ABSTRACT BACKGROUND/OBJECTIVES To measure the endothelial cell density (ECD) of the in toto pre-stripped endothelial Descemet membrane lamellae (EDML) and to describe the impact of pre- and

intraoperative endothelial cell loss (ECL) on postoperative midterm clinical outcome. SUBJECTS/METHODS The ECD of 56 Corneoscleral Donor Discs (CDD) was first measured with an inverted

specular microscope (t0pre). The measurement was then repeated non-invasively after the preparation of the EDML (t0post). DMEK was performed the next day using these grafts. Follow-up

examinations took place 6 weeks, 6 months and 1 year postoperatively where the ECD was assessed. In addition, the impact of ECL 1 (during preparation) and ECL 2 (during surgery) on the ECD,

and 512 ± 37, respectively The ECL 1 (9% on average) had no significant impact on the main outcome measures after 6 months and 1 year (p > 0.11). The ECL 2 correlated significantly with

the ECD and the pachymetry at 1 year postop (_p_ < 0.02). CONCLUSION Our results indicate that the non-invasive ECD measurement of the prestripped EDML roll before its transplantation is

feasible. Despite significantly decreasing ECD up to 6 months postoperatively, visual acuity further improved and thickness further decreased up to 1 year postoperatively. You have full

access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS THINNING RATE OVER 24 MONTHS IN ULTRATHIN DSAEK Article 15 March 2022 THE PREDICTABILITY OF

GRAFT THICKNESS FOR DESCEMET’S STRIPPING AUTOMATED ENDOTHELIAL KERATOPLASTY USING A MECHANICAL MICROKERATOME SYSTEM Article Open access 23 December 2022 OPTIMIZED LABORATORY TECHNIQUES FOR

ASSESSING THE QUALITY OF PRE-STRIPPED DMEK GRAFTS Article Open access 04 March 2025 INTRODUCTION From its introduction in 2006 [1] up to now, Descemet-Membrane Endothelial Keratoplasty

(DMEK) has become the gold standard therapy for corneal endothelial diseases [2]. This is due to its good visual outcomes and low post-operative complication rates in comparison to

penetrating keratoplasty (PKP) and is since reflected in its increasing frequency in Germany [3]. In the literature the reported Endothelial Cell Loss (ECL) after DMEK varied a lot between

different studies [4,5,6]. In order to reduce the ECL to a minimum, an accomplished, minimally invasive preparation method of the endothelial Descemet membrane lamellae (EDML) is necessary

[7]. The measurement of the ECL caused by the preparation of the EDML could be an additional screening method to monitor the quality of the grafts preoperatively. Unfortunately, already

existing measurement methods could not provide precise results due to two potential problems: (1) The measurement of the ECD after preparation was invasive because of unrolling and fixating

the EDML in order to count endothelial cells. This may lead to an overestimation of the ECL [7,8,9,10,11]. (2) The measurement protocol was not applied after a simulation of the entire

preparation protocol and, therefore, led to an underestimation of ECL [12,13,14]. A reproducible, non-invasive measurement of ECL was introduced in 2020 [15]. Safi et al. analysed 30 rolled

endothelial Descemet membrane lamellae (EDML) directly after their preparation and counted their endothelial cell density (ECD) by means of inverted light microscopy. In contrast to former

methods, the performed measurements were non-invasive and took place after the complete preparation procedure with no further manipulation of the EDML. For reproducibility and accuracy

purposes, each measurement was repeated 5 times, each time a minimum of 3 pictures were taken (two in the periphery, one in the centre of the EDML). This new, non-invasive measurement

protocol showed a high reproducibility (standardised Cronbach’s alpha of 0,924) and satisfying results in terms of ECL [15]. In order to provide a better understanding of the impact of the

ECL caused by the preparation and due to the transplantation itself on the midterm clinical outcome, the purpose of this study was to correlate this pre- and intraoperative ECL with ECD, VA

and pachymetry at 6 months and 1 year after DMEK. MATERIALS & METHODS STUDY SET-UP Since 2013, more than 1100 DMEKs have been performed by 5 microsurgeons in the Department of

