TECHNIQUES
Influence of Donor Characteristics on Descemet Membrane Endothelial Keratoplasty Sonja Heinzelmann, MD, Silja Hüther, Daniel Böhringer, MD, Philipp Eberwein, MD, Thomas Reinhard, MD, and Philip Maier, MD
Purpose: Penetrating keratoplasty is being replaced by posterior lamellar techniques like Descemet stripping automated endothelial keratoplasty or Descemet membrane endothelial keratoplasty (DMEK) for the surgical treatment of patients with endothelial insufficiency. Although DMEK leads to the best visual results, Descemet stripping automated endothelial keratoplasty is still the standard procedure for many surgeons because it is technically more standardized. Here, we investigated how donor characteristics may influence DMEK surgery.
Methods: After in vitro preparation of DMEK grafts (n = 28), we measured the width of the graft roll, which we correlated to various donor characteristics. In 31 DMEK cases, we measured the intraoperative time from implantation to attachment of the graft, which we correlated to the respective donor characteristics and endothelial cell loss. We used Pearson’s method and a multifactorial linear model for the statistical assessments. Results: We found a statistically significant correlation between donor age (P , 0.001) and endothelial cell density (P , 0.05), and the width of the DMEK rolls. That is, older donors and grafts with higher endothelial cell densities formed broader graft rolls. Donor age also showed a trend to directly influence the unfolding time that took longer using younger grafts. Furthermore, the relative endothelial cell loss increased with longer unfolding times. Conclusions: We found that donor age and endothelial cell density influence the properties of DMEK grafts, and thereby the duration of the surgical procedure. Increased unfolding times result in higher endothelial cell loss. Therefore, it seems reasonable to accept preferably older donors with high endothelial cell densities for DMEK, which may be particularly true for inexperienced surgeons or complex clinical situations. Received for publication December 12, 2013; revision received January 31, 2014; accepted February 3, 2014. Published online ahead of print March 26, 2014. From the Eye Center, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. The authors have no funding or conflicts of interest to disclose. All authors have equally contributed to conception and design, or acquisition of data, or analysis and interpretation of data; and drafting the article or revising it critically for important intellectual content; final approval of the version to be published. S. Heinzelmann and P. Maier had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Reprints: Philip Maier, University Eye Hospital Freiburg, Killianstr 5, 79106 Freiburg, Germany (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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Key Words: cornea, endothelial keratoplasty, DMEK (Cornea 2014;33:644–648)
D
uring the last few years, different techniques for posterior lamellar keratoplasty have been developed to selectively replace the corneal endothelium in patients with endothelial insufficiency.1,2 Of these techniques, Descemet stripping automated endothelial keratoplasty (DSAEK) has become the standard procedure because graft preparation and graft unfolding are well standardized and reproducible. However, not all patients reach the visual acuity that may be optically possible.3,4 The reason for this might be irregularities and incongruent collagen fibers of the posterior corneal stroma in the interface between donor and host stroma,5 changes of higher-order aberrations,6–8 remnants of the recipient’s Descemet membrane, or irregularities of the posterior curvature.9 The more recently introduced Descemet membrane endothelial keratoplasty (DMEK) provides faster and more complete visual rehabilitation than DSAEK.3,10,11 However, graft preparation and unfolding are less standardized. Therefore, many surgeons still prefer DSAEK because they fear higher endothelial cell loss and a higher graft failure rate after DMEK. Yet, recent studies have shown that the endothelial cell loss and the graft failure rate after DMEK is approaching that of DSAEK.3,10,12,13 Furthermore, the risk for endothelial immune reactions seems to be much lower after DMEK than after DSAEK or penetrating keratoplasty.14 In this study, we investigate whether donor characteristics influence the properties of DMEK grafts and thereby the surgical procedure and the final outcome. Therefore, we correlated donor characteristics, firstly, to the width of DMEK grafts measured experimentally at the end of the preparation, and secondly, to the duration of graft unfolding and postoperative endothelial cell loss to find out whether this technically challenging technique may be simplified by donor selection.
MATERIALS AND METHODS Donor Preparation (Experimental Series) All grafts were stored in organ culture at 34°C according to the guidelines of the European Eye Bank Association. All DMEK grafts were prepared in a standardized way using an 8-mm trephine as described elsewhere.15 In 28 experimental cases with grafts unsuitable for transplantation due to equivocal or positive serological tests, we measured the width of the roll Cornea Volume 33, Number 6, June 2014
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Descemet Membrane Endothelial Keratoplasty
TABLE 2. Donor/Recipient Characteristics of our Clinical Series Fuchs dystrophy/pseudophakic bullous keratopathy Donor age
84% (n = 26)/16% (n = 5)
Total storage time Storage deswelling time Preoperative endothelial cell count
FIGURE 1. The DMEK grafts rolls were photographed at the end of the preparation. The width of these rolls was measured and related to the length of the grafts, so that the real width of the grafts could be calculated.
that the DMEK graft spontaneously formed when it was placed into buffered saline solution (Fig. 1). The graft characteristics of this experimental series are summarized in Table 1.
