Accepted Manuscript Endothelial cell changes as an indicator for upcoming allograft rejection following Descemet membrane endothelial keratoplasty Claire Monnereau , Marieke Bruinsma , Lisanne Ham , Lamis Baydoun , Silke Oellerich , Gerrit R.J. Melles PII:

S0002-9394(14)00302-X

DOI:

10.1016/j.ajo.2014.05.030

Reference:

AJOPHT 8936

To appear in:

American Journal of Ophthalmology

Received Date: 12 November 2013 Revised Date:

22 May 2014

Accepted Date: 22 May 2014

Please cite this article as: Monnereau C, Bruinsma M, Ham L, Baydoun L, Oellerich S, Melles GRJ, Endothelial cell changes as an indicator for upcoming allograft rejection following Descemet membrane endothelial keratoplasty, American Journal of Ophthalmology (2014), doi: 10.1016/j.ajo.2014.05.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Draft:

18 (May 20th, 2014)

Journal:

Am J Ophthalmol

Word-file:

WBSO.2002.001.2012.194 - 1212 Endothelial changes preceding allograft rejection

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ACCEPTED MANUSCRIPT

Endothelial cell changes as an indicator for upcoming allograft rejection following

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Descemet membrane endothelial keratoplasty

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Claire Monnereau;1,2 Marieke Bruinsma;1-3 Lisanne Ham;1-3 Lamis Baydoun;1,2 Silke Oellerich;1 Gerrit R.J. Melles1-3

Netherlands Institute for Innovative Ocular Surgery, Rotterdam, The Netherlands; 2Melles Cornea

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Clinic Rotterdam, The Netherlands; 3Amnitrans EyeBank Rotterdam, The Netherlands

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Correspondence to: Gerrit R.J. Melles, MD, PhD, Netherlands Institute for Innovative Ocular Surgery, Rotterdam, The Netherlands, tel no: +31 10 297 4444, fax no: +31 10 297 4440, e-mail: [email protected], website www.niios.com

Short running title: Endothelial cell changes before allograft rejection

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ABSTRACT

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Purpose. To report early, specific changes in donor endothelial cell morphology as a predictor of an upcoming allograft rejection after Descemet membrane endothelial keratoplasty (DMEK).

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Design. Retrospective, observational case series.

Methods. Out of a cohort of 500 eyes that underwent DMEK at a tertiary referral

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center, seven eyes developed typical clinical signs of an allograft rejection. Specular microscopy images prior to, during, and after the rejection episode were analyzed and compared with a case control group of 49 asymptomatic DMEK eyes that matched baseline characteristics of the rejection group. Endothelial cell morphology was evaluated by subjective scoring [range 1–5] in

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a masked fashion as well as by an objective comparison of endothelial cell density, cell size, coefficient of variation, and hexagonality in rejection versus control eyes. Results. Subjective scores (median) were higher before and after rejection (2.5 and 5,

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respectively) than in the DMEK control group (2.0 and 2.5, respectively) at comparable time points (P=0.0230 and P=0.0005, respectively). Endothelial cell density also differed before

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(P=0.0106) and after rejection (P=0.0240), while hexagonality differed before (P=0.0499), but not after rejection (P=0.1767). Conclusion. Our study suggests that allograft rejection may not be an acute event, but rather a slow onset immune response. Early, specific changes in endothelial cell morphology were found to ‘announce’ an upcoming allograft rejection. If so, monitoring donor endothelium after DMEK or other forms of keratoplasty may be used to anticipate a rejection episode and/or to prevent an allograft rejection from clinically manifesting itself.

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KEYWORDS: Allograft rejection, immune response, Descemet membrane endothelial keratoplasty, endothelial keratoplasty, posterior lamellar keratoplasty, corneal transplantation,

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endothelium

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INTRODUCTION Allograft rejection has been reported to occur in up to 15-30% of cases after traditional

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penetrating keratoplasty.1-6 Lower incidences have been described after endothelial keratoplasty, in which 5-12% of cases showed a rejection in a larger series of eyes that underwent ‘Descemet stripping (automated) endothelial keratoplasty’ (DSEK/DSAEK), and in about 1-5% of eyes

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that underwent ‘Descemet membrane endothelial keratoplasty’ (DMEK).7-13

These percentages may suggest that thinner grafts, carrying a lower antigen load,

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decrease the risk of rejection. However, an allograft rejection can still occur after DMEK, which may present with a Khodadoust line and/or keratic precipitates.10,13 There may not be a known diagnostic predictor for a keratoplasty episode, so that allograft rejection is commonly diagnosed only after the eye shows clinical signs like redness and anterior uveitis, and the

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patient notices subjective complaints, like a drop in visual acuity, and/or ocular discomfort. However, while performing routine specular microscopy evaluations in our entire cohort of DMEK patients, we observed characteristic endothelial changes in eyes that later developed a

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clinically manifest allograft rejection. Therefore, the aim of the current study was to evaluate which early endothelial cell changes could be detected by retrospective analysis of sequential, in

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vivo specular microscopy images at standardized time intervals before a rejection became clinically apparent in seven DMEK eyes with a manifest allograft rejection. In addition, cell morphology in the rejection group was compared to that in a randomly selected group of asymptomatic DMEK control eyes.

