Since in 2002 we introduced Descemet-endothelial keratoplasty (DEK or DMEK)^1,2 and in view of the good visual and anatomical results obtained, we have standardized the technique^3-5 to facilitate its reproducibility and learning for novice surgeons who want to start with it. In this chapter we describe our standardized "no-touch" technique, along with other additional techniques to facilitate the unfolding of the graft in certain situations⁶.

CARE PRIOR TO SURGERY

Before starting surgery, it is important to make sure you get a hypotonic eye to avoid intraoperative complications. For this, we position the stretcher in anti-Trendelenburg position and, after the retrobulbar injection (4-5 ml of ropivacaine 10 mg/ml, mixed with 150 i.u. of hyaluronidase), we perform a manual ocular massage for 2-3 minutes, followed by other 10-15 minutes with a Honan’s balloon. Two weeks before surgery, we perform a superior iridotomy using Nd:YAG laser to all patients, to prevent increases in postoperative intraocular pressure^3,4.

INCISIONS AND DESCEMETORHEXIS

We first mark the main incision of 3 mm wide at 12 o'clock in the limbus and perform three paracenteses at 10:30, 1:30 and 7:30 hours (right eye) or 4:30 hours (left eye) with a scalpel. Then, with a 30 G cannula, we completely fill the anterior chamber (AC) with air and with an inverted Sinskey’s hook (DORC International, Zuidland, The Netherlands) pre-mark Descemet’s membrane (DM) in its 360° and we perform the descemetorhexis, sometimes with the help of a DM scraper (DORC)^1-4. It is important to ensure that no fragment of DM remains in the recipient, which could compromise graft adhesion or visual results. Once DM is detached, we tunnel the main incision to a length of 3 mm, through which we extract DM. To avoid the hydration of the stroma, we leave the AC filled with air until inserting the donor tissue.

PREPARATION AND INSERTION OF GRAFT

In our center, the DM-endothelium disc (DME) for the graft, 9.5 mm in diameter, is prepared in the eye bank one or two weeks before surgery and stored in a glass vial with culture medium (CorneaMax, Eurobio, Courtaboeuf, France) at 31°C⁵. During the intervention, we pour the contents of the vial into a glass container and with a glass pipette (DMEK Set, DORC) aspirate the culture medium and rinse the DME roll with balanced saline solution (BSS, Alcon) to remove the remains of the culture medium. Then, we dye it with 0.06% trypan blue (VisionBlue^TM, D.O.R.C.) for 1-2 minutes, then rinse it and re-dye it to ensure good coloration. By direct flow of BSS over the tissue we try to create a "double roll" to facilitate its unfolding in the AC. Next, we aspirate it in a glass injector of our own manufacture (Melles’ Injector, DMEK Set DORC), perform a complete exchange of air-BSS in the AC and insert the graft by pressure in the injector (Figure 1)^3,4.

DEPLOYMENT OF THE GRAFT

Once we have the graft in the AC in the form of a "double roll" of DME, we can deploy it by small touches on the surface of the cornea between the two curls. Then we check its correct orientation – with the curls facing the corneal stroma – by means of the Moutsouris’ sign (Figure 2)^3,4. If it is inverted, we will rotate it by soft irrigation with BSS through one of the paracenteses. Once properly oriented, we can inject a small bubble of air between the two rolls and enlarge it little by little to continue the deployment of the graft. This would be the standard "no touch" technique (Figure 3)^3,4,6. In cases where this does not occur, special techniques may be applied:

a) Dirisamer’s technique. If it is not possible to obtain the "double roll" or if the graft has a greater tendency to roll up again – it is "tighter", something more frequent in young donors – we can open it by means of two parallel cannulas on the corneal surface (Figure 4), one that fixes one end of the graft and the other unrolls the rest.

b) Dapena’s maneuver. It is also possible to unfold a "tight" graft by mobilizing the air bubble, through indentation of the corneal surface with a cannula (Figure 5).

c) Sliding technique with a cannula. On the other hand, "looser" grafts – what occurs more with elderly donors – that tend to open when injected in AC, can be unwound using a single cannula without injecting air (Figure 6)⁶.

CENTERING AND FIXING THE GRAFT

As the DME disc is opened, we center it by small touches with a cannula in the limbus or with BSS. If it is not possible indirectly, it is possible to center it manually with a cannula. Once unfolded and centered, we enlarge the air bubble in front of the graft to flatten it against the iris. We aspirate the air immediately and inject a bubble behind the transplant to elevate it and position it against the receiving stroma. In case there is a fold in the periphery, we will flatten it by indentation of the cornea with the cannula. To obtain a good fixation of the graft we keep the patient 60 minutes with the AC filled with air at about 20 mmHg. After this time, we perform a partial air-BSS exchange, leaving a 50% bubble in pseudophakic patients or 30% in phakic patients^3,4.