All DMEK (n = 31) were performed under general anesthesia by 3 experienced corneal surgeons at the Eye Center, Albert-Ludwigs-University of Freiburg according to the technique described by Melles et al16 as a consecutive case series. All grafts were punched with an 8-mm trephine and descemetorhexis was done approximately according to an 8-mm marking on the corneal surface. All surgeries were video recorded and we measured the time interval from the time point when the graft was placed into the anterior chamber to the time point when it was first attached to the posterior stroma of the host (= unfolding time). This time frame includes also the time it takes to center the graft after it has been unfolded and before it is attached to the posterior stroma of the recipient. All graft characteristics of this clinical series are summarized in Table 2. The study was approved by the local ethics committee and all patients gave their informed consent that their clinical data may be used for scientific analyses.
Statistical Analysis We used Pearson’s correlation method to assess the bivariate interdepencies of donor age, width of the DMEK
TABLE 1. Donor Characteristics of our Experimental Series 74.8 22.0 2.1 2383 23.7 2.01
6 6 6 6 6 6
12.6 yrs 5.3 d 1d 173 cell/mm2 15.5 h 0.45 mm
Donor characteristics of our experimental series (n = 28) presented as mean 6 SD.
Ó 2014 Lippincott Williams & Wilkins
9.1 6 8.2 (range, 2.7–41.8; median, 7.0; lower quartile, 3.4; upper quartile, 12.4) minutes
Donor/recipient characteristics of our clinical series (n = 31) presented as mean 6 SD.
rolls, and endothelial cell densities. In the second step, we fitted a multifactorial linear regression analysis to declare influencing factors on the unfolding time.
Descemet Membrane Endothelial Keratoplasty (Clinical Series)
Donor age Total storage time Storage deswelling time Preoperative endothelial cell count Time from death to corneoscleral excision Width of DMEK graft
Time from death to corneoscleral excision Unfolding time
69.1 6 12.7 (range, 34–91; median, 71; lower quartile, 64; upper quartile, 74) years 21.4 6 6.0 d 1.6 6 0.8 d 2397 6 223 (range, 2117–2920; median, 2336; lower quartile, 2190; upper quartile, 2555) cell/mm2 20.0 6 11.2 h
RESULTS Donor Characteristics and Width of DMEK Graft In our experimental series of 28 graft preparations, we found a statistically significant influence of donor age (P , 0.001) and endothelial cell density (P , 0.05) on the width of the DMEK graft. The older the donor or the higher the endothelial cell count the broader were the rolls Descemet membrane spontaneously formed when it was placed into buffered saline solution (Figs. 2A, B). In our clinical series of 31 DMEK, we found that donor age had a statistically significant influence on the unfolding time (P , 0.01). The younger the donor the longer it took to unfold the DMEK graft in the anterior chamber (Fig. 3). However, as seen in Figure 3, there was one outlier with a very long unfolding time of more than 40 minutes with a graft from a young donor below 40 years of age. Although the long unfolding time in this case was according to the surgeon (P.M.) mainly influenced by the tightness of the graft roll, there were further complicating factors like status after DSAEK and tube surgery for glaucoma treatment that may have further increased the time to unfold the graft. Yet, if this outlier is removed from the statistical analysis, the influence of donor age on the unfolding time shows a trend but does not reach statistical significance anymore. To find out how the unfolding time influences the fate of the graft by means of postoperative endothelial cell loss, we correlated the difference between the preoperative endothelial cell density from organ culture to the early postoperative central endothelial cell density measured at a mean 104 days after surgery (range, 19–347 days; median, 103 days; lower quartile, 69 days; upper quartile, 130 days). We www.corneajrnl.com |
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FIGURE 2. Influence of donor age (A) and graft’s endothelial cell density (B) on the graft width. Pearson’s correlation revealed a statistically significant increase of graft width with increasing donor age (P , 0.01, A) and increasing endothelial cell density (P , 0.05, B).
found a statistically significant correlation between the relatively early postoperative endothelial cell loss and the unfolding time with increasing endothelial cell loss by longer unfolding time (Fig. 4).
DISCUSSION In the experimental series of this study, we found that donor age and donor endothelial cell density have a statisti-
FIGURE 3. Influence of donor age on the unfolding time. The multifactorial linear regression analysis revealed a statistically significant influence of donor age (P , 0.01) on the unfolding time; longer unfolding times occurred using grafts from younger donors.
