CLINICAL SCIENCE
Diabetes Mellitus Increases Risk of Unsuccessful Graft Preparation in Descemet Membrane Endothelial Keratoplasty: A Multicenter Study Mark A. Greiner, MD,*† Jordan J. Rixen, MD,* Michael D. Wagoner, MD, PhD,*† Gregory A. Schmidt, BS, CEBT,† Christopher G. Stoeger, MBA, CEBT,‡ Michael D. Straiko, MD,§ M. Bridget Zimmerman, PhD,* Anna S. Kitzmann, MD,*† and Kenneth M. Goins, MD*†
Purpose: The aim of this study was to evaluate preparation outcomes of tissue prepared for Descemet membrane endothelial keratoplasty (DMEK) from diabetic and nondiabetic donors.
Key Words: Descemet membrane endothelial keratoplasty, diabetes mellitus, eye banking, corneal edema (Cornea 2014;33:1129–1133)
Methods: In this nonrandomized, consecutive case series, DMEK grafts were prepared from diabetic and nondiabetic donors by experienced technicians in 2 eye banks using slightly different, modified submerged manual preparation techniques to achieve “prestripped” graft tissue. Graft preparation results were analyzed retrospectively. The main outcome measure was the rate of unsuccessful (failed) DMEK graft preparations, defined as tears through the graft area that prevent tissue use.
Results: A total of 359 corneas prepared from 290 donors (114 diabetic and 245 nondiabetic) were included in the statistical analysis of graft preparation failure. There were no significant differences between diabetic and nondiabetic donor tissue characteristics with respect to donor age, death to preservation time, death to preparation time, endothelial cell density, percent hexagonality, or coefficient of variation. DMEK tissue preparation was unsuccessful in 19 (5.3%) cases. There was a significant difference in the site-adjusted rate of DMEK preparation failure between diabetic [15.3%; 95% confidence interval (CI), 9.0–25.0] and nondiabetic donors (1.9%; 95% CI, 0.8– 4.8), and the corresponding site-adjusted odds ratio of DMEK graft preparation failure in diabetic donor tissue versus nondiabetic donor tissue was 9.20 (95% CI, 2.89–29.32; P = 0.001). Conclusions: Diabetes may be a risk factor for unsuccessful preparation of donor tissue for DMEK. We recommend caution in the use of diabetic tissue for DMEK graft preparation. Further study is needed to identify what subset of diabetic donors is at risk for unsuccessful DMEK graft preparation. Received for publication March 8, 2014; revision received August 1, 2014; accepted August 1, 2014. Published online ahead of print September 15, 2014. From the *Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, IA; †Iowa Lions Eye Bank, Coralville, IA; ‡Lions VisionGift, Portland, OR; and §Devers Eye Institute, Portland, OR. Drs K. M. Goins, M. A. Greiner, A. S. Kitzmann, and M. D. Wagoner received salary support for eye bank medical directorships. The remaining authors have no funding or conflicts of interest to disclose. Reprints: Mark A. Greiner, MD, Department of Ophthalmology and Visual Sciences, Cornea and External Diseases, University of Iowa Hospitals and Clinics, 200 Hawkins Dr, Iowa City, IA 52242 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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escemet membrane endothelial keratoplasty (DMEK) represents an increasingly popular transplant technique that achieves superior visual results and maximizes the speed of visual recovery in the treatment of corneal endothelial dysfunction from Fuchs corneal dystrophy and pseudophakic bullous keratopathy.1–6 Graft preparation has been a barrier to the widespread adoption of DMEK surgery because of the delicate nature of the tissue and the risks of tissue destruction during the preparation process. The graft tissue, a 15-mm-thick monolayer of corneal endothelial cells and their adherent Descemet membrane (DM), can tear during preparation, rendering the tissue unsuitable for transplantation and challenging the surgeon to confront risks of tissue wastage, financial loss, and surgery cancellation.7–10 Several eye banks in the United States have responded to this challenge by offering surgeons “prestripped” tissue,10,11 shipped after the endothelial cell-DM monolayer (EDM) has been separated manually from 80% to 90% of the stromal area of attachment and laid back down in its native position. When punched to the desired size, this preparation yields donor endothelial tissue that the surgeon can grasp and prepare for insertion with relative ease at the time of surgery. Because of technical complexity, the risks of tissue loss from DMEK graft preparation are substantial. The outcomes of domestic eye bank–prepared DMEK tissue have not been assessed. Experienced eye bank technicians at Iowa Lions Eye Bank [(ILEB), Coralville, IA] began preparing corneal tissue for DMEK surgery in 2012 and observed anecdotally that DMEK tissue preparation resulted in tears and was more difficult to perform when the donor had a history of diabetes mellitus. We hypothesized that diabetes mellitus may cause functional abnormalities at the interface between the EDM and posterior stroma that become manifest during DMEK graft preparation. Based on this observation and hypothesis, ILEB halted DMEK preparation of tissue from diabetic donors and reached out to other eye banks that prepare DMEK tissue to determine whether others shared similar results. In collaboration with Lions VisionGift [(LVG), www.corneajrnl.com |
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Portland, OR], we performed this retrospective investigation to evaluate our concerns about the preparation of diabetic donor tissue for DMEK surgery.
MATERIALS AND METHODS We performed a retrospective study of eye bank records of tissue from diabetic and nondiabetic donors prepared for DMEK surgery by experienced technicians at 2 Eye Bank Association of America accredited facilities (ILEB and LVG). The main outcome measure was the rate of unsuccessful (failed) DMEK preparations. The secondary outcome measure was the difficulty of DMEK preparation. The Institutional Review Boards at the University of Iowa and Legacy Health Systems determined that approval was not required for this study.
Donor Corneal Tissue Corneoscleral tissue was obtained, inspected, and stored in Optisol-GS (Bausch & Lomb, Irvine, CA) at 4°C in accordance with the Eye Bank Association of America– compliant protocols at ILEB and LVG. Donor tissue characteristics including donor age, death to preservation time, and death to preparation time were extracted from eye bank records. Before preparation, noncontact specular microscopy (using a KeratoAnalyzer EKA-10; Konan Medical USA, Inc, Irvine, CA) was performed to determine endothelial cell density (ECD), hexagonality, and coefficient of variation, which were recorded as the average of 2 measurements.
