wait, the cornea becomes clearer and eventually he develops 20/40 vision and cornea clears.

Why this clears? Now we have an evidence that the mesenchymal cells, which are part of the limbal biopsy, are the ones which keep working and they clear the cornea over a period of time. Now we have enough scientific evidence, publications. And we, in fact, started doing mesenchymal stem cell therapy for corneal scars and prevention of corneal scars.

Another unique application of SLET is in patients with OSSN. When you have a small, less than three o’clock hours tumor, you don’t need anything. Because they usually don’t develop stem cell deficiency. If you have more than two o’clock hours OSSN at the time of excision, you should do a SLET, otherwise you’ll definitely develop stem cell deficiency. As seen here in this picture, case two on top.

Now, this is another example of doing an excision of large OSSM, combining with primarily SLET, an excellent outcome. And this now has been published by Dr. Swathi Kelki from LVP in AJO, a couple of years back.

This is pediatric limbal stem cell deficiency, a case published by Vikas Mittal from Ambala. And you see this in 2010, LSCD, this is on top, is the face pictures and bottom is the eye picture. And you can see how the eye or the cornea clears over a period of time. This is a patient done by us. A two-year-old boy with a toilet cleaner. Underwent a SLET in the acute case and I would recommend if you have a child, do a SLET in the acute case. Don’t hesitate, you will save the eye and you prevent amblyopia.

And this is the study that we had published of 125 eyes in “Journal Ophthalmology” in 2016. Where we showed a well-defined criteria for success and failure. We showed that the success rate in adults and children are exactly the same. And remember I told in CLET, we are not able to achieve this. Number two, the visual acuity improved significantly in both the groups. And number three, the graph is there in the paper, but not here, that the technique, the learning curve is not very difficult. We compared the outcome of cases done by me and my junior faculty and by our fellows. And surprisingly, all three groups did very well.

This is the immunohistochemistry of excised corneas or few corneas in the initial stage when we had to do a PK or a DALK, and it shows the phenotypic restoration of corneal phenotype after the SLET.

And these are further replication of these kinds of results by other groups. This is a multi-centered trial, wherein people from different countries got together and shown the outcome which we have shared in the paper in “Ophthalmology.”

And another interesting point is that this technique can be modified by different surgeons based on what they think, for what indication. Someone described a customized SLET or mini-SLET for pterygium, or a sandwich technique for LSED.

Now, if you look at this summary, our first paper was published in 2012. To date, more of them in the peer-reviewed literatures, there are 450 patients who have received SLET. And based out of from eight countries. There are 30 publications of SLET as of two months back, and for the last five years there have been no single publication about CLET. We have done several workshops to teach surgeons from different countries and these are the graphs showing the cases and the publication over a period of time.

And this is the comparison of three techniques, CLAU, CLET and SLET. And if you look at the success rate in anatomical and visual gains are very comparable. Very, very much comparable in all techniques. So it is just the safety of which technique and which is affordable. When we compared the three different techniques on these seven parameters, what turns out is SLET is better or equal in most of these parameters of safety and efficacy. And affordability and repeatability.

And this is the summary of CLET outcomes. You will notice that most of this work is done by a group in Italy and another group by us in India. Other players are very small number of publications or small number of eyes.

This is a very important slide where you are seeing what I mean that it’s cost-effectiveness. This is the recent data which is yet to be published, where we are seeing the cost of SLET in India, in LVP, the answer of is $700. Cost of CLET is $7,000. Similarly, in Europe and now in U.S. also, a Holoclar-based technique or CLET-based technique called Holoclar, which costs about 85,000 euro. So obviously, such an expensive treatment cannot be afforded, even if the government pays for it.

Now, just a summary of differences. I’m sure there’s repetition, but it’s worth it. The SLET is a single step procedure, CLET is two step. It’s more expensive, not replicable because there’s hardly any groups who have replicated this procedure. There’s no risk of contamination in the SLET. No culture related, no secondary biopsy required. And the long-term outcomes, modifications, we are taught all of them.

The question four. For autologous SLET, what is not indicated in post operative period? Remember this is autologous SLET. Topical antibiotics and steroids, bandage contact lens, systemic immunosuppressive therapy, oral analgesic an and when required. So the question, what is not indicated? Lovely. Very good, audience, you got it! Thank you very much.

