Observational Study Medicine ® OPEN Risk factors for graft failure after penetrating keratoplasty Rafael Ignacio Barraquer, PhD, MDa,b,c, Luis Pareja-Aricò, MDa,d, Alba Gómez-Benlloch, MDa,b, ∗ Ralph Michael, PhDa,b, Abstract The objective of our study was to define principal risk factors for graft failure in patients who underwent penetrating keratoplasty (PK). Retrospective data obtained from a cohort of 895 penetrating keratoplasties performed between 2001 and 2006 were analysed. Recipient related factors, graft characteristics, and surgical technique were assessed in a univariate analysis and with a multivariate proportional hazard model to detect principal risk factors for definitive graft failure. Multivariate analysis showed clear significance for diagnosis and number of previous grafts and border line significance for the oldest donor age group. Patients with keratoconus had the best 10-year survival estimate (95%), followed by endothelial and stromal Downloaded from http://journals.lww.com/md-journal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 02/11/2022 dystrophies (both 55%), infectious leukomas (49%), trauma (33%) and chemical burns (14%). Primary PK grafts had a survival rate of 81%, second grafts of 33% and third or more grafts of 16%. Overall 10-year survival estimate based on univariate analysis was found to be 65%. In conclusion, we found that primary diagnosis and previous graft failures in the recipient are the most important risk factors of graft failure after a PK. Abbreviations: HLA = human leukocyte antigen, PK = penetrating keratoplasty. Keywords: cornea, graft failure, multivariate analysis, penetrating keratoplasty, risk factors 1. Introduction the upward trend of the previous year. Of the 7511 corneas obtained, 34% (2577) were rejected, 31% (794) due to donor Penetrating keratoplasty (PK) is a 110-year-old procedure whose problems and 69% (1783) during processing.[2] effectiveness and safety has led to be the most frequently performed This type of transplant differs in that the cornea is an avascular type of transplant worldwide. Even with the consolidation of new, tissue with blood supply only at the limbus and it is less invasive, lamellar techniques, in 2012 PK still accounted for immunologically privileged which makes it different from other about 70% of corneal transplantations.[1] transplanted organs and therefore has a much lower rejection The corneal transplant, the oldest of all, is indicated in those rate. Several recent studies on PK outcomes focused on specific diseases that damage the original cornea, making it opaque, with indications, such as Fuchs’s dystrophy and pseudophakic corneal the consequent loss of vision (infections, traumatisms, burns, etc.). edema[3–5] or focused on endothelial cell loss.[6] In our study, we The first corneal transplant in our country was carried out in 1940 aim to identify both donor and recipient principal risk factors for at the Centro de Oftalmologia Barraquer in Barcelona. Nowadays, PK failure, based on graft survival estimates and considering all in Spain there are 180 accredited hospitals to perform this type of indications for PK. Similar global studies date back to the transplant and 112 centres authorized to obtain corneas, 1990s.[7] Our analysis includes univariate as well as multivariate distributed throughout the Autonomous Communities in Spain. techniques. We consider recent recommendations for transparent During 2016 there were 3862 donors of which 7511 corneas were reporting of a multivariable prediction model for individual obtained. A total of 4187 transplants were performed, continuing prognosis and diagnosis (TRIPOD).[8] This includes careful and methodical estimation of sample size for avoiding over fitting Editor: Juan Carlos Serna-Ojeda. effects and an accurate data management through multiple The authors have no conflicts of interest to disclose. imputation and pooling handling of missing values. a Institut Universitari Barraquer, Universitat Autònoma de Barcelona, b Centro de Oftalmología Barraquer, c Universitat Internacional de Catalunya, Barcelona, d Hospital Clinico Universitario Valladolid, Universidad de Valladolid, Valladolid, 2. Materials and methods Spain. ∗ 2.1. Study protocol Correspondence: Ralph Michael, Centro de Oftalmologia Barraquer, Muntaner 314, 08021 Barcelona, Spain (e-mail: ralphm@barraquer.com). Clinical records of all patients who underwent a penetrating Copyright © 2019 the Author(s). Published by Wolters Kluwer Health, Inc. keratoplasty due to any cause at the Centro de Oftalmologia This is an open access article distributed under the terms of the Creative Barraquer between 2001 and 2006 were considered for our Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC- ND), where it is permissible to download and share the work provided it is retrospective cohort study (n = 966). Cases with missing primary properly cited. The work cannot be changed in any way or used commercially diagnosis were excluded (n = 42). Children aged 11 years old or without permission from the journal. below were also excluded (n = 23) because the cumbersome Medicine (2019) 98:17(e15274) examinations may lead to the possibility of misdiagnosing the Received: 10 September 2018 / Received in final form: 4 March 2019 / outcome of interest.