Donor Diabetes Status The donor medical history, including a diagnosis of diabetes mellitus, was recorded at the time of tissue donation. The diagnosis of diabetes mellitus was determined using current eye bank donor screening protocols at ILEB and LVG to identify the donor medical history, including donor medical records review and donor risk assessment interview with an individual who knew the donor well. Additional information about a donor’s diabetes status, including the duration of disease, insulin dependence status, and previous hemoglobin A1c readings, was not available routinely at the time of tissue preparation. All medical history information was recorded in an electronic donor data management system at each eye bank. The electronic databases were queried for the presence of diabetes mellitus in the medical history.
DMEK Graft Preparation Experienced eye bank technicians (2 at ILEB and 4 at LVG) prepared corneal tissue for DMEK surgery using a technique based on the submerged cornea using backgrounds away method.5 Both sites modified the original submerged cornea using backgrounds away technique to adapt tissue processing to the eye bank setting. Using aseptic technique, the tissue was vacuum mounted on a suction trephine (Barron punch trephine system; Katena Products Inc, Denville, NJ) to stabilize the cornea, with the endothelial
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layer exposed. At LVG, a cystotome was used to score the tissue; at ILEB, a 9.5-mm Barron trephine was tapped lightly to perform a partial-thickness trephination through DM into the posterior stroma. At both sites, the tissue was then stained with VisionBlue (DORC International, the Netherlands) for better visualization of the scored edge and rinsed gently with balanced salt solution (Alcon, Fort Worth, TX) or OptisolGS. EDM tissue, peripheral to the scoring groove, was removed with forceps to prevent interference of this tissue with graft preparation. Under a small amount of Optisol-GS to keep the endothelium from drying, the EDM complex was peeled away from the underlying stroma with 1 tying forceps, to free approximately 80% of the graft. The membrane was then placed back in its normal anatomic location using surgical sponges to remove fluid and encourage anatomical positioning. At ILEB, eye bank technicians performed an additional smaller diameter trephination of the graft if requested by the surgeon. After successful prestripping, suction was released, the corneoscleral donor tissue was placed back into Optisol-GS in the viewing chamber, and then the tissue was evaluated postpreparation using slit lamp and specular microscopy. Total graft preparation time was recorded as the time when the tissue was removed from Optisol-GS until it was returned to the storage medium.
DMEK Graft Preparation Failure Unsuccessful DMEK graft preparation (failure) was defined as one in which a tear occurred while separating the EDM from the stroma that rendered the tissue unusable for transplantation. In some cases, tears occurred in the peripheral EDM during the stripping process, which could be eliminated by a second trephination either at the eye bank or by the receiving surgeon. These tears were not considered preparation failures. If a tear was large enough to extend into the graft despite a second trephination, the preparation was deemed a failure. For the statistical analysis of graft preparation failure, all corneas prepared for DMEK at ILEB between December 18, 2012, and July 19, 2013, were included. All donor corneas prepared at ILEB after July 19, 2013, were excluded from the statistical analysis because this was the date when eye bank leadership made the decision to stop preparing DMEK tissue from diabetic donors. All corneas prepared for DMEK at LVG between October 9, 2011, and October 17, 2013, were included with the exception of donor tissue imported from outside eye banks because the diabetic status of the donor was not tracked.
DMEK Graft Preparation Difficulty The degree of difficulty in DMEK graft preparation was defined as the time out of the storage medium during preparation for all tissues prepared successfully. Failed DMEK preparations were excluded from this portion of the analysis. All DMEK grafts prepared successfully at ILEB between December 18, 2012, and July 19, 2013, and at LVG between October 9, 2011, and September 1, 2013, were used for this portion of the analysis because of the equivalency of the Ó 2014 Lippincott Williams & Wilkins
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Diabetes and DMEK Graft Preparation
preparatory steps. On September 1, 2013, LVG began marking tissue with an inked “S” stamp after EDM stripping to aid in orientation during surgery. Because of this additional step, tissue prepared by LVG after this date was excluded from analysis.
Statistical Analysis Our statistical analysis was designed to account for data from the 2 sites. A generalized linear model for binary response was used to test the effect of diabetes on DMEK graft preparation failure adjusted for site. By including site and diabetes as fixed effects in our logistical model, we were able to calculate the odds ratio (OR) of preparation failure for diabetic donors relative to nondiabetic donors adjusted for site effect. Because the dependent variable for processing failure may come from the eyes obtained from the same donor, the generalized estimating equation method12,13 was used to estimate the parameters of the generalized linear model and account for the correlation of the outcome variable of eyes from the same donor. The generalized linear model for a continuous variable fitted by the generalized estimating equation method was used to make a site-adjusted comparison of donor age, death to preservation, death to preparation, ECD, preparation time, percent hexagonality, and coefficient of variation between diabetic and nondiabetic donor eyes. From the fitted model, the site-adjusted mean rate of graft preparation failure with a 95% confidence interval (CI) for eyes with and without diabetes and the site-adjusted effect of diabetes on graft preparation failure (expressed as the OR of DMEK graft preparation failure) were computed. Additionally, from the fitted model, the site-adjusted mean processing time and SEM for eyes with and without diabetes were computed.
RESULTS A total of 359 donor corneas prepared for DMEK surgery from 290 donors were included for analysis (Table 1). This included 114 corneas from diabetic donors and 245 corneas from nondiabetic donors. A total of 295 DMEK grafts, prepared successfully from 241 donors, were included in the statistical analysis of graft preparation difficulty. This included 84 corneas from diabetic donors and 211 corneas from nondiabetic donors.
Donor Tissue Characteristics The tissue characteristics of diabetic and nondiabetic donor corneas are included in Table 2. There were no statistically significant differences between the 2 groups with respect to donor age, death to preservation time, death to preparation time, ECD, hexagonality, or coefficient of variation before preparation.