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METHODS A total of 500 consecutive eyes that underwent DMEK for Fuchs endothelial dystrophy,

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pseudophakic bullous keratopathy, or endothelial graft failure, were included in our study. Of these 500 eyes, operated on between October 2007 and September 2012, seven eyes of six patients (4 male, 2 female; with a mean age of 62 ±18 years) developed an allograft rejection 4 to 42

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months after DMEK (Tables 1 and 2). From the remaining 493 eyes, 49 asymptomatic eyes of 49 patients (35 male, 14 female; with a mean age of 64 ±11 years) were selected as a control

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group, representing eyes without detectable clinical signs of allograft rejection throughout their overall mean follow-up time of 22 ± 17 months [range, 6–52 months] (Tables 1 and 3). Per eye from the rejection group (n=7), seven control eyes (thus, n=49 control eyes in total) were selected such that the baseline characteristics ‘recipient age’, ‘surgery indication’, ‘donor age’,

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‘preoperative endothelial cell density’ and available ‘follow-up times’ for Timepoint 1 (before rejection) and Timepoint 3 (after rejection) matched (Table 1). Control eyes were selected without knowing the clinical outcome (visual acuity, endothelial cell density) for the respective eye.

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The retrospective study of prospectively collected data with patient informed consent for research participation was conducted in compliance with the Institutional Review Board and

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Informed Consent requirements, in adherence to the tenets of the Declaration of Helsinki, at the Netherlands Institute for Innovative Ocular Surgery. The study was submitted to http://www.clinicaltrials.gov (Study registration NCT00521898).

Donor tissue and surgery From donor globes, corneo-scleral buttons were excised less than 36 hours post mortem and stored by organ culture at 31oC (CorneaMax EuroBio, Courtaboeuf, France).14,15 After one

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week of culture, endothelial cell morphology and viability were evaluated and DMEK grafts were prepared as described previously.15 Each Descemet–roll was then stored ‘free-floating’ in organ

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culture medium until the time of transplantation. ‘Descemetorhexis’ of 9.0 mm diameter in the recipients’ eyes and ‘standardized’ DMEK

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surgery were performed as described in detail previously.16,17

Postoperative medication

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Postoperative medication included Chloramphenicol 0.5% drops for two weeks, and a steroid regime of Dexamethasone 0.1% drops four times daily for four weeks, followed by Fluorometholone (FML) drops four times daily, tapered to once daily until one year postoperatively. Thereafter, patients took an FML drop once daily or once every other day.

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Except for case 1 (rejection at 30 months postoperatively), who stopped topical medication by himself, all other cases with a rejection episode stated to be compliant to the standard steroid regime (Table 2).

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Within the control group, the same standard steroid regime was applied and for Timepoint 1, only one patient within this group received a different steroid regime

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(Dexamethasone drops 0.1% twice daily at eight month follow-up) and for Timepoint 3, three patients had a different steroid regime (two cases with FML once every other day at seven months and nine months follow-up, respectively, and one patient taking Dexamethasone drops 0.1% once to twice daily at 18 months follow-up).

Postoperative analysis

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Patients were evaluated at 1 day, 1 week, and 1, 3, 6, 9 and 12 months after DMEK, and at 6 months time intervals thereafter. Best corrected visual acuity (BCVA) and

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postoperative complications were recorded and/or collected in a MySQL database. Sequential specular microscopy imaging was routinely performed to monitor endothelial cell density (Topcon SP3000, Topcon Europe Medical, The Netherlands) at similar time intervals from

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one month onwards. Endothelial cell morphology of 56 transplanted corneas (7 corneas that developed an allograft rejection after DMEK, and 49 asymptomatic DMEK control eyes)

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were retrospectively analyzed, by grading the endothelial cell morphology objectively and subjectively. Objective grading of the central endothelial cell images was performed by using the Topcon SP3000 IMAGEnet software (with manual correction of misassigned cell borders), and these morphometric values included endothelial cell density, coefficient of

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variantion, hexagonality, and cell size (Table 3).