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cally significant impact on the width of the DMEK graft rolls. Because the thickness of Descemet membrane increases with age at about 1 mm per decade,17 it might be the thickness that affects the extent of how tight Descemet membrane rolls up when it is placed into buffered saline solution after being peeled off the posterior corneal stroma. Murphy et al18 found that Descemet membrane prenatally shows a fast growth in thickness from a very thin ordinary basement membrane and differentiates to a lamellar structure consisting of at least 30 layers by the end of gestation. Later, the membrane becomes thicker (from about 3 mm at birth to up to 18 mm at 98 years of
FIGURE 4. Correlation of unfolding time and early postoperative relative endothelial cell loss. Longer unfolding times lead to a statistically significant higher endothelial cell loss (P , 0.01, Pearson correlation). Ó 2014 Lippincott Williams & Wilkins
Cornea Volume 33, Number 6, June 2014
age) during life in an exponential manner by deposition of nonlamellar material posterior to the original prenatal layer. Beside the increase in thickness with aging, Kabosova et al19 found differences in the composition of Descemet membrane between infants and adults. In their histochemical analyses, the stromal side of Descemet membrane stained for laminin332, tenascin-C, and fibrillin-1 in infant but not in adult corneas, possibly having an impact on the elasticity of Descemet membrane. So Descemet membrane may also lose elasticity with aging. However, beside these data on the composition of Descemet membrane, we are not aware of any data in the literature on its elasticity. Thus, this needs further experimental clarification. Increased thickness and decreased elasticity may explain why older Descemet membranes show less tendency to roll tightly and vice versa. Although we did not investigate the preparation process itself, peeling off grafts from older donors seems to be easier as well in our experience. The reason for this could also be the increased thickness, because in this study, older Descemet membranes seem to tear less frequently than younger ones. However, in the context of Descemet tears during graft preparation, the cleavage plane of Descemet membrane may be an even more important factor than the thickness of Descemet membrane. From electron microscopic studies, we know that the stromal attachment of Descemet membrane is achieved by the so-called interfacial matrix.20,21 When stripping off Descemet membrane, Schloetzer-Schrehardt et al21 found that the cleavage plane is consistently between this interfacial matrix and the posterior corneal stroma. However, they found adherent stromal collagen fibers projecting into Descemet membrane, but only focal irregularities caused by breaks and ruptures of the interfacial matrix zone, which were, however, not associated with donor age or other donor characteristics. To our knowledge, there are as yet no studies that correlate the thickness of Descemet membrane to the endothelial cell density. However, one could speculate, because Descemet membrane primarily is the basement membrane of the corneal endothelium and therefore a product of corneal endothelial cells, it might also be thicker in individuals with high endothelial cell densities where many cells produce the membrane compared with individuals with low endothelial cell densities given the equal time of deposition. Whether the density of endothelial cells also influences the composition or the elasticity of Descemet membrane remains unclear and needs to be clarified in the future. In the context of these specific characteristics of the donor’s Descemet membrane, there may also be differences between the storage methods of the corneal grafts. All grafts included in this study had been stored in organ culture that is the preferred method in Europe. However, the characteristics of Descemet membrane may be different when cold storage is used for corneal preservation, because beside the difference in storage temperature, the storage time is shorter and there is no swelling and deswelling of the donor cornea. Therefore, the investigations presented here need to be repeated for cold storage donor corneas to find out whether Descemet membrane shows the same behavior for both storage methods. In the second clinical part of this study, we found that donor age showed a trend to influence the unfolding time, that Ó 2014 Lippincott Williams & Wilkins
Descemet Membrane Endothelial Keratoplasty
is, the time from placing the graft in the anterior chamber to the first attachment of the graft to the posterior stroma, where most manipulations of the graft occur. The unfolding of the graft took longer when grafts from younger donors were used. Because we did not have the possibility to measure the width of the DMEK grafts in our clinical series, we could not directly correlate the width of DMEK grafts to the unfolding time. However, it seems likely that increased unfolding times with younger grafts are a result of the reduced width of these grafts because it is technically more challenging to unfold tighter grafts. Finally, it is not surprising that we also found a statistically significant correlation between the unfolding time and the early postoperative endothelial cell loss as a result of the intensive manipulations of the graft during the unfolding procedure. Therefore, according to our experience, it seems helpful to use grafts from older donors (eg, above 65 years of age) and with high endothelial cell counts (eg, above 2300 cells per square millimeter) to improve the success rate of DMEK surgery, especially during the learning curve. In this context, eye banks may also accept older donors because these grafts may be better for DMEK than