[9,10] Furthermore, we excluded 6 cases with Accepted: 25 March 2019 donor age below 21 years because the cornea in children is not fully http://dx.doi.org/10.1097/MD.0000000000015274 developed and such donations are a rare exception at our eye bank, 1 Barraquer et al. Medicine (2019) 98:17 Medicine with the large majority of donations between age 50 and 90 years. 2.3. Statistical methods This resulted in a final number of 895 cases to be analysed. All donor 2.3.1. Survival. Considering previous prognostic studies, the corneas were obtained from enucleated eyes and were provided by survival rate of penetrating keratoplasty at 5 and 10 years is the Banco de Ojos para Tratamientos de la Ceguera, Barcelona. around 74% and 63%, respectively.[7,12] In order to avoid the Ethical approval is currently not required in Spain for retrospective effect of overfitting,[14,15] we estimated a sample size of 900 to studies which are based only on reviews of clinical records. 1000 cases (around 240 failures) in order to achieve an event per After surgical intervention, all patients received a corticoid-based variable ratio of 20 to 30. therapy for preventing early graft rejection. However, the dosage of Data were censored at the time of the last visit if there was no anti-inflammatory medications or the prescription of additional failure event. Association of recipient, graft, and surgical technique therapies such as antibiotics or immunosuppressants depended on factors with the occurrence of a failure event were assessed in the background and clinical status of each patient. Donor cornea univariate and multivariate proportional hazards models. endothelial cells density lower limit was 2200 cell/mm2. 2.3.2. Univariate. A starting unadjusted univariate analysis was Outcome of interest was graft failure, defined as the loss of performed using Kaplan Meier statistics and log rank tests (log graft transparency capable of compromising vision for at least 3 rank test for trend when relevant), identifying statistically months despite maximum anti-inflammatory therapy. For significant differences (P < .05) between individual subgroups. declaring a graft failure, photographical or written description Life table analysis was used to compute the 10-year survival of the affected graft was checked in the clinical history. If during estimates of the graft. the follow-up an eye lost its visual acuity because of another reason but the graft was still transparent, it was not being 2.3.3. Multivariate. Then, Cox proportional hazards regression considered as a failure. Graft failure assessment was made in each model and pooling of imputed data were performed by a forced clinical consultation by a corneal surgeon or general ophthal- entry method of all variables. Multiple imputation method[16,17] mologist. Frequency of consultation was not standardized but at addressed to fulfil missing data of predictors. Around 6 out of the least 1 consultation every year had been performed. 12 studied variables were included in the multiple imputation model. Five imputed data sets were created as part of multiple imputation, and then combined to produce an overall estimate of 2.2. Variables each regression coefficient. Sparse categories of nominal Twelve variables were extracted for each patient to develop the variables, such as diagnosis or type of combined surgery, were model. This selection was made according to convincing or probable grouped into a single “other” category. risk factors identified in previous studies.[7,11,12] Recipient related All analyses were made using IBM SPSS Statistics 22 software. variables considered were sex, age, primary indication/diagnosis for PK, vascularization of corneal bed and number of previous PK 3. Results performed. Graft-related variables were donor’s age and sex, preservation status of the graft, time from donor’s death to Altogether 895 PK from 778 patients were included in the study. enucleation and from donor’s death to transplantation. Graft Mean follow-up time was 5.8 years. Among these cases, 29% (n = diameter and the presence or not of a combined surgery were 258) were identified as a graft failure. Overall 10-year survival considered as surgery technique related variables. estimate based on univariate analysis was found to be 65%. Continuous variables (age of the donor, age of the recipient, time from death to enucleation, and time from death to transplant) were 3.1. Recipient related factors categorized in discrete intervals. Cutting points for categorization of continuous variables were selected based on clinical expertise Univariate analysis revealed a significant influence related to and previous studies regarding various donor and receipt primary diagnosis, number of previous grafts and vascularisa- characteristics that lead to an increased risk of graft failure.