Subjective grading of the specular microscopy images was performed by two masked observers (MB and LH), with long term experience in evaluating in vivo specular microscopy

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images. For each eye, two central specular microscopy images were evaluated, and the overall endothelial cell morphology was graded on a scale from 1 to 5: (1) “Quiet” endothelial cell

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layer with a regular cell morphology and distribution, i.e. a cell layer of inactivated cells with no nuclei visible and a hexagonal cell pattern without exhibiting polymorphism and polymegatism; (2) Slightly irregular endothelial cell morphology and/or distribution, but without any sign of cellular activation, i.e. visibility of cellular nuclei and/or increased cellular reflectivity; (3) Mild to moderate irregular endothelial cell morphology and/or distribution, and mild to moderate appearance of cellular activation; (4) Severe irregular endothelial cell morphology and/or distribution, and clear presence of cellular activation with

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enlarged cell nuclei; (5) Extreme irregular endothelial cell morphology and/or distribution, and presence of highly activated cells (Figure 1).

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To evaluate the change in endothelial cell morphology before and after the clinical manifestation of an allograft rejection, three different ‘Time Points’ were chosen for each cornea that developed an allograft rejection. Time Point 1 = Before rejection, no clinical

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signs, changes in endothelial cell morphology; Time Point 2 = Clinically manifest allograft rejection; Time Point 3 = After rejection, no clinical signs, persistent changes in endothelial

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cell morphology (Figure 2; Tables 2 and 3). On average, Time Point 1 occurred at 10 ±8 months [range, 1-24 months] after DMEK, being 6 ±7 months [range, 1-18 months] prior to rejection; Time Point 2 represented the rejection episode at 17 ±13 months [range, 4-42 months] after DMEK; and Time Point 3 averaged 23 ±17 months [range, 6-48 months] after

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DMEK, being 6 ±6 months [range, 2-18 months] after rejection (Figure 2, Table 3). Because no specific event could be defined in control DMEK eyes (by definition: no clinical signs of an allograft rejection), two Time Points were chosen in control eyes that

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correspond to Timepoint 1 and 3 of the rejection group. Hence, in DMEK control eyes, Time Point 1 averaged 10 ±8 months [range, 1-26 months], and Time Point 3 averaged 22 ±17

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months [range, 6-52 months] after DMEK (Figure 2, Table 1).

Statistical analysis

Repeated measures ANOVA tests were used to test if there was a difference in (subjective) endothelial cell morphology scores between eyes that later developed an allograft rejection and DMEK control eyes; the 95% confidence interval bars were calculated using Morey's method.18 Multi-variant analysis of the different cell morphologies prior to the

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rejection episode was attempted, but no valid results could be obtained because of the small sample size.

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The differences between medians of the objective and subjective morphometric values as examined with the specular microscope were analyzed using bootstrap median hypothesis tests using R (version 2.15.3).19,20 A classification tree analysis was performed to identify

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parameters associated with a higher probability of allograft rejection.

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RESULTS In our cohort of 500 DMEK eyes with an overall mean follow-up of 34 ± 18 months,

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seven eyes developed an allograft rejection at 4 to 42 months after surgery (Table 2). In five eyes, rejection was associated with subjective complaints and objective clinical signs (ocular discomfort, redness, corneal edema, anterior uveitis, and/or a drop in visual acuity). In the

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remaining two eyes, rejection was diagnosed at the slit-lamp evaluation during a scheduled follow-up examination. Allograft rejection subsided after intensified topical steroid therapy in

requiring a re-DMEK (Table 2).

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all eyes, except for two eyes of the same patient who developed a secondary graft failure

Changes in endothelial cell morphology preceding allograft rejection

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At Time Point 1, eyes that later developed an allograft rejection showed a difference in subjective scoring (P=0.0230), endothelial cell density (P=0.0106), and hexagonality (P=0.0499) compared to control eyes. The latter indicates that despite the absence of clinical

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signs of rejection, specific changes in endothelial cell morphology could already be observed (Figure 3, 4; Table 3). The rejection and control group did not differ in terms of the

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coefficient of variation (P=0.2855). The changes in endothelial cell morphology were visible 6 ± 7 months [range, 1-18 months] before detectable subjective and/or objective clinical signs of rejection (Table 2).

Changes in endothelial cell morphology during allograft rejection At Time Point 2, all eyes investigated showed one or more of the typical signs of an allograft rejection (reduced visual acuity, ocular discomfort, red eye, corneal edema, keratic

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precipitates, anterior uveitis) (Table 2). Owing to corneal edema, specular microscopy images of the endothelium could only be made in three eyes, so that this limited data set was not

Changes in endothelial cell morphology after allograft rejection

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included in the statistical analyses (Table 3).