grafts from young individuals. Because the effect of donor age on the width of DMEK grafts and the unfolding time was much stronger than the effect of the endothelial cell density, older donors with a medium endothelial cell count may be more suitable for DMEK surgery than young donors with high endothelial cell counts, where the intraoperative endothelial cell loss may be much higher because surgery may be much more challenging. However, the unfolding time is primarily influenced by the experience of the surgeon and the individual technique used for DMEK, and with increasing experience, tighter grafts can also be unfolded without significantly more manipulations than those for the unfolding of broader grafts. This may also be true for the usage of further techniques like external corneal compressions instead of using air bubbles for unfolding of the graft. From our experience, a lower age limit of about 60 to 65 years for the donors seems to be advisable for surgeons starting with DMEK. This age limit may be reduced by increasing experience with the surgical technique to 45 to 50 years. However, experts in DMEK surgery may also accept younger donors depending on their own experience. In this study, all surgeons were well experienced in DMEK, although the techniques regarding this new, technically demanding surgical method are still being developed. So the results presented here may be especially important for the learning curve regarding this surgical technique to reduce the rate of primary graft failures. Beside the experience of the surgeon, the recipient characteristics also play an important role in DMEK because it may be very challenging to unfold the graft in complex clinical situations (eg, patients with aniridia or aphakia but also in patients with high myopia or after vitrectomy resulting in a very deep anterior chamber). In such cases, it may also be helpful to use grafts from older donors forming broader graft rolls so that unfolding in the anterior chamber is technically less demanding and the success rate of DMEK may be increased. www.corneajrnl.com |
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In conclusion, the results of this study suggest that grafts from older donors with high endothelial cell counts are most suitable for DMEK. This might especially be important for surgeons who still fear the technical demands of DMEK compared with DSAEK, so that DMEK may be easier to learn if donors older than 65 years are accepted in the beginning. This may also be true for experienced DMEK surgeons performing DMEK in complex clinical situations. REFERENCES 1. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea. 2006;25:879–881. 2. Heinzelmann S, Maier P, Reinhard T. Perspectives of posterior lamellar keratoplasty. In search of the perfect lamella [in German]. Ophthalmologe. 2011;108:825–832. 3. Guerra FP, Anshu A, Price MO, et al. Endothelial keratoplasty: fellow eyes comparison of descemet stripping automated endothelial keratoplasty and descemet membrane endothelial keratoplasty. Cornea. 2011; 30:1382–1386. 4. Heinzelmann S, Maier P, Böhringer D, et al. Visual outcome and histological findings following femtosecond laser-assisted versus microkeratome-assisted DSAEK. Graefes Arch Clin Exp Ophthalmol. 2013;251:1979–1985. 5. Heinzelmann S, Böhringer D, Maier PC, et al. Correlation between visual acuity and interface reflectivity measured by pentacam following DSAEK. Acta Ophthalmol. 2013;92:e1–e4. 6. Hindman HB, Huxlin KR, Pantanelli SM, et al. Post-DSAEK optical changes: a comprehensive prospective analysis on the role of ocular wavefront aberrations, haze, and corneal thickness. Cornea. 2013;32: 1567–1577. 7. Rudolph M, Laaser K, Bachmann BO, et al. Corneal higher-order aberrations after Descemet’s membrane endothelial keratoplasty. Ophthalmology. 2012;119:528–535. 8. Muftuoglu O, Prasher P, Bowman RW, et al. Corneal higher-order aberrations after Descemet’s stripping automated endothelial keratoplasty. Ophthalmology. 2010;117:878–884.e6.
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9. Dirisamer M, Parker J, Naveiras M, et al. Identifying causes for poor visual outcome after DSEK/DSAEK following secondary DMEK in the same eye. Acta Ophthalmol. 2013;91:131–139. 10. Tourtas T, Laaser K, Bachmann BO, et al. Descemet membrane endothelial keratoplasty versus descemet stripping automated endothelial keratoplasty. Am J Ophthalmol. 2012;153:1082–1090.e2. 11. Maier P, Reinhard T, Cursiefen C. Descemet stripping endothelial keratoplasty–rapid recovery of visual acuity. Dtsch Arztebl Int. 2013;110: 365–371. 12. Khor WB, Mehta JS, Tan DT. Descemet stripping automated endothelial keratoplasty with a graft insertion device: surgical technique and early clinical results. Am J Ophthalmol. 2011;151:223–232.e2. 13. Phillips PM, Phillips LJ, Much JW, et al. Descemet stripping endothelial keratoplasty: six-month results of the first 100 consecutive surgeries performed solo by a surgeon using 1 technique with 100% follow-up. Cornea. 2012;31:1361–1364. 14. Anshu A, Price MO, Price FW Jr. Risk of corneal transplant rejection significantly reduced with Descemet’s membrane endothelial keratoplasty. Ophthalmology. 2012;119:536–540. 15. 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. 16. Melles GR, Ong TS, Ververs B, et al. Descemet membrane endothelial keratoplasty (DMEK). Cornea. 2006;25:987–990. 17. 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. 18. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci. 1984;25: 1402–1415. 19. Kabosova A, Azar DT, Bannikov GA, et al. Compositional differences between infant and adult human corneal basement membranes. Invest Ophthalmol Vis Sci. 2007;48:4989–4999. 20. 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. 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.