Ophthalmology, University Saarland Medical Center in Homburg/Saar (UKS) [16]. For this retrospective study, 56 Corneoscleral Donor Discs (CDD) were dissected from 43 donors with a mean age

of 75 ± 10 (range 54–95) years and transplanted into 56 eyes of 55 DMEK recipients from July 2019 to February 2021. Since, we are not performing non-invasive specular microscopy of the donor

roll on a routine basis, we originally planned on having 70 consecutive grafts for the present study, what seemed to be enough for a clinically significant statement. However, 14 patients

were lost to follow-up for unknown reasons. The indication for all the DMEK operations performed in this study was Fuchs endothelial corneal dystrophy. For the presented study, donor corneas

were provided in house by the _Klaus Faber Center for Corneal Diseases, incl. LIONS-Eye Bank Saar-Lor-Lux, Trier/Westpfalz_. The study followed the tenets of the 1964 Declaration of

Helsinki and was approved by the Ethics Commission of the German Medical Association (Identification-Number: BU217/20).N All the transplanted grafts had passed a strict quality control in

the eye bank including negative serology, negative swap for bacteria and a sufficient ECD > 2200 cells/mm2. Therefore, they fulfilled the criteria of a typical DMEK operation. The CDD

were originally stored in organ culture medium I (Biochrom AG, Berlin, Germany) without dextran. ECD measurements were performed on the CDD before the preparation of the EDML (t0pre) and

then after the preparation on the rolled EDML (t0post). DMEK follow-up examinations at 6-weeks, 6-months and 1-year included: ECD, visual acuity (VA), intraocular pressure (IOP) and

pachymetry. TISSUE PREPARATION Following the inhouse guidelines of Seitz et al. [16, 17], an experienced surgeon performed the preparation of the EDML from the CDD in the operating theatre

one day before DMEK surgery. Briefly, the first step included the fixation of the CDD epithelium-down on a suction block (‘Hanna trephination system’, Moria Surgical, Antony, France) and

then staining it for 30 s with Blue Color Caps (BCC) (Croma GmbH, Leobendorf, Austria) to optimise visualisation. After carefully rinsing the BCC with organ culture medium I, a circular mark

of 7.5 mm diameter was applied on the endothelium using the ‘Moria descemet’s stripping automated endothelial keratoplasty (DSAEK) trephination system’. Then, using a razor blade, 1–1.5 mm

incisions were made in a hexagonal shape outside the existing 7.5 mm mark. Afterwards, a slow detachment of the entire EDML from the periphery to the centre was attempted via a small, blunt

forceps. 120-degree rotations of the underlying suction block with repetition of the previous step helped to detach the EDML step by step until leaving only a small central part of the EDML

attached to the remaining stroma. After that, complete trephination of the EDML with the 7.5-mm trephine was applied resulting in a curvilinear edge. Then 3 asymmetric half-circle marks at

the edge of EDML according to Bachmann et al. [18] to avoid upside-down attachment of the graft in the patients’ eye later. The remaining small central part of EDML attached to the stroma

could now be completely and safely detached from the stroma and by means of another larger forceps carefully put into a well plate containing organ culture medium I without dextran, where

the EDML took a rolled configuration. One day after the EDML preparation, DMEK took place, in which the EDML was inserted into the anterior chamber by means of a Geuder glass cartridge. The

graft was then attached to the recipient’s stroma by sulphur hexafluoride (SF6 20%) gas injection [16]. MEASUREMENT OF ENDOTHELIAL CELL DENSITY (ECD) Before the preparation of the EDML, ECD

of the CDD was measured by an inverted light microscope (model 090-135.001; Leica Microsystems, Wetzlar, Germany). To ensure precise and comparable ECD measurements before and after the EDML

preparation, preoperatively, one image was taken in the centre of the CDD, and two were taken in the middle peripheral region (5–7 mm away from the centre). Around 5 h after preparation,

the rolled EDML with a diameter of 7.5 mm was placed under the same microscope to measure the ECD again. Without any further manipulation, three pictures were again taken, two in the

periphery (again 5–7 mm away from centre) and one in the centre of the EDML. A specialised software first automatically calculated ECD by using a 5.5 cm sided square (=116.599 micrometre of

the EDML) within which endothelial cells were counted. The automated cell counting was then manually corrected by an experienced medical technician to achieve the most accurate ECD. The

total ECD was achieved by taking the average of the three taken pictures. Around 20–40 cells are usually counted in one region of interest of an endothelial image. The reliability,

