The rise in lamellar keratoplasty techniques has prompted the revision of the criteria for the selection of corneal tissue and the development of new tissue processing techniques in eye banks. The selective replacement of only a part of the corneal layers requires a specific donor tissue preparation for each type of keratoplasty. This can be done by the surgeon immediately before starting the operation, or in the eye bank by specialized personnel and beforehand. Next, we will describe the techniques most used today to prepare the donor tissue used in anterior and posterior lamellar keratoplasty.

PREPARATION OF THE CORNEAL TISSUE FOR ANTERIOR LAMELLAR KERATOPLASTY

The preparation of the donor cornea for anterior lamellar keratoplasty will vary depending on whether it is a superficial lamellar graft (SALK) or a deep lamellar graft (DALK).

Preparation for superficial anterior lamellar keratoplasty (SALK)

In the case of SALK, the same surgeon usually prepares the donor, since the thickness will depend in each case on the depth of the lamellar dissection that has previously been performed on the recipient. The graft can be obtained manually, by using a microkeratome or the femtosecond laser. The use of anterior discs for SALK cases due to corneal herpes obtained together with the carving of a posterior lenticule for endothelial lamellar keratoplasty (DSAEK) has been described¹. This shows the possibility of using a cornea donor for two receptors. Although the option of preserving isolated anterior discs has been described with the use of glycerin², Optisol-GS³ or cryopreservation⁴, it is not an extended practice in eye banks. The routine use of the femtosecond laser would theoretically allow to store pre-cut tissue with the desired diameter and depth, although the suboptimal visual results obtained with this technique, the scarce demand and its high cost have limited the development of this option.

Preparation for deep anterior lamellar keratoplasty (DALK)

The surgeon usually performs the preparation of the donor for DALK during the surgical act, once the dissection of the receiving stroma from the posterior lamina (Descemet/Dua) with integrity of the same has been achieved. For this technique, the donor cornea does not require a good endothelium, but transparency and other properties of the stroma are important. After trepanning the donor cornea in all its thickness, its endothelial face is stained with trypan blue to achieve a better visualization of the Descemet’s membrane (DM). This is then removed (together with the endothelium) by gentle scraping with dry cellulose sponges (Figure 1) or by pulling from its perimeter with forceps. The latter is preferable because it is less traumatic for deep stromal layers. The button thus prepared is placed in the receiving window and sutured in place.

In the case of DALK, the work of the eye bank consists of preserving and storing corneal tissue with good characteristics in its anterior layers (especially the stroma in all its thickness), specifically for surgeons who request tissue of these characteristics. To this end, corneas with a low endothelial count are usually preserved and sent to the surgeon without manipulation. This will practice the removal of the donor endothelium. With the increase in Descemet-endothelial keratoplasties (DMEK), the residual donor cornea (to which the Descemet-endothelium has been removed) has been preserved in an isolated form in cell culture medium. This increases tissue availability for DALK, while no significant alteration in the results has been observed⁵.

PREPARATION OF THE CORNEAL TISSUE FOR ENDOTHELIAL LAMELLAR KERATOPLASTY (DSEK/DSAEK)

Endothelial lamellar keratoplasty involves a graft that includes the endothelium, DM and a thin lamina of posterior stroma. This tissue can be obtained by manual or "automated" dissection with a microkeratome. In both cases, an artificial anterior chamber (AAC) is required for dissection, when starting from a corneoscleral segment. With an entire globe, the microkeratome could be used directly, although the corneoscleral cap is often separated previously because the other technique is more standardized. To ensure correct fixation and pressurization in the AAC, the donor segments must have a minimum diameter of 17 mm. For this reason, donation teams routinely use this diameter at present. The 14 mm trephines that were used previously give rise to segments clearly insufficient for this use.

Manual technique of Melles

Through a limbal incision at 300-350 μm deep, we proceed to dissect the cornea with spatulas that follow its curvature. This creates a deep stromal pocket (Figure 2). Subsequently, the posterior lamellar disc (the graft) is obtained by trepanation from the endothelial side. This technique does not require complex or expensive instruments but obtaining a uniform and regular dissection plane is technically demanding. In addition, the regularity of the bed obtained will depend on the skill of the surgeon and determines the visual results⁶.