Ó 2014 Lippincott Williams & Wilkins
Influence of Donor Characteristics on Descemet Membrane Endothelial Keratoplasty Sonja Heinzelmann, MD, Silja Hüther, Daniel Böhringer, MD, Philipp Eberwein, MD, Thomas Reinhard, MD, and Philip Maier, MD
Purpose: Penetrating keratoplasty is being replaced by posterior lamellar techniques like Descemet stripping automated endothelial keratoplasty or Descemet membrane endothelial keratoplasty (DMEK) for the surgical treatment of patients with endothelial insufficiency. Although DMEK leads to the best visual results, Descemet stripping automated endothelial keratoplasty is still the standard procedure for many surgeons because it is technically more standardized. Here, we investigated how donor characteristics may influence DMEK surgery.
Methods: After in vitro preparation of DMEK grafts (n = 28), we measured the width of the graft roll, which we correlated to various donor characteristics. In 31 DMEK cases, we measured the intraoperative time from implantation to attachment of the graft, which we correlated to the respective donor characteristics and endothelial cell loss. We used Pearson’s method and a multifactorial linear model for the statistical assessments. Results: We found a statistically significant correlation between donor age (P , 0.001) and endothelial cell density (P , 0.05), and the width of the DMEK rolls. That is, older donors and grafts with higher endothelial cell densities formed broader graft rolls. Donor age also showed a trend to directly influence the unfolding time that took longer using younger grafts. Furthermore, the relative endothelial cell loss increased with longer unfolding times. Conclusions: We found that donor age and endothelial cell density influence the properties of DMEK grafts, and thereby the duration of the surgical procedure. Increased unfolding times result in higher endothelial cell loss. Therefore, it seems reasonable to accept preferably older donors with high endothelial cell densities for DMEK, which may be particularly true for inexperienced surgeons or complex clinical situations. Received for publication December 12, 2013; revision received January 31, 2014; accepted February 3, 2014. Published online ahead of print March 26, 2014. From the Eye Center, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. The authors have no funding or conflicts of interest to disclose. All authors have equally contributed to conception and design, or acquisition of data, or analysis and interpretation of data; and drafting the article or revising it critically for important intellectual content; final approval of the version to be published. S. Heinzelmann and P. Maier had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Reprints: Philip Maier, University Eye Hospital Freiburg, Killianstr 5, 79106 Freiburg, Germany (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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Key Words: cornea, endothelial keratoplasty, DMEK (Cornea 2014;33:644–648)
D
uring the last few years, different techniques for posterior lamellar keratoplasty have been developed to selectively replace the corneal endothelium in patients with endothelial insufficiency.1,2 Of these techniques, Descemet stripping automated endothelial keratoplasty (DSAEK) has become the standard procedure because graft preparation and graft unfolding are well standardized and reproducible. However, not all patients reach the visual acuity that may be optically possible.3,4 The reason for this might be irregularities and incongruent collagen fibers of the posterior corneal stroma in the interface between donor and host stroma,5 changes of higher-order aberrations,6–8 remnants of the recipient’s Descemet membrane, or irregularities of the posterior curvature.9 The more recently introduced Descemet membrane endothelial keratoplasty (DMEK) provides faster and more complete visual rehabilitation than DSAEK.3,10,11 However, graft preparation and unfolding are less standardized. Therefore, many surgeons still prefer DSAEK because they fear higher endothelial cell loss and a higher graft failure rate after DMEK. Yet, recent studies have shown that the endothelial cell loss and the graft failure rate after DMEK is approaching that of DSAEK.3,10,12,13 Furthermore, the risk for endothelial immune reactions seems to be much lower after DMEK than after DSAEK or penetrating keratoplasty.14 In this study, we investigate whether donor characteristics influence the properties of DMEK grafts and thereby the surgical procedure and the final outcome. Therefore, we correlated donor characteristics, firstly, to the width of DMEK grafts measured experimentally at the end of the preparation, and secondly, to the duration of graft unfolding and postoperative endothelial cell loss to find out whether this technically challenging technique may be simplified by donor selection.
MATERIALS AND METHODS Donor Preparation (Experimental Series) All grafts were stored in organ culture at 34°C according to the guidelines of the European Eye Bank Association. All DMEK grafts were prepared in a standardized way using an 8-mm trephine as described elsewhere.15 In 28 experimental cases with grafts unsuitable for transplantation due to equivocal or positive serological tests, we measured the width of the roll Cornea Volume 33, Number 6, June 2014
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Descemet Membrane Endothelial Keratoplasty
TABLE 2. Donor/Recipient Characteristics of our Clinical Series Fuchs dystrophy/pseudophakic bullous keratopathy Donor age
84% (n = 26)/16% (n = 5)
Total storage time Storage deswelling time Preoperative endothelial cell count
FIGURE 1. The DMEK grafts rolls were photographed at the end of the preparation. The width of these rolls was measured and related to the length of the grafts, so that the real width of the grafts could be calculated.
that the DMEK graft spontaneously formed when it was placed into buffered saline solution (Fig. 1). The graft characteristics of this experimental series are summarized in Table 1.
