Purpose
To investigate the effect of culture conditions of donor tissue on functional outcome after Descemet membrane endothelial keratoplasty.
Design
Retrospective, single-center, consecutive case series.
Methods
Descemet membrane endothelial keratoplasty was performed routinely in 82 eyes of 82 consecutive patients using corneal donor tissue prestored in either short-term culture (Optisol-GS; Bausch & Lomb) at 4 C (group A; n = 37) or organ culture (Dulbecco Modified Eagle Medium [Biochrom]; CorneaMax Medium [Eurobio]) at 34 C (group B; n = 45) in a randomized fashion. Main outcome measures included the number of air injections necessary for graft attachment as well as best-corrected visual acuity (in logarithm of the minimal angle of resolution [logMAR] units), central corneal thickness, and endothelial cell density at 1, 3, and 6 months after surgery.
Results
Best-corrected visual acuity increased from 0.69 ± 0.53 logMAR and 0.67 ± 0.31 logMAR before surgery to 0.33 ± 0.21 logMAR and 0.28 ± 0.18 logMAR after 1 month ( P < .05), to 0.24 ± 0.16 logMAR and 0.18 ± 0.16 logMAR after 3 months ( P < .05), and to 0.18 ± 0.12 logMAR and 0.15 ± 0.10 logMAR after 6 months (n.s.) in groups A and B, respectively. Endothelial cell density decreased from 2647 ± 236 cells/mm 2 and 2515 ± 249 cells/mm 2 before surgery to 1499 ± 277 cells/mm 2 and 1526 ± 205 cells/mm 2 after 1 month ( P < .05), to 1441 ± 213 cells/mm 2 and 1443 ± 316 cells/mm 2 after 3 months (n.s.), and to 1587 ± 366 cells/mm 2 and 1457 ± 285 cells/mm 2 after 6 months (n.s.) in groups A and B, respectively. Central corneal thickness declined from 664 ± 89 and 662 ± 107 μm before surgery to 529 ± 92 μm and 517 ± 62 μm after 1 month ( P < .05), to 511 ± 46 μm and 510 ± 46 μm after 3 months ( P < .05), and to 529 ± 68 μm and 507 ± 50 μm after 6 months (n.s.) in groups A and B, respectively. Best-corrected visual acuity, endothelial cell density, and central corneal thickness values showed no significant differences between both groups at any time point after surgery. However, a significantly higher total number of air injections was necessary in group A (n = 34) compared with group B (n = 26) to obtain graft attachment ( P < .05).
Conclusions
These findings suggest that donor tissue culture conditions have no significant effect on functional outcome, but may influence graft adhesion and rebubbling rate after Descemet membrane endothelial keratoplasty surgery.
New techniques for lamellar corneal surgery recently have been described allowing for selective intervention at the level of the corneal endothelium. In routine corneal surgery, deep lamellar endothelial keratoplasty, small-incision deep lamellar endothelial keratoplasty, and Descemet stripping endothelial keratoplasty or Descemet stripping automated endothelial keratoplasty (DSAEK) all have been used to replace the diseased endothelium and Descemet membrane (DM) of a host with posterior corneal stroma, DM, and endothelium of a donor. Several large studies have shown that in the hands of an experienced surgeon, DSAEK is superior to penetrating keratoplasty (PK) with respect to postoperative astigmatism and postoperative function. However, transplantation of the posterior stroma leads to an increase in corneal thickness, and data from a small series of patients suggest that functional outcome is correlated inversely with central corneal thickness after DSAEK. Furthermore, the configuration of the posterior corneal surface may be affected adversely by the addition of a posterior stromal lamella, which can lead to increased higher-order aberrations. Recently, Descemet membrane endothelial keratoplasty (DMEK) was described allowing transplantation of isolated endothelium-Descemet membrane layer (EDM) without adherent corneal stroma. Initial results with this technique are equivalent to or even superior to that of DSAEK possibly because there is minimal alteration of the posterior surface of the cornea.