So now let us quickly review what is the mechanism of limbal regeneration post-SLET. This is, you have seen in that video, and this is a beautiful work published by Vikas Mittal, of amazing view of his patient, every other day, removing the contact lens, staining and you can see that clearing or epithelialization is indicated by no staining. And in about two weeks, you get almost complete healing. And this you can compare. When healing pattern is very similar to the pattern of cell growth in CLET. On the left hand side panel, this is the cells growing in the lab from his explant, and correspondingly you can see the similar pattern in SLET in vivo in human eyes. And in about day 10 to 14 days, roughly, you get monolayer. And then the stratification happens.

If you compare the healing pattern with the CLAU, which is from Dr. Kenyon and Tseng’s paper in “Ophthalmology” in 1989, you see the central cornea which is the most critical part heal the last. You’re likely to get scarring, you’re likely to get VED. While in the SLET, each explant is like a mini-limbus. There’s multi-directional epithelial growth. And all of the cornea, all parts of the cornea equally heal fast. And there is no lag.

How does SLET work? Each of these implants or explants have growing cells all around. MSCs which are part of the explants are success for contributing to the success and their exosomes secreted by mesenchymal cells. The limbal explants, in SLET they stay longer on the surface and they continue to show as a mother cells clearing and sending the epithelial daughter cells. And these implants are like mini limbus with all cellular and ECM elements. Extracellular matrix elements.

This is another amazing study we did, still not published, but high definition AS-OCT of these patient serially. If you look at on day one, day five, day eight, you see the flattening of these explant or transplants or whatever you call implants. And as you progress on day 11, you can see the epithelialization and the amniotic membrane thinning and start disintegrating. And in about six weeks, you see stratification. In six months you may not be able to identify. There may be a rare patient who still have an explant. And in this patient you can make out there is a white spot. And this is compared with a normal cornea.

So in summary, the SLET is an effective procedure which restores corneal surface and improves vision. It is replicable because it has been reported by other groups. It is repeatable, it is affordable, eliminates the needs for stem cell lab or no regulatory requirements. It’s reliable, it works very well in children and adults, and there are further applications like in OSSN, recurrent pterygium, persistent epithelial defect, acute chemical burns. And open platform technology. It is not owned or protected by a patent. Therefore, surgeons can innovate depending on the situation, depending on the indication, what they are encountering.

Thank you very much for your kind attention.

The first question, got a 6 ,7-year-old patient with conjunctiva on the lower side of the cornea due to distichiasis. Should this patient be perfect for start and also in Indonesia we only have a dry amniotic membrane, can it work? Well, I think in this first case, I would suggest that you first try to rectify the distichiasis. And if it is due to SAS or if other problems, then this technique is not good. So understand why there’s stem cell deficiency. And dry amniotic membrane, probably is okay if you have the glue.

Anonymous attendee, “Thank you for the excellent lecture. Why is the absence of symblepharon a prerequisite of SLET?” The reason it’s not a prerequisite, but it is easy for people who are learning. Because once you have symblepharon that means there is a more serial problem and you need to be skilled enough and maybe you need to do surgery in stages, rather than combining. What you saw in my hand, I had been doing it for almost 25 years and I am quite okay. But many of complex cases I also do in a staged manner.

Dr. Uslove, “So all cases are not recommended, except after traumatic or chemical injury?” By and large, it’s correct. Only for the beginners. What I said for beginners, start in chemical burns. Aniridia, GVSD, OCP, Stevens-Johnson, all these kind of cases that you should take once you are very comfortable and familiar.

“Can we use femtolaser to get through the pannus?” Well, this is like you can, if you have this. But I have not tried, so I don’t know. If you’re able to, please let me know because it might be a very good quantified way of cutting this.

“What are the prognosis of this technique?” Prognosis, as I’ve shown you, is excellent. About 78 to 82% success rate and now we have five year followup as well.

“Please repeat the harvesting of donor tissue once more.” Well, I can save some of the videos and maybe Cybersight will have this lecture, so you can watch these videos or you can reach me through Cybersight about any specific request. I’ll be happy to share more videos or answer more questions.

“When do you do pannus dissection? How does the cones come back?” As I’ve shown in the videos, you start the pannus dissection from periphery, you remove the cones comes back from all 360 degrees, which we have cut margin. From there the cones come back and by then the limbus and the cornea has healed so it prevents the cones coming over to the cornea.

“What about the orientation of donor pieces?” Well, don’t worry about orientation. Epithelial side up or down, I don’t think it matters. Even though we have written in the paper that the epithelial side should be up, it really doesn’t matter.

“Do you recommend SLET for large pterygium as a primary?” Yes, I do. I recommend SLET for a pterygium but for the conjunctival part, I will still use a conjunctival autograft, if there’s a large pterygium or double headed pterygium.

