Article Text

DMEK lenticule preparation from donor corneas using a novel ‘SubHyS’ technique followed by anterior corneal dissection
  1. Gianni Salvalaio,
  2. Mohit Parekh,
  3. Alessandro Ruzza,
  4. Stefano Ferrari,
  5. Davide Camposampiero,
  6. Diego Ponzin
  1. International Center for Ocular Physiopathology (ICOP), The Veneto Eye Bank Foundation, Zelarino, Venice, Italy
  1. Correspondence to Diego Ponzin, The Veneto Eye Bank Foundation, Padiglione Rama, Via Paccagnella 11, Zelarino, Venice 30174, Italy; diego.ponzin{at}fbov.it

Abstract

Purpose To describe a novel submerged hydro-separation (SubHyS) technique followed by anterior corneal dissection to prepare a Descemet endothelial graft (DEG) for Descemet's membrane endothelial keratoplasty from human donor corneas.

Methods 30 human donor corneas were immersed in liquid (organ culture (OC) storage medium). Using a 25-gauge needle, approximately 0.3 mL of OC was injected (SubHyS) in the posterior stroma to create a liquid bubble. The bubbled cornea was mounted onto a modified artificial chamber with the epithelial side facing the air. The endothelium was protected with a viscoelastic solution. The anterior cornea was excised with a Barron radial vacuum trephine and the residual peripheral stroma was removed manually using micro-scissors. The DEG was dismounted and washed. The endothelial cell density (ECD) and mortality of the prepared DEG was determined. All the DEGs were preserved in deturgescent medium for 7 days using a cornea claw which was fixed to the sclera. ECD and mortality were checked post preservation.

Results Complete detachment of Descemet's membrane and stroma was achieved in all 30 cases. Bubble burst was observed in five cases (excluded from the study) due to overfilling of the liquid. The average diameter of the excised DEG was 10.96 mm. The average endothelial cell loss post preservation was 27.69%. Histological analysis confirmed elimination of the residual stroma (n=13).

Conclusions The DEGs can be preserved in a deturgescent medium for up to 7 days. The procedure provides a standardised, pre-validated (quality assured), pre-separated, no-touch, ready-to-use tissue and also reduces the preparation time. Further, the tissues can be trephined as per the surgeon’s convenience and can either be rolled or a contact lens could be used for final delivery of the DEG using a surgical glide.

Keywords
  • DMEK
  • pre-cut tissues

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Keywords

Introduction

Endothelial keratoplasty (EK) has become a popular form of corneal transplantation in patients with diseased endothelium. The most widely practiced form of EK is Descemet's stripping automated EK (DSAEK), which is used to transplant a layer of donor stroma in addition to Descemet's membrane (DM) and endothelium. DSAEK is a more standardised and optimised method which is the primary choice of EK for many surgeons due to its ease of harvest, tissue manipulation, transplantation and postoperative visual outcomes.

Descemet's membrane endothelial keratoplasty (DMEK) is a recent surgical technique which allows the replacement of a diseased endothelium with a healthy donor DM and endothelial layer. It does not involve a layer of stroma as DSAEK. This recent development is gaining popularity in terms of graft survival and early rehabilitation rate, as only the damaged layer is replaced while the rest of the cornea is left intact, unlike penetrating keratoplasty (PK). The tissue separation using the big bubble technique has been a gold standard primarily used for deep anterior lamellar keratoplasty (DALK), but has now been used to separate the Descemet endothelial graft (DEG) for DMEK.1