PREPARATION OF THE RECIPIENT

The Descemet-endothelial keratoplasty technique (DEK or DMEK) (video 7.2.2.1) begins with the preparation of the recipient. An incision of 3.2 mm at 300 μm depth is made in the temporal transparent corneal limbus, plus two paracenteses in the upper and lower quadrants (Figure 1). In pseudophakic eyes, the pupil contracts with acetylcholine (Miochol, Bausch & Lomb, Rochester, NY). In combined procedures ("triple") of DEK plus cataract surgery the pupil is dilated with minimal cycloplegia (or none) and phacoemulsification and intraocular lens implant are performed through the same main incision that will then be used for the DEK. For all the maneuvers of this phase, the anterior chamber (AC) is filled with a cohesive viscoelastic (Healon, Abbott Medical Optics, Santa Ana, Ca.).

A peripheral inferior iridectomy is performed with a 30 G needle whose bent tip is passed through the pupil under the iris to the peripheral position, and with a Sinskey’s hook (Bausch & Lomb Surgical, St. Louis, Mo.) is scratched on said point to make a hole (Figure 2). This is extended by stretching it with these instruments and its edge is cut with intraocular scissors, removing some stroma and pigment of the iris to ensure its permeability.

The AC is reformed with cohesive viscoelastic, and a central area of ​​8.0 mm in diameter is peeled off and removed from the Descemet’s membrane (DM) (Figure 3). Peripheral scraping of the stroma is not performed, but care should be taken that there are no fragments of the DM of the recipient in that area. The viscoelastic is removed by mechanized irrigation-aspiration. Myosis is induced with acetylcholine and the intraocular pressure is normalized.

PREPARATION OF GRAFT

The microscope is brought to the donor's table. The cornea has been processed by the technicians at our local eye bank (Lions VisionGift, Portland, OR) by pre-dissection of the DM-endothelium (DME) of the donor stroma, leaving a hinge of 10% to maintain the DME stable and bound in the transport vial filled with Optisol (Bausch & Lomb). The position of the hinge is identified with a notch that the technician practices in the sclera, who also marks an "S" with dry ink on the stromal side of the DME, to avoid that a graft put in reverse goes unnoticed before fixing it with the gas bubble.

The graft is stained with trypan blue (VisionBlue, Dutch Ophthalmic USA, Exeter, NH) to also identify its edges. The donor corneoscleral cap is placed on a die-cut base (Moria, Doylestown, PA) with the endothelial side up and centered according to the edges of the DME flap (Figure 4). The trephine-die is applied – usually 7.50 or 7.75 mm in diameter – under direct visualization and the DME is cut with alternating pressure and percussion. The membrane portion peripheral to the cut is removed. The graft is coated with balanced salt solution (BSS) and taken gently with a pair of forceps to complete the dissection in the hinge area. The concavity of the corneoscleral cap is filled with trypan blue and the tissue is deposited again in said dye bath for 4 minutes for its staining.

The injector is prepared with 15 mm of a French single lumen nasogastric catheter #14 (Covidien, Mansfield, MA) as an assembler. One end of that catheter is attached to the Straiko-Jones modified glass tube (Gunther Weiss Scientific Glassblowing, Portland, OR) and the other one is attached to a 3 or 5 ml syringe, both filled with BSS. The effective action of the injector is tested, first with the tissue previously peeled from the donor, with maneuvers of injection and aspiration of the same in a Petri dish filled with BSS.

After these 4 minutes, we verify that donor tissue is well stained by means of progressive infusions of BSS that dilute the bath of trypan blue and the excess of liquid is absorbed with surgical sponges. Once the tissue, a deep blue, is easily visualized in the almost transparent solution, the tip of the Straiko-Jones tube is immersed in the well with the bevel facing upwards and the tissue roll is sucked up to a just proximal position to the tip. The injector is brought to the operative field.

INSERTION, DEPLOYMENT AND FIXING OF THE GRAFT

The pressure in the AC is reduced to almost zero and the tip of the injector is inserted in the AC through the 3.2 mm incision. The tip is rotated to ensure that the tissue will be injected in the correct direction. We inject the tissue and through the paracentesis liquid is released so that the pressure drops, the AC narrows and captures the tissue. We remove the tip of the injector taking care to avoid the tissue following it towards the outside, by means of external compression with a cannula on the central incision to the tip (Figure 5). The incision is closed with a single independent nylon 10-0 suture.