One of the factors determining the success of DMEK is the functional integrity of the grafted endothelium. Endothelial cell loss occurs after both PK and lamellar keratoplasty. The magnitude of the decrease of endothelial cell counts in the early phase after surgery has been described to be significantly higher after both DMEK and DSAEK than that after PK. Interestingly, cell counts seem to stabilize several months after DSAEK surgery, and midterm cell counts after penetrating and lamellar surgery are equivalent. The mechanisms controlling survival and function of endothelial cells after both PK and lamellar surgery currently are not understood. For lamellar surgery, it has been speculated that tissue manipulation during donor preparation (eg, endothelial stripping) as well as manipulation of the tissue during surgery may be responsible for endothelial cell loss.
One of the factors that determine endothelial viability is the effect of culture conditions during storage of donor tissue. How culture conditions affect the outcome after posterior lamellar surgery in general and after DMEK in particular has not been investigated previously. Storage conditions vary widely between continents: whereas short-term storage at 4 C is the method of choice in the United States and most of Asia, long-term organ culture at 34 C is preferred by European eye banks. Currently, most large series describing endothelial function after DSAEK are based on short-term storage at 4 C, whereas most available data on DMEK stem from organ culture at 34 C. It is likely that not only cellular functions of the endothelium, but also structural and biophysical properties of DM, which are important for graft adhesion, are affected by the culture method. Based on the European experience with tissue banking, we tested the hypothesis that organ culture at 34 C not only supports endothelial cell viability in the early postoperative phase after DMEK, but also enhances graft adhesion.
Methods
Patients
DMEK was performed in 82 eyes of 82 consecutive patients (43 males and 39 females) between 40 and 89 years of age (mean age ± standard deviation [SD], 68.1 ± 10.4 years). Indications for surgery were Fuchs endothelial dystrophy (n = 74), bullous keratopathy after failed DSAEK (n = 6), bullous keratopathy after trabeculectomy with 5-fluorouracil (n = 1), and bullous keratopathy in Fuchs heterochromic cyclitis (n = 1). In our study, donor tissue was assigned to patients according to the availability of tissue by our eye bank. Based on the ophthalmologic examination, patients with a similar state of endothelial dysfunction were assigned to fixed slots on a waiting list in the order of their appearance. From the top of this list, patients were called for surgery consecutively and were assigned randomly to either an organ-cultured graft or a graft from short-term storage by the eye bank technician. For analysis, the patients were assigned to 2 groups. Patients in group A (n = 37 eyes) received an EDM prepared from a corneoscleral button that had been stored in Optisol-GS (Bausch & Lomb, Irvine, California, USA) at 4 C. Patients in group B (n = 45 eyes) received an EDM from a corneoscleral button that had been stored in Dulbecco modified eagle medium containing streptomycin and penicillin (Biochrom, Berlin, Germany) as well as fetal calf serum (Linaris, Bettingen am Main, Germany) or in CorneaMax Medium (Eurobio, Les Ulys, France) containing streptomycin and penicillin as well as fetal calf serum at 34 C. Depending on the status of the lens, DMEK was combined with phacoemulsification and implantation of an Acri.Tec single-piece acrylic intraocular lens 46 S AcriSmart (Acri.Tec GmbH, Hennigsdorf, Germany) in 15 of 37 patients in group A and in 15 of 45 patients in group B.