DMEK Graft Preparation Failure DMEK tissue preparation was unsuccessful in 19 (5.29%) cases (Table 3). There were no statistically significant differences in the preparation failure rate between the 2 sites when adjusted for the prevalence of diabetes (ILEB, 7.7%; 95% CI, 3.8–15.2; LVG, 4.1%; 95% CI, 2.0–8.1; P = 0.251). Adjusting for a donor history of diabetes mellitus, there was no significant difference in the rate of preparation failures between technicians performing tissue preparation. However, there was a significantly higher site-adjusted rate of DMEK preparation failure in diabetic donors (15.3%; 95% CI, 9.0–25.0) compared with that in nondiabetic donors (1.9%; 95% CI, 0.8–4.8) (Fig. 1), and the corresponding site-adjusted OR of DMEK graft preparation failure in diabetic donor tissue versus that of nondiabetic donor tissue was 9.20 (95% CI, 2.89–29.32; P = 0.001). To demonstrate that inclusion of corneal tissue from the same donor was not a confounding variable, statistical analysis was repeated on a subset of 217 donor corneas prepared for DMEK surgery (70 diabetic and 147 nondiabetic), excluding all cases in which paired corneas were obtained from the same donor. This continued to show a significantly higher site-adjusted rate of DMEK preparation failure in diabetic donors (15.8%; 95% CI, 8.3–28.1) compared with that in nondiabetic donors (2.2%; 95% CI, 0.7–6.7), and the corresponding siteadjusted OR of DMEK graft preparation failure in diabetic donor tissue versus that of nondiabetic donor tissue excluding paired corneas was 8.29 (95% CI, 2.2–31.4; P = 0.0005).
DMEK Graft Preparation Difficulty There was a statistically significant difference in the site-adjusted processing time for successful DMEK tissue preparation between diabetic (25:36 6 0:48) and nondiabetic (22:42 6 0:36) donor tissue (P = 0.009) (Table 3).
TABLE 1. Corneal Tissue Prepared for DMEK No. ILEB Donor corneas With diabetes mellitus Without diabetes mellitus Total Donors With diabetes mellitus Without diabetes mellitus Total
LVG
Total
TABLE 2. Donor Corneal Tissue Characteristics in Diabetic Versus Nondiabetic Tissues Mean (SEM)
23 50 73
91 195 286
114 245 359
19 45 64
74 152 226
93 197 290
Ó 2014 Lippincott Williams & Wilkins
Diabetes Donor age (yrs) Death to preservation time (h) Death to preparation time (d) ECD before preparation (cells/mm2) Hexagonality (%) Coefficient of variation
65.0 10:48 4.3 2730 57 34
(1.1) (0.6) (0.2) (35.0) (0.8) (0.5)
No Diabetes 64.1 10:30 4.4 2755 56 35
P
(0.6) (0.4) (0.1) (18.1) (0.5) (0.4)
0.424 0.589 0.598 0.515 0.277 0.318
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TABLE 3. DMEK Graft Preparation Outcomes in Diabetic Versus Nondiabetic Tissues Graft preparation failure Successful preparations, n (%) Failed preparations, n (%) Site-adjusted preparation failure rate, % (95% CI) Graft preparation difficulty Mean processing time, min (SEM)
Diabetes
No Diabetes
P
99 (86.84)
241 (98.37)
—
15 (13.16)
4 (1.63)
—
15.3 (9.0–25.0)
1.9 (0.8–4.8)
0.001
25:36 (0:48)
22:42 (0:36)
0.009
DISCUSSION Our multicenter study demonstrates that diabetes is a significant risk factor for unsuccessful DMEK graft preparation. The rate of unsuccessful tissue preparation was 15.3% for diabetic donor tissue. A donor history of diabetes and the 9-fold increase in the odds of processing failure that it conferred in our study should heighten awareness among surgeons and eye banks that stripping diabetic tissue is more likely to result in damaging the graft. In addition, DMEK grafts may be more difficult to prepare when tissue comes from a diabetic donor. In contrast, our eye banks prepared DMEK tissue successfully from the vast majority (98.4%) of nondiabetic donor corneas. These findings may serve as a useful tool for eye banks and surgeons to guide tissue selection for DMEK graft preparation and underscore the importance of investigating risk factors and basic mechanisms governing tissue loss and dysfunction. We feel that the strong association between tissue preparation failure and diabetes represents a functional manifestation of molecular alterations because of chronic
FIGURE 1. DMEK tissue preparation failure rate for diabetic and nondiabetic donor tissues. The mean preparation failure rate (y axis) for diabetic donors was 15.3% (95% CI, 9.0–25.0) and that for nondiabetic donors was 1.9% (95% CI, 0.8–4.8). Siteadjusted OR of preparation failure in diabetic versus nondiabetic donors was 9.20 (95% CI, 2.89–29.32; P = 0.001).