Short jets of BSS are used to move the graft and check the orientation of the roll (Figure 6). The AC remains very narrow during deployment. We deploy and center the roll using a modified Yoeruek’s percussion technique, based on small blows on the corneal surface and limbus that generate waves in the fluid and move the tissue. Short flows of fluid through the paracentesis or the main incision are also allowed to escape selectively to manipulate the tissue.

Once deployed, the tissue is raised to its position by a slow injection of 20% sulphur hexafluoride gas (SF₆), with a 27 G cannula through a paracentesis on the surface of the iris and under the donor DME. Care must be taken not to inject until the tip of the cannula is visualized below the center of the graft and the eye looks straight ahead. At first, we inject only a bubble of 8 or 9 mm. Once it has been verified that all the edges of the disc are unrolled and safe, we completely fill the AC and pressurize it with SF₆ at 20%. We leave it like this for a few minutes and, if necessary, suture with nylon 10-0 any paracentesis that is not hermetic. Then some of the gas is let out and the AC is reformed with BSS to leave a bubble of 80%, so that it does not cover the inferior iridotomy in the supine position.

THE POSTOPERATIVE PERIOD

A 12-hour dissolution collagen shield (Oasis, Glendora, CA) impregnated with antibiotics and corticosteroids is applied on the cornea. We leave the eye bandaged until the next day. The patient is kept for one hour in the supine position in the preoperative area after which he is sent home. Since the gas bubble does not cover the lower iridotomy, the eye is not examined before discharge. The patient is instructed so that, once at home, he remains lying in the supine position as long as possible, although he can be incorporated during periods of 20 minutes for meals and grooming. He is allowed to sleep that night in whatever position he is comfortable. The patients are controlled on days 1, 6, 14 and 30 of the postoperative period. Our bubble reforming rate is less than 10% and that of primary graft failure has been reduced to zero since we use the "S" mark to avoid inverted grafts. The average endothelial cell loss at 6 months is 28% and more than 75% of eyes without comorbidities reach a vision of 20/20 or better.

I have been doing Descemet-endothelial keratoplasty (DEK or DMEK) since 2008, and over 2000 cases later, I have a technique that works very nicely for me. However, the technique continues to change and evolve, therefore the title: “My Technique as of May 2016”. I should also add that the technique I use varies depending on the case, and the method I will describe will be for an uncomplicated case (video 7.2.3.1).

PREPARATION OF RECIPIENT

Over half the cases we do now for Fuchs’ Dystrophy are combined with phako and IOL implantation. In those cases, we use the same 2.8 mm incision for both phako and graft insertion. There is no reason that we could not be using 2.2 mm incisions, except that I already have a number of 2.8 mm diamond blades. Once the cataract surgery is completed and the IOL is in position, we strip the recipient Descemet’s membrane (DM) with viscoelastic in the anterior chamber (AC). After stripping we use the irrigation-aspiration (I&A) mode to remove the viscoelastic from around the IOL and AC, and then also vacuum off the posterior stroma in the area of stripping to minimize postoperative haze or difficulties with the graft sticking.¹ Miostat™ (carbacol) is irrigated into the AC to constrict the pupil. Trypan blue (VisionBlue™) is next injected – this is essential to help identify any residual tags of DM that were not removed or loose stromal fibers that may need to be trimmed off –. I use a bimanual I&A to remove the tags of DM, as well as to provide irrigation while I make an inferior iridotomy with 23 G intra-ocular scissors. The I&A is then used to verify a patient peripheral iridotomy (PI) and to remove any pigment epithelium from the posterior iris that could block the PI. The wounds are hydrated (the keratome incision and the two stab incisions 45 degrees to each side of the keratome incision).

PREPARATION OF THE DONOR

We prepare all of our own donor tissue either on the day of surgery, or one or two days before. Our preferred donor preparation technique is a modified SCUBA technique², in which we strip the entire donor DM off except for a small central area. The donor DM is trephined, usually at 8mm. Then instead of placing the donor tissue on the corneoscleral rim back into the storage solution to totally strip it off, we now stain it with trypan blue, decant off the extra dye, and then place it back on the cutting block. On the cutting block we use the trifold technique described by Massimo Busin.³ We then completely detach the donor from the stroma, pull it into the cartridge of an IOL injector, fill the tip with storage solution, then plug both ends of the cartridge, and load it into the IOL inserter.

INSERTION, DEPLOYMENT AND FIXING OF THE GRAFT

Unlike Busin, I just inject the donor into the eye and do not typically pull it in (Figure 1a). Once it is in the eye, I place a 10-0 nylon suture in the keratome incision, lightly fill the AC with Balanced Salt Solution (BSS), and verify the orientation of the donor DM either with intraocular OCT or a hand-held slit beam. If not oriented correctly (Figure 1b), I use BSS to gently flip it over, and then recheck the orientation (Figure 1c).