[13] tion. Recipient sex showed no significant influence (Table 1). Table 1 Summary of results from univariate analysis (Kaplan–Meier) and multivariate proportional hazard model (Cox regression). Kaplan–Meier Kaplan–Meier ∗ Missing data Log rank test Log rank test for trend† Cox regression Diagnosis 0 <.001 Keratoconus against all others P < .001 Recipient age 0 <.001 <.001 Not significant Recipient sex 0 .688 Not significant Vascularisation 23 (2.6%) <.001 <.001 Not significant Number previous grafts 0 <.001 <.001 First against all others P < .001 Donor age 0 .519 .220 >80 against 61–80 P = .043 Donor sex 2 (0.2%) .365 Not significant Preservation 91 (10.2%) .001 Not significant Time death enucleation 125 (14.0%) .415 .186 Not significant Time death transplant 61 (6.8%) .003 <.001 Not significant Graft diameter 3 (0.3%) .013 .304 Not significant Combined surgery 0 <.001 Not significant Total number of cases n = 895. ∗ The log rank test gives equal weights to the contribution of each failure time. † Tests the probability that there is a trend in survival scores across the groups. 2 Barraquer et al. Medicine (2019) 98:17 www.md-journal.com Primary diagnosis 1.0 Chemical burns (n = 32) Endothelial dystrophies (n = 170) 0.8 Infectious leukomas (n =180) Cumulative survival Keratoconus (n = 341) 0.6 Others (n = 78) Stromal dystrophies (n = 55) 0.4 Trauma (n= 39) 0.2 0.0 0 5 10 15 Time (years) Figure 1. Kaplan–Meier plots of graft survival in patients with different primary diagnoses. The 10-years estimates showed the difference of prognosis between keratoconus and other procedures. Keratoconus was also the most frequent diagnosis (n = 341), followed by infectious leukomas (n = 180) and endothelial dystrophies (n = 170). Considering primary diagnosis, patients with keratoconus 3.3. Surgical technique. Both graft diameter and effect of showed the best 10-year survival estimate (95%), followed by combined surgery were significant in univariate analysis endothelial and stromal dystrophies (both 55%), infectious (Table 1). Graft diameter between 7.0 and 7.4 mm as well as leukomas (49%), trauma (33%), and chemical burns (14%); between 8.0 and 8.4 mm showed the best 10-years survival other diagnoses resulted in a 10-year survival rate of 37% (Fig. 1). estimate (70% and 69%), followed by diameters of 7.5 to 7.9 mm Primary PK grafts had the best 10-year survival estimate (60%) (Fig. 4). No combined surgery resulted in a 10-year (81%), followed by second grafts (33%) and third or more grafts survival estimate of 67% and a combination with vitrectomy (16%). Avascular recipient corneas had the best 10-year survival displayed 26%. The combination with other surgeries (like, for estimate (74%), followed by vascularisation in 1 to 3 quadrants instance, Flieringa ring implantation or cataract surgery) it was (37%) and vascularisation in all 4 quadrants (28%). Recipient 61% (graph not shown). However, multivariate analysis revealed with age younger than 50 years showed 10-year survival estimate no-significance for graft diameter and combined surgery (Table 1, between 83% and 73%, which compared to estimates between Fig. 3). 47% and 44% for recipients older than 50 years (Table 1, Fig. 2). Multivariate analysis for recipient related factors showed 4. Discussion significant results only for primary diagnosis and number of previous grafts. Compared with keratoconus, stromal dystro- Correlation between the different analysed characteristics is a phies and endothelial dystrophies showed a mean hazard ratio of foreseeable event capable of leading to confusion biases in failure of 4.6 and 6.0, respectively, followed by infectious univariate analysis. For example, eyes with chemical burns or leukomas (7.4), trauma (10.0), and chemical burn (11.9); trauma were more likely than keratoconus to have vasculariza- other diagnoses resulted to have a mean hazard ratio of 9.5, tion in one or more quadrants. With the goal of identifying compared with keratoconus. Second grafts had 2.6 times and independent risk factors, multivariate analysis with Cox third and subsequent grafts 3.8 times higher risk of failure proportional hazard regression was performed. Figure 3 shows (Table 1, Fig. 3). 