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hyperglycemia, involving DM and its zone of attachment with the posterior stroma. Excess glucose in diabetics is known to bind to an assortment of proteins through nonenzymatic glycosylation.14 With time, advanced glycation end products (AGEs) are formed, which consist of a glucose derivative bound covalently to a protein amino group. The altered structure of proteins containing AGEs can effect protein function or lead to covalent crosslinking of nearby proteins.15 Kaji et al16 demonstrated the accumulation of AGEs in human diabetic corneas. Although their investigation failed to detect immunoreactivity of an anti–AGE antibody in DM in the 8 corneas examined, the DM–stroma interface was not investigated explicitly and AGEs are known to create crosslinks between various amino acid residues in diabetic corneal collagen.17 In successful DMEK preparations, separation of the graft occurs at a cleavage plane between the posterior stroma and the interfacial matrix (IFM), anterior to the fetal anterior banded layer of DM.18–22 Adhesive glycoproteins between the IFM and posterior stroma seem to help mediate adhesion between DM and stroma. In their 2013 review of 350 manually prepared DMEK grafts, Schlötzer-Schrehardt et al found that 7 of the 350 grafts were prepared unsuccessfully (2.0%) because of very strong adhesions between the IFM and posterior stroma, resulting in tears within DM that were posterior to the IFM. Six of the 7 failed DMEK preparations in that series had thickened interfacial matrices on electron microscopy, with more intense staining for adhesive glycoproteins in that zone on immunohistochemistry. We suspect that glycation products from chronic hyperglycemia may form and deposit in the IFM, to create stronger adhesion and increased tendency for tearing during DMEK graft preparation, as was observed. Wide-spaced collagen found throughout DM and stroma in diabetic corneas23,24 may also have a deleterious effect on the tensile strength of DM. We feel that further investigation of the molecular composition and function of diabetic posterior corneal tissue is warranted. Additionally, we did not detect a morphometric difference in the corneal endothelium between diabetic and nondiabetic donor tissue in our study cohort. These findings reinforce the fact that the problems observed with diabetic DMEK graft preparation are most likely molecular in origin, and point to a need for further study at the level of cellular metabolism to better explain the differences between diabetic and nondiabetic tissues. DMEK is becoming more popular,3 and our study also calls attention to the implications of diabetes on the supply of donor corneal tissue for DMEK. The number of US citizens with diabetes mellitus has been increasing steadily.25 Nearly one third of the donors in this study had a history of diabetes. With the combination of increasing prevalence and earlier mortality related to this disease, it is reasonable to anticipate that the donor pool will be enriched with diabetic donors over the coming years. There may be a reduction in the quantity of tissue available for DMEK, whether diabetic donors fail on preparation or are shunted toward alternate cornea transplant techniques such as Descemet stripping automated endothelial keratoplasty or penetrating keratoplasty. Although we hope that studies such as ours will help sustain efforts at the local and national levels to protect the supply of donor corneal Ó 2014 Lippincott Williams & Wilkins
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tissue, we did not undertake this analysis to advocate for or against a specific eye bank policy. Our study has important limitations that stem from the retrospective nature of the investigation. We used a historical method to determine a donor’s diabetic status based on the review of medical records and interviews with individuals who knew the donor. Although it is used commonly, this approach is limited by the availability and accurate interpretation of medical records at the time of review, and the accuracy of reporting from nonmedical sources including the next of kin and other family members. Importantly, the historical method cannot identify donors with undiagnosed diabetes, an important public health25 and study design concern, which further limits accurate identification of diabetes in donors. Future studies would be strengthened by a prospective design that uses a practical historical definition of diabetes mellitus in a donor, supplemented by the use of ancillary methods for determining undiagnosed disease such as postmortem hemoglobin A1C testing. Additionally, technicians who prepared DMEK tissue were not masked to the donor’s diabetic status in this study. Although the lack of masking was not likely a source of bias in this study because diabetes was not yet known to be a potential risk for tissue damage during preparation, it is possible that the lack of masking may become a source of bias in future studies given the findings we report. Future prospective studies on diabetes and keratoplasty would benefit from the masking of technicians and surgeons to the donor’s diabetes status before tissue preparation and surgery. Our analysis was also limited by difficulties inherent in accessing premortem data that indicate disease severity, such as serial hemoglobin A1C values, from families and individuals who consent for tissue transplantation rather than research. It is likely that diabetes does not convey an equivalent risk of DMEK preparation failure for all corneal tissues. It would be ideal to identify those tissues at the highest risk for preparation failure. By examining DMEK preparation failures prospectively on the basis of disease severity including hemoglobin A1C, duration of diabetes, type of diabetes, and insulin dependence status, it would be possible to conduct a stratified assessment of the risk conferred by diabetes to better use our available donor tissue. In conclusion, our study shows that DMEK graft preparation may be more likely to fail when the donor has a history of diabetes mellitus. Even when successful, tissue preparation from these donors may be more difficult and may have implications on subsequent graft survival that warrant further investigation. We believe that these findings may be attributable to functional manifestations of molecular alterations in diabetic DM that affect normal adhesion to the posterior stroma. For these reasons, we advise caution in the use of diabetic tissue for DMEK graft preparation and recommend avoidance of such tissue whenever possible. Based on these data, surgeons and eye banks should have a heightened awareness related to the increased risk of tissue loss associated with a donor’s history of diabetes. Further study is needed to identify what subset of diabetic donors is at risk for unsuccessful DMEK graft preparation. Ó 2014 Lippincott Williams & Wilkins
Diabetes and DMEK Graft Preparation
REFERENCES 1. Dapena I, Ham L, Droutsas K, et al. Learning curve in Descemet’s membrane endothelial keratoplasty: first series of 135 consecutive cases. Ophthalmology. 2011;118:2147–2154. 2. Dirisamer M, Ham L, Dapena I, et al. Efficacy of Descemet membrane endothelial keratoplasty: clinical outcome of 200 consecutive cases after a learning curve of 25 cases. Arch Ophthalmol. 2011;129:1435–1443. 3. Eye Bank Association of America. 2013 Eye Banking Statistical Report. Washington, DC: Eye Bank Association of America; 2013. 4. Guerra FP, Anshu A, Price MO, et al. Descemet’s membrane endothelial keratoplasty: prospective study of 1-year visual outcomes, graft survival, and endothelial cell loss. Ophthalmology. 2011;118:2368–2373. 5. Price MO, Giebel AW, Fairchild KM, et al. Descemet’s membrane endothelial keratoplasty: prospective multicenter study of visual and refractive outcomes and endothelial survival. Ophthalmology. 2009;116: 2361–2368. 6. Tourtas T, Laaser K, Bachmann BO, et al. Descemet membrane endothelial keratoplasty versus Descemet stripping automated endothelial keratoplasty. Am J Ophthalmol. 2012;153:1082.e2–1090.e2. 7. Kruse FE, Laaser K, Cursiefen C, et al. A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty. Cornea. 2011;30:580–587. 