Once it is correctly oriented, I slightly shallow the AC. The tissue is flattened and unfolded more by pushing on the cornea with a cannula tip, and a small bubble is placed under the donor (Figure 2a). The donor is then unfolded with gentle taps and pressure application to the corneal surface (Figures 2b,c), often with moving the eye in different directions to help control the positioning of the air bubble. Next “golf swings” with an irrigating cannula are used to position the donor into the exact area where stripping of the recipient was performed (Figures 3a,b). Next a 90% air fill is applied to push the donor up against the recipient (Figure 3c).

THE POSTOPERATIVE PERIOD

The patient is sent to the recovery area for one hour, and then checked in our clinic to make sure that the PI is patent, the IOP is not too high, and that there is a fluid meniscus over the PI. If all is OK, then the patient is released. If not, then some air is removed if the air fill is total. If the IOP is elevated but the air fill is correct, and fluid is over an intact PI, then topical glaucoma drops are applied. Rarely a paracentesis might be done for high pressures not controlled with drops.

Under what circumstances does this technique vary?

In pseudophakic eyes we do not use viscoelastic in the AC; we merely remove the recipient DM with air filling the anterior chamber. No Miostat™ is used, but we still use VisionBlue™. These patients receive preoperative pilocarpine 1% to constrict the pupil during surgery. For eyes remaining phakic, we strip DM with viscoelastic in the AC, essentially the same as the procedure described above.

Young donors less than 50 years of age, and occasionally some in the 50’s, have donor DM that curls very tightly. In those cases, we typically uncurl the donor with a very shallow AC and it can still be challenging. We still verify the correct orientation with either a slit beam or intraoperative OCT before completely unfolding the tissue. Rarely will I use a bubble on top of the donor to help it unfold unless the donor is in the 30’s, or in some cases when the recipient has had a PPV and scleral fixated IOL with no tendency for the AC to shallow.

The most challenging part of DEK (DMEK) surgery is often the post-operative period. An eye with an irregular posterior stromal surface, such as a previous penetrating keratoplasty, may require repeated air injections to get the graft to fixate around and over the old graft wound. Eyes with glaucoma filters can also require repeat air injections if the eye is either soft or the air quickly escapes into the filter. Air can occasionally cause blockage of a trabeculectomy with elevation of the IOP. In those cases, air may need to be removed earlier than usual.

All of our cases are done with topical anesthesia and intravenous sedation. We do not use blocks or general anesthesia. We have never had posterior synechia occur in our cases, although I have been told that can happen with use of long acting gases other than air and with miotics.

In our center, we use an original bimanual technique to introduce and position the Descemet’s membrane and endothelium (DME) graft for Descemet-endothelial keratoplasty (DEK or DMEK)^1,2.

PREPARATION OF GRAFT

The donor DME can be prepared from the day before to minutes before the surgery, as is usually our case. One can also use dissected tissue from the eye bank. We use the technique described by Kruse et al³. The corneoscleral segment is positioned at the base of a Barron’s tread-punch. The cut is marked and stained with 0.6% trypan blue (VisionBlue^TM). The most frequent diameter is 8.0 mm, but it can vary according to the characteristics of the recipient. To access the margin of the DME, a strip of tissue between 1.0-1.5 mm is removed outside the 8 mm mark with a 45° knife. Next, the proximal margin is raised with a blunt knife and peeled using forceps without teeth (Figure 1). Once the peeling is complete, the graft is cut with the trephine and extracted bimanually with the same forceps. The DME disc folds spontaneously in a roll form, and we dye it again with trypan blue.

Finally, the graft is placed in the cartridge of an injector for 1.8 mm (Medicell ViscoJECT) for intraocular lenses (IOL) (Fig. 2), filled with balanced saline solution (BSS), and advanced gently with a 25 G silicone tip (3221 FlexTip) cannula. A small air bubble is injected behind the rolled graft, whose purpose is not to assist in the deployment and orientation of the DME but to reduce the surface tension between it and the injector and prevent it from adhering to the plunger. No type of viscoelastic is used, since it can reduce the adherence of the graft to the posterior stroma.

PREPARATION OF THE RECIPIENT

Under retrobulbar anesthesia, the corneal epithelium is removed in those areas where it is edematous, to facilitate visualization and also to improve epithelialization after the intervention. We mark the limbal incision of 2.8 mm wide at 11 o’clock and perform 3 paracenteses of 25 G at 10, 6, and 1.30 o’clock. A 25 G beveled infusion cannula is connected through it, connected to the Constellation^® pump (Alcon, Fort Worth, TX, USA). This allows a continuous control of the air pressure or of the infusion liquid necessary in each step of the surgery. To extract the recipient’s DME, air is injected at 20 mmHg.