4 forest plots that summarize the results found on this approach using multiple imputation combined dataset. Table 1 further, 3.2. Graft characteristics. Considering univariate analysis, compares the statistically and non-statistically significant differ- preservation status and time between death and transplant ences between the results of the multivariate and univariate showed a significant effect for PK (Table 1). No preserved corneas analysis. showed a 10-year survival estimate of 71% as compared to Our study was of retrospective nature with different follow- preserved corneas with 56% (Fig. 4), whereas time between death up times and possible differential loss to follow-up which might and transplant of <24 hours between 73% and 70%, a time have introduced bias. Data had to be obtained from clinical between 24 and 36 hours of 61%, and more than 36 hours of records, relying on photographic and written descriptions. 52%, all estimates at 10 years post PK (graph not shown). Donor Another limitation might be the grouping of diagnoses and age, donor sex, and time between death and enucleation showed the different sizes of these diagnosis groups. Primary diagnoses no significant influence (Table 1). The P value for donor sex was like keratoconus or infectious leukomas were overrepresented just at the limit with.05 after rounding to 2 digits (Fig. 3). in comparison to others like chemical burns or trauma. We have Multivariate analysis showed border line significance (P tried to find a balance between grouping of similar diagnoses = .043) for one donor age group. Preservation status was just and the size of these groups. All of the about 1000 cases not significant (P = .096) and time between death and enucleation considered were done in a single center by 6 different surgeons, and time between death and transplant had clearly no significant following the same techniques with the same instruments. influence (Table 1, Fig. 3). Data collection had been standardized with clear guidelines 3 Barraquer et al. Medicine (2019) 98:17 Medicine Primary versus subsequent grafts 100% 1.0 80% Case distribution 0.8 Cumulative survival 1st 60% 2nd 0.6 40% 3rd or more 20% 0.4 0% 0.2 s L es En om euk a s us s as er ie rn AL m hi on do al d om h th us rau bu ia trop he rop oc O al T t t ra ys ys ic l m Ke 0.0 ld io C el ct th fe r St In 0 5 10 15 Time (years) Vascularization 100% 1.0 90% 80% Case distribution 0.8 70% Cumulative survival Avascular 60% 1 to 3 quadrants 50% 0.6 40% 4 quadrants 30% 20% 0.4 10% 0% 0.2 s L es uk a es us s as er rn AL m hi hi on do al d om th au bu ia trop he rop oc O Tr al t t ra ys ro s le ys ic m 0.0 Ke ld u io m C el ct th fe St 0 5 10 15 In En Time (years) Recipient age 100% 1.0 80% Case distribution 11-30 years 0.8 Cumulative survival 31-50 years 60% 51-70 years 40% 0.6 > 70 years 20% 0.4 0% 0.2 s L s En om euk a es Tr s s as ie er nu rn AL m hi do al d om h th au bu o ia trop he rop oc O al t t ra ys ys ic l m Ke ld us 0.0 io C el ct th fe r St In 0 5 10 15 Time (years) Figure 2. Kaplan–Meier plots of graft survival in patients segmented by number of previous PK, quadrants of vascularization and recipient’s age. Stacked bar charts of each one of these variables segmented by diagnosis are also displayed. PK = penetrating keratoplasty. and some training, but was done by 20 different ophthalmolo- 4.1. Diagnosis as principal risk factor for graft failure gist, which may have introduced some variations, for instance with respect to the decision on the exact time point of graft The principal aim of this study was to assess independent risk failure. factors for graft failure in PK. Our overall estimated graft failure We had performed a similar study with univariate analysis of rate from all causes after a 10 years follow-up was 35%. This 2886 cases of PK at our clinic between 1956 and 1987.[11,18] We value is consistent with previous studies with similar populations will refer to this as “our previous study” in the following and evaluation times which found 40% at 10 years follow-up.