reproducibility and precision of this non-invasive counting method has been verified earlier [15]. This method to detect the ECD was used before as well as around 5 h after the preparation,

to allow for a certain degree of stabilisation of the endothelial cell layer after migration and redistribution of EC, in order to achieve a more valid ECD. Until their transplantation on

the next day, the EDML were stored in an incubator at 36 °C. The postoperative ECD measurements in all patients were performed using a clinical specular microscope _(Tomey specular

microscope EM-3000__©__; Tomey GmbH, Erlangen, Germany)_ at 6-weeks, 6-months and 1-year intervals. All measurements were performed by a well-trained, medical technician using the automatic

cell count modality but without manual correction. ADDITIONAL MAIN OUTCOME MEASURES Corneal pachymetry was performed preoperatively, 6-weeks, 6-months, and 1-year after DMEK with an anterior

segment optical coherence tomograph (AS-OCT) _(CASIA2; Tomey GmbH, Erlangen, Germany)_ and was measured in μm. Additionally, VA and IOP measurements took place preoperatively, 6-weeks,

6-months, and 1-year after DMEK and were retraced by means of digital patients’ records. VA results are shown in logMAR and IOP was measured by Goldmann applanation tonometry in mmHg.

STATISTICAL ANALYSIS Excel (Microsoft, Redmond, Washington) and SPSS software (IBM, Armonk, New York) were used for analysing all statistical data. All descriptive statistics were shown as

mean ± standard deviation (SD) and median. The evolution of ECD, pachymetry, VA and IOP over time were presented in a ‘Related-samples Friedman’s Two-Way Analysis of Variance by Ranks’ with

an alpha set to be 0.05. For each multivariate test, Pillai’s trace was significant. All significant values (_p_ < 0.05) have been adjusted by the Bonferroni correction for multiple

tests. The Spearman Rank correlation test was used for correlation of pre- and intraoperative ECL with clinical main outcome measures. RESULTS QUANTIFICATION OF ENDOTHELIAL CELL LOSS (ECL)

Preoperatively, the mean ECD at t0pre was 2584 ± 200 c/mm2. The ECD was decreasing significantly during follow-up (Table 1, Fig. 1). Using ECD values, the ECL at each step was calculated

(ECL 1: Difference in ECD between t0pre and t0post; ECL 2: Difference in ECD between t0post and 6-weeks after DMEK; ECL 3: Difference in ECD between 6-weeks and 6-months after DMEK; ECL 4:

Difference in ECD between 6-months and 1-year after DMEK). All results were statistically significant except for ECL 4 (Table 2). PACHYMETRY Pachymetry was assessed from t0pre to 1-year

after DMEK during follow-up. Preoperatively at t0pre the mean pachymetry was 597 ± 63 μm. Corneal thickness was decreasing significantly during follow-up (Table 1, Fig. 1). There was a

significant decrease in the corneal thickness (CT) at all time points (_p_ < 0.001) except between 6-weeks and 6-months after DMEK (_p_ = 0.74) (Fig. 1). VISUAL ACUITY (LOGMAR) Visual

acuity increased significantly during follow-up (Table 1, Fig. 1). The course of the VA over time showed a significant improvement with a _p_ < 0.001 when comparing the results of t0preop

and 6-weeks after DMEK, and _p_ = 0.003 when comparing 6-months and 1-year after DMEK. There was only a small insignificant improvement of the VA between 6-weeks and 6-months with _p_ = 

0.23. INTRAOCULAR PRESSURE (IOP) A mean of 13 (±3) mmHg was measured at t0preop. IOP remained stable during each follow-up appointment. CORRELATIONS OF PRE- AND INTRAOPERATIVE ENDOTHELIAL