A central descemetorhexis of 9-10 mm in diameter is made – to be 1-2 mm larger than the donor disc – with an inverted Sinskey’s hook (Price Endothelial Keratoplasty Hook, Moria SA, France), followed by complete detachment of the DME with the same hook or a DME scraper. It is important to carefully monitor the progress of the descemetorhexis to ensure that no DME fragments are left in place, as this could limit the final visual acuity (VA) and/or adhesion of the graft to the stroma. Performing this maneuver under air improves the visualization of the DME and facilitates its complete elimination (Figure 3). Once the DME is completely dislodged, we perform the 2.8 mm tunnel-borne corneal main incision, extract the DME and replace the infusion of air with liquid, also at 20 mmHg. Finally, we inject acetylcholine and perform a peripheral iridectomy through the 6 o’clock incision with vitreous-retinal surgery forceps (Figure 4).

INSERTION AND POSITIONING OF THE GRAFT

The most complex and crucial step of the DEK (DMEK) is the insertion of the graft and its positioning in the proper orientation so that it adheres correctly to the stroma of the recipient. If the endothelium is facing the stroma, the graft will be dislodged in the early postoperative period, even if initially it is attached.

First, the infusion fluid pressure is reduced to 5 mmHg to prevent the DME roll from exiting through the incision after its injection due to reflux of fluid. The first 3 mm of the tip of the injector are introduced into the anterior chamber (AC) through the main incision. The DME is then injected gently. It is important that the incision allow some leakage of the BSS to prevent the AC from becoming too pressurized as this will push the roll back into the cartridge or then eject it from the AC. Once the roll is inserted (Figure 5) and before removing the injector from the incision, the infusion cannula is removed. This step is essential to prevent the graft from being expelled. The incision is sutured with 1 to 3 10-0 nylon sutures to ensure that it is tight and maintain an adequate AC amplitude in the postoperative period.

Next, a Gills’ cannula connected to the mechanized irrigation-aspiration system is introduced through the main incision to maintain the stability of the AC. The infusion flow must be low to center the graft while maintaining proper orientation with the rolled edges facing up. Frequently this step is not necessary since some touches on the cornea may be enough to open a properly positioned DME disc. When the corneal transparency allows it, we place a 30 G cannula above the DME (Figure 6) and under one of the peripheral curls to check its orientation according to the Moutsouris’ sign (see chapter 7.2.1). If the graft is inverted, we rotate it by means of a soft irrigation with BSS and check its orientation again.

When the donor is very young and tends to create very tight curls, some surgeons gently inject an air bubble into the DME roll to unroll it. Once correctly positioned and deployed, the infusion is maintained at low pressure and a cannula is inserted through the paracentesis on the left. An air bubble is injected slowly so that the DME disk is left open and applied to the iris. The proper centering of the graft is checked again and the bubble is removed in front of the graft, as well as the infusion, and a relatively narrow AC remains.

Currently we only execute this step with very young donors; in the rest we perform soft touches on the cornea with a cannula – like those described by the group of Melles et al⁴ – until the complete deployment of the graft and its proper position is achieved, without the need to inject air (Figure 7). Those transplants that are slightly off-center but cover the central corneal area are acceptable, since if there is a space between the graft and the DME of the recipient, it will be repopulated by migration of endothelial cells from the donor or recipient. Decentering does not seem to be related to the final VA, so excessive manipulation of the graft should be avoided if centering is not perfect.

Finally, we inject 20% sulphur hexafluoride (SF₆) behind the DME disc, starting at the center of the pupil, until the AC is almost completely filled and leaving only a peripheral meniscus of 1 mm (Figure 8). In comparison with air, we observe with the SF₆ a significant decrease in the reinjection rate in the postoperative period.

Until recently, two main techniques of graft preparation for Descemet-endothelial keratoplasty (DEK or DMEK) had been published. The most popular is the dissection or peeling of the Descemet’s membrane (DM) from the corneal periphery with the help of forceps^1,2,3. The graft thus obtained rolls on itself with the endothelial cells on the external face, which does not facilitate its placement without traumatizing it. Other authors have described the dissection of DM by injection of air into the deep stroma, in a way similar to the big bubble technique⁴. With this method it is difficult to obtain grafts >7 mm in diameter.

We have described a third technique that seems easier, even for less experienced surgeons⁵. It consists of trepanning the DM incompletely and detaching it by hydro-dissection, with the cornea mounted in the anterior chamber (AC) with the artificial endothelium upwards: hence the name "inverted". The technique also has the advantages that the graft rolls with the endothelium inwards and this facilitates its marking to control orientation.