Donor Tissue
Corneal donor tissue was obtained either from eye banks in Europe (Germany and The Netherlands) or the United States. Forty-five corneoscleral buttons of European origin were organ cultured, whereas 37 corneoscleral buttons from United States eye banks were stored short term. All 37 corneoscleral buttons obtained from United States eye banks were stored in Optisol-GS at 4 C for 226 ± 74 hours (range, 24 to 336 hours; median, 240 hours; mode, 240 hours). Corneoscleral buttons in organ culture were preserved in Dulbecco modified eagle medium (n = 44) or in CorneaMax Medium (n =1). Corneoscleral buttons were cultured at 34 C for 487 ± 179 hours (range, 70 to 868 hours; median, 503 hours; mode, 240 hours). There was no significant difference concerning donor age between corneas that were organ cultured (range, 45 to 84 years; mean ± SD, 68.3 ± 8 years) and corneas from short-term culture (range, 49 to 84 years; mean ± SD, 66 ± 11 years; P = n.s., t test). There was, however, a significant difference concerning death to preservation time between corneas that were organ cultured (range, 3 to 64 hours; mean ± SD, 16 ± 13 hours) and corneas from short-term culture (range, 2 to 20 hours; mean, 9 ± 4.5 hours; P < .05, t test). Endothelial cell density (ECD) of all donor corneas was quantified by the respective eye bank before culture. ECD of corneoscleral buttons in organ culture was quantified a second time at the end of the culture period after transfer into deswelling medium, which is normally 1 to 3 days before surgery. The mean ± SD preoperative ECD of donor corneas in the organ culture group was 2515 ± 249 cells/cm 2 (range, 2050 to 2948 cells/cm 2 ; n = 45). The mean ± SD preoperative ECD of donor corneas in the short-term culture group was 2647 ± 236 cells/cm 2 (range, 2064 to 3122 cells/cm 2 ; n = 37). The comparison of preoperative ECD between both groups showed a significant difference ( P = .017, t test).
Surgery
DMEK procedures were performed by 2 experienced surgeons (F.E.K., C.C.) using the same technique. One day before surgery, all patients underwent neodymium:yttrium–aluminum–garnet laser iridotomy. Immediately before transplantation, the EDM was stripped from the donor corneal stroma by a technique published by Kruse and associates that includes several modifications from the original technique described by Melles and associates. After mounting the corneoscleral buttons on a suction block (Hanna Trephination System; Moria Instruments, Antony, France), the endothelium was marked by gentle touch with an 8.0-mm trephine and was stained with 0.06% trypan blue (Vision Blue; D.O.R.C. Deutschland GmbH, Berlin Germany) for 60 seconds. The submerged EDM peripheral to the mark was removed with a razor blade. The central edge first was lifted with a round blade and then grasped with 2 forceps. By simultaneous centripetal movement of the 2 forceps, the EDM was detached incompletely, followed by trephination with an 8-mm trephine and marking the edges with a 1-mm trephine, as described previously. For better visualization, EDM was stained again and stripping was completed. Transfer of the graft into the patient’s eye and unfolding was achieved by a standardized technique. Because of the elastic properties of DM, the EDM roll, which formed spontaneously, was transferred into the same type of standard intraocular lens injector cartridge (Acri.Tec GmbH) that had been used in patients receiving an intraocular lens implant. Recipient preparation was carried out through the same 2.5-mm corneal tunnel at the 12-o’clock position, regardless of simultaneous phacoemulsification, and the patient’s EDM was removed under air using an inverted hook (Price Hook; Moria). The graft was injected into the anterior chamber maintaining the volume of the anterior chamber with the irrigation hand piece (diameter, 0.6 mm; Geuder AG, Heidelberg, Germany). The EDM was positioned centrally using short bursts of balanced salt solution and unfolded by injecting a series of subsequent small air bubbles. By enlarging the air bubble, the EDM then was flattened out on the surface of the iris and air was removed completely, resulting in shallowing of the anterior chamber. Air was injected underneath the graft until the anterior chamber was filled completely with air and left in place for 30 minutes. On completion of the surgery, air was reduced to approximately 50% of the anterior chamber volume.
Postoperative Fllow-up
The clinical outcome after DMEK was assessed by the number of air injections necessary for graft attachment, best-corrected visual acuity (BCVA; logarithm of the minimal angle of resolution [logMAR] units), central corneal thickness (CCT), and endothelial cell density (ECD) at 1, 3, and 6 months after surgery. BCVA (logMAR) was determined before as well as 1, 3, and 6 months after surgery. CCT was quantified before as well as 1, 3, and 6 months after transplantation by Scheimpflug imaging (Pentacam; Oculus, Wetzlar, Germany). ECD was evaluated 1, 3, and 6 months after surgery by in vivo confocal microscopy (SeaEagle; HAI Laboratories, Lexington, Kentucky, USA).