“What is the success rate of allo-SLET?” Allo-SLET I have done fairly okay and I would say success rate with good immunosuppressant is excellent. Like CLET, allo-CLET, which we have published several years back in AJO, success rate at five year was 72-75%.

“After injury, how much time interval is needed for SLET surgery?” I would wait until the epithelium heals or do the amniotic membrane transplant acute phase. If it’s a pediatric eye, then you can do the SLET. Or you can also do cadaveric SLET where you don’t need immunosuppressant for epithelialization and you can do along with the amniotic membrane.

“The implanted stem cell on top of amniotic?” Yes. Because the amniotic membrane provides the scaffold on which the cells grow. You can put directly on the cornea, I have not done so. And I think the explants might move and it’s just the amniotic membrane. Since I have been growing the cells on the amniotic membrane, for CLET, I felt that using replicated the same technique in the eye would be a good thing to do. That’s why I have not attempted the explants on the cornea directly without an amniotic membrane.

“How many pieces are needed for the cornea?” 10 to 12 pieces on average.

“How long should the contact lens?” About 10 to 14 days, until the epithelium heals. Steroids I would do a slow taper over six to eight weeks.

Success rate of SLET is about 80% in chemical burns.

“After gluing the amniotic membrane, you put the stem cells?” Why do these keep moving? Yes, you will put the fibrin glue over each of the explants.

“What is the success rate of SLET as primary,” we talked about. “How long does it take to complete whole procedure?” In my head it takes about 30 to 40 minutes.

“Side of graft that should be up?” It really does not matter which side up.

“We prepared amniotic membrane ourselves in our eye bank.” And that I have been doing in the RPI eye bank as well.

“I have been doing allogenic SLET?” Yes, Farad, I have been doing allo-SLET for quite some time now.

“Can SLET be used for corneal scarring without LSA?” No, I don’t think that’s a very good idea. Mysochiamel cells yes, they can. But in the Mitchell scar they may not also work. In the acute stage, yes.

“In case of chemical injury with corneal mal, granuloma, would you do a stepwise approach?” Yes. I would be step wise.

Lars Dermoid, I would do SLET definitely in that area of the corneal and limbal side.

“How many times SLET can be take?” Okay, I did not understand the question, but I have done SLET two times, three times and in case of SLET and CLET I have done even four times.

The amniotic membrane side, the epithelial side of the amniotic membrane is up, the stromal side is down towards the cornea.

“Please repeat the steps, why the amniotic membrane side up?” Because the epithelial side is up because putting the explant, manipulating them is much easier while the sticky side you want on the cornea so that it sticks nicely on the corneal surface.

“What is the time it would take for us to learn this technique?” Anders, it depends on your program. Anyone who’s familiar with the surface cells can be taught this technique.

“Can SLET be done without AMG?” Well, you can try, I have not done, but if you have the fibrin glue and then maybe try? I have not done, but I am developing a synthetic or a bioengineered BLV membrane with a company in the U.K.

“Is suturing with a graft and alternate fiber is not available?” The amniotic membrane you can suture, it’s very easy, I can show you or share this video if you want later on, or through the Cybersight portal. But if you don’t have fibrin glue, maybe use the whole blood. And use these blood drops as a part of the glue and allow them to clot and then put the amniotic membrane. That might work, I have not done myself.

Mohammed Syed, “Thank you very much. Is there any limit of interval after tumor doing SLET? Generally if the other eye’s okay, and if the patient is not pediatric you should wait for at least six to eight month. Until the surface is not inflamed and healed completely.

Yes, I answered that. “What can we do SLET in old chemical.” I have done SLET as old as 20-30 years post chemical burns. And my oldest patient of CLET was 40-year-old chemical burn in both eyes.

“In acute stage, what do SLET?” Well, I’m not talking about in acute stage AMG. I prefer AMG alone. But there is a paper and an experience by Dr. Guitai from Center Italy, where they used cadaveric SLET as a procedure for epithelial healing. Sometimes the amniotic membrane, the acute phase, disintegrates very rapidly and you need to repeat very quickly. So for that reason.

“If there’s no fibrin glue available, where do you recommend to use for suture?” Well, Herbert, if you don’t have the fibrin glue, I suggest you use the whole blood, rather than suturing these expanded. Could be very technically difficult.

“Any particular donor tissue orientation?” Donor tissue orientation really does not matter. And I would not pay much attention.

“Mucous membrane grafting?” Mucous membrane grafting I generally reserve for patients who have ditmars and keratinization. Or symblepharon which is not amenable with cones or you don’t have enough cones.