8. Lie JT, Birbal R, Ham L, et al. Donor tissue preparation for Descemet membrane endothelial keratoplasty. J Cataract Refract Surg. 2008;34: 1578–1583. 9. Melles GR, Ong TS, Ververs B, et al. Descemet membrane endothelial keratoplasty (DMEK). Cornea. 2006;25:987–990. 10. Terry MA. Endothelial keratoplasty: why aren’t we all doing Descemet membrane endothelial keratoplasty? Cornea. 2012;31:469–471. 11. Cursiefen C. Descemet membrane endothelial keratoplasty: the taming of the shrew. JAMA Ophthalmol. 2013;131:88–89. 12. Hanley JA, Negassa A, Edwardes MD, et al. Statistical analysis of correlated data using generalized estimating equations: an orientation. Am J Epidemiol. 2003;157:364–375. 13. Rosner B, Milton RC. Significance testing for correlated binary outcome data. Biometrics. 1988;44:505–512. 14. Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosylation and the pathogenesis of diabetic complications. Ann Intern Med. 1984;101: 527–537. 15. Stitt AW. Advanced glycation: an important pathological event in diabetic and age related ocular disease. Br J Ophthalmol. 2001;85:746–753. 16. Kaji Y, Usui T, Oshika T, et al. Advanced glycation end products in diabetic corneas. Invest Ophthalmol Vis Sci. 2000;41:362–368. 17. Sady C, Khosrof S, Nagaraj R. Advanced Maillard reaction and crosslinking of corneal collagen in diabetes. Biochem Biophys Res Commun. 1995;214:793–797. 18. Johnson DH, Bourne WM, Campbell RJ. The ultrastructure of Descemet’s membrane. I. Changes with age in normal corneas. Arch Ophthalmol. 1982;100:1942–1947. 19. Levy SG, McCartney AC, Moss J. The distribution of fibronectin and P component in Descemet’s membrane: an immunoelectron microscopic study. Curr Eye Res. 1995;14:865–870. 20. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci. 1984;25: 1402–1415. 21. Schlötzer-Schrehardt U, Bachmann BO, Laaser K, et al. Characterization of the cleavage plane in DESCemet’s membrane endothelial keratoplasty. Ophthalmology. 2011;118:1950–1957. 22. Schlötzer-Schrehardt U, Bachmann BO, Tourtas T, et al. Reproducibility of graft preparations in Descemet’s membrane endothelial keratoplasty. Ophthalmology. 2013;120:1769–1777. 23. Akimoto Y, Sawada H, Ohara-Imaizumi M, et al. Change in longspacing collagen in Descemet’s membrane of diabetic Goto-Kakizaki rats and its suppression by antidiabetic agents. Exp Diabetes Res. 2008;2008:818341. 24. Latour G, Kowalczuk L, Savoldelli M, et al. Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy. PLoS One. 2012;7:e48388. 25. Centers for Disease Control and Prevention. Diabetes data & trends. Available at: http://apps.nccd.cdc.gov/DDTSTRS/default.aspx. Accessed December 7, 2013.
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Diabetes Mellitus Increases Risk of Unsuccessful Graft Preparation in Descemet Membrane Endothelial Keratoplasty: A Multicenter Study Mark A. Greiner, MD,*† Jordan J. Rixen, MD,* Michael D. Wagoner, MD, PhD,*† Gregory A. Schmidt, BS, CEBT,† Christopher G. Stoeger, MBA, CEBT,‡ Michael D. Straiko, MD,§ M. Bridget Zimmerman, PhD,* Anna S. Kitzmann, MD,*† and Kenneth M. Goins, MD*†
Purpose: The aim of this study was to evaluate preparation outcomes of tissue prepared for Descemet membrane endothelial keratoplasty (DMEK) from diabetic and nondiabetic donors.
Key Words: Descemet membrane endothelial keratoplasty, diabetes mellitus, eye banking, corneal edema (Cornea 2014;33:1129–1133)
Methods: In this nonrandomized, consecutive case series, DMEK grafts were prepared from diabetic and nondiabetic donors by experienced technicians in 2 eye banks using slightly different, modified submerged manual preparation techniques to achieve “prestripped” graft tissue. Graft preparation results were analyzed retrospectively. The main outcome measure was the rate of unsuccessful (failed) DMEK graft preparations, defined as tears through the graft area that prevent tissue use.
Results: A total of 359 corneas prepared from 290 donors (114 diabetic and 245 nondiabetic) were included in the statistical analysis of graft preparation failure. There were no significant differences between diabetic and nondiabetic donor tissue characteristics with respect to donor age, death to preservation time, death to preparation time, endothelial cell density, percent hexagonality, or coefficient of variation. DMEK tissue preparation was unsuccessful in 19 (5.3%) cases. There was a significant difference in the site-adjusted rate of DMEK preparation failure between diabetic [15.3%; 95% confidence interval (CI), 9.0–25.0] and nondiabetic donors (1.9%; 95% CI, 0.8– 4.8), and the corresponding site-adjusted odds ratio of DMEK graft preparation failure in diabetic donor tissue versus nondiabetic donor tissue was 9.20 (95% CI, 2.89–29.32; P = 0.001). Conclusions: Diabetes may be a risk factor for unsuccessful preparation of donor tissue for DMEK. We recommend caution in the use of diabetic tissue for DMEK graft preparation. Further study is needed to identify what subset of diabetic donors is at risk for unsuccessful DMEK graft preparation. Received for publication March 8, 2014; revision received August 1, 2014; accepted August 1, 2014. Published online ahead of print September 15, 2014. From the *Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, Iowa City, IA; †Iowa Lions Eye Bank, Coralville, IA; ‡Lions VisionGift, Portland, OR; and §Devers Eye Institute, Portland, OR. Drs K. M. Goins, M. A. Greiner, A. S. Kitzmann, and M. D. Wagoner received salary support for eye bank medical directorships. The remaining authors have no funding or conflicts of interest to disclose. Reprints: Mark A. Greiner, MD, Department of Ophthalmology and Visual Sciences, Cornea and External Diseases, University of Iowa Hospitals and Clinics, 200 Hawkins Dr, Iowa City, IA 52242 (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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escemet membrane endothelial keratoplasty (DMEK) represents an increasingly popular transplant technique that achieves superior visual results and maximizes the speed of visual recovery in the treatment of corneal endothelial dysfunction from Fuchs corneal dystrophy and pseudophakic bullous keratopathy.1–6 Graft preparation has been a barrier to the widespread adoption of DMEK surgery because of the delicate nature of the tissue and the risks of tissue destruction during the preparation process. The graft tissue, a 15-mm-thick monolayer of corneal endothelial cells and their adherent Descemet membrane (DM), can tear during preparation, rendering the tissue unsuitable for transplantation and challenging the surgeon to confront risks of tissue wastage, financial loss, and surgery cancellation.7–10 Several eye banks in the United States have responded to this challenge by offering surgeons “prestripped” tissue,10,11 shipped after the endothelial cell-DM monolayer (EDM) has been separated manually from 80% to 90% of the stromal area of attachment and laid back down in its native position. When punched to the desired size, this preparation yields donor endothelial tissue that the surgeon can grasp and prepare for insertion with relative ease at the time of surgery. Because of technical complexity, the risks of tissue loss from DMEK graft preparation are substantial. The outcomes of domestic eye bank–prepared DMEK tissue have not been assessed. Experienced eye bank technicians at Iowa Lions Eye Bank [(ILEB), Coralville, IA] began preparing corneal tissue for DMEK surgery in 2012 and observed anecdotally that DMEK tissue preparation resulted in tears and was more difficult to perform when the donor had a history of diabetes mellitus. We hypothesized that diabetes mellitus may cause functional abnormalities at the interface between the EDM and posterior stroma that become manifest during DMEK graft preparation. Based on this observation and hypothesis, ILEB halted DMEK preparation of tissue from diabetic donors and reached out to other eye banks that prepare DMEK tissue to determine whether others shared similar results. In collaboration with Lions VisionGift [(LVG), www.corneajrnl.com |
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Portland, OR], we performed this retrospective investigation to evaluate our concerns about the preparation of diabetic donor tissue for DMEK surgery.