[19] discussion. In our previous study, we had a total overall graft failure rate of 4 Barraquer et al. Medicine (2019) 98:17 www.md-journal.com < .001 Trauma vs KC Recipient sex male vs female .219 < .001 Infectious leukomas vs KC Recipient age 31-50 vs 11-30 years .741 Stromal distrophies vs KC < .001 Recipient age 51-70 vs 11-30 years .378 Endothelial distrophies vs KC < .001 Recipient age >70 vs 11-30 years .563 Chemical burns vs KC < .001 Vascularisation 4 quadrants vs no .165 Other vs KC < .001 Vascularisation 1 to 3 quadrants vs no .079 0 5 10 15 20 25 30 Second vs first graft < .001 (KC = Keratoconus) Hazard ratio Third or more vs first graft < .001 0 1 2 3 4 5 6 Donor sex male vs female .046 Hazard ratio Donor age 21-40 vs 61-80 years .208 Donor age 41-60 vs 61-80 years .949 Donor age > 80 vs 61-80 years .043 Preserved vs not presered .096 < 7.0 vs 8.0-8.4 mm .388 ------------------------------------ .887 ----------------------------- < 2 vs 2-4 hours 7.0-7.4 vs 8.0-8.4 mm .739 Graft diameter enucleation Time death 4-6 vs 2-4 hours .214 7.5-7.9 vs 8.0-8.4 mm .453 6-8 vs 2-4 hours .345 8.5-8.9 vs 8.0-8.4 mm .951 > 8 vs 2-4 hours .684 > 9.00 vs 8.0-8.4 mm .063 --------------------- < 12 vs 12-24 hours .587 Vitrectomy vs no other surgery .218 Time death transplant 24-36 vs 12-24 hours .712 Other surgeries vs no other surgery .795 > 36 12-24 hours .721 0 1 2 3 4 0 1 2 3 Hazard ratio Hazard ratio Figure 3. Forest plots displaying multivariate analysis and hazard ratios associated with PK failure. Hazard ratios larger than one indicate a higher risk of graft failure. PK = penetrating keratoplasty. 35% at 5 years follow-up, which compares to 26% in our present could be the status of recipient’s corneal endothelial cells, which study, which is a substantial clincally relavant improvement. conserve their integrity even in severe cases of keratoconus. It is Among all 12 studied variables considered to be potential risk possible that peripheral endothelial cells maintained after a factors for definitive rejection, primary diagnosis other than standard PK procedure contribute significantly to the long-term keratoconus was shown as the strongest predictor in multivariate maintenance of the graft. Considering only our 314 cases with analysis. Our data reveals in fact, that the graft survival in PK for keratoconus, the significance of our evaluated factors with keratoconus is 95% at 10 years. Very similar keratoconus univariate analysis were similar to the results for all diagnoses survival rates (94% and 100%) at 10 years have been reported by together, except for preservation type and graft diameter which several researchers.[20,21] A plausible explanation for this result were not significant for this subgroup. This was in agreement Preservation status Graft diameter 1.0 Not preserved < 7.0 mm 1.0 Preserved 7.0-7.4 mm 0.8 7.5-7.9 mm 0.8 Cumulative survival 8.0-8.4 mm Cumulative survival 8.5-9.0 mm 0.6 > 9.0 mm 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 5 10 15 0 5 10 15 Time (years) Time (years) Figure 4. Kaplan–Meier plots and 10-years PK survival estimates in patients with different graft diameters and preservation status of corneal button. PK = penetrating keratoplasty. 5 Barraquer et al. Medicine (2019) 98:17 Medicine Primary vs. subsequent grafts for different diagnoses 1.0 All cases 1st graft Survival at 10 years 0.8 2nd or more grafts 0.6 0.4 0.2 0.0 s s s a s ns as er ie ie nu m r m ph ph th au bu co o O tro ro uc Tr to al st le ra ys ic dy m Ke ld os he al ia iu m C el ct ro th fe St do In En Figure 5. Estimated survival at 10 years after PK for different diagnoses comparing primary versus second or more grafts. with our multivariate analysis (Table 1). The relative effect on type and graft diameter were not significant. The 10 year survival graft survival for first, second, third and more grafts was similar for chemical burns and endothelial dystrophies were similar after for keratoconus as compared to all diagnoses together, just with a first grafts compared to second or more grafts. All other higher percentages of survival due to the overall better prognosis diagnoses showed a large difference between first and second or for keratoconus. more grafts (Fig. 5). As could have been expected, 10 year For the infectious leukoma category, we found a failure rate of survival after second or more grafts was independent of 51% as 10-year survival estimate. We have to consider that this vascularization before the first PK. Recipient age had less impact category combined corneal ulcers with different infectious on second or more grafts when compared to first grafts. aetiologies which in most cases are not known at the time of the PK surgery. Out of our 180 cases in this category, 35 were 4.3. Neovascularization. A larger meta-analysis, combining caused by herpes simplex virus, 2 by trachoma and for all other studies with univariate or multivariate analysis or both found a cases were nonspecified infectious leukomas. Different types of significant graft failure increase associated with the number of keratitis may have a specific influence on long term prognosis of corneal quadrants affected by neovascularization before PK.