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At Time Point 3, after the clinical symptoms had subsided following intensified

steroid treatment, rejection eyes still showed a difference in subjective scoring (P=0.0005),

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and endothelial cell density (P=0.0024), while hexagonality did not show a difference (P=0.1767) compared to control eyes (Figures 3, 4; Table 3).

Overall, it is noteable that the higher subjective scores in the rejection group appear

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not to be simply caused by a low endothelial cell density, since also the control group contained eyes with low endothelial cell density. These eyes usually showed a regular

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hexagonal cell pattern without any signs of cellular activation (Figure 5).

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DISCUSSION In the current study, we retrospectively evaluated specular microscopy images of seven

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DMEK eyes that developed an allograft rejection. Surprisingly, up to several months before the rejection became clinically apparent, characteristic endothelial cell changes could already be observed. Compared to asymptomatic DMEK control eyes, that showed a quiet endothelial

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monolayer (Figure 4), DMEK eyes with an upcoming rejection showed a distinctive activation of the endothelial cells over time (Figure 3). While control eyes showed a ‘dormant’ cell type

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with homogenous reflectivity, fairly regular cell shape and distribution, and an invisible cell nucleus, eyes with a developing allograft rejection showed a clearly different image with disorganized cell shape, size and distribution, high reflectivity, and/or pronounced cell nuclei. The occurrence of such specific cellular changes preceding a rejection episode may not

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have been described before. There seem to be no earlier reports in the literature on the use of specular microscopy to describe the condition of the corneal endothelium prior to allograft rejection after lamellar keratoplasty. When analyzing morphometric measures, a low endothelial

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cell density has been correlated with an increased risk of pentrating keratoplasty graft failure,21 but not with a higher risk of immune response or tissue rejection. The reason that an association

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between endothelial morphology and allograft rejection may not have been recognized before, might be explained by the fact that in most centers, specular microscopy is not routinely performed after keratoplasty, but limited to events or complications associated with endothelial dysfunction. As a result, both doctors and technicians may rarely have observed these characteristic changes preceding an allograft rejection, since specular microscopy would most commonly have been performed after the event to evaluate endothelial damage induced by the immune response. In our cohort of DMEK eyes, specular microscopy is performed regularly at

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standardized time intervals before and after surgery, which may have enabled us to become familiar with the natural course in postoperative endothelial wound healing, and to also

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recognize an abnormal change in cell morphology prior to the actual clinical manifestation of an allograft rejection.10,13

Interestingly, although allograft rejection quickly resolved in all eyes after intensified

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steroid treatment, the endothelial cell morphology did not return to normal during the study period (Figure 3). This would suggest irreversible damage induced by allograft rejection.

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Hence, when the endothelial structure shows detectable changes between sequential examinations, it may be advocated to schedule shorter follow-up examinations, to monitor the development of an allograft rejection, and potentially intensify the steroid regime. The observed endothelial cell changes prior to the actual clinical manifestation of an allograft rejection,

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suggest a phase in which the immune response already slowly compromises the endothelial cell layer. If so, this would indicate that an allograft rejection is not an acute event (as often perceived clinically), but a much slower immunological process, building up to a ‘sudden’

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ophthalmic crisis. Furthermore, early detection of an upcoming immune response might enable prevention of a full blown allograft rejection, when treatment could be started before clinical

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symptoms occur.

It should be noted, however, that owing to the low percentage of allograft rejections occurring after DMEK, the study group is relatively small and it would be interesting to test our findings on a larger rejection group. If future studies would consistently show that an endothelial cell activation level of ≥2.5 is associated with an upcoming allograft rejection (as observed for 5 out of 7 eyes with a rejection in the current study), this value could be used as a cut-off point, to decide whether the patients should be monitored more frequently or whether a change in treatment

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is indicated. However, as with most tests or imaging techniques, there was some overlap in our study between eyes at risk and control eyes, so that any decision on intervention should

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preferably still be taken on a case-by-case basis. Furthermore, our model of endothelial cell activation may have limitations, because recognizing cellular changes preceding an allograft rejection may require experience in evaluating

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endothelial cell morphology, and will be subject to inter- and intraobserver bias. In addition, scoring error due to poor image quality may occur, although it should be noted that poor image

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quality most often has its own cause, for example, the presence of corneal edema. As such, these eyes would clinically already be ‘suspicious’ and monitored more frequently. In conclusion, our study showed that allograft rejection after DMEK is not an acute immune response, but a rather slow onset immunological process, that seems to build up to a

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‘sudden’ clinical crisis, with all known subjective and objective clinical signs. If so, it could open the door to early detection and possible prevention of full outbreak of allograft rejection, which is known as a major complication after keratoplasty. Biannual screening with specular

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microscopy could be performed to monitor the condition of the endothelium, in order to detect early changes in endothelial morphology. Future studies may be directed toward intensifying the

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steroid regime in eyes with suspected morphologic changes, which could enable prevention of a full blown allograft rejection if treatment is started before clinical symptoms occur.