CELL LOSS WITH ECD, PACHYMETRY AND VA AT 6 MONTHS AND 1 YEAR POSTOPERATIVELY The ECL 1 due to preparation was 9% on average. This did not correlate with any of the postoperative clinical

main outcome measures (_p_ > 0.11). Intraoperative ECL 2 (42% on average) correlated significantly with ECD at 1 year postoperatively (_p_ = 0.027, Fig. 2) and with pachymetry at 1 year

postoperatively (_p_ = 0.019, Fig. 2). DISCUSSION The non-invasive method of counting the ECD on the EDML, introduced in 2020 by Safi et al. [15], can be used directly before DMEK in order

to exactly know the ECL caused by the EDML preparation itself. Our results demonstrated that measuring the ECD on the rolled EDML is feasible and revealed on average a 9% ECL attributed to

the preparation procedure itself. Despite a rather high ECL rate during the transplantation, pachymetry, VA and intraocular pressure showed favourable results. The intraoperative ECL was

correlated with ECD and pachymetry after 1 year of follow-up. Even though DMEK has become the gold standard therapy for treating corneal endothelial dysfunction in Germany [2, 3], a high ECL

postoperatively may finally result in graft failure. In a large literature review, it was shown that ECL varies between different studies from 19% to 44% 6 months postoperatively [19]. In

2020, Basak et al. [20] for instance presented 27.2% and 33.5% ECL after 6 and 12 months postoperatively. In addition, Birbal et al. (2020) [5] stated an ECL of 37% at 6 months and 40% at 1

year postoperatively. All these different studies have one common ground, which is a steep loss of endothelial cells (EC) early after the operation and a subsequent flattening of the ECL

curve. Overestimation of the ECD by the eye banks might be a potential explanation for this initial, large loss of EC. However, a high ECL during the preparation could also explain this

problem [21,22,23,24]. We assume that there are at least 2 causes of this rapid ECL. First, the manipulation and stripping of the tissue itself during the preparation may cause immediate

ECL. The second cause of this rapid ECL shortly (around 5 h) after the preparation might be a migration and redistribution of the endothelial cells. It is believed that corneal endothelial

cells close the wound gap mainly via migration and increased cell spreading (Inoda et al. 2020). In vitro, endothelial cell migration phenomenon from Quarter-DMEK grafts was proven and seem

to occur along the radial cut edges of the graft (Miron et al. 2018). Although we do not have enough information, this could be one of the reasons for the measured ECL after preparation. But

since the time period between stripping and repeat measurement is very short (around 5 h), this concept of explanation seems to be of minor impact. Another source of this high ECL might be

the 1 day storage after the preparation and before surgery took place. As mentioned in Safi et al. 2020, an average of 8% additional loss was detected on day 1 after preparation of the

tissue. Therefore, it might be advisable to plan tissue preparation and surgery on the same day when it is logistically doable. Again, further scientific research concerning this issue seems

to be necessary. Since there were only either invasive and overestimating methods for ECD measurement after the preparation [7,8,9,10,11], or methods which only involved a partial

preparation and therefore an underestimation of the ECL [12,13,14], it was necessary to find a non-invasive and reproducible method to count the ECD preoperatively on the rolled DMEK graft

[15]. Mayko et al. [12] for instance had a different and incomplete preparation technique, whereby after stripping the Descemet membrane, a peripheral hinge was maintained and not stripped,

and was used as an anchor for the EDML. The EDML was then spread again in its placed on the CDD (which was used as a support system), in contrast to our preparation method in which the EDML

was totally stripped from the CDD and placed in the culture medium as a roll simulating the exact preparation method used in a typical DMEK operation. On the other hand, in our study we used

an inverse light microscope to measure the ECD, whereas in the study performed by Mayko et al. [12], a clinical specular microscope was used. Due to the differences between the preparation

and measurement methods, different results may have been obtained. Giving another example of a study which contradicts our ECL findings due to tissue preparation, Böhm et al. [25] reported a

similar lack of change in ECD for preloaded DMEK rolls. Though again in their study a clinical specular microscope was used for quality evaluation after preparation. Whereas our

measurements were not only carried out by an inverse light microscope and automatically analysed by the software for ECD within the square, but also manually screened by a trained technician

to provide the most accurate results. In addition, Rickmann et al. [22] showed that there is a significant difference between inverse light microscopy and specular microscopy in terms of

ECD measurements, concluding that automated light microscopy showed a higher ECD of 31.85% compared to automated specular microscope, and manual light microscopy measurements showed 10.51%

higher ECD compared to specular microscopy. Therefore, both methods cannot be used interchangeably and the early postoperative rapid decrease of ECD might be partly artefactual. To our

understanding regarding the technical basic aspects, there is no fundamental difference between the functioning of inverted light microscopy (ILM) and specular microscopy (SM). Though the

main difference is that inverted light microscopy is suitable for corneas stored in organ culture, while specular microscopy is mostly suitable for corneas stored in hypothermic storage.