[28] PK. For example, herpes simplex keratitis may cause a higher rate This meta-analysis showed that the loss of corneal angiogenic of graft failure due a higher risk of corneal neovascularization privilege after PK increases the risk of graft failure, becoming and and the risk of recurrence of the herpetic disease in the graft.[22,23] independent risk factor of poor prognosis outcome of penetrating Trauma and chemical burns related PK deserves also a special keratoplasty procedures. There is evidence suggesting that the use mention, since they proved to be the indications with the worst of antiangiogenic pharmacologic agents such as Bevacizumab prognosis; 10-year survival rates of 33% and 14% and failure may be able to improve survival on high risk neovascularized hazard ratios of 10.0 and 11.9 respectively. This makes PK a corneas if used prior to the PK.[29] Our univariate analysis was merely short term, temporary solution to the underlying disease. also significant with respect to neovacularization, but not our Results with regard to primary diagnosis in the present study multivariate analysis (Table 1). We think that the significant were very similar to our previous study, except for chemical burn, result in our univariate analysis is due to the strong relation which was 16% in our previous study and 34% in our present between certain primary diagnoses and neovascularization in the study at 5 years follow-up. present study. As can be seen in Figure 2, diagnosis like keratoconus have minimal corneal neovascularization presence before PK, while chemical burns or trauma were more likely to 4.2. Influence of a repeated keratoplasty in graft prognosis have vascularization in one or more quadrants. As expected in accordance with our previous and other studies[11,18,24–26], repeated keratoplasties demonstrated a 4.4. Donor age and sex. Donor age was not significant with poorer prognosis that initial keratoplasty, with a clear increasing univariate analysis and showed borderline results with multivar- trend of risk in both univariate and multivariate analysis. This is iate analysis. Grafts from donors aged 80 years and older were at probably due to deteriorated condition of the corneal bed, a slightly higher risk of failure in comparison to patients under 80 probability of increased intraocular pressure during previous years. keratoplasty, and the violation of anterior chamber immunologi- The Cornea Donor Study (Mannis 2013) found evidence of a cal privilege in patients with more than one PK procedure.[27] donor age effect at the extremes of the age range. The success rate Evaluating first grafts and second or more grafts separately, the was higher for donors from 12 to 33 years (96%) and lower for significance of our evaluated factors with univariate analysis was donors from 72 to 75 years (62%); the study did not included similar to the results for all cases together, however, preservation donors older than 75 years. The Australian Corneal Graft 6 Barraquer et al. Medicine (2019) 98:17 www.md-journal.com Registry reported no significant association of donor age with graft failure in multivariate analysis but PPV did in univariate failure; considering donors up to 97 years of age.[19] analysis. A possible explanation for this could be explained Donor sex revealed no statistical significant results, both with because of the low number of combined surgeries in our sample univariate and multivariate analysis, although our multivariate and the strong interdependency between PK-PPV procedure and results were near our significance limit of .05 after rounding to patients with trauma as primary diagnosis (who had a worse two digits. The literature is also not consistent in this respect with prognosis than other diagnoses). confliction results.[3,28,30,31] In summary, long term prognosis of PK depends mostly on the Some studies suggest that male grafts can be subject to primary diagnosis and the number of previous graft failures in the alloimmune reactivity in female recipients as antigens of the Y recipient. Patients with keratoconus had the best 10-year survival genes are only expressed in males and not in females.[32,33] estimate (95%), followed by endothelial and stromal dystrophies However, earlier investigations on human penetrating kerato- (both 55%), infectious leukomas (49%), trauma (33%) and plasty did not demonstrate any effect of gender matching.[34] The chemical burns (14%). Primary PK grafts had a survival rate of Collaborative Corneal Transplant Study showed that matching 81%, second grafts of 33% and third or more grafts of 16%. for human leukocyte antigen (HLA): HLA-A, -B and -DR had no In conclusion, our study could not confirm that corneal significant effect on overall graft survival, the incidence of neovascularization in itself is a risk factor for corneal irreversible rejection or the incidence of rejection episodes.