Corneal donation for research versus for transplantation: A-year prospective study of acceptance rates in a French University Hospital.

Fresh corneal donation is essential for basic and preclinical research, but more unknown to public and the medical teams than donation for transplantation: it may raise concerns. We prospectively compared the acceptance rates and the characteristics of targeted corneal donation for research versus donation for transplantation during one year. The Agence de la Biomédecine authorized us to procure fresh corneas targeted for research, only from the donors with medical contraindications for transplantation, in order not to increase grafts shortage. Three nurses from the hospital coordination team of Saint-Etienne University Hospital, obtained consent for research and transplantation in parallel, screening all intra-hospital deaths cases, following standard protocol to check no refusal from families, despite the French opt-out system. They contacted 127 families for research and 244 for transplantation, in 71% of cases by telephone. Consent was obtained in 62% of cases for research and 54% for transplantation (P = 0.135). The main contraindication for transplantation was the cognitive disorders (66%) followed by the blood cancers (8%). This new specific activity, providing new source of fresh corneas for research immediately usable without any eyebank storage steps, didn't reduce the number of corneas procured for transplantation versus previous years (P = 0.998). Donors in the research group were 10 years older (P<0.001) without difference regarding endothelial cell quality (P = 0.071), allowing maximal clinical relevance for protocols using these fresh human scientific corneas provided by targeted donation.

Introduction

Organ and tissue donation is essential for fundamental and applied research. A state as close as in-vivo is equally important to ensure reliability of the results and clinical relevance. Needs are steadily increasing while availability is decreasing [1-Transplantation. 2017 ">3]. Legal and cultural differences between nations regarding donation in general and those for research complicate comparisons [4-7]. However, quantitative data are lacking as well as concrete solutions to correct imbalance.

In ophthalmology, cornea is no exception. A growing shortage of tissue for research is well documented in the USA [8, 9]. Worldwide, shortage also concerns donation for transplantation. Cornea is by far the most transplanted tissue, but in 2015 only 1/70 patients waiting worldwide could benefit from a corneal graft each year [10].

In France, corneal procurement is governed by the Bioethics Laws revised in 2018, based on the European standards [11]. Cornea is procured from a deceased donor, then necessarily stored in an authorized eyebank which carries out tissue quality and microbiological safety tests. Donation chain is an opt-out system with an online national refusal register (NRR). In absence of refusal expressed during his/her lifetime, any deceased person may legally be procured. Routinely, the hospital coordination team systematically seeks to ask relatives (without precise guidelines on degree of kinship) about the intentions of their deceased if no instruction (written or oral) is left by deceased and the relatives themselves refuse, the coordinators trust them and the procurement is never imposed.

The scientific human corneas used in France have two different origins: they come mainly from waste products of transplantation donation and a minority from body’s donation to science. The first are corneas discarded by quality controls in eyebanks. They represent about 50% of 11,000 corneas procured [12], but infected corneas must be withdrawn and only those that don’t have refusal for scientific use can be used. They are mostly poorer quality corneas to those required for transplantation. Besides these corneas are only available after several days of storage and are no longer fresh tissue. The second, resulting from donation in anatomy laboratories of medicine faculties, represents only a few hundred per year in France. They are the only fresh scientific corneas currently available, provided that death-to-procurement time is <24hours when body arrives in laboratory. According to legislative framework, distribution of these corneas for research are free of charge in France.

Among our research axes aims we developed an active storage machine for corneal grafts, which restores equivalent of intraocular pressure and renews storage medium. After testing it on porcine corneas [13], preclinical validation of this device was performed on a large series of human fresh corneas [14, 15]. To avoid all biases related to discarded corneas from eyebanks, we requested from the Agence de la Biomédecine (ABM, whose mission is to supervise, evaluate, promote procurement and transplantation) opportunity to procure fresh scientific human corneas, for this preclinical study and all other protocols using our device, and for protocols aiming at make evolve eyebanking, corneal imaging and bioengineering. We were concerned that donation for research could raise concerns because of its novelty and specificity. We therefore conducted a-year prospective study comparing characteristics of targeted donation for research and usual donation for transplantation.