OBTAINING THE INVERTED GRAFT

The donor corneoscleral segment is placed first in the concave base of a special trepan-die (Muraine's Punch, Moria, France). The circular blade of this instrument is interrupted in two opposite sectors of 4 mm, so that it cuts the tissue in only 330°. It is also calibrated so that only the DM and the posterior stroma are trephined in a non-perforating manner.

Once this partial cut is made, the cornea is mounted with the endothelium outward in an artificial AC. When closed and pressurized just enough with air, the shape of the cornea is reversed, with the pre-cut DM in the convexity. It is verified that the cut is incomplete, with two opposite DM zones unharmed. It is easy to detach the DM between the corneal periphery and the trepanned areas with Troutman forceps and so that there are two small fins in the non-trepanned sectors. We slide one of the branches of the forceps under said wing to initiate the plane of cleavage of the graft. The separation of the latter is completed by hydro-dissection towards the sides with a 27-gauge blunt cannula, which first detaches the periphery of the graft from the trephination zone in the 360° and later the rest of the graft in its entirety. The same operation is performed in the front flap, which allows to obtain very quickly the complete isolation of a DM-endothelium graft with a diameter of 8 mm.

A drop of methylcellulose is deposited on the central part of the graft and rolled up on itself, with the endothelial side inwards. The DM exposed to the outside is dried with a hemostat sponge in order to mark it with a violet marker to facilitate its location in the AC. The graft is transferred to a plastic cartridge for intraocular lens implantation (IOL) through a 2.4 mm incision, which has been previously filled with culture medium.

At present the graft is obtained complete in 99% of the corneas. This figure is comparable to that of more experienced groups and much higher than the rate published by those in the learning phase. Thus, Price et al published in 2009 failures in 12 cases out of 72 (17%)⁶. The endothelial analysis shows that this technique is very little traumatic, with less than 5% of dead cells after 3 days of cultivation of the cut graft⁵. These results are comparable to those of the Melles’ group, who report a decrease from 2,701 to 2,604 cells/mm² (3.6%) after the dissection⁷. However, the loss seems more important after the big bubble type dissection as reported by Krabcova et al in their study⁸.

INSERTION, DEPLOYMENT AND FIXING OF THE GRAFT

The deployment of the endothelial graft is another difficult stage of the procedure that carries a high risk of cell damage. Some use a glass tube to insert the DM roll into the AC, but we prefer an IOL injector as the graft cannot rotate inside. With experience we realized that it was preferable to use tissue from relatively older donors (55-60 years), since those from young donors are thinner and tend to coil very vigorously.

Several techniques have been described to deploy the graft in AC, but none is really standard. In which we have developed, the tissue rolls with the endothelial cells inward, which probably protects them. In practice, we find it much easier to deploy the disc and press it on the receiving stroma with this orientation. The real problem, especially if the cornea is edematous, is to ensure that the graft is placed in the correct position, that is, with the endothelium facing the iris. Otherwise, it will not be functional and generally ends up being discarded. One possibility is to examine the orientation of the curls at the periphery of the graft, either with a slit lamp or with surgical OCT. The simplest and most demonstrative is, however, to mark it before its introduction in the AC. In our technique, the stromal side of the donor tissue is exposed to the outside and can be easily and safely stained with a violet marker (a letter "F"). Once in the AC, even if the cornea is cloudy, it is easy to confirm the correct orientation thanks to the mark, before pressing the graft on the recipient stroma with an air bubble.

THE POSTOPERATIVE PERIOD

The most common complication after a DEK (DMEK) is the partial detachment of the graft, with relatively high air reinjection rates, from 10% to 63% according to the studies^9,10. We believe that this problem is mainly due to a lack of correspondence between the posterior corneal curvature of the receiver and that of the donor. When this happens, the graft is so thin that it quickly coils around the periphery towards the receiving cornea. It is necessary to inject a new bubble of air in the AC with relative urgency, in order to flatten the graft well and prevent it from rolling up permanently.

(For reasons of space, only the specific aspects of the technique are included here. Prof. Dua's original is available on the SEO website: https://www.oftalmoseo.com)

The evolution of endothelial keratoplasty has been rapid. The different aspects of the ELK (DSAEK) and DEK (DMEK) techniques are developed in the preceding chapters of this work. Although the second one is positioning itself as the procedure with the best visual results, the first one is still the most practiced. This is partly due to the long learning curve, both for obtaining the donor tissue and for the surgical technique.