As DMEK decreases the unwanted damage to the corneal interface, it may also optimise and enhance the postoperative visual outcome.2 DMEK may be advantageous with respect to complete visual rehabilitation, increase in graft survival rates, and more importantly, it can be less expensive with minimal surgical instruments required for the preparation.3 ,4 Although various methods of mechanical dissection of the donor corneal tissue have been described,1 ,5–11 DMEK is not considered as one of the best options because of the high surgical skills required for harvesting and transplanting the donor endothelium. It was found that such challenging transplantations can lead up to 16% of tissue wastage and endothelial cell loss of >8% just after donor tissue preparation.5–7 Moreover, the handling of such a delicate tissue adds another challenge.1 ,7 ,8 Therefore, there is still room for further refinements aimed at standardising the technique, which could be easier and more importantly validated.12 Also, compared with PK, DMEK provides faster visual recovery with less astigmatism.13 However, it has been found that the visual outcomes often do not meet the expected visual potential, with visual acuity often limited to 20/40.7 However, pneumatic dissection and most peeled DMEK grafts do not have any adherent stroma which is an issue with air bubble prepared grafts.1 ,14–16 It is important to understand whether the stromal residues are helping the attachment of the graft or the very thin residues are responsible for graft detachment. With all these issues, improvement in donor tissue preparation, validation and transplantation has become the next challenge for DMEK surgery.

Thus, as there are no reported methods of a pre-standardised/validated or ready-to-use tissue, we describe a method that could potentially be used for preparation of a DMEK tissue, and its effect in a deturgescent medium for 7 days of preservation which could be further used for transportation. This is a novel technique which could be used by eye banks to provide a pre-cut tissue to reduce tissue manipulation prior to surgery.

Methods

Collection of samples and pre-evaluation

Thirty human donor corneas unsuitable for transplantation (low endothelial cell count, ≤2200 cells/mm2) were collected from the Veneto Eye Bank Foundation (Venice, Italy). A written consent from the donor's family was obtained for the use of the corneal tissues for research. The average endothelial cell density before the experiments was found to be 1920 (±223.45) cells/mm2. The average donor's age was 64.83 (±9.34) years and the male:female ratio was 19:11. The corneas were preserved in organ culture (OC) medium for 16.50 (±8.95) days before the experiments. The endothelial cell density and viability were determined using trypan blue stain (0.25%). The corneas were then exposed to 1.8% sucrose (hypotonic environment) and the cells were visualised using a 10× magnification lens of an Axiovert 25 inverted light microscope (Carl Zeiss, Germany). The endothelial cell density and mortality were determined throughout the cornea manually using a 10×10 reticule mounted in the eye piece of the microscope.

Submerged hydro-separation

All the procedures were carried out under sterile conditions. The corneal tissues were completely submerged in a sterile small basin (approximately 7.8 cm diameter and 1 cm height) containing 15–20 mL of sterile OC medium, as shown in figure 1A. OC medium was composed of 2% new-born calf serum with MEM-Earle as a base medium along with 25 mM Hepes buffer, 26 mM sodium bicarbonate, 1 mM pyruvate, 2 mM glutamine, 250 ng/mL amphotericin B, 100 IU/mL penicillin G and 100 µg/mL streptomycin. The tissue was held at the sclera with stainless steel forceps. A 25-guage needle (bent with bevel up) connected to a 1 mL syringe filled with OC medium (liquid) (media constituents as listed above) was inserted into the peripheral cornea through the sclera using the trabecular meshwork as a point of reference (figure 1B). The needle was further moved radially beneath the endothelium in the posterior stroma for approximately 3 mm towards the central cornea. Around 0.3 mL of the liquid was injected in the tissue to separate the DEG and the stroma. The liquid primarily filled the posterior stroma and later cleaved the stroma at its weak point further entering into the stroma-DM phase. A small clear bubble was visible at the mid-periphery (figure 1C, C.1) with the initiation of the process, which ensured that the procedure was accurate. The bubble was enlarged to achieve >10 mm diameter (measured using vernier calipers), as shown in figure 1D (see online supplementary video clip 1).

Figure 1

Initial steps of the submerged hydro-separation technique and bubble formation. (A) Immersion of the corneal tissue in organ culture medium, (B) insertion of the needle to facilitate the initiation of the liquid bubble, (C) expansion of the liquid bubble to the maximum diameter possible (approximately 10 mm), (C.1) top view of the injected needle in the cornea, and (D) complete bubble and removal of the needle.