Material and methods

Ethical considerations

All procedures conformed to the tenets of the Declaration of Helsinki for biomedical research involving human subjects. The ABM specifically authorized corneas procurement for research (PFS15-008 & PFS16-010) and study was approved by local Institutional Review Board “Ethics Committee of the CHU de Saint-Etienne, Research Commission of Terre d’Ethique” (IORG0007394, N°IRBN272016/CHUSTE). The ABM asked us to select for research only persons with medical contraindication to transplantation, based on the European standards, so as not to reduce corneas number for waiting recipients (). All other inclusion criteria didn’t differ from those usually used for corneal transplantation donation.

Study design

We collected prospectively during a-year, all data concerning corneal donation for research and transplantation in the Saint-Etienne university hospital. Main objective was to compare acceptance rates between both groups.

Secondary objectives were to analyze whether differences exist between both groups in donor profiles or corneas procured. We compared: 1/ consent seeking process data: obtaining consent methods (face-to-face or telephone), answer delay (immediate or delayed to think about); 2/ reasons for refusal; 3/ methods of expressing opposition to donation (NRR, instructions left to relatives); 4/ donors and corneas characteristics: endothelial cell density (ECD) measured 48hours after procurement (main quantitative quality criterion in eyebanks: done by the Saint-Etienne eyebank technicians for transplantation, and a unique skilled operator (TG) for research) and cataract operated eye (potentially with lower corneal quality); 5/ acceptance rate according to hospital coordinators experience to potentially optimize interview protocol.

Finally, to verify if this new research task impacted transplantation activity, we compared this year’s study to previous 13 years' activity carried out in the same hospital environment with the same eyebank connection.

Hospital coordination team missions

Three nurses of the hospital coordination team were involved. One was very experienced (20 years of experience and about 2000 interviews), one was experienced (15 years of experience and about 1500 interviews), the third (devoting 50% of her time to administrative-regulatory tasks) had 5 years of experience and about 500 interviews.

Regular mission consisted in obtaining consent to organ and tissue donation for transplantation. In France donation chain follows an opt-out system. More specifically for the corneas, they daily screened all intra-hospital deaths. Following French guidelines health authorities, maximum death-to-procurement time was 24 hours. Were recommended but not mandatory: death-to-body-refrigeration time <4hours, if not death-to-procurement time should be <12hours. In parallel, coordinators screened deceased persons with medical contraindication to donation for transplantation. Interviews were conducted according to standard written procedures, either face-to-face or by telephone, as we reported in 2002 [16]. For the research group, coordinators explained to the relatives of the eligible deceased that corneal donation wasn’t possible for transplantation due to medical contraindication, but for local medical research: so the coordinators asked the relatives to consent to cornea donation directly for research. General purpose of the corneal grafts research axes conducted by same team of surgeons and researchers was explained, if the relatives so wished. When clarifications were required, a detailed explanatory letter from department’s head (PG) could be provided to the relatives, just as follow-up support if needed. The type of the consent depended of each eligible deceased: written or oral refusal during lifetime for deceased, or oral consent or refusal (by telephone or face-to-face) from relatives contacted and informed.

Corneas procurement

All corneas were procured by 3 trained ophthalmology residents. Fresh human corneas were procured by in situ excision with the same settings whatever the target, transferred to our laboratory within 20 minutes (for research) or to eyebank as usual (for transplantation). Costs of sterile single-use instruments and organoculture medium for the research group were covered by our research laboratory funding. The ABM authorized us to procure only fresh scientific corneas: the remaining eye tissue was left on the deceased as procurements for transplantation, with tegumentary reconstruction ad integrum.

Statistics

Normality of continuous data distribution was analyzed with Shapiro-Wilk test with non-normality threshold of 5%. Normal distribution data were described by mean±SD, [min-max]. When the variable followed normal distribution, an unpaired Student t-test was used to compare donors’ characteristics of both groups. Chi2 test was used to compare percentages. Statistical significance was set at P<0.05, with two-tailed tests (unless specified one-tailed) and adjusted with Tukey technique when multiple tests were performed. Analyses were performed with SPSS 25.0 (IBM Corp, Armonk, NY).

