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Femtosecond laser and microkeratome-assisted Descemet stripping endothelial keratoplasty: first clinical results
  1. Andreia Martins Rosa1,2,3,
  2. Maria Fátima Silva4,
  3. Maria João Quadrado1,2,3,
  4. Esmeralda Costa1,3,
  5. Inês Marques1,
  6. Joaquim Neto Murta1,2,3
  1. 1Ophthalmology Unit, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal
  2. 2Centro Cirúrgico de Coimbra, Coimbra, Portugal
  3. 3Faculty of Medicine, University of Coimbra, Coimbra, Portugal
  4. 4Visual Neuroscience Laboratory, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
  1. Correspondence to Professor Joaquim Neto Murta, Ophthalmology Unit, Centro Hospitalar e Universitário de Coimbra, Praceta Mota Pinto, Coimbra 3049, Portugal; jmurta{at}


Aim To perform Descemet stripping automated endothelial keratoplasty (DSAEK) using a novel technique to obtain very thin (<100 µm) posterior corneal disks.

Methods Twenty five DSAEK grafts were prepared with two sequential cuts: the first cut, of variable thickness, was made with a femtosecond laser and the second with a 300 µm microkeratome head. Spectacle corrected visual acuity, endothelial cell density evaluation with specular microscopy and anterior segment optical coherence tomography to measure central and peripheral graft thickness was performed preoperatively and postoperatively at 1, 3 and 6 months.

Results There were no irregular cuts or perforations during tissue preparation. Central graft thickness was 79.6 µm (SD±14.5; range 54–98) and 69.3 µm (SD±14.2; range 49–96) at 3 and 6 months. Corrected distance visual acuity improved from 0.91 logMAR preoperatively to 0.11 logMAR at 6 months. Donor endothelial cells averaged 2675 cells/mm2 preoperatively and 1729 cells/mm2 at 6 months. There were no graft detachments.

Conclusions This new technique consistently yielded very thin grafts (<100 µm), excellent visual acuity results and good endothelial cell counts. No donor tissue was wasted.

  • Cornea
  • Treatment Surgery
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Descemet stripping automated endothelial keratoplasty (DSAEK) has become the standard procedure for Fuchs dystrophy and other endothelial dysfunction disorders.1 DSAEK consists of stripping a patient's diseased endothelium and replacing it with healthy endothelium, Descemet's membrane and a layer of stroma prepared from a donor cornea with an automated microkeratome.2 ,3

One limitation of DSAEK is that some eyes do not achieve good visual acuity despite a clear cornea and minimal residual astigmatism. This may be due to interface irregularity, the presence of donor posterior stroma or a thick endothelial graft.4–6 The influence of endothelial graft thickness on visual acuity in DSAEK is controversial, with some authors reporting better results with thinner grafts and others finding no correlation.6–11 However, in studies where no correlation was found, grafts were relatively thick (>160 µm).8–10 Indeed, in none of these studies was graft thickness less than 100 µm. The procedure to obtain thin grafts carries a higher risk of perforation. Our group has previously reported a technique that combines a femtosecond laser with a microkeratome to create grafts of less than 100 µm in eye bank eyes (Murta et al, 2nd Eucornea Congress, 2011).

We present below the first clinical outcomes of DSAEK performed with this new approach.

Materials and methods


This prospective study comprised 25 eyes from 25 patients with Fuchs endothelial dystrophy (14 eyes) or pseudophakic bullous keratopathy (11 eyes). Exclusion criteria were coexisting non-corneal abnormalities, such as macular degeneration and advanced glaucoma, and history of previous corneal surgery or visually significant corneal scarring. There were no relevant coexisting systemic diseases. Institutional review board approval was obtained and patients were provided with informed consent after the possible consequences of participation had been explained.

Surgical technique

Donor corneas were obtained from Fondazione Banca Degli Occhi (Venice, Italy) preserved in organ culture medium at 31°C. Each cornea was mounted on an artificial anterior chamber (ALTK, Moria SA, Antony, France) filled with balanced salt solution (BSS, Alcon, Fort Worth, Texas, USA). Central pachymetry was performed with an ultrasonic pachymeter (CorneoGage Plus 50 MHz; Sonogage, Cleveland, Ohio, USA) after removal of the epithelium. Five readings were averaged. The BSS bottle was elevated to 220 cm and the tubing clamped at 60 cm from the anterior chamber. Two cuts were made. An Intralase FS60 femtosecond laser (Abbott Medical Optics, Santa Ana, California, USA) was used for the first cut and a Moria CBm microkeratome with a 300 µm cutting head was used for the second. The thickness of the first cut was calculated as follows:Embedded Image

*This figure is the sum of the theoretical microkeratome cut thickness (300 µm) plus the desired final graft thickness (110 µm).

Femtosecond settings were full lamellar cut, diameter 9.5 mm, raster energy 1.5 µJ and anterior side cut at 90°. No suction ring was used and docking was straightforward. The first anterior stromal lenticule was easily removed following the femtosecond cut. In no case was manual dissection needed. The second cut was performed immediately afterwards with the 300 µm microkeratome head, keeping the manual rotation speed constant and with total duration of approximately 5 s. The tubing was unclamped from the BSS bottle at 150 cm.