Anterior corneal dissection

A modified polytetrafluoroethylene (PTFE) artificial chamber (cornea mounting space of 13 mm diameter with 6 mm depth) (figure 2A–C) was custom built for this study. The endothelium was protected using a surgical viscoelastic solution, Viscoat (Alcon, Italy), to prevent the DEG from any mechanical damage (figure 3A). The centre of the bubbled cornea (epithelial side) was marked using a skin marker and was mounted onto the PTFE artificial chamber with the epithelial surface facing the air. Two ‘tissue non tissue’ curved patches were used to prevent tight adhesion between the sclera and the stopper to preserve the cornea from later damage during dismounting. The internal pressure inside the artificial chamber was maintained in a range of 15–25 mm/Hg (figure 3B). The corneal tissue was then fixed with a stopper. The pressure was increased slightly (figure 3B). A Barron radial vacuum trephine (Altomed, UK) was used to trephine approximately 8.5 mm of the anterior cornea (epithelium and stroma), as shown in figure 3C. This exposed the fluid completely. The stromal residue and the peripheral stroma were cut using micro-scissors, thus exposing the fluid up to 10 mm diameter or as much as possible (figure 3D–G). The stopper was opened securely ensuring no damage was caused to the corneal endothelium. The fluid was then removed by picking the cornea up vertically and draining the liquid down. The endothelium was washed gently using sterile phosphate-buffered saline (PBS) to remove all the viscoelastic solution, as shown in figure 3H. A very thin, clear, transparent naked DEG attached to the sclera was observed (figure 4A). The endothelial morphology, mortality and endothelial cell density were checked after the DEG preparation using trypan blue (n=30) and alizarin red (n=8) (see online supplementary video clip 2).

Figure 2

Modified artificial anterior chamber. (A) Standard design of an artificial anterior chamber made with polytetrafluoroethylene, (B) final working model with the stopper on, (C) the depth and diameter of the new modified model.

Figure 3

Graft preparation. (A) Preservation of the endothelial cells using a viscoelastic solution, (B) mounting of the cornea on the artificial anterior chamber after closing the stopper and maintaining the pressure, (C) trephining the anterior cornea using Barron radial vacuum trephine, (D) cutting the extra remnants of the anterior cornea using micro-scissors, (E) complete opening of the anterior cornea and exposure of the internal liquid, (F) excision of the peripheral stromal residues to increase the diameter of the cornea, (G) the graft after cutting the anterior cornea, (H) washing of the final graft using phosphate-buffered saline to remove the viscoelastic solution completely.

Figure 4

Final graft. (A) The final graft after preparation and endothelial viability check, (B) the graft fixed at sclera with a corneal claw for preservation in transport medium.

Preservation

The excised tissues were preserved using a corneal claw (to keep the tissue afloat) in deturgescent medium containing 6% dextran T500 along with the OC medium for 7 days (figure 4B) (see online supplementary video clip 3). The viable endothelial cell density was determined post preservation.

Mimicking shipping and trephination

The shipping was not simulated in real life but was mimicked using a rocking table. The tissues were punched using vacuum trephine with the endothelial side facing upwards and were rolled using sterile PBS. A contact lens could be used for trephining when the surgeon is more comfortable with this technique.

Histology

Periodic acid-Schiff staining was performed on 13 samples from different donors to check the variability in tissue selection and bubble performance.

Results

Complete detachment of the DEG and stroma was achieved in all the samples. Twenty-eight cases required single injection (adjusting the needle inside the tissue was excluded) whereas two cases required two injections with the needle being inserted at a different site. The average diameter of the acquired bubble was found to be approximately 10.96 (±0.09) mm. Sixteen bubbles appeared from the periphery whereas 14 bubbles initiated centrally. Bubble burst was observed in five cases due to overfilling of the liquid. Histology showed that the detached layer purely consisted of endothelium and the underlying DM without any stromal residues for all the tissues that were analysed (n=13) regardless of the donor age (figure 5A, B). At the end of the storage period (7 days), the average loss in endothelial cell density was 27.69%, as shown in figure 6A–D. Higher mortality outside the optic zone was observed in some cases at the far periphery.