All DMEK (n = 31) were performed under general anesthesia by 3 experienced corneal surgeons at the Eye Center, Albert-Ludwigs-University of Freiburg according to the technique described by Melles et al16 as a consecutive case series. All grafts were punched with an 8-mm trephine and descemetorhexis was done approximately according to an 8-mm marking on the corneal surface. All surgeries were video recorded and we measured the time interval from the time point when the graft was placed into the anterior chamber to the time point when it was first attached to the posterior stroma of the host (= unfolding time). This time frame includes also the time it takes to center the graft after it has been unfolded and before it is attached to the posterior stroma of the recipient. All graft characteristics of this clinical series are summarized in Table 2. The study was approved by the local ethics committee and all patients gave their informed consent that their clinical data may be used for scientific analyses.
Statistical Analysis We used Pearson’s correlation method to assess the bivariate interdepencies of donor age, width of the DMEK
TABLE 1. Donor Characteristics of our Experimental Series 74.8 22.0 2.1 2383 23.7 2.01
6 6 6 6 6 6
12.6 yrs 5.3 d 1d 173 cell/mm2 15.5 h 0.45 mm
Donor characteristics of our experimental series (n = 28) presented as mean 6 SD.
Ó 2014 Lippincott Williams & Wilkins
9.1 6 8.2 (range, 2.7–41.8; median, 7.0; lower quartile, 3.4; upper quartile, 12.4) minutes
Donor/recipient characteristics of our clinical series (n = 31) presented as mean 6 SD.
rolls, and endothelial cell densities. In the second step, we fitted a multifactorial linear regression analysis to declare influencing factors on the unfolding time.
Descemet Membrane Endothelial Keratoplasty (Clinical Series)
Donor age Total storage time Storage deswelling time Preoperative endothelial cell count Time from death to corneoscleral excision Width of DMEK graft
Time from death to corneoscleral excision Unfolding time
69.1 6 12.7 (range, 34–91; median, 71; lower quartile, 64; upper quartile, 74) years 21.4 6 6.0 d 1.6 6 0.8 d 2397 6 223 (range, 2117–2920; median, 2336; lower quartile, 2190; upper quartile, 2555) cell/mm2 20.0 6 11.2 h
RESULTS Donor Characteristics and Width of DMEK Graft In our experimental series of 28 graft preparations, we found a statistically significant influence of donor age (P , 0.001) and endothelial cell density (P , 0.05) on the width of the DMEK graft. The older the donor or the higher the endothelial cell count the broader were the rolls Descemet membrane spontaneously formed when it was placed into buffered saline solution (Figs. 2A, B). In our clinical series of 31 DMEK, we found that donor age had a statistically significant influence on the unfolding time (P , 0.01). The younger the donor the longer it took to unfold the DMEK graft in the anterior chamber (Fig. 3). However, as seen in Figure 3, there was one outlier with a very long unfolding time of more than 40 minutes with a graft from a young donor below 40 years of age. Although the long unfolding time in this case was according to the surgeon (P.M.) mainly influenced by the tightness of the graft roll, there were further complicating factors like status after DSAEK and tube surgery for glaucoma treatment that may have further increased the time to unfold the graft. Yet, if this outlier is removed from the statistical analysis, the influence of donor age on the unfolding time shows a trend but does not reach statistical significance anymore. To find out how the unfolding time influences the fate of the graft by means of postoperative endothelial cell loss, we correlated the difference between the preoperative endothelial cell density from organ culture to the early postoperative central endothelial cell density measured at a mean 104 days after surgery (range, 19–347 days; median, 103 days; lower quartile, 69 days; upper quartile, 130 days). We www.corneajrnl.com |
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FIGURE 2. Influence of donor age (A) and graft’s endothelial cell density (B) on the graft width. Pearson’s correlation revealed a statistically significant increase of graft width with increasing donor age (P , 0.01, A) and increasing endothelial cell density (P , 0.05, B).
found a statistically significant correlation between the relatively early postoperative endothelial cell loss and the unfolding time with increasing endothelial cell loss by longer unfolding time (Fig. 4).
DISCUSSION In the experimental series of this study, we found that donor age and donor endothelial cell density have a statisti-
FIGURE 3. Influence of donor age on the unfolding time. The multifactorial linear regression analysis revealed a statistically significant influence of donor age (P , 0.01) on the unfolding time; longer unfolding times occurred using grafts from younger donors.