Results

Medical wards of pre-selected deceased

In 12 months, on 1442 intra-hospital deaths, coordinators sought absence of opposition to donation for 371 deceased eligible to potential corneal donation: 127 for research, 244 (232 in circulatory arrest, 12 from multi-organ donor) for transplantation. For each group, deceased were mainly from 3 services: emergency (21%), geriatrics (20%) and intensive care (16%) for research, and intensive care (28%), emergency (12%) and pneumology (9%) for transplantation.

Methods of interviewing the relatives and answer delay

Seventy-one percent of interviews were conducted by telephone, without difference between both groups (72% for research, 70% for transplantation P = 0.389). Answer was immediate in 69% (257/371) of cases and didn’t differ between both groups (64% for research, 72% for transplantation P = 0.540). Relatives answered more often immediately during face-to-face interview (79% of cases) than by telephone (66% of cases) (P = 0.016), without difference between both groups (immediate answer was done: a/during face-to-face interview 74% for research, 81% for transplantation P = 0.393; b/ by telephone 61% for research, 68% for transplantation P = 0.218).

Acceptance rates

Consent was obtained in 62% (79/127) of cases for research and 54% (132/244) for transplantation (P = 0.135), providing 158 fresh corneas for research and 264 for eyebank respectively. Acceptance rates weren’t influenced by gender of eligible donors, with 57% (123/216) of acceptance among males and 57% (88/155) among females (P = 0.974) respectively for overall series, without difference between both groups: 62% of females gave their consent for research versus 52% for transplantation (P = 0.212); respectively 62% of males for research versus 55% for transplantation (P = 0.357). details acceptance rates within and between both groups, according to interview type and answer delay by relatives: no significant differences were found. Telephone made it possible to procure 110 corneas for research and 172 for transplantation (i.e. 67% of all corneas).

Acceptance rates varied significantly with coordinator's experience: the most experienced obtained 71% and 62% of consent, respectively for research and transplantation, the one with intermediate experience 55% and 52%, the 3rd less experienced 43% and 39% (P = 0.003 for overall comparison between coordinators, both groups combined). None of them had more success for research than for transplantation (P = 0.253, P = 0.730, P = 0.824 respectively for the 3 coordinators). Coordinator's experience was directly correlated to acceptance rates whatever target: r = 0.9966 for research (P = 0.026), r = 0.9972 for transplantation (P = 0.024) (unilateral tests).

Analysis of opposition to donation

No opposition was found on NRR. Clear (oral or written) opposition from subject prior to death, left to the relatives, was found in 19% (9/48) of cases for research versus 37.5% (42/112) for transplantation (P = 0.045). In other cases, refusal was a relatives’ decision (from one person if alone, or from a collegial decision). There wasn’t any influence of the gender or relationship of the person(s) providing consent (P = 0.342 and P = 0.254 respectively). Regardless target (79% for research, 80% for transplantation), the relatives mainly didn’t provide any justification (P = 0.036). When refusal was mentioned, it was: "Donation isn’t priority" in 10% of cases for research and 11% for transplantation (P = 0.999); "Our relative suffered too much, was too sick or too old" in 8% of cases for research and 6% for transplantation (P = 0.999); relatives didn’t call coordinators back in 3% of cases for research and 3% for transplantation (P = 0.999).

Donors characteristics and corneal cells quality

Medical contraindications that allowed selection in the research group were mainly the cognitive disorders in 66% (52/79) of cases, followed by the blood cancers (8%). The details were provided in . Donors in the research group were significantly more likely to be females (P = 0.004) and older by an average of 10 years (P<0.001); their bodies were also refrigerated quicker (P<0.001). However, there wasn’t significant difference in death-to-procurement time (P = 0.911), percentage of eyes with cataract surgery (P = 0.120) and ECD (P = 0.071). Twelve pairs of corneas (15%) in the research group were available for experimentation within 6 hours or less after death, and 24 (30%) within 12 hours or less, with an average death-to-experimentation in laboratory time 15h15±6hours [0h55;24h08]. Further details of these characteristics were noted in .

The medical contraindications that allowed procurement in the research group.