Figure 5

(A, B) Histological analysis of the final graft using periodic acid-Schiff (PAS) staining at 10× and 400× magnifications. The Descemet endothelial graft was separated completely without any stromal residues. The donor age had no influence on graft preparation. Dua's layer was not identified using PAS.

Figure 6

Trypan blue staining analysis at 100× magnification. (A) Post Descemet endothelial graft (DEG) preparation—there was no or very little mortality seen after the preparation. (B) Post DEG preservation (7 days)—scattered mortality was observed after preservation near the periphery. (C, D) Post DEG preservation (7 days)—very little to no mortality was observed in the central or optic zone of the DEG.

Discussion

Air injection for DEG separation is the method which is routinely performed for lamellar keratoplasty (DALK/DMEK).1 However, the success rate of creating the big bubble relies on several factors, including the skills of the operator, mainly because of the manual manipulation required is high. Preparation of the donor tissue has therefore been a challenge. A new method is introduced here to reduce the risk of tissue preparation failure in the operating theatre. As per our experience, the most crucial step in the DMEK graft preparation is the bubble formation. Therefore the aim of this study is to supply a pre-cut tissue (DEG graft) to eliminate this issue from the surgical theatre. The only manipulation required by the corneal surgeon would be to trephine the DEG before transplantation.

In our study we observed that the liquid filled the stromal lamellae and expanding throughout the posterior stroma. It further cleaved the posterior stroma generating the bubble from mid-periphery towards the periphery or directly from the periphery in some cases. The liquid flows evenly throughout the tissue and it accommodates itself into the stromal lamellae. A complete bubble was created with further injection of the liquid. In some cases, the bubble was formed when the injection site was between the stroma and DM without undergoing any stromal filling (right plane of cleavage). In addition, we also found that along with the liquid bubble there was a relatively low generation of small bubbles which did not render the tissue opaque as usually seen with the air bubble. This poses a major advantage and generates high yield. Although bubble bursting is a risk in type II bubbles where the DM is not supported by the Dua's layer (DL),17 there was no tissue wastage seen in our study using liquid bubble. However, it is necessary to control the amount of liquid injected just like air.

Dua et al suggested that a pre-Descemetic layer is one of the causes for different types of bubble formation. Therefore, our hypothesis with the liquid bubble is the following: initially, the liquid expands evenly in the posterior stroma (when the needle insertion plane is not the stroma-DM phase) and then it starts generating a bubble at mid-periphery/periphery where the pressure cleaves the posterior stroma near the insertion site of the needle, due to the high force or overflow of the incoming liquid or on the other sites because of a weak stromal point. Whereas, when the needle is in the right plane of cleavage (stroma-DM phase), a clear bubble is visible. The bubble further enlarges towards the periphery thus entering the DM-Stroma phase or DM-DL (Dua's Layer) phase, which is still undetermined. However, we believe that the viscosity, pressure and the insertion site still remain issues with respect to different types of bubble formation. This further supports the hypothesis that the tissues obtained using this method were free from stromal residues as the bubble was formed in the DM-Stroma phase after the posterior stromal cleavage. While using air as the medium of separation, it is observed that the small air bubbles which are generated in the posterior stroma render the tissue opaque further leading to irregular air expansion and a loss of the tissue. Also, we believe that the air pressure may not be enough to penetrate and separate the posterior stroma and enter the stroma-DM phase thus leading to a tissue with residual stromal remnants. Usually, it is observed that most of the surgeons create the bubble initiating from the posterior stroma as it is slightly tricky to get the right plane of injection, therefore this technique will help to reduce the tissue loss drastically which is normally observed with air bubble as the liquid has a benefit of creating a cleavage and further expanding through the stroma-DM phase. Histologically, we also determined that the 13 lenticules that underwent histologic examination did not possess any stroma or DL. However, the above hypothesis warrants further investigation.