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ACKNOWLEDGEMENTS / DISCLOSURE a. Funding/Support: Dr. Baydoun received a World Ophthalmology Congress Travel

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Grant for the World Ophthalmology Congress 2014 in Tokyo, Japan. b. Financial Disclosures (including none): Dr. Melles is a consultant for DORC

International/Dutch Ophthalmic USA. All other authors have no potential conflict of interest

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to disclose.

c. Contributions to Authors in each of these areas: Design of the study (CM, SO, GM);

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Conduct of the study (CM, MB, LH, SO, GM), Data analysis and interpretation (CM, MB, LH, LB, SO, GM), writing (CM, GM) and critical revision of the manuscript (CM, MB, LH, LB, SO, GM).

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d. Other Acknowledgments: None

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REFERENCES 1. Thompson RW Jr, Price MO, Bowers PJ, Price FW jr. Long-term graft survival after

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penetrating keratoplasty. Ophthalmology 2003;110(7):1396-1402. 2. Dua HS, Azuara-Blanco A. Corneal allograft rejection: risk factors, diagnosis, prevention, and treatment. Indian J Ophthalmol 1999;47(1):3-9.

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3. Maguire MG, Stark WJ, Gottsch JD, et al. Risk factors for corneal graft failure and rejection in the Collaborative Corneal Transplantation Studies. Ophthalmology

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1994;101(9):1536-1547.

4. Claesson M, Armitage WJ, Fagerholm P, Stenevi U. Visual outcome in corneal grafts: a preliminary analysis of the Swedish Corneal Transplant Register. Br J Ophthalmol 2002;86(2):174-180.

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5. Williams KA, Muehlberg SM, Lewis RF, Coster DJ. How successful is corneal transplantation? A report from the Australian Corneal Graft Register. Eye 1995;9(2):219-227.

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6. Coster DJ, Williams KA. The impact of corneal allograft rejection on the long-term outcome of corneal transplantation. Am J Ophthalmol 2005;140(6):1112-1122.

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7. Guerra FP, Anshu A, Price MO, Giebel AW, Price FW jr. Descemet’s membrane endothelial keratoplasty; prospective study of 1-year visual outcome, graft survival, and endothelial cell loss. Ophthalmology 2011;118(12):2368-2378. 8. Price MO, Jordan CS, Moore G, Price FW jr. Graft rejection episodes after Descemet stripping with endothelial keratoplasty: part two: the statistical analysis of probability and risk factors. Br J Ophthalmol 2009;93(3):391-395.

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9. Price MO, Gorovoy M, Benetz BA, et al. Descemet’s stripping automated endothelial keratoplasty outcomes compared with penetrating keratoplasty from the Donor

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Cornea Study. Ophthalmology 2010;117(3):438-444. 10. Anshu A, Price MO, Price FW jr. Risk of corneal transplant rejection significantly reduced with Descemet's membrane endothelial keratoplasty. Ophthalmology

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2012;119(3):536-540.

11. Hjortdal J, Pedersen IB, Bak-Nielsen S, Ivarsen A. Graft rejection and graft failure

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after penetrating keratoplasty or posterior lamellar keratoplasty for Fuchs endothelial dystrophy. Cornea 2013;32(5):e60-63.

12. Lee WB, Jacobs DS, Musch DC, Kaufman SC, Reinhart WJ, Shtein RM. Descemet’s stripping endothelial keratoplasty: safety and outcomes. Ophthalmology

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2009;116(9):1818–1830.

13. Dapena I, Ham L, Netukova M, van der Wees J, Melles GRJ. Incidence of early allograft rejection after Descemet membrane endothelial keratoplasty. Cornea

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2011;30(12):1341-1345.

14. Ham L, Dapena I, van Luijk, van der Wees J, Melles GRJ. Descemet membrane

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endothelial keratoplasty (DMEK) for Fuchs endothelial dystrophy. Review of the first 50 consecutive cases. Eye 2009;23(10):1990-1998. 15. Lie JT, Birbal R, Ham L, van der Wees J, Melles GR. Donor tissue preparation for Descemet membrane endothelial keratoplasty. J Cataract Refract Surg 2008;34(9):1578-1583. 16. Melles GRJ, Wijdh RH, Nieuwendaal CP. A technique to excise the descemet membrane from a recipient cornea (descemetorhexis). Cornea 2004;23(3):286-288.