The parallel development of deep anterior lamellar keratoplasty showed that (it was possible to cleanly separate the Descemet’s membrane (DM) and the endothelium from the rest of the stroma by pneumo-dissection or big bubble (BB) .The study of this maneuver gave rise to the description of the predescemetic layer of Dua (PDL)¹ (see chapter 5.2), which called attention to its role in lamellar corneal surgery, not only the anterior but also the endothelial one⁽¹⁾. From experiments performed in vitro, Dua et al proposed the use of a graft composed of PDL and DM-endothelium as an option for endothelial keratoplasty, and together with Agarwal⁴ published the first clinical results in humans, the procedure was called "Pre-Descemet’s Endothelial Keratoplasty" (PDEK)⁽²⁾.

HOW TO ENSURE THE OBTAINING OF A BIG BUBBLE TYPE 1?

Regardless of the type of bubble that forms (1, 2 or mixed), a feature of the intrastromal air circulation during injection is its leakage through one or more points in the periphery of the cornea adjacent to the trabecular meshwork⁶ (Figure 1A). Occasionally the air leak points are located in the scleral ring, presumably through the collector channels of the aqueous humor. The variability of these escape patterns makes it difficult to obtain a consistent intrastromal air pressure that generates a BB type 1 predictably. On the other hand, BB type 2 usually begins in the corneal periphery, where PDL presents multiple fenestrations (Figure 1B) that mark the beginning of its transition to the fibers that continue towards the trabecular meshwork. There are between 15 and 20 groups of such fenestrations with an average size of 20 μm each in the PDL around the corneal circumference. The air leak through it immediately passes into the space between the PDL and the DM⁵.

To ensure the creation of a BB type 1 in a consistent manner, it would be important to occlude these fenestrations. To this end, Dua and Said have developed a fixing ring (PDEK-clamp, Janach, Italy)⁶ consisting of two rings of 9 mm in diameter and 1 mm in width. When a corneoscleral segment is fixed with this system, the inner ring occludes the fenestrations by compressing the peripheral corneal tissue along its entire circumference. It has a lateral route that allows the passage of a needle to perform pneumatization of the stroma (Figure 2). In this way, all the injected air is retained in the stroma inside the inner diameter of the ring⁶ and we obtain a BB type 1 with less than 1 ml of air. Even young donors, with excellent endothelial counts and in whom a correct dissection for DEK (DMEK) is difficult, can be used without problems for PDEK⁷.

PRE-DESCEMET-ENDOTELIAL GRAFT PREPARATION

Fresh corneoscleral buttons can be used, preserved in Optisol^® or in hot tissue culture medium. The stromal edema produced by the culture medium can be an advantage when inserting the needle without perforating anteriorly or posteriorly and facilitating the dispersion of the air inside the stroma. To facilitate the procedure, we suggest not performing the usual drying of the donor in a dextran medium, although this needs to be validated.

Use of the Dua-Said fixation ring

Although in the initial cases of PDEK the tissue was obtained without the fixation ring, we recommend its use for the reasons stated above. Under an operating microscope, the donor corneoscleral segment is placed with the endothelium upwards on the completely open fixation ring. In order for it to correctly occlude the peripheral fenestrations in the PDL, centering is fundamental. This prevents the formation of a type 2 BB, which would only allow the tissue to be used for a DEK instead of PDEK. A 30 G needle is inserted, folded 135° and with the bevel upwards, into the middle stroma, mounted in a 5 ml syringe filled with air. It is entered from the scleral ring that surrounds the cornea (Figure 3A). A sterile, dark posterior base can be used to improve visualization and contrast while inserting and advancing the needle tip. Once it reaches a central position, the air is injected slowly but steadily to pneumatize the stroma (Figure 3B) and continues until the formation of a type 1 BB is achieved (Figure 3C), which can be enlarged 0.5-1.0 mm in diameter advancing the tip of the needle inside and injecting a small amount of air (Figure 3D). Special care must be taken to avoid excessive pressure that could cause the bubble to explode – on average at 500 mmHg – which produces a characteristic sound.

Resection of the donor tissue disc

Once the BB has been formed, we have two options: aspirate the air with the same syringe and collapse the bubble or remove the needle without emptying it. We open the fixation ring and release the tissue. If we have collapsed the bubble, we can use a trepan of adequate size to obtain the donor disc. In some peripheral areas adhesions can persist that will need to be cut. If we have left the BB formed, we incise with a 15° knife in the most peripheral zone of the same (Figure 3E). The donor tissue is then cut circumferentially with scissors along the entire BB (Figure 3F). The visualization of these maneuvers can be improved by using trypan blue stain (VisionBlue^®). The tissue thus obtained is moistened with a few drops of balanced saline solution (BSS) or culture medium and placed again flat on the posterior stroma of the donor corneo-scleral disc.