[35] In transplantation as it appeared in other studies. We confirmed our study we found a very small tendency of a lower 10 year that penetrating keratoplasty survival and long-term prognosis is survival estimate for male donor to female recipients (62%) as strongly diagnosis related. This could have implications for compared to the other three matching options (65 to 66%), but future patient selection, taking into account another therapeutic far from being statistically significant (P = .758; log rank test for or surgical approaches in those cases where the prognosis is Kaplan–Meier analysis). worse. Future research should be prospective with fixed follow- up times, balanced grouping of the different diagnoses and 4.5. Eye banking practices. Factors associated with eye banking include lamellar procedures of corneal transplants. practices, including preservation status, time between death and enucleation (considering 12 hours the upper limit) and death to Acknowledgments transplant time, were not significant with multivariate analysis. We can find similar outcomes in the bibliography, where retrieval of We are very grateful to the residents who collected all data from the donor cornea after brain-death or cardiac-death, death to the clinical history files: Luis Camacho, Jorge Fernández enucleation time (within standard limits), type of corneal storage Engroba, Marta Jerez, Hugo Medín, Jessica Botella, Laura medium used within the eye bank, or death to graft time (within Fernández de Cotero, Carlos Fernández-Vega, Pablo Sanjuan, approved limits for the preservation medium used) did not Belén Torres, Maddi Alonso, Andrés Fernández-Vega, Andrea influence corneal graft survival significantly.[19] None of the factors Laiseca, Andrea Llovet, Maximiliano Olivera, Gonzalo García de related to the processing of the tissues (post-mortem interval, Oteyza, Sarah Karam, María Montserrat Martos, and Ferran length of storage and endothelial cell density) were associated with Vilaplana. We thank Gemma Julio for reviewing the manuscript any increased risk of graft failure in Cornea Donor Study[36] and her valuable comments. neither for the results from the Veneto Eye Bank.[37] Author contributions 4.6. The graft diameter influence Conceptualization: Rafael I. Barraquer, Ralph Michael. Large diameter penetrating keratoplasties are evaded due to Data curation: Luis Pareja-Aricò, Alba Gómez-Benlloch. closeness of limbar vasculature because the proximity to Formal analysis: Luis Pareja-Aricò. conjunctival vessels and therefore to antigenic material.[38] In Investigation: Luis Pareja-Aricò, Alba Gómez-Benlloch, Ralph our present study, statistically significant differences were found Michael. when comparing graft diameters in univariate, but not with Methodology: Rafael I. Barraquer, Luis Pareja-Aricò, Alba multivariate analysis. Larger than 9 mm grafts had the lowest 10 Gómez-Benlloch, Ralph Michael. years survival estimate and were just not significant compared Project administration: Ralph Michael. with 8.0 to 8.4 mm grafts (P = .063). The literature has varied Resources: Rafael I. Barraquer. conclusions with studies reporting no association between graft Software: Luis Pareja-Aricò. size and the incidence of allograft rejection and lower allograft Supervision: Rafael I. Barraquer. rejection rates with larger[39] or smaller[40] graft sizes. Bidaut- Validation: Luis Pareja-Aricò, Alba Gómez-Benlloch, Ralph Garnier et al[41] found a no significant difference in graft Michael. diameter, dividing the sample in < 8 mm or > 8 mm. Visualization: Ralph Michael. Writing – original draft: Luis Pareja-Aricò, Alba Gómez- 4.7. Controversy about combined surgery procedures Benlloch, Ralph Michael. Writing – review & editing: Rafael I. Barraquer, Luis Pareja- Unlike the proved evidence regarding the association between Aricò, Alba Gómez-Benlloch, Ralph Michael. repeated PK and an increased risk of graft failure, combined keratoplasty with pars plana vitrectomy (PPV) or other surgeries References remains controversial.[24,42] Fasolo et al[37] showed a 2.8-fold greater risk of graft failure after PK and PPV, however Sugar [1] Gain P, Jullienne R, He Z, et al. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol 2016;134:167–73. et al[42] and Yu et al[43] did not find an increased risk of graft [2] Organización Nacional de Transplantes—Memoria de Actividad de failure after combined procedures. In our present study, neither Tejidos 2016 Available at: http://www.ont.es/infesp/Memorias. Accessed vitrectomy PPV nor other surgeries had influence in the risk of June 11. 2018. 7 Barraquer et al. Medicine (2019) 98:17 Medicine [3] Stulting RD, Sugar A, Beck R, et al. 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