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17. Dapena I, Moutsouris K, Droutsas K, Ham L, van Dijk K, Melles GRJ. Standardized ‘no touch’ technique for Descemet membrane endothelial keratoplasty (DMEK).

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Arch Ophthalmol 2011;129(1):98-94. 18. Morey RD. Confidence intervals from normalized data: a correction to Cousineau (2005). Tutorial in quantitative methods for psychology 2008;4(2);61‐64.

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19. Efron B, Tibshirani RJ. Hypothesis testing with the bootstrap. In: Efron B, Tibshirani RJ, editors. An introduction to the bootstrap .Florida: Chapman & Hall / CRC, 1993:

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220-237.

20. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. 2013 Vienna, Austria. Available at http://www.R-project.org/. Accessed November 12, 2013.

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21. Benetz BA, Lass, JH, Gal RL, et al. Endothelial morphometric measures to predict endothelial graft failure after penetrating keratoplasty. JAMA Ophthalmol

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2013;131(5):601-608.

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FIGURE CAPTIONS

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Figure 1. Reference specular microscopy images. The images display the sequential stages used to subjectively score the endothelial cell morphology on a scale from 1 to 5: (1) Quiet endothelial cell layer with a regular cell morphology and distribution, without any sign of

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cellular activation; (2) Slightly irregular endothelial cell morphology and/or distribution, but without any sign of cellular activation; (3) Mild to moderate irregular endothelial cell

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morphology and/or distribution, and mild to moderate appearance of cellular activation. Note the increased cellular reflectivity (black arrow) with detectable cell nuclei (white arrow); (4) Severe irregular endothelial cell morphology and/or distribution, and clear presence of cellular activation with enlarged cell nuclei; (5) Extreme irregular endothelial cell

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morphology and/or distribution, and presence of highly activated cells.

Figure 2. Relevant time points after Descemet membrane endothelial keratoplasty.

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Diagram displaying the postoperative time after Descemet membrane endothelial keratoplasty (DMEK) at which allograft rejection occurred (upper line, thin bars), and Time Point 1 (upper

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line, thick bars,postoperative time interval at which endothelial changes preceding allograft rejection were detectable), Time Point 2 (postoperative time interval at which allograft rejection manifested clinically), and Time Point 3 (postoperative time interval at which postrejection measurements were taken). The lower line represents the mean 'pre-event' and 'post-event' time intervals in DMEK control eyes, i.e. the endothelial morphology values used for comparison to those in eyes with an allograft rejection.

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Figure 3. Specular microscopy images after Descemet membrane endothelial keratoplasty (rejection group). The images show the central (upper images) and peripheral (lower images)

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donor endothelium at various time intervals after Descemet membrane endothelial keratoplasty (DMEK) (Case 3). The eye suffered from a rejection episode at 42 months after surgery. (Upper left and lower left image) Note that at 6 months after DMEK, specular microscopy shows a

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normal quiescent endothelial cell layer with a regular hexagonal pattern. (Upper second left and lower second left image) At 24 months after DMEK, however, in the complete absence of any

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clinical signs of an allograft rejection, the overall cell morphology has changed: prominent cell nuclei are visible as well as a disorganized cell mosaic. (Upper third and lower third image) At 42 months after DMEK, the patient requested a consultation on his own initiative for ocular discomfort and a subjective drop in visual acuity, and the eye is diagnosed with an allograft

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rejection. (Upper right and lower right image) After intensified steroid therapy, the rejection subsided clinically, but the aberrant changes in endothelial cell morphology persisted in the long term, causing progressive central corneal edema (so that further specular microscopy imaging

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was not possible).

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Figure 4. Specular microscopy images of donor endothelium at various time intervals after Descemet membrane endothelial keratoplasty (control group). Images show the endothelium of a 65-year old pseudophakic patient of the control group after Descemet membrane endothelial keratoplasty . Note that both the central (upper images) and peripheral (lower images) endothelial cell layer consistently shows a quiescent morphology throughout the follow-up period.

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Figure 5. Specular microscopy images after Descemet membrane endothelial keratoplasty. Images show the endothelium of three eyes of the control group after Descemet membrane

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endothelial keratoplasty. (Left) Image taken at 9 months postoperatively of a 41-year old patient. Endothelial cell density (ECD) was 930 cells/mm2. (Middle) Image taken at 18 months

postoperatively of 58-year old patient. ECD was 770 cells/mm2. (Right) Image taken at 30

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months postoperatively of a 83-year old patient. ECD was 580 cells/mm2.

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ACCEPTED MANUSCRIPT Table 1. Baseline characteristics of the rejection group showing endothelial cell changes before an upcoming allograft rejection after Descemet membrane endothelial keratoplasty and the control group.