Note that cognitive disorders, first source of targeted corneas for research, include any neurodegenerative disease (such as Alzheimer, Parkinson disease, Multiple Sclerosis, Amyotrophic lateral sclerosis…). These diseases represent contraindications for corneal procurement and transplantation, to ensure safety of the graft: the principle is to rule out any degenerative disease presumed to be transmissible via corneal transplantation. These contraindications follow a precautionary principle.

Comparison of transplantation activity with previous years

With 244 families contacted for transplantation during this study year, activity wasn’t different from that of previous 13 years, which had concerned an average of 227±24 [181 to 264] families (P = 0.994). Similarly, number of corneas procured for transplantation (264) didn’t differ from the average of previous 13 years (284±24 [from 223 to 330] (P = 0.998)).

Discussion

Corneal donation for research is still too little-known to public and must be encouraged as much as that for transplantation, both of which are essential to fight against blindness. We showed that it is possible to specifically target deceased to procure fresh corneas for research, in parallel with transplantation activity, without putting them in competition.

Acceptance rate didn’t differ between the research and the transplantation groups, whereas we feared that donation for research might have raised more reluctance [17, 18]. Several mutual and non-exclusive explanations can be formulated: 1/ people who accept idea of corneal donation don’t oppose research and transplantation; 2/ some may think that they help even more people via research [19] than transplantation, which treats traditionally only two patients; 3/ older age (10 years) in the research group can make higher consent rate [20]; 4/ great experience of the coordinators in presenting legislation and research issues with empathy plays a key role [3, 21, 22]; 5/ longstanding [16] involvement of the coordinators in research projects, directly related here to improve corneal grafts storage keeps motivation over time; 6/ general public’s knowledge of our health region and team's research works (dedicated mainly but not only to corneal eyebanking and transplantation) through regular follow-up support for the relatives may help to make meaning of donation. But in practice relatives didn’t ask the coordinators for so much details about research aims.

Our high acceptance rate for transplantation was significantly higher than those reported for transplantation in literature [18, 21, 23] where rates exceeding two-thirds acceptance are exceptional [16, 24, 25]. Comparison between the teams is highly complex because it questions donation regulation, overall organization of the teams, donors screening and experience of the coordinators evolving over time. Our findings result certainly from combination of an opt-out system and a small well-structure, highly motivated experienced team. Key role of "human factor" in success of this deeply personal approach was reported before [25-27].

Our study further confirms crucial role of telephone interview, reported regularly [20, 28, 29]. In 2002 [16], we showed that for transplantation, telephone accounted for 58% of interviews and 48% of the corneas procured. Here for transplantation, its role is even more important with 71% of interviews and 67% of corneas procured. While in 2002 telephone was less efficient than face-to-face interviews, this difference wasn’t significant in 2017: communication resources evolution with the smartphones pervasive in daily life, is probably involved.

Refusals analysis shows that NRR is probably widely underused. The donation opponents tell probably rarely their relatives why, with a potential memory bias, as the justifications collected by our coordinators seem to indicate. Whatever target, donation legislative framework like presence of NRR aren’t well known to general public. Generally speaking, question of donation isn’t still sufficiently addressed in families, even for favorable people with only 50% of them having informed their relatives [22, 30]. At time of death, when relatives have to decide in place of their deceased, fear of doing wrong or assuming responsibility can lead to refusal.

The medical contraindications to corneal donation for transplantation are numerous, based mainly on precautionary principle solely and deprive recipients of potentially great intrinsic quality corneas: only rabies (constantly fatal), herpes simplex virus, bacteria, fungi and retinoblastoma have been formally demonstrated to be transmissible via the cornea. So, targeting these contraindications allows researchers to benefit from them. Some sporadic cases of prion disease were reported in recipients, and circumstantial evidence has implicated corneal transplantation as a mechanism of transmission of iatrogenic prion disease: transplantation is presumed (but not formal) to be the source of prion disease in recipients [31-34]. Although we could be concern about disease transmission or research bias induced by these contraindications, it clearly depends on the research aim(s). In this study, every cornea was procured by in situ excision with single-use sterile instrument, without any contact with retina or optic nerve, which are be considered as specific risk factors to possible iatrogenic spread of sporadic and variant prion disease [35, 36]. We did risk analysis before the use of those corneas to develop our active storage machine to make evolve eyebanking, with assessment of their baseline intrinsic quality (endothelial cell density [37-40], transparency [41], presence of scar or not, presence of previous refractive surgery or not) and safety (no infection [42-44]), following tests used in daily routine in eyebanks. Furthermore, with these common medical contraindications people may wish to give but feel excluded from donation: donation for research can be a way to give them back this opportunity. However many people are interested in donating their corneas for research but aren’t aware that it was possible to do so [45]. Thus, they leave more often no instruction to their relatives concerning procurement for research.