Unlike other methods, the technique described in this paper provides a validated, standardised high quality tissue preparation. In fact, it is difficult to validate the performance of a tissue when it is rolled or manually scraped and preserved. As the DEG tissue of this study is attached to the sclera, the rolling during preservation and transportation is prevented, thus reducing unwanted endothelial cell loss and preventing the upside-down transplantation,18 as the endothelial side can be determined by the scleral anatomy. However, once the tissue is trephined and injected inside the eye the orientation is lost which is another limitation of this study considering the post transplantation effect of the implanted graft. Furthermore, being the endothelium completely exposed, a continuous sufficient supply of preservation (transport) medium is provided to the endothelial side. As per the conventional OC protocol, the tissues were preserved in the deturgescent medium. However the tissues could also be stored and transported in other media that could preserve the corneal endothelial integrity.

The corneal tissues used in this study were unsuitable for human transplant which is a limitation of this study as it does not represent the tissues that would actually be used by a surgeon, therefore this process will need a further validation with transplant quality tissue. This technique provides a DEG but the stroma is usually lost due to oedema and therefore only a DMEK tissue but not the DALK tissue can be used which is another known limitation to this study.

Earlier studies have reported preparations that include tissue handling with instruments that can create mechanical damages to the endothelium, thus potentially leading to post-surgery graft failure.1 ,5 ,7 ,8 ,18–20 The technique described here is a no-touch method, as the sharp instruments normally used for dissections do not have any contact with the endothelium, thus minimising the damage. Even after the increased manipulation time and preparatory measures, the endothelial cell loss observed in our study was comparable to others.1 The DEG prepared here has some advantages in terms of grafts generated (yield), bubble formation, diameter, tissue layer detachment (figure 5A, B), as compared to the other mechanical techniques.

The submerged hydro-separation (SubHyS) method described here does not allow the endothelium to dry. A possible reason for the graft viability and higher success rates with this procedure is that the liquid bubble gives the endothelium a cushion bed for further cutting. The liquid helps to keep the tissue intact in the artificial chamber. If an air bubble is used instead of liquid, the DEG sticks to the stroma as soon as the anterior cornea is cut/trephined.

The SubHyS technique described here does not require high surgical skills. In fact, it is easier to manipulate the tissue using liquid as a medium of separation considering the plane of cleavage. The technique allows a DEG to be obtained from the eye bank ready for transplantation. It also decreases the time required by the surgeon for preparing the donor DEG and reduces requests for spare tissues in case of failure during bubble formation. Furthermore, it allows the transplantation of a pre-validated tissue, thus leading to a higher quality assured graft transplant, which is an important parameter for standards of quality and safety as outlined by the European Commission Directives (Directive 2004/23/EC). Validation of the DEG is a concern when DEGs are prepared in the surgical theatre and therefore a pre-validated tissue would be advantageous to study and report the postoperative results more accurately.

However, despite all these benefits, the efficacy of this technique will only be seen after the clinical transplantation of the pre-prepared grafts. Graft dislocation, detachment or failure will have to be evaluated after a clinical trial has been carried out. The standardisation of such a technique will help to reduce the debate of whether graft rejection, failure or dislocation is due to graft preparation or surgical error.

In conclusion, this procedure provides the surgeon with a standardised, pre-validated, pre-separated, no-touch, ready-to-use tissue. It reduces the time required for preparation of the DEG before surgery and decreases the amount of damage while preparing a graft during surgery. Moreover, this technique can be used according to the surgeon's needs: it could be trephined and used with a stromal handle, rolled, or a contact lens or other device could be employed to facilitate the transplantation using a surgical glide. Further, clinical validation needs to be evaluated. We expect that the above advantages and the way the preparation technique is carried out would facilitate widespread use of the DMEK procedure and take it to the next step of corneal transplantation.

References

Supplementary materials

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Footnotes

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.