Control eyes (n=49) 64.1 (±10.8, 38-83)

2/5

14/35

5

35

1

7

Recipient gender (F/M)

Surgery Indication Fuchs endothelial dystrophy Bullous Keratopathy

676 (±126, 568-929)

Average FU time for Time Point 1 (months) (±SD, range)

685 (±125, 516-1235)

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Preoperative pachymetry (µm) (±SD, range)

0.6255 -

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1 Failed DSEK

P-value

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Recipient age (years) (±SD, range)

Rejection eyes (n=7) 61.7 (±18.2, 31-78)

0.8536

10.4 (±7.7, 1-24)

10.3 (±7.8, 1-26)

0.8147

22.7 (±17.2, 6-48)

22.3 (±17.4, 6-52)

0.9546

61.1 (±9.4, 49-75)

63.8 (±8.9, 43-84)

0.4746

2534 (±105, 2380-2700

2536 (± 195, 1900-2910

0.9807

1/6

16/33

-

3 (43%)

27 (55%)

1 (14%)

5 (10%)

3 (43%)

12 (27%)

-

2 (4%)

-

2 (4%)

Death-preservation time (hours) (±SD, range)

21 (±6, 15-32)

22 (±7, 9-36)

0.3524

Death-to-DMEK preparation time (days) (±SD, range)

7 (±3, 4-12)

7 (±4, 2-13)

0.4646

Death-to-surgery time (days) (±SD, range)

15 (±4, 9-18)

14 (±4, 8-23)

0.3996

Average FU time for Time Point 3 (months) (±SD, range) Donor age (years) (±SD, range)

TE D

Preoperative Donor ECD (cells/mm2) (±SD, range) Donor gender (F/M) Donor death cause Cardio/Stroke

Cancer Trauma

AC C

Other

EP

Respiratory

DSEK: Descemet stripping endothelial keratoplasty FU: Follow-up SD: Standard deviation F: Female M: Male ECD: Endothelial cell density

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ACCEPTED MANUSCRIPT TABLE OF CONTENTS STATEMENT

AC C

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Incidence of allograft rejection after Descemet membrane endothelial keratoplasty is low, and an allograft rejection is commonly diagnosed after the eye shows clinical signs. However, characteristic endothelial changes seemed to occur before allograft rejection clinically manifested. This observation may open the door to early detection and possible prevention of clinically manifested allograft rejection, and its associated complications.

ACCEPTED MANUSCRIPT Monnereau et al. Endothelial changes before graft rejection

Average follow up time (m) Time point 1 No clinical signs, changes in endothelial cell morphology

Control eyes Median [IQR]

P-value

49

--

10 [Range, 1 – 24]

Subjective score (1 – 5)

2.5 [2.0 – 3.25]

ECD (cells/mm2)

1102 [869 – 1420]

28.0 [26 – 32]

2.0 [2.0 – 2.5]

0.0230

1616 [1378 – 1931]

0.0106

0.2855

64 [58.5 – 66.0]

56.3 [52 - 61]

0.0499

7 (3 analyzed, 4 n.a.)

49

--

17 [Range, 4 – 42]

n.r.

--

n.a.

n.r.

--

880 [Range, 325 – 1391]

n.r.

--

1140 [Range, 720 – 3235]

n.r.

--

26 [Range, 23 – 31]

n.r.

--

60 [Range, 49 – 65]

n.r.

--

7 (4 analyzed, 3 n.p.)

48

--

23 [Range, 6 – 48]

22 [6. – 52]

Subjective score (1 – 5)a

5.0 [2.25 – 5.0 ]

2.5 [1.5 – 2.5]

0.0005

ECD (cells/mm2)

880 [840 – 1000]

1401 [1195 – 1747]

0.0240

Coefficient of variation (%)

27.5 [24.2 – 29.8]

27.0 [24.0 – 29.2]

0.3653

Hexagonality (%)

62.5 [49.0 – 66.5]

59.0 [52.0 – 62.0]

0.1767

n= Average follow up time (m)

ECD (cells/mm2) Cell size (µm2)

TE D

Subjective score (1 – 5)

EP

Coefficient of variation (%) Hexagonality (%)

AC C

n=

M AN U

Hexagonality (%)

Average follow up time (m)

Time point 3 No clinical signs, persistent changes in endothelial cell morphology

--

27.1 [23.7 – 30.0]

Coefficient of variation (%)

Time point 2 Clinically manifest allograft rejection

10 [1 – 26]

SC

n=

Rejection eyes Median [IQR] 7 (7 analyzed)

RI PT

Table 3. Evaluation of endothelial cell morphology in the rejection and control group at different time points after Descemet membrane endothelial keratoplasty.