MATERIALS AND METHODS We performed a retrospective study of eye bank records of tissue from diabetic and nondiabetic donors prepared for DMEK surgery by experienced technicians at 2 Eye Bank Association of America accredited facilities (ILEB and LVG). The main outcome measure was the rate of unsuccessful (failed) DMEK preparations. The secondary outcome measure was the difficulty of DMEK preparation. The Institutional Review Boards at the University of Iowa and Legacy Health Systems determined that approval was not required for this study.
Donor Corneal Tissue Corneoscleral tissue was obtained, inspected, and stored in Optisol-GS (Bausch & Lomb, Irvine, CA) at 4°C in accordance with the Eye Bank Association of America– compliant protocols at ILEB and LVG. Donor tissue characteristics including donor age, death to preservation time, and death to preparation time were extracted from eye bank records. Before preparation, noncontact specular microscopy (using a KeratoAnalyzer EKA-10; Konan Medical USA, Inc, Irvine, CA) was performed to determine endothelial cell density (ECD), hexagonality, and coefficient of variation, which were recorded as the average of 2 measurements.
Donor Diabetes Status The donor medical history, including a diagnosis of diabetes mellitus, was recorded at the time of tissue donation. The diagnosis of diabetes mellitus was determined using current eye bank donor screening protocols at ILEB and LVG to identify the donor medical history, including donor medical records review and donor risk assessment interview with an individual who knew the donor well. Additional information about a donor’s diabetes status, including the duration of disease, insulin dependence status, and previous hemoglobin A1c readings, was not available routinely at the time of tissue preparation. All medical history information was recorded in an electronic donor data management system at each eye bank. The electronic databases were queried for the presence of diabetes mellitus in the medical history.
DMEK Graft Preparation Experienced eye bank technicians (2 at ILEB and 4 at LVG) prepared corneal tissue for DMEK surgery using a technique based on the submerged cornea using backgrounds away method.5 Both sites modified the original submerged cornea using backgrounds away technique to adapt tissue processing to the eye bank setting. Using aseptic technique, the tissue was vacuum mounted on a suction trephine (Barron punch trephine system; Katena Products Inc, Denville, NJ) to stabilize the cornea, with the endothelial
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layer exposed. At LVG, a cystotome was used to score the tissue; at ILEB, a 9.5-mm Barron trephine was tapped lightly to perform a partial-thickness trephination through DM into the posterior stroma. At both sites, the tissue was then stained with VisionBlue (DORC International, the Netherlands) for better visualization of the scored edge and rinsed gently with balanced salt solution (Alcon, Fort Worth, TX) or OptisolGS. EDM tissue, peripheral to the scoring groove, was removed with forceps to prevent interference of this tissue with graft preparation. Under a small amount of Optisol-GS to keep the endothelium from drying, the EDM complex was peeled away from the underlying stroma with 1 tying forceps, to free approximately 80% of the graft. The membrane was then placed back in its normal anatomic location using surgical sponges to remove fluid and encourage anatomical positioning. At ILEB, eye bank technicians performed an additional smaller diameter trephination of the graft if requested by the surgeon. After successful prestripping, suction was released, the corneoscleral donor tissue was placed back into Optisol-GS in the viewing chamber, and then the tissue was evaluated postpreparation using slit lamp and specular microscopy. Total graft preparation time was recorded as the time when the tissue was removed from Optisol-GS until it was returned to the storage medium.
DMEK Graft Preparation Failure Unsuccessful DMEK graft preparation (failure) was defined as one in which a tear occurred while separating the EDM from the stroma that rendered the tissue unusable for transplantation. In some cases, tears occurred in the peripheral EDM during the stripping process, which could be eliminated by a second trephination either at the eye bank or by the receiving surgeon. These tears were not considered preparation failures. If a tear was large enough to extend into the graft despite a second trephination, the preparation was deemed a failure. For the statistical analysis of graft preparation failure, all corneas prepared for DMEK at ILEB between December 18, 2012, and July 19, 2013, were included. All donor corneas prepared at ILEB after July 19, 2013, were excluded from the statistical analysis because this was the date when eye bank leadership made the decision to stop preparing DMEK tissue from diabetic donors. All corneas prepared for DMEK at LVG between October 9, 2011, and October 17, 2013, were included with the exception of donor tissue imported from outside eye banks because the diabetic status of the donor was not tracked.
DMEK Graft Preparation Difficulty The degree of difficulty in DMEK graft preparation was defined as the time out of the storage medium during preparation for all tissues prepared successfully. Failed DMEK preparations were excluded from this portion of the analysis. All DMEK grafts prepared successfully at ILEB between December 18, 2012, and July 19, 2013, and at LVG between October 9, 2011, and September 1, 2013, were used for this portion of the analysis because of the equivalency of the Ó 2014 Lippincott Williams & Wilkins
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Diabetes and DMEK Graft Preparation
preparatory steps. On September 1, 2013, LVG began marking tissue with an inked “S” stamp after EDM stripping to aid in orientation during surgery. Because of this additional step, tissue prepared by LVG after this date was excluded from analysis.