Therefore, using ILM it is possible to better differentiate between alive and dead endothelial cells by staining the cells for example with trypan blue. In opposite to SM, ILM does not tend

to overestimation of endothelial cell density by including dead endothelial cells, which cannot be identified as exact (Jirsova et al. 2017) [26], but allows a more precise measurement of

ECD, resulting in values, which reflect the actual number of EC closer to reality. Therefore, we used in our study an inverted light microscope. We are currently performing a study to

compare the accuracy of both types of microscopes and to better investigate their differences, the result of which are still pending. Our ECL values directly after tissue preparation are in

line with the results of Tran et al. [11] who showed an ECL of 9.3%, and the results of Krabcova et al. [27] showing around 5% ECL. More dramatic ECL values after tissue preparation were

also mentioned by Jardine et al. [9], Schallhorn et al. [10], and Downes et al. [28] with respective values of 22.5%, 27% and 29.2%. Despite the rather high level of intraoperative/early

postoperative ECL, all the other clinical outcome measures in the present study were very satisfying. Thus, the sole importance of a high level of ECD postoperatively is questionable. This

hypothesis is supported by the findings of Hammer et al. [29], who came to the conclusion that rather the functionality or quality of all the endothelial cells is crucial for better visual

outcomes than solely the quantity of endothelial cells. An additional source of inaccuracy between different studies regarding the ECL might be the usage of different models of specular

microscopes postoperatively, which are known to vary immensely between manufacturers [30]. Huang et al. [31] discourage to fully trust specular microscopes because of their inaccuracy in

terms of exact ECD counting in the eyes with high polymegethism and/or large cell size, which is unfortunately often the case after DMEK. This indicates that the low postoperative ECD level

might at least in part be artificial, caused by the inaccuracy of specular microscopes themselves. The endothelial cell count was realised during regular clinical follow-ups by well-trained

technicians using the automatic cell count but without manual correction. Clinical specular microscopy in general seems to find a significantly lower ECD (around 32% lower) than transmitted

light microscopy [22], which may lead not only to relatively low postoperative ECD values, but also to the high ECL perioperative. In addition, a standard diameter of our EDML of 7.5 mm as

well as the inclusion of eyes after pars-plana vitrectomy (Aljundi et al. [32]) may contribute to this finding. One issue that might have affected the preoperative accuracy of the ECD

measurement might be the tightness of the EDML roll. This was not tackled by neither Safi et al. [15] nor by our study. However, in our studies we figured that generally ECD measurement of a

loose roll is technically easier than that of a tight roll. And typically, the rolls tend to be loose directly after preparation. In terms of evolution of pachymetry, the literature

described quite different results. Borgardts et al. [2] for instance found an increase in the CT from the pre- to post-operative state, while the majority of the scientific articles stated a

significant decrease in the CT postoperatively [33, 34]. The results of the present study are in line with most of the studies performed for this parameter and showed a significant

reduction of the CT after transplantation, reaching a 10% difference in the early postoperative follow-up and a 14% difference after 1 year postoperatively compared to the preoperative

level. It is worth noting that a preoperative CT > 625 μm was correlated with worse postoperative CT and VA results [35]. Therefore, early treatment of endothelial corneal pathologies

with DMEK is recommended at our Department of Ophthalmology. Our results also showed a significant drop in the CT, between 6 months (539 μm) and 1 year (512 μm) postoperative in contrast to

a publication by Chamberlain et al. [36]. DMEK is known for its good clinical outcomes, especially in terms of fast VA recovery [5]. Starting with an average VA of 0.50 preoperatively, VA

improved to an average of 0,06 at 1 year postoperatively. No events of ocular hypertension were detected postoperatively in the present study, only small variations of the IOP within normal

ranges were observed throughout our study. Indeed, that there are numerous reports of the ECL post DMEK and its impact on intermediate term outcomes. For instance, Hayashi et al. [37] and

Shahnazaryan et al. [38], which as well as our study only treated patients with Fuchs endothelial corneal dystrophy. Not only the indication for DMEK, but also the clinical outcomes after