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cally significant impact on the width of the DMEK graft rolls. Because the thickness of Descemet membrane increases with age at about 1 mm per decade,17 it might be the thickness that affects the extent of how tight Descemet membrane rolls up when it is placed into buffered saline solution after being peeled off the posterior corneal stroma. Murphy et al18 found that Descemet membrane prenatally shows a fast growth in thickness from a very thin ordinary basement membrane and differentiates to a lamellar structure consisting of at least 30 layers by the end of gestation. Later, the membrane becomes thicker (from about 3 mm at birth to up to 18 mm at 98 years of
FIGURE 4. Correlation of unfolding time and early postoperative relative endothelial cell loss. Longer unfolding times lead to a statistically significant higher endothelial cell loss (P , 0.01, Pearson correlation). Ó 2014 Lippincott Williams & Wilkins
Cornea Volume 33, Number 6, June 2014
age) during life in an exponential manner by deposition of nonlamellar material posterior to the original prenatal layer. Beside the increase in thickness with aging, Kabosova et al19 found differences in the composition of Descemet membrane between infants and adults. In their histochemical analyses, the stromal side of Descemet membrane stained for laminin332, tenascin-C, and fibrillin-1 in infant but not in adult corneas, possibly having an impact on the elasticity of Descemet membrane. So Descemet membrane may also lose elasticity with aging. However, beside these data on the composition of Descemet membrane, we are not aware of any data in the literature on its elasticity. Thus, this needs further experimental clarification. Increased thickness and decreased elasticity may explain why older Descemet membranes show less tendency to roll tightly and vice versa. Although we did not investigate the preparation process itself, peeling off grafts from older donors seems to be easier as well in our experience. The reason for this could also be the increased thickness, because in this study, older Descemet membranes seem to tear less frequently than younger ones. However, in the context of Descemet tears during graft preparation, the cleavage plane of Descemet membrane may be an even more important factor than the thickness of Descemet membrane. From electron microscopic studies, we know that the stromal attachment of Descemet membrane is achieved by the so-called interfacial matrix.20,21 When stripping off Descemet membrane, Schloetzer-Schrehardt et al21 found that the cleavage plane is consistently between this interfacial matrix and the posterior corneal stroma. However, they found adherent stromal collagen fibers projecting into Descemet membrane, but only focal irregularities caused by breaks and ruptures of the interfacial matrix zone, which were, however, not associated with donor age or other donor characteristics. To our knowledge, there are as yet no studies that correlate the thickness of Descemet membrane to the endothelial cell density. However, one could speculate, because Descemet membrane primarily is the basement membrane of the corneal endothelium and therefore a product of corneal endothelial cells, it might also be thicker in individuals with high endothelial cell densities where many cells produce the membrane compared with individuals with low endothelial cell densities given the equal time of deposition. Whether the density of endothelial cells also influences the composition or the elasticity of Descemet membrane remains unclear and needs to be clarified in the future. In the context of these specific characteristics of the donor’s Descemet membrane, there may also be differences between the storage methods of the corneal grafts. All grafts included in this study had been stored in organ culture that is the preferred method in Europe. However, the characteristics of Descemet membrane may be different when cold storage is used for corneal preservation, because beside the difference in storage temperature, the storage time is shorter and there is no swelling and deswelling of the donor cornea. Therefore, the investigations presented here need to be repeated for cold storage donor corneas to find out whether Descemet membrane shows the same behavior for both storage methods. In the second clinical part of this study, we found that donor age showed a trend to influence the unfolding time, that Ó 2014 Lippincott Williams & Wilkins
Descemet Membrane Endothelial Keratoplasty
is, the time from placing the graft in the anterior chamber to the first attachment of the graft to the posterior stroma, where most manipulations of the graft occur. The unfolding of the graft took longer when grafts from younger donors were used. Because we did not have the possibility to measure the width of the DMEK grafts in our clinical series, we could not directly correlate the width of DMEK grafts to the unfolding time. However, it seems likely that increased unfolding times with younger grafts are a result of the reduced width of these grafts because it is technically more challenging to unfold tighter grafts. Finally, it is not surprising that we also found a statistically significant correlation between the unfolding time and the early postoperative endothelial cell loss as a result of the intensive manipulations of the graft during the unfolding procedure. Therefore, according to our experience, it seems helpful to use grafts from older donors (eg, above 65 years of age) and with high endothelial cell counts (eg, above 2300 cells per square millimeter) to improve the success rate of DMEK surgery, especially during the learning curve. In this context, eye banks may also accept older donors because these grafts may be better for DMEK than grafts from young individuals. Because