Statistical Analysis Our statistical analysis was designed to account for data from the 2 sites. A generalized linear model for binary response was used to test the effect of diabetes on DMEK graft preparation failure adjusted for site. By including site and diabetes as fixed effects in our logistical model, we were able to calculate the odds ratio (OR) of preparation failure for diabetic donors relative to nondiabetic donors adjusted for site effect. Because the dependent variable for processing failure may come from the eyes obtained from the same donor, the generalized estimating equation method12,13 was used to estimate the parameters of the generalized linear model and account for the correlation of the outcome variable of eyes from the same donor. The generalized linear model for a continuous variable fitted by the generalized estimating equation method was used to make a site-adjusted comparison of donor age, death to preservation, death to preparation, ECD, preparation time, percent hexagonality, and coefficient of variation between diabetic and nondiabetic donor eyes. From the fitted model, the site-adjusted mean rate of graft preparation failure with a 95% confidence interval (CI) for eyes with and without diabetes and the site-adjusted effect of diabetes on graft preparation failure (expressed as the OR of DMEK graft preparation failure) were computed. Additionally, from the fitted model, the site-adjusted mean processing time and SEM for eyes with and without diabetes were computed.
RESULTS A total of 359 donor corneas prepared for DMEK surgery from 290 donors were included for analysis (Table 1). This included 114 corneas from diabetic donors and 245 corneas from nondiabetic donors. A total of 295 DMEK grafts, prepared successfully from 241 donors, were included in the statistical analysis of graft preparation difficulty. This included 84 corneas from diabetic donors and 211 corneas from nondiabetic donors.
Donor Tissue Characteristics The tissue characteristics of diabetic and nondiabetic donor corneas are included in Table 2. There were no statistically significant differences between the 2 groups with respect to donor age, death to preservation time, death to preparation time, ECD, hexagonality, or coefficient of variation before preparation.
DMEK Graft Preparation Failure DMEK tissue preparation was unsuccessful in 19 (5.29%) cases (Table 3). There were no statistically significant differences in the preparation failure rate between the 2 sites when adjusted for the prevalence of diabetes (ILEB, 7.7%; 95% CI, 3.8–15.2; LVG, 4.1%; 95% CI, 2.0–8.1; P = 0.251). Adjusting for a donor history of diabetes mellitus, there was no significant difference in the rate of preparation failures between technicians performing tissue preparation. However, there was a significantly higher site-adjusted rate of DMEK preparation failure in diabetic donors (15.3%; 95% CI, 9.0–25.0) compared with that in nondiabetic donors (1.9%; 95% CI, 0.8–4.8) (Fig. 1), and the corresponding site-adjusted OR of DMEK graft preparation failure in diabetic donor tissue versus that of nondiabetic donor tissue was 9.20 (95% CI, 2.89–29.32; P = 0.001). To demonstrate that inclusion of corneal tissue from the same donor was not a confounding variable, statistical analysis was repeated on a subset of 217 donor corneas prepared for DMEK surgery (70 diabetic and 147 nondiabetic), excluding all cases in which paired corneas were obtained from the same donor. This continued to show a significantly higher site-adjusted rate of DMEK preparation failure in diabetic donors (15.8%; 95% CI, 8.3–28.1) compared with that in nondiabetic donors (2.2%; 95% CI, 0.7–6.7), and the corresponding siteadjusted OR of DMEK graft preparation failure in diabetic donor tissue versus that of nondiabetic donor tissue excluding paired corneas was 8.29 (95% CI, 2.2–31.4; P = 0.0005).
DMEK Graft Preparation Difficulty There was a statistically significant difference in the site-adjusted processing time for successful DMEK tissue preparation between diabetic (25:36 6 0:48) and nondiabetic (22:42 6 0:36) donor tissue (P = 0.009) (Table 3).
TABLE 1. Corneal Tissue Prepared for DMEK No. ILEB Donor corneas With diabetes mellitus Without diabetes mellitus Total Donors With diabetes mellitus Without diabetes mellitus Total
LVG
Total
TABLE 2. Donor Corneal Tissue Characteristics in Diabetic Versus Nondiabetic Tissues Mean (SEM)
23 50 73
91 195 286
114 245 359
19 45 64
74 152 226
93 197 290
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Diabetes Donor age (yrs) Death to preservation time (h) Death to preparation time (d) ECD before preparation (cells/mm2) Hexagonality (%) Coefficient of variation
65.0 10:48 4.3 2730 57 34
(1.1) (0.6) (0.2) (35.0) (0.8) (0.5)
No Diabetes 64.1 10:30 4.4 2755 56 35
P
(0.6) (0.4) (0.1) (18.1) (0.5) (0.4)
0.424 0.589 0.598 0.515 0.277 0.318
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TABLE 3. DMEK Graft Preparation Outcomes in Diabetic Versus Nondiabetic Tissues Graft preparation failure Successful preparations, n (%) Failed preparations, n (%) Site-adjusted preparation failure rate, % (95% CI) Graft preparation difficulty Mean processing time, min (SEM)
Diabetes
No Diabetes
P
99 (86.84)
241 (98.37)
—
15 (13.16)
4 (1.63)
—
15.3 (9.0–25.0)
1.9 (0.8–4.8)
0.001
25:36 (0:48)
22:42 (0:36)
0.009
DISCUSSION Our multicenter study demonstrates that diabetes is a significant risk factor for unsuccessful DMEK graft preparation. The rate of unsuccessful tissue preparation was 15.3% for diabetic donor tissue. A donor history of diabetes and the 9-fold increase in the odds of processing failure that it conferred in our study should heighten awareness among surgeons and eye banks that stripping diabetic tissue is more likely to result in damaging the graft. In addition, DMEK grafts may be more difficult to prepare when tissue comes from a diabetic donor. In contrast, our eye banks prepared DMEK tissue successfully from the vast majority (98.4%) of nondiabetic donor corneas. These findings may serve as a useful tool for eye banks and surgeons to guide tissue selection for DMEK graft preparation and underscore the importance of investigating risk factors and basic mechanisms governing tissue loss and dysfunction. We feel that the strong association between tissue preparation failure and diabetes represents a functional manifestation of molecular alterations because of chronic
FIGURE 1. DMEK tissue preparation failure rate for diabetic and nondiabetic donor tissues. The mean preparation failure rate (y axis) for diabetic donors was 15.3% (95% CI, 9.0–25.0) and that for nondiabetic donors was 1.9% (95% CI, 0.8–4.8). Siteadjusted OR of preparation failure in diabetic versus nondiabetic donors was 9.20 (95% CI, 2.89–29.32; P = 0.001).