Microkeratome technique

Obtaining the lenticule or donor disk for DSAEK with the help of a microkeratome is the most widely used technique nowadays, due to its safety, reproducibility and relative technical simplicity. In addition, most corneal surgeons are familiar with the use of the microkeratome in refractive surgery.

The corneoscleral segment is mounted on an AAC, which is connected to an infusion system with air or balanced salt solution (BSS) to maintain adequate tension during the passage of the microkeratome (Figure 3). It is recommended to eliminate all the corneal epithelium before cutting to improve visibility, avoid the risk of implanting epithelial cells in the donor-recipient interphase, as well as reducing the initial corneal thickness by 50-70 μm, which allows a thinner graft. The head of the microkeratome is chosen according to the initial corneal thickness (measured with an ultrasonic pachymeter) and the thickness desired for the graft. The center of the cornea is marked with a gentian violet marker to facilitate subsequent centering at the time of endothelial trepanation.

For the preparation of the DSAEK graft, automated or manual microkeratomes can be used, although the "A" of the English acronym refers to the former. They can also be single-use or multiuse (table 1). With manual microkeratomes, the depth of cut depends, apart from the head used, on the speed of passage. The slower the passage, the greater the depth of cut. The term "automated" applied to a microkeratome refers to having a mechanized advance, so its cutting speed is constant. This limits the variability in the depth of the graft, although this also depends on other factors such as the pressure in the AAC.

"Ultra-fine" grafts are those with a thickness <100 μm. They allow ultra-thin endothelial lamellar keratoplasty (UT-DSAEK) with intermediate characteristics between classical DSAEK and DMEK. Depending on the initial thickness of the corneoscleral cap and the available heads, it will sometimes be necessary to perform two steps with the microkeratome, with different heads, to obtain such ultrafine grafts⁷.

Once the cut is made, the anterior lamellar disc is placed on the posterior stroma again. Usually marks are added on the edges of the cut to verify that both pieces are correctly assembled and do not move. The corneoscleral cap thus cut is stored again in the culture medium. Through an adapter, an optical coherence tomography (OCT) of the anterior segment can be performed, which allows to accurately measure the depth of the cut obtained without extracting the tissue from the medium during the entire conservation period.

Once it is trephined, the tissue prepared with the microkeratome usually has a profile of meniscal lenticule, being finer in the center than in the periphery. This morphology facilitates its handling with the instruments, since the disc tends to maintain the convex shape of the cornea, but also induces a hyperopic refractive change that must be considered when determining the power of the lens to be implanted (if done in the same intervention). The finer the donor tissue, the more difficult it will be to manipulate it, but the induced refractive change will also be lower, and, in theory, the final visual acuity will be better. The bed obtained with microkeratome is more regular and homogeneous than with the manual technique, which improves the visual prognosis of patients.

Femtosecond laser technique

The procurement technique is simple: the donor corneo-sclera is placed in an AAC, at a pressure of approximately 20 mmHg. The femtosecond laser equipment is programmed with the desired dimensions for the vertical and laminar sections of the lenticule, according to the central and peripheral pachymetries previously obtained. The optical coupling between the laser and the donor cornea is then carried out, either by applanation lens or by a liquid interphase according to the laser model. The cutting is performed; the anterior disc is delaminated with a spatula, repositioned on the bed, and reintroduced into the culture medium.

The femtosecond laser allows creating dissection planes at a very exact depth. However, studies using scanning electron microscopy show that the surfaces obtained with laser in deep corneal planes are more irregular than those obtained with microkeratome⁸. The lower compaction of collagen fibers in the posterior stroma (compared to the anterior one) would be a cause of the loss of precision and regularity of laser cutting⁹. With the femtosecond laser, discs of corneal tissue with parallel faces are theoretically obtained and therefore without refractive effect. But their intraoperative management can be more laborious because they do not maintain so much the corneal convexity. Apart from its higher cost, this technique has not proven to obtain better visual results than the others for this application.