Targeted donation for research has another advantage: it allows the researchers to dispose the fresh tissues immediately after procurement, without passing through the eyebanks. Several positive consequences can be stated: it provides short circuit, without extra-work for the eyebanks and less charges for the researchers at the end of the chain (variations occur in different countries, such as the USA where procurements are done by paid technicians); it removes the damaging processes on cornea quality induced by storage itself.

Our specific approach imposed by the ABM made it possible to procure a large quantity of fresh scientific corneas over a short period of time, without difference quality from those grafted, ensuring optimal clinical relevance for the research protocols in eyebanking. Through people giving their bodies to science, we found that we could just procure 41±17 fresh scientific corneas per year over last 3 years in our faculty of medicine: it would have taken almost 4 times longer to obtain same number of fresh corneas versus our study with 158 corneas a-year. Without these fresh tissues, preclinical validation of our active storage machine [14] couldn’t have been performed under same conditions as its future use, like the other following protocols to improve corneal storage using our device with the extensions of authorizations by the ABM. Death-to-refrigeration time difference between groups was explained by difference in the services from which deceased came, not involving same post-death logistics. Donors age was logically higher, with more females in the research group, since we were asked to select potential donors for research on the diseases that were more frequent with aging and females live classically longer. It didn’t impact research protocols, since donor age isn’t a significant factor in survival of corneal grafts [46, 47].

Concerning coordinators, despite their additional workload to obtain targeted donation for research, there wasn’t negative impact on already important transplantation activity, neither on number of the families interviewed nor on number of corneas procured. That underlines possibility to have clinical and research processes coordinated without penalizing each other. The coordinators increased their efficiency without additional working time, didn’t receive bonus, made and have pursued it with belief: whatever research and clinical endeavors, they did both tasks without any conflict. Donation chain may vary in different countries. Regarding cost effectiveness of targeted corneal donation, we believe it may be applied whatever medico-economic system. In France, our laboratory had to pay only materials. But if needed, extra-cost from people in charge of obtaining consent or/and procurements, must be implemented at chain’s end for researchers. Anyway, without passing through the eyebanks, researchers will have less fees and better-quality fresh corneas, immediately available for experimentations, with this targeted corneal donation.

Despite being prospective and designed to minimize potential bias, our study presents some limitations: 1/ center-effect with highly motivated coordinators that have strong and long-lasting links with medical-research team for two decades. However, we believe that extrapolation to the other research teams motivated to obtain exceptional quality tissues, could have similar results. 2/ net inter-coordinator effect. Despite using standard protocol, some variations were intrinsically linked to age-related experience: more experienced coordinator probably adapts better to each family profile [26, 28] whatever target donation.

Several proposals have recently been made to improve acceptance of corneal donation for research. In the USA, Williams et al. advocates for scientific donation [9], by proposing creation of an eye donation registry for research [48]; collaborations between the eyebanks and the research institutes to recover corneas unsuitable for transplantation [49], as we have implemented since almost 20 years to procure discarded grafts from our eyebank. Besides, [50] proposed the creation of an online portal, under the aegis of the Association for Research in Vision and Ophthalmology, to specifically link the eyebanks to researchers in need of eye tissue.