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n=6

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n.a.: Not available; n.p.: Objective analysis of specular microscopy images not possible n.r. : Not relevant ECD: Endothelial cell density IQR: Interquartile range m: Month

ACCEPTED MANUSCRIPT Monnereau et al. Endothelial changes before graft rejection

Table 2. Demographics and clinical details of eyes presenting with an allograft rejection after Descemet membrane endothelial keratoplasty

Age/Gender

OD/OS

6

7

45/M

77/F

61/M

62/M

31/F

78/M

78/M

OS

OS

OS

OD

OD

OD

OS

‘Failed’ DSEK

FED

FED

phakic

pseudophakic

pseudophakic

pseudophakic

phakic

pseudophakic

pseudophakic

Time after DMEK

18m

3m

24m

9m

12m

1m

6m

Time before rejection

12m

1m

18m

1m

6m

6m

1m

607 µm

559 µm

573 µm

552 µm

508 µm

503 µm

441 µm

Slit-lamp

Clear cornea

Clear cornea

Clear cornea

Clear cornea

Clear cornea

Clear cornea

Clear cornea

Remarks

-

-

History of high myopia and retinal detachment

History of high myopia and retinal detachment

-

-

-

30m

4m

42m

10m

18m

7m

7m

622 µm

700 µm

628 µm

n.a.

505 µm

598 µm

n.a.

Discontinued steroids 24m after DMEK

FML drops 3x daily

Slit-lamp

Remarks

FML drops 1x daily

FML drops 1x daily

FML drops 3x daily

FML drops 3x daily

Cornea edematous whitish spots in the interface, mild cellular reaction in AC

Clear cornea with keratic precipitates

Corneal edema with keratic precipitates

Keratic precipitates, cells and flare in AC

Clear cornea with dispersed keratic precipitates

Diffuse corneal edema with Khodadoust line

No subjective complaints

Subjective drop in VA, ocular redness, photophobia

Subjective slow drop in VA, ocular discomfort

Subjective ocular discomfort

No subjective complaint

Subjective drop in VA, ocular redness

Subjective ocular redness and discomfort

6m

48m

12m

27m

9m

9m

4m

6m

2m

9m

2m

2m

818 µm

637 µm

-

493 µm

452 µm

FML 4x daily, tapered to 1x daily over 7m

Predn. 8x daily and DexM ointment (overnight) tapered to Predn 1x daily over 2m, then DexM 1x daily on the long-term

Predn. 8x daily and DexM ointment (overnight) tapered over 1m, then DexM 3x daily tapered to 1x daily over 2m

Clear cornea, quiet AC

Clear cornea, quiet AC

Clear cornea, quiet AC

-

-

-

48m

Time after rejection

18m 610 µm

597 µm

Steroid regime to treat rejection

DexM 4x daily, tapered over 3m, then FML 1x daily

DexM 6x daily for 10d, then Predn. 6x daily tapered to 3x daily over 1m; then DexM 3x daily tapered over 6m to 1x daily

Slit-lamp

Clear cornea, quiet AC

Clear cornea, quiet AC

Remarks

-

-

Case 3 and 4 represent both eyes of the same patient

AC: Anterior chamber BCVA: Best corrected visual acuity BK: Bullous keratopathy

FML drops 1x daily

Clear cornea with wihitish keratic precipitates and mild cellular reaction in AC

Time after DMEK

Pachymetry

M AN U

Steroid regime

TE D

Pachymetry (µm)

SC

FED

Pachymetry (µm)

a

5

FED

Time after DMEK

Time Point 3 No clinical signs, persistent changes in endothelial cell morphology

4

FED

Lens status

Time Point 2 Clinically manifest allograft rejection

3

BK (after phakic IOL removal)

Indication for surgery

Time Point 1 No clinical signs, changes in endothelial cell morphology

a

2

EP

Patient data

a

1

RI PT

Case #

AC C

Eyes with allograft rejection after DMEK

Predn. 6x daily tapered over 3m, than FML 1x daily

Predn. 6-8 x daily tapered to 3x daily over 4m, then FML 4x daily

Diffuse corneal decompensation Re-DMEK 56m after initial DMEK

Diffuse corneal decompensation Re-DMEK 16m after initial DMEK

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CF: Counting fingers DexM: Dexamethasone drops 0.1% DMEK: Descemet membrane endothelial keratoplasty DSEK: Descemet stripping endothelial keratoplasty FED: Fuchs endothelial dystrophy FML: Fluorometholone drops HM: Hand movement m: months n.a.: not available Predn.: Prednisolone drops

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