“All cases use fibrin glue before AMP?” Fibrin glue is essential for sticking the membrane to the ocular surface. But not absolute essential. You can use the sutures, sten or monofilament sutures. And then for sticking the explant, the fibrin glue’s required. Because I don’t know what other way you can stick the explants on the membrane.

“In large strabismus you take the donor from the same eye?” Yes, if there’s two to three o’clock hours limbus available I will be happy to take. And we have a nice paper published about ipsilateral SLET and ipsilateral CLET.

“What can be used in place of amniotic membrane?” At the moment, I really don’t know. Because I don’t have a synthetic membrane. If you don’t have the membrane, maybe try cleaning the pannus and use the explant directly on the cornea and use fibrin glue. If you don’t have fibrin glue try whole blood. Patient’s own blood.

“Can we use amniotic membrane rather than pieces?” That’s absolutely fine. That is what Guillermo Amescua from Bascom Palmer did. He was scared that the explant will fall so he used another membrane on top of the explant. That’s perfectly fine and it works very well.

“Does MMC application interest give advantage?” Clearly not necessary, unless you are very severe symblepharon, Aziza. You have symblepharon fibrosis, you can use MMC and wash it completely and that should be fine.

“Have you experienced any complication when you revolve the BCL?” Not really. Because BCL comes generally free, there’s no adhesion, there’s no sticking. And there’s no problem.

“If it is failed during that, can we repeat SLET?” Yes, you can. But take the biopsy from at least two-three clock hours away from the initial place of biopsy.

“Can we use industrial glue?” Muhammad, not a good idea. Industrial glue or what we use in the construction industry is very toxic and the stem cells will not tolerate that.

“How to get amniotic membrane, if we’re allowed?” It’s very simple, those of you who don’t have the amniotic membrane, please contact me through Cybersight. I’ll share my video of how to prepare the amniotic membrane in any situation. And in India it is available commercially from different sources.

“Which is placed first, AM or limbal stem cell pieces?” As I showed in the video, after cleaning the pannus, first do the gentle cautery, achieve homeostasis, then amniotic membrane and fibrin glue. Cut off the excess membrane, then the limbal pieces on top of the amniotic membrane, and then fibrin glue again, and BCL.

Maria is asking, “When you put glue over the BCL, don’t get?” No, they don’t stick. That’s why, after putting the explant and then the glue, wait for about a minute. And even if you don’t wait, fibrin glue is wet. The contact lens will not stick because it is plastic and it’s very smooth. You do not, the glue does not stick to the contact lens.

“What happens to a transplanted amniotic over time?” The amniotic membrane gets disintegrated by the enzymes in the tear film and the leukocytes present in the tear film.

In rare cases you may get integrated on the cornea but not very often. And that was a paper published by Harminder Dua several years ago in “Ophthalmology” that he found in some cases the amniotic membrane collagen in the cornea.

“If we don’t have fibrin glue, can we put the donor stem cell first and then stick the explants on?” Yes, that may be a good idea. Yes. I think that might be a good idea, actually. That if you don’t have the fibrin glue, you could put the fibrin glue directly on the ocular surface, I mean the explants. Then use blood and then amniotic membrane on top and you can suture the amniotic membrane. Please try and let me know if it works.

Sahtik Bafna, “Is there any chance to do procedure without amniotic membrane.” Just talked about, yes. Thank you mother Smitha. Paulo Denta, “I’m a huge fan of your work.” Thank you very much for your kind words.

“How do you compare SLET with CLET in long-term ocular surface?” Well, just shown you the only problem with CLET is the amount of tissue you take. Other than that there’s no problem. And if you’re happy with the CLET and the results, then you really don’t need to do SLET. Because the amount of tissue you are taking in SLET is considerably less. And in my videos you may have realized that I am still taking much more than what’s needed because these are old videos. Now I just take a very tiny amount.

“Do you have any specific plan for the simple limbal cell pieces? How about central perimeter?” Yes. Very important question that I did not touch up on. So let us understand. If the central perimeter of the cornea is very clear, then you should not put the explants on the visual axis because the opacity and the presence of the explant can distort visual acuity. So I will avoid those and distribute only in the mid-periphery and just about two or three millimeter inside the limbus and not the central cornea. If there is scarring or the cornea is not clear, then don’t worry about it.

Tali, “Thank you so much.” Thank you, Tali.

“Would you recommend using SLET for failed PKP?” Yes, I would. Failed PKP, if there is a pannus. Failed PKP with normal limbus, I’ll do a PKP again.

And thank you, everyone.