The decision for additional air injections was based on slit-lamp biomicroscopy and the results of examinations using the slit-lamp optical coherence tomography device (Heidelberg Engineering, Heidelberg, Germany). Slit-lamp optical coherence tomography examination was performed routinely every third day within the first 2 weeks after surgery and included at least 2 sections in each of the 4 quadrants. Rebubbling was performed in all patients with significant corneal edema over more than 3 clock hours in conjunction with a detachment of the EDM of more than 100 μm in depth. Air was injected under topical anesthesia, filling the chamber completely for 1 hour and removing the air bubble to 50% of the volume of the anterior chamber. Special care was taken to ensure correct positioning of the bubble immediately after surgery by keeping the patients in a supine or otherwise fixed position in the hospital for 24 hours after the procedure.
Statistical Evaluation
Statistical evaluation was performed using SPSS software version 17.0 (SPSS, Inc, Chicago, Illinois, USA). The normal distribution of all tested values was assessed by using the Kolmogorov-Smirnov method. In normally distributed samples, differences between groups were evaluated by t test for independent samples, and differences between samples within each group were assessed by t test for paired samples. In not normally distributed samples, differences between groups were assessed by the Mann–Whitney U test. The significance level was set at P = .05. In addition, Bonferroni correction was applied by multiplying the results of the initial statistical evaluations ( t test) by the number of tests (n = 8). The correlation between rebubbling rate and storage time in the short-term culture group was assessed using the Spearman test.
Results
Visual Outcome
The BCVA achieved after DMEK is shown in Figure 1 . For the final analysis, 13 patients (4 in group A and 9 in group B) were excluded because of pre-existing conditions limiting visual acuity, for example, macular degeneration (n = 6), epiretinal gliosis with cystoid macular edema (n = 1), advanced glaucomatous optic atrophy (n = 3), and previous retinal surgery (retinal detachment, macular hole, endophthalmitis; n = 3). Mean BCVA ± SD in group A was 0.69 ± 0.53 logMAR before surgery (n = 33) and increased to 0.33 ± 0.21 logMAR at 1 month (n = 33), to 0.24 ± 0.16 logMAR at 3 months (n = 26), and to 0.18 ± 0.12 logMAR at 6 months (n = 20) after surgery. The increase in BCVA within the first month and between 1 month and 3 months was statistically significant ( P ≤ .005, t test). During a follow-up period of up to 6 months, no further statistically significant increase was noted ( Figure 1 ). Mean BCVA ± SD in group B was 0.67 ± 0.31 logMAR before surgery (n = 36) and increased to 0.28 ± 0.18 logMAR at 1 month (n = 35), to 0.18 ± 0.16 logMAR at 3 months (n = 27), and to 0.15 ± 0.10 logMAR at 6 months (n = 21) after surgery. The increase in BCVA within the first month and between 1 month and 3 months was statistically significant ( P ≤ .005, t test). During a follow-up period of up to 6 months, no further statistically significant increase was noted ( Figure 1 ). The comparison of BCVA between both groups A and B showed no significant difference at any time point ( P = n.s., t test; Table 1 ).