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hyperglycemia, involving DM and its zone of attachment with the posterior stroma. Excess glucose in diabetics is known to bind to an assortment of proteins through nonenzymatic glycosylation.14 With time, advanced glycation end products (AGEs) are formed, which consist of a glucose derivative bound covalently to a protein amino group. The altered structure of proteins containing AGEs can effect protein function or lead to covalent crosslinking of nearby proteins.15 Kaji et al16 demonstrated the accumulation of AGEs in human diabetic corneas. Although their investigation failed to detect immunoreactivity of an anti–AGE antibody in DM in the 8 corneas examined, the DM–stroma interface was not investigated explicitly and AGEs are known to create crosslinks between various amino acid residues in diabetic corneal collagen.17 In successful DMEK preparations, separation of the graft occurs at a cleavage plane between the posterior stroma and the interfacial matrix (IFM), anterior to the fetal anterior banded layer of DM.18–22 Adhesive glycoproteins between the IFM and posterior stroma seem to help mediate adhesion between DM and stroma. In their 2013 review of 350 manually prepared DMEK grafts, Schlötzer-Schrehardt et al found that 7 of the 350 grafts were prepared unsuccessfully (2.0%) because of very strong adhesions between the IFM and posterior stroma, resulting in tears within DM that were posterior to the IFM. Six of the 7 failed DMEK preparations in that series had thickened interfacial matrices on electron microscopy, with more intense staining for adhesive glycoproteins in that zone on immunohistochemistry. We suspect that glycation products from chronic hyperglycemia may form and deposit in the IFM, to create stronger adhesion and increased tendency for tearing during DMEK graft preparation, as was observed. Wide-spaced collagen found throughout DM and stroma in diabetic corneas23,24 may also have a deleterious effect on the tensile strength of DM. We feel that further investigation of the molecular composition and function of diabetic posterior corneal tissue is warranted. Additionally, we did not detect a morphometric difference in the corneal endothelium between diabetic and nondiabetic donor tissue in our study cohort. These findings reinforce the fact that the problems observed with diabetic DMEK graft preparation are most likely molecular in origin, and point to a need for further study at the level of cellular metabolism to better explain the differences between diabetic and nondiabetic tissues. DMEK is becoming more popular,3 and our study also calls attention to the implications of diabetes on the supply of donor corneal tissue for DMEK. The number of US citizens with diabetes mellitus has been increasing steadily.25 Nearly one third of the donors in this study had a history of diabetes. With the combination of increasing prevalence and earlier mortality related to this disease, it is reasonable to anticipate that the donor pool will be enriched with diabetic donors over the coming years. There may be a reduction in the quantity of tissue available for DMEK, whether diabetic donors fail on preparation or are shunted toward alternate cornea transplant techniques such as Descemet stripping automated endothelial keratoplasty or penetrating keratoplasty. Although we hope that studies such as ours will help sustain efforts at the local and national levels to protect the supply of donor corneal Ó 2014 Lippincott Williams & Wilkins
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tissue, we did not undertake this analysis to advocate for or against a specific eye bank policy. Our study has important limitations that stem from the retrospective nature of the investigation. We used a historical method to determine a donor’s diabetic status based on the review of medical records and interviews with individuals who knew the donor. Although it is used commonly, this approach is limited by the availability and accurate interpretation of medical records at the time of review, and the accuracy of reporting from nonmedical sources including the next of kin and other family members. Importantly, the historical method cannot identify donors with undiagnosed diabetes, an important public health25 and study design concern, which further limits accurate identification of diabetes in donors. Future studies would be strengthened by a prospective design that uses a practical historical definition of diabetes mellitus in a donor, supplemented by the use of ancillary methods for determining undiagnosed disease such as postmortem hemoglobin A1C testing. Additionally, technicians who prepared DMEK tissue were not masked to the donor’s diabetic status in this study. Although the lack of masking was not likely a source of bias in this study because diabetes was not yet known to be a potential risk for tissue damage during preparation, it is possible that the lack of masking may become a source of bias in future studies given the findings we report. Future prospective studies on diabetes and keratoplasty would benefit from the masking of technicians and surgeons to the donor’s diabetes status before tissue preparation and surgery. Our analysis was also limited by difficulties inherent in accessing premortem data that indicate disease severity, such as serial hemoglobin A1C values, from families and individuals who consent for tissue transplantation rather than research. It is likely that diabetes does not convey an equivalent risk of DMEK preparation failure for all corneal tissues. It would be ideal to identify those tissues at the highest risk for preparation failure. By examining DMEK preparation failures prospectively on the basis of disease severity including hemoglobin A1C, duration of diabetes, type of diabetes, and insulin dependence status, it would be possible to conduct a stratified assessment of the risk conferred by diabetes to better use our available donor tissue. In conclusion, our study shows that DMEK graft preparation may be more likely to fail when the donor has a history of diabetes mellitus. Even when successful, tissue preparation from these donors may be more difficult and may have implications on subsequent graft survival that warrant further investigation. We believe that these findings may be attributable to functional manifestations of molecular alterations in diabetic DM that affect normal adhesion to the posterior stroma. For these reasons, we advise caution in the use of diabetic tissue for DMEK graft preparation and recommend avoidance of such tissue whenever possible. Based on these data, surgeons and eye banks should have a heightened awareness related to the increased risk of tissue loss associated with a donor’s history of diabetes. Further study is needed to identify what subset of diabetic donors is at risk for unsuccessful DMEK graft preparation. Ó 2014 Lippincott Williams & Wilkins
Diabetes and DMEK Graft Preparation
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