the effect of donor age on the width of DMEK grafts and the unfolding time was much stronger than the effect of the endothelial cell density, older donors with a medium endothelial cell count may be more suitable for DMEK surgery than young donors with high endothelial cell counts, where the intraoperative endothelial cell loss may be much higher because surgery may be much more challenging. However, the unfolding time is primarily influenced by the experience of the surgeon and the individual technique used for DMEK, and with increasing experience, tighter grafts can also be unfolded without significantly more manipulations than those for the unfolding of broader grafts. This may also be true for the usage of further techniques like external corneal compressions instead of using air bubbles for unfolding of the graft. From our experience, a lower age limit of about 60 to 65 years for the donors seems to be advisable for surgeons starting with DMEK. This age limit may be reduced by increasing experience with the surgical technique to 45 to 50 years. However, experts in DMEK surgery may also accept younger donors depending on their own experience. In this study, all surgeons were well experienced in DMEK, although the techniques regarding this new, technically demanding surgical method are still being developed. So the results presented here may be especially important for the learning curve regarding this surgical technique to reduce the rate of primary graft failures. Beside the experience of the surgeon, the recipient characteristics also play an important role in DMEK because it may be very challenging to unfold the graft in complex clinical situations (eg, patients with aniridia or aphakia but also in patients with high myopia or after vitrectomy resulting in a very deep anterior chamber). In such cases, it may also be helpful to use grafts from older donors forming broader graft rolls so that unfolding in the anterior chamber is technically less demanding and the success rate of DMEK may be increased. www.corneajrnl.com |
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Heinzelmann et al
In conclusion, the results of this study suggest that grafts from older donors with high endothelial cell counts are most suitable for DMEK. This might especially be important for surgeons who still fear the technical demands of DMEK compared with DSAEK, so that DMEK may be easier to learn if donors older than 65 years are accepted in the beginning. This may also be true for experienced DMEK surgeons performing DMEK in complex clinical situations. REFERENCES 1. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea. 2006;25:879–881. 2. Heinzelmann S, Maier P, Reinhard T. Perspectives of posterior lamellar keratoplasty. In search of the perfect lamella [in German]. Ophthalmologe. 2011;108:825–832. 3. Guerra FP, Anshu A, Price MO, et al. Endothelial keratoplasty: fellow eyes comparison of descemet stripping automated endothelial keratoplasty and descemet membrane endothelial keratoplasty. Cornea. 2011; 30:1382–1386. 4. Heinzelmann S, Maier P, Böhringer D, et al. Visual outcome and histological findings following femtosecond laser-assisted versus microkeratome-assisted DSAEK. Graefes Arch Clin Exp Ophthalmol. 2013;251:1979–1985. 5. Heinzelmann S, Böhringer D, Maier PC, et al. Correlation between visual acuity and interface reflectivity measured by pentacam following DSAEK. Acta Ophthalmol. 2013;92:e1–e4. 6. Hindman HB, Huxlin KR, Pantanelli SM, et al. Post-DSAEK optical changes: a comprehensive prospective analysis on the role of ocular wavefront aberrations, haze, and corneal thickness. Cornea. 2013;32: 1567–1577. 7. Rudolph M, Laaser K, Bachmann BO, et al. Corneal higher-order aberrations after Descemet’s membrane endothelial keratoplasty. Ophthalmology. 2012;119:528–535. 8. Muftuoglu O, Prasher P, Bowman RW, et al. Corneal higher-order aberrations after Descemet’s stripping automated endothelial keratoplasty. Ophthalmology. 2010;117:878–884.e6.
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9. Dirisamer M, Parker J, Naveiras M, et al. Identifying causes for poor visual outcome after DSEK/DSAEK following secondary DMEK in the same eye. Acta Ophthalmol. 2013;91:131–139. 10. Tourtas T, Laaser K, Bachmann BO, et al. Descemet membrane endothelial keratoplasty versus descemet stripping automated endothelial keratoplasty. Am J Ophthalmol. 2012;153:1082–1090.e2. 11. Maier P, Reinhard T, Cursiefen C. Descemet stripping endothelial keratoplasty–rapid recovery of visual acuity. Dtsch Arztebl Int. 2013;110: 365–371. 12. Khor WB, Mehta JS, Tan DT. Descemet stripping automated endothelial keratoplasty with a graft insertion device: surgical technique and early clinical results. Am J Ophthalmol. 2011;151:223–232.e2. 13. Phillips PM, Phillips LJ, Much JW, et al. Descemet stripping endothelial keratoplasty: six-month results of the first 100 consecutive surgeries performed solo by a surgeon using 1 technique with 100% follow-up. Cornea. 2012;31:1361–1364. 14. Anshu A, Price MO, Price FW Jr. Risk of corneal transplant rejection significantly reduced with Descemet’s membrane endothelial keratoplasty. Ophthalmology. 2012;119:536–540. 15. 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. 16. Melles GR, Ong TS, Ververs B, et al. Descemet membrane endothelial keratoplasty (DMEK). Cornea. 2006;25:987–990. 17. 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. 18. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci. 1984;25: 1402–1415. 19. Kabosova A, Azar DT, Bannikov GA, et al. Compositional differences between infant and adult human corneal basement membranes. Invest Ophthalmol Vis Sci. 2007;48:4989–4999. 20. 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. 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.
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