The maximum diameter of a BB type 1 is on average 8.5 mm, which gives a donor disc for PDEK somewhat smaller. Ex vivo studies show that endothelial counts are similar in discs for PDEK or DEK if they are obtained by pneumodisection⁸ and that the tissue obtained for PDEK can be stored in storage medium for up to a week without significant cell loss being observed⁹. The tissue is slightly thick (about 5-10 μm) in the preservation medium, but progressive dehydration is observed in the postoperative period and its thickness at one month is, according to OCT, approximately 30 μm¹⁰.

PREPARATION OF THE RECIPIENT

In the receiving eye, preparation for PDEK is initiated in a similar way to the other endothelial keratoplasties. An upper corneal tunnel and two or three paracenteses are performed. If the pull-through technique is used, a paracentesis should be placed 180° from the main incision. The DM area to be removed on the epithelial side is marked and descemetorhexis is performed. An anterior chamber (AC) maintainer may or may not be used according to the surgeon's preference.

INSERTION OF GRAFT

The donor is re-stained with VisionBlue^® for one minute. This tissue, when containing PDL, tends to roll less than DM-endothelium, and more than isolated PDL⁸. This makes it easier to deploy the graft in the recipient eye and would help reduce endothelial cell loss. It is possible to manipulate it without risk of damaging the endothelium by means of gentle spatula strokes on the stromal surface of the PDL. It is thus folded with the endothelium towards the inside of the roller, in such a way that the lateral thirds overlap in the center (Figure 3G). A standard cartridge for intraocular lens implant filled with BSS can be used to insert it into the AC. The folded tissue is taken with blunt forceps and placed in the groove of the cartridge with the PDL in contact with the floor of the cartridge and the folded tissue facing upwards, so that by rotating the injector 180° for insertion, the endothelium is facing the plane of the iris (Figure 3H). We gently push the tissue towards the mouthpiece (Figure 4A), insert it into the corneal incision, and either inject the tissue (Figure 4B) or drag it gently into the AC with forceps from the opposite paracentesis.

DEPLOYMENT AND FIXING OF THE GRAFT

When the PDEK roll is inserted into the AC, usually the endothelium is placed downward and the face of the PDL toward the recipient corneal stroma (Figure 4C). Although the natural tendency of PDEK tissue is to coil with the endothelium outward like that of DEK (DMEK), if it has been folded as described, the endothelium remains inside the roll. The tissue is deployed laterally by small percussions (taps) on the cornea while maintaining a relatively narrow AC, and even a small air bubble can be used to facilitate deployment (Figure 4D). Once completely opened, it is applied against the cornea by means of an air bubble (Figures 4E and 4F). If necessary, we can center the disc by tapping on the cornea or with the help of a blunt spatula through a paracentesis, placing it between the donor PDL and the posterior face of the recipient. The AC is left completely filled with air with the incisions closed with sutures or by hydration according to the surgeon's protocol.

In the immediate postoperative period, corticosteroids, antibiotics and topical mydriatics are administered. As in any lamellar procedure in which a large air bubble is left in the AC, it is important to place the patient in the supine position and perform a pressure control after one hour. If there is hypertension, we can use oral or intravenous acetazolamide, intravenous mannitol or drain a small amount of air through a paracentesis.

RESULTS

The initial results described by a series of surgeons are promising and similar to the DEK (Figures 5A and B, 6A and B), which makes the PDEK a viable option in endothelial keratoplasty. Undoubtedly there is a learning curve until surgeons master the principles of the technique and acquire the skills to perform it. The fixation ring has made obtaining the donor tissue an easy, safe and reproducible procedure. The presence of the PDL allows a simpler manipulation of the tissue, facilitating its intraocular deployment and its centering. The thickness of the graft is reduced by 5-10 μm during the first month (Figure 5C and 6C), which represents the dehydration of the PDL. The visual results are as good as in DEK (DMEK) and definitely superior to ELK (DSAEK). Patients in whom ELK has been practiced in the first eye and PDEK in the other eye prefer the visual quality obtained with this last procedure.

(1) Some authors have described the separation of DM in donor corneas since 2010 by means of an air bubble^2,3 (Editor's note).

(2) When the first draft of the article on PDEK was written, Dr. Amar Agarwal suggested Prof. Dua the denomination of Dua’s Layer Endothelial Transplant (DLET). Prof. Dua responded that, given the controversy that had arisen regarding this layer and the coincidence with the acronym previously used by Melles (DLET: Deep Lamellar Endothelial Transplant), the new procedure was called pre-Descemet’s-endothelial keratoplasty (PDEK).