Techniques combined with excimer laser

Recently, modifications of the UT-DSAEK have been described, being called MELEK (Microkeratome and Excimer Laser-assisted Endothelial Keratoplasty) and FELEK (Femtosecond and Excimer Laser-assisted Endothelial Keratoplasty). In the first, after cutting with a microkeratome, the ablation of the bed is performed with an excimer laser to reduce its thickness¹⁰. The other combines a femtosecond laser cut followed by ablation with excimer laser: this would achieve an excellent quality surface of the bed or interface¹¹.

PREPARATION OF THE CORNEAL TISSUE FOR DESCEMET-ENDOTELIAL (DMEK) KERATOPLASTY

The graft for Descemet-endothelial keratoplasty (DMEK) consists only of DM and endothelium, so its dissection is a very delicate maneuver, with risk of damaging the endothelium or tearing the tissue. This difficulty explains the variety of techniques proposed.

Immersion dissection technique (SCUBA)

Price et al described in 2007 the technique of obtaining the graft for DMEK called «SCUBA technique» (Submerged Cornea Using Backgrounds Away)¹². This technique consists of carrying out all the maneuvers of separating the DM from the endothelium in "underwater" conditions, that is, with the donor tissue submerged in the preservation medium (Optisol GS) or in BSS. With a Sinskey's hook, small tears are made all around (360°) of the peripheral area of ​​the posterior surface of the cornea. With a blunt instrument the 2/3 of the Descemet are partially delaminated and then cut with a trephine-die with a diameter slightly inferior to the area of ​​the detached Descemet. This results in a donor membrane in the form of a thin disk, perfectly circular and with continuous edges. All maneuvers are performed in the concavity of the corneoscleral segment filled with the aforementioned liquid to facilitate the flotation of tissue. This prevents the formation of folds due to gravity with the consequent endothelial cell damage.

"Non-contact" technique

The Melles group around 2011^13,14 developed the “non-contact” technique. In it, the contact of donor endothelial tissue with the instruments is avoided at all times, neither on its edges nor on its bed (Figure 4). For this, the periphery of the DM is first detached from its edge, acceding with a Desmarres' type blade, just in front of the scleral spur, in the 360° until finding the plane of dissection. The peripheral ring thus detached is pulled with forceps, which allows dissection of the DM in its entirety. Next, the Descemet-endothelial graft is trephined, which, after being mobilized with BSS, is rolled with the endothelial surface out. In this way it is ready to be injected into the recipient's anterior chamber.

Muraine’s technique

The corneoscleral segment is fixed at the base of the special trephine-die designed by Muraine for this technique¹⁵. The die makes an incomplete circular cut that leaves two sectors or "hinges" diametrically opposed without cutting. The corneo-sclera is mounted on an AAC with the endothelial side facing up and the corneal concavity is evoked by air injected into the AAC. The endothelial surface is stained with trypan blue and the DM is dissected on the hinges outside the trephination area until a plane of dissection is found. With a 27G cannula, a hydrodissection is performed, with BSS or preservation medium, which raises the DM, first in the direction of the corneal apex and then towards the edges of the trephination until the DM completely comes off and the Descemet-endothelial graft roll is obtained.

Pre-Descemet-endothelial keratoplasty (PDEK)

In this technique, a thin membrane is transplanted, which, in addition to the endothelium and Descemet, includes the Dua’s layer of the donor¹⁶. Dua showed that the incorporation of this thin layer of collagen to the Descemet-endothelial graft facilitates its surgical manipulation and deployment in the anterior chamber, since the graft tends to roll less¹⁷. To prepare this tissue we proceed with a technique analogous to that of the "big bubble" of Anwar for the DALK, but with the cornea inverted. The corneo-scleral cap is mounted with the endothelial side facing up and a 30 G needle is inserted with a 5-cc syringe from the middle periphery to the corneal center, where air is injected. Two types of bubbles, type 1 and type 2, can be obtained, according to the plane in which they are formed (Figure 5). The first is smaller and does not reach the limbus; it is formed between the posterior stroma and the Dua’s layer. The type 2 is larger, it expands to the limbus and is formed between the Dua’s layer and the DM, making it finer and brittle. In principle, an attempt is made to obtain a type 1 bubble. Once the separation is obtained, the trepanation is performed from the endothelial side, and the tissue disc is released to inject into the recipient.