| Before Surgery | 1 Month after Surgery | 3 Months after Surgery | 6 Months after Surgery | |
|---|---|---|---|---|
| Group A (short-term culture) | 0.69 ± 0.53 (0.15 to 3.0), n = 33 | 0.33 ± 0.21 (0.1 to 1.0), n = 33 | 0.24 ± 0.16 (0.0 to 0.7), n = 26 | 0.18 ± 0.12 (0.0 to 0.5), n = 20 |
| Group B (organ culture) | 0.67 ± 0.31 (0.3 to 1.6), n = 36 | 0.28 ± 0.18 (0.0 to 0.8), n = 35 | 0.18 ± 0.16 (−0.1 to 0.7), n = 27 | 0.15 ± 0.1 (0.0 to 0.4), n = 21 |
| P Value | n.s. | n.s. | n.s. | n.s. |
Endothelial Cell Density
The mean ± SD preoperative ECD of donor corneas in group A was 2647 ± 236 cells/cm 2 (n = 37). After 1 month, mean ECD had decreased to 1499 ± 277 cells/cm 2 (n = 32). This decrease was statistically significant when compared with preoperative values ( P < .005, t test). At 3 months, a further decrease of ECD to 1441 ± 213 cells/cm 2 (n = 28) was observed. The mean ECD ± SD at 6 months was 1587 ± 373 cells/cm 2 (n = 22). The decrease of ECD between 1 and 3 months as well as that between 3 and 6 months were not statistically significant ( Figure 2 ). The mean preoperative ECD of donor corneas in group B was 2515 ± 249 cells/cm 2 (n = 45). After 1 month, ECD had decreased to 1526 ± 205 cells/cm 2 (n = 37). This decrease was statistically significant when compared with preoperative values ( P < .005, t test). At 3 months, a further decrease of ECD to 1443 ± 316 cells/cm 2 (n = 32) was observed. The ECD after 6 months was 1457 ± 285 cells/cm 2 (n = 25). The decrease of ECD between 1 and 3 months as well as that between 3 and 6 months were statistically not significant ( Figure 2 ).
The comparison of ECD between group A and B showed a significantly lower preoperative ECD in group B ( P = .017, t test). After surgery, no significant differences in ECD were observed at any time point between both groups ( Table 2 ).
| Before Surgery | 1 Month after Surgery | 3 Months after Surgery | 6 Months after Surgery | |
|---|---|---|---|---|
| Group A (short-term culture) | 2647 ± 236 (2064 to 3122), n = 37 | 1499 ± 277 (905 to 2222), n = 32 | 1441 ± 213 (1024 to 1832), n = 28 | 1587 ± 373 (926 to 2382), n = 21 |
| Group B (organ culture) | 2515 ± 249 (2050 to 2948), n = 45 | 1526 ± 205 (1001 to 206), n = 37 | 1443 ± 316 (586 to 316), n = 32 | 1457 ± 285 (1001 to 285), n = 25 |
| P value | ≤ .05 | n.s. | n.s. | n.s. |
Central Corneal Thickness
Before surgery, the mean CCT ± SD in group A was 664 ± 89 μm (n = 32). At 1 month after surgery, CCT decreased to 529 ± 92 μm (n = 31) and to 511 ± 46 μm (n = 25) at 3 months, and measured 529 ± 68 μm (n = 20) after 6 months. The decrease of CCT within the first month was statistically significant ( P ≤ .005, t test). The decrease between 1 and 3 months and that between 3 and 6 months were statistically not significant ( Figure 3 ). Before surgery, the mean CCT ± SD in group B was 662 ± 107 μm (n = 41). At 1 month after surgery, mean CCT ± SD decreased to 517 ± 62 μm (n = 34), to 510 ± 46 μm after 3 months (n = 31), and to 507 ± 50 μm after 6 months (n = 25). The decrease of CCT within the first month and that between 1 and 3 months were statistically significant ( P ≤ .005, t test). In the follow-up until 6 months, no further statistically significant decrease was noted ( Figure 3 ). The comparison of CCT in both groups A and B showed no statistically significant differences at any time point ( P = n.s., t test; Table 3 ).
| Before Surgery | 1 Month after Surgery | 3 Months after Surgery | 6 Months after Surgery | |
|---|---|---|---|---|
| Group A (short-term culture) | 664 ± 89 (527 to 1024), n = 32 | 529 ± 92 (396 to 935), n = 31 | 511 ± 46 (427 to 603), n = 25 | 529 ± 68 (437 to 681), n = 20 |
| Group B (organ culture) | 662 ± 107 (491 to 1127), n = 41 | 517 ± 62 (374 to 703), n = 34 | 510 ± 46 (367 to 593), n = 31 | 507 ± 50 (387 to 618), n = 25 |
| P value | n.s. | n.s. | n.s. | n.s. |
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