DMEK – a significant improvement in visual acuity and a significant reduction in terms of pachymetry – unite those mentioned studies with ours. As for Hayashi et al., they even used a quite

similar preparation technique with the very same ‘Moria suction block’. In addition, the postoperative ECD measurements were at the same time 6 months and 1 year after DMEK and performed

with the same clinical specular microscope (‘Tomey specular microscope EM-3000’). However, the essential novelty of our study is, that the non-invasive method of measuring the ECD on the

EDML, introduced in 2020 by Safi et al. [15], was now used in a clinical context. It showed that it can be used directly before DMEK to differentiate the exact ECL caused by the EDML

preparation itself from the intraoperative ECL. This allows us to not only state the ECL from before preparation to after DMEK and its influence on postoperative outcomes, but also to assess

the impact of the preparation of each tissue itself on the postoperative clinical outcomes by noninvasively quantifying the ECL it causes. Neither study Hayashi et al. [37] nor Shahnazaryan

et al. [38] contains this intermediate measurement step, which allows a more accurate understanding of ECL in the whole pre- and postoperative period of DMEK therapy. We noticed a rapid and

significant improvement concerning visual acuity and pachymetry 6 weeks postoperatively, this improvement showed no further statistically significant changes in the following 6 months. Only

after that timepoint and until one year postoperatively did these parameters further significantly improve. The mechanism behind this pattern of improvement remains unexplainable, and

further investigations with confocal microscopy and/or anterior segment OCT might be helpful in the future. In conclusion, we presented in this study a new safe and effective method of

counting the ECD on the rolled, non-manipulated DMEK-grafts preoperatively. Only during preparation, there was an endothelial cell loss of 9%. From 6 weeks to 6 months, the ECD continued to

decrease significantly, only to stabilise after 1 year postoperatively. The higher the early endothelial loss after surgery was, the lower was ECD and the higher was central corneal

thickness at 1 year of follow-up. SUMMARY WHAT WAS KNOWN BEFORE * Descemet-Membrane Endothelial Keratoplasty has become a gold standard therapy for corneal endothelial diseases with good

visual outcomes and low post-operative complication rates. * There is a high variation of reported endothelial Cell Loss after Descemet-Membrane Endothelial Keratoplasty between different

studies. WHAT THIS STUDY ADDS * This study showed a new safe and effective method of counting the endothelial cell density on the rolled, non-manipulated DMEK-grafts preoperatively and was

now used in a clinical context. * It presented only during preparation, there was an endothelial cell loss of 9%. * It showed the higher the early endothelial loss after DMEK-surgery was,

the lower was endothelial cell density and the higher was central corneal thickness at 1 year of follow-up. DATA AVAILABILITY Data is available from the corresponding author on reasonable

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2020;218:1–6. Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank the Klaus Faber Center for Corneal Diseases, LIONS Eye Bank Saar-Lor-Lux,

Trier/Westpfalz for their help in preserving and measuring the donor corneas. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Ophthalmology, Saarland University Medical Center,

Homburg, Germany Kolja Berg, Tarek Safi, Berthold Seitz & Loay Daas Authors * Kolja Berg View author publications You can also search for this author inPubMed Google Scholar * Tarek Safi

View author publications You can also search for this author inPubMed Google Scholar * Berthold Seitz View author publications You can also search for this author inPubMed Google Scholar *

Loay Daas View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS KB was responsible for writing the manuscript, conducting the search, screening

potentially eligible studies, extracting and analysing data, interpreting results and updating reference lists. TS was responsible for the preclinical trial (published earlier), research and

feedback. BS and LD provided their professional support and feedback to the corresponding author. CORRESPONDING AUTHOR Correspondence to Kolja Berg. ETHICS DECLARATIONS COMPETING INTERESTS

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Berg, K., Safi, T., Seitz, B. _et al._ Non-invasive endothelial cell density measurement of in toto pre-stripped DMEK-roll – impact of

pre- and intraoperative endothelial cell loss on postoperative midterm clinical outcome. _Eye_ 37, 2956–2962 (2023). https://doi.org/10.1038/s41433-023-02450-x Download citation * Received:

15 May 2022 * Revised: 13 January 2023 * Accepted: 10 February 2023 * Published: 22 February 2023 * Issue Date: October 2023 * DOI: https://doi.org/10.1038/s41433-023-02450-x SHARE THIS

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