Donor tissue was removed by gently pulling the scleral rim from the top of the anterior chamber and was transferred to an 8.5 mm Hessburg-Barron trephine (Katena Products, Denville, New Jersey, USA).

Recipient preparation and donor insertion were performed as previously described.12 In brief, the procedure consisted of stripping Descemet's membrane, performing an inferior iridectomy to prevent pupillary block, transferring the graft using a Busin glide (Moria SA) and filling the anterior chamber almost completely with air underneath the graft.

Seventeen patients were pseudophakic preoperatively. Concurrent cataract surgery was performed in eight cases, before the Descemet stripping. Surgeries were performed by one surgeon (JNM). Postoperative medication consisted of ofloxacin 3 mg/ml and dexamethasone 1 mg/ml six times a day for 2 weeks and then tapered according to clinical response.

Outcome analysis

Graft thickness was measured at 1, 3 and 6 months using anterior segment optical coherence tomography (Spectralis Anterior Segment Module, Heidelberg, Germany). The scanning line was positioned on the 180° axis into the corneal vertex and measurements were taken at the vertex and at 3 mm on either side to obtain peripheral thickness (figure 1).

Figure 1

Anterior segment optical coherence tomography performed 1 month after Descemet stripping automated endothelial keratoplasty (femtosecond and microkeratome assisted) showing a posterior lamellar graft with a central thickness of 60 µm, temporal thickness of 88 µm and nasal thickness of 81 µm. Access the article online to view this figure in colour.

Corrected distance visual acuity (CDVA) with subjective refraction was recorded preoperatively and at 1, 3 and 6 months after surgery. Baseline donor endothelial cell density (ECD) was provided by the eye bank and was measured postoperatively with specular microscopy (Tomey EM-3000, Nagoya, Japan).

Statistical analysis was performed with IBM SPSS Statistics V.19 (SPSS Inc, Chicago, USA). Repeated measures analysis of variance with Bonferroni correction for multiple comparisons was used after verifying that data did not significantly deviate from normal distribution (Kolmogorov–Smirnov test). All results with p<0.05 were considered statistically significant.


A total of 25 patients (17 female and 8 male subjects) aged between 48 and 88 years (mean age 65.0±13.4 years) were included. Average postoperative follow-up was 6 months (range 5–7 months). Tissue was prepared without perforations, irregular cuts or buttonholes.

Central preoperative donor corneal thickness after epithelium removal was 542.4 µm (range: 505–600 µm). Central graft thickness decreased continuously from 83.1 µm at the first month to 79.6 and 69.3 µm at 3 and 6 months (table 1 and figure 2).

Figure 2

Improvement in best corrected visual acuity (BCVA) (logMAR) and reduction in mean central graft thickness (μm) is observed during a 6-month follow-up.

Table 1

Central and peripheral DSAEK graft thickness measured with anterior segment OCT at 1, 3 and 6 months postoperatively

Multiple comparisons showed statistically significant differences between the first and sixth month central graft thickness and between the third and sixth month central graft thickness (F(2,44)=21.39; p<0.001; ηp2=0.493, p<0.001 in both comparisons). The same analysis was performed for peripheral DSAEK graft thickness but the differences were not significant (F(2,44)=0.189; p=0.828; ηp2=0.009).

The linear Pearson correlation coefficient was used to evaluate the relationship between the obtained graft thickness and the original donor corneal thickness. No statistically significant correlation was found at any time point (1 month: rPearson=−0.39, p=0.33; 3 months: rPearson=−0.54, p=0.17 and for 6 months: rPearson=−0.69, p=0.13).

CDVA continuously improved from 0.91 logMAR preoperatively to 0.15 and 0.11 logMAR at 3 and 6 months postoperative (table 2 and figure 2). Statistically significant differences were found for CDVA between preoperative and at 3 and 6 months (F(2,42)=11.41; p=0.015; ηp2=0.65; p=0.017 and p=0.013, respectively, multiple comparisons). No differences in CDVA were found between 3 and 6 months postoperatively (p=0.56). At 3 and 6 months, 60.0% and 85.7% of eyes achieved CDVA of 20/30 or more. CDVA was 20/20 in 10% of eyes at 3 months and 14.3% at 6 months. Spherical equivalent increased from −1.31D preoperatively to −0.7D at 6 months.

Table 2

Preoperative and postoperative best corrected visual acuity (CDVA in Snellen and logMAR) and spherical equivalent (D)

Average precut ECD was 2675 cells/mm2 (SD±251) and 1729 cells/mm2 (SD±296) at 6 months, representing a 35.4% cell loss (table 2). There was a main effect for ECD (F(2,42)=39.40; p<0.001; ηp2=0.85) with statistically significant differences between donor ECD and ECD at 3 and 6 months (multiple comparisons; p=0.001 and p<0.001, respectively) while the ECD decline between 3 and 6 months was not significant (p=0.99).

In order to evaluate the influence of concomitant cataract surgery in terms of visual acuity, graft thickness and endothelial cell loss, we also compared patients undergoing only DSAEK with those having concurrent cataract surgery. In terms of visual acuity at 3 and 6 months, there were no statistically significant differences between the two groups (DSAEK group: 0.14±0.12 logMAR and 0.11±0.09 logMAR at 3 and 6 months vs the concurrent cataract surgery group: 0.16±0.06 logMAR and 0.14±0.06 logMAR; independent samples t test: p=0.71 and p=0.13 at 3 and 6 months). There were no differences in central graft thickness (DSAEK group: 80.8±16.4 µm (mean±SD) and 70.0±17.4 µm at 3 and 6 months vs the concurrent cataract surgery group: 76.8±9.8 and 68.3±9.7 µm; p=0.66 and p=0.86 at 3 and 6 months). The same was true for peripheral graft thickness (p=0.45 and p=0.38 at 3 and 6 months, respectively) and ECD (p=0.97 and p=0.25 at 3 and 6 months, respectively).

Grafts unfolded successfully in the anterior chamber in all surgeries. There were no graft detachments, pupillary blocks or primary graft failures.


The influence of endothelial graft thickness on visual acuity in DSAEK is controversial.7–10 ,13 However, studies on this subject have focused either on relatively thick grafts (160–170 µm) or on grafts prepared by manual dissection.8–10 ,13 ,14 Assessment of grafts of less than 130 µm shows a positive correlation between thickness and visual acuity,6 ,7 suggesting that an effect may exist under a certain thickness. Even though other factors may also influence visual acuity, such as the extension and duration of preoperative corneal oedema and the irregularity of the anterior cornea surface, graft thickness seems to be an important factor.

Few techniques for creating grafts thinner than 100 µm have been presented.15–17 Therefore, to evaluate the influence of graft thickness on visual acuity we must have a technique that will consistently yield thin grafts.

The technique we describe in this paper involves performing a femtosecond laser first to prevent the variability of a double microkeratome cut and reduce the risk of perforation. Avoidance of a double femtosecond cut reduces the risk of a rough stromal bed and endothelial damage.18 ,19

Despite aiming for grafts of less than 100 µm, a target graft thickness figure of 110 µm was used in the formula for safety reasons. Thus, approximately 410 µm of corneal tissue is cut with a 300 µm microkeratome head. Because the microkeratome we use usually cuts more than 300 µm,20 we found that having 110 µm as the target would actually yield grafts thinner than 100 µm without perforation. Final graft thickness is obtained by programming the laser to cut at a customised depth for each cornea, depending on the initial pachymetry, in order to always leave the same thickness (approximately 410 µm) for the microkeratome cut. Accordingly, there was no correlation between initial donor thickness and graft thickness at any time point because the first cut essentially makes all corneas equal in terms of thickness. This avoids using nomograms and performing pachymetry between cuts, and means only one microkeratome head is needed, here a 300 µm head, but other heads will probably give a good result as well. Since the microkeratome cut depth depends on several factors, such as intrachamber pressure, tissue thickness and manual rotation speed, these factors are kept as constant as possible to reduce variability. An important aspect of this technique is that the thinner cut is performed first, leaving a thick cornea for the microkeratome. Because the anterior stroma is more compact, this approach avoids irregular cuts and buttonholes during the microkeratome cut.21 A further advantage is that equipment already available in many cornea centres can be used. One limitation of this technique is that donor corneas of less than 500 µm cannot be cut because the Intralase has a minimum ablation depth of 90 µm.

This new approach shows endothelial cell loss comparable with that reported for standard DSAEK and visual results similar to published DMEK results.8 ,22–28 In general, endothelial cell loss can result from overmanipulation. However, we used the same DSAEK technique as we did previously with thick grafts; there were no complications, and the grafts unfolded successfully in the anterior chamber in all procedures. Moreover, it is also known that organ cultured corneas systematically carry non-viable endothelial cells that are implicated in cell death and go undetected when trypan blue staining is used.29 There were no differences in terms of visual acuity, graft thickness and endothelial cell loss between patients undergoing DSAEK alone and those having concurrent cataract surgery. Concerning visual acuity, patients without concurrent cataract surgery were already pseudophakic, which could explain why no differences were found. Concerning graft thickness and endothelial cell loss, because cataract surgery is performed before Descemet stripping and graft insertion it is not likely to influence these parameters.

In conclusion, grafts of less than 100 µm could be safely and consistently obtained. Although the initial results are very promising, further studies with larger patient cohorts and longer follow-up times are necessary. Studies should also clarify the ideal graft thickness for DSAEK, in terms of both visual recovery and endothelial cell loss.


We thank Robert Van Velze for his help on setting the parameters of this study.


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  • Contributors AMR, conception and design, analysis and interpretation of data, manuscript writing; MJQ, EC and IMIPM, analysis and interpretation of data; MFS, manuscript writing and revising it critically for important intellectual content; JNM, conception and design, manuscript revision and approval of the version to be published.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval Ethics approval was obtained from the local ethics board.

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

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