To make this work useful to other teams, we may suggest some ways to increase number of donation for research: 1/ to develop privileged relationships between the research laboratories and the team in charge of donation in local hospital. Exposing her regularly aims, advances and results of researches, is probably crucial to induce lasting links; 2/ to encourage companionship between the novice and the experienced coordinators; 3/ to integrate in donation promotion campaigns, awareness raising to donation legislative framework and possibility to donate for research; 4/ to increase the resources allocated to the coordinators and ensure that all eligible relatives of deceased can be contacted in a timely manner: pressure comes from legislative framework and bodies transportation from hospital mortuary to private funeral homes, explaining mainly gap between number of intrahospital deaths and deceased eligible contacted in this study. Respectively 413 families were not contacted due to legislative framework with death-to-procurement time >24h (which discards deceased from eligibility to for corneal donation for research or transplantation); 411 families were not contacted due to fast transport of the deceased body from hospital mortuary to private funeral homes or conservative care (which prevent from interviewing families for research or transplantation). The third cause is that sometimes the coordinators didn’t have enough time to treat every dossier of eligible deceased (n = 243 families) for research or for transplantation in their daily multi-tasks: they share time between corneas, other tissues (vessels, bone), organs (mainly kidney).

In summary, in parallel with transplantation activity, by targeting donors with medical contraindication to corneal donation for transplantation, we can obtain many fresh scientific corneas of similar quality to those grafted, immediately available for the researchers, without increasing shortage for waiting recipients or incurring extra-fees by passing through the eyebanks. This targeted corneal donation could be a potential solution to make research advance better and faster.

List of the medical contraindications to corneal donation for transplantation in effect at the time of the study (2017).

(DOCX)

Click here for additional data file.

13 Feb 2020

PONE-D-19-35417

Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital

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Reviewer #1: The study is technically sound with a valid scientific question. The results were interesting and surprising. We note that the paper did not mention whether the research cornea donors were tested for relevant communicable diseases and what the disposition of the corneas would be should the results come back positive. Was there any concern that some of the cognitive disorders were due to prion disease?

Our other query would be the consent taking process for biomedical research, perhaps this can be mentioned in the paper? Will this be different from the consent for clinical transplant purposes? Or can we assume that ABM has already provided the necessary authorization and therefore no further consent is required?

The manuscript is easily understood but there is room for improvement with regard to overall sentence construction, grammatical flow and word choices.

Agree with the author’s view that the high consent rates may be a single centre effect and may not represent the situation in most other institutions around the world. However, it echoes the need for a consistent and sustained messaging to the general public about cornea donation, whether it be for therapy, research or education.

Reviewer #2: In this prospective study, the authors compared the donation acceptance rate for cornea transplantation (deceased patients with no medical contraindication for corneal transplantation) and for research (deceased patients with contraindication for corneal transplantation) and evaluated if the acceptance rate for transplantation donation remained stable over a year.

General comments

Obtaining fresh corneas for research is highly challenging and a main concern for worldwide researchers. While it is highly interesting to see that acceptance rates for research and for transplant were comparable, this paper opens several questions regarding research regulations of corneas. Indeed, in France, historically, ineligible corneas for transplant were being used for research, in consequence, research corneas came from patients without medical contraindication for transplant.

In the present study, corneas from deceased patients with medical contraindication for transplant (including cognitive disorders, blood cancers, neurologic disorder, uncontrolled infection, tumor of CNS, severe influenza and hemodilution) were used for research to increase the number of fresh corneas needed for research purpose.

The main question here might be “should we use tissues from cognitive disorders, blood cancers, neurologic disorder, tumor of CNS, uncontrolled infections… for our research?” Indeed, the authors explained that one of the main focus of their lab was to develop an active storage machine for corneal graft. We could be concern about both disease transmission and induced research bias which is not at all discussed in the paper.

Thus, the discussion should address those points which can’t be only resumed in one sentence (Line 279-281) “The medical contraindications to corneal donation for transplantation are numerous, based mainly on precautionary principle solely and deprive recipients of potentially great intrinsic quality corneas”.

Was any risk analysis performed before the use of those corneas?

Point by point comments

Some additional points need to be clarified and are listed below.

Results

Line 153 the authors reported 1442 intra-hospital deaths but only 371 deceased eligible. Can the authors explain the reasons of the gap since cornea for research are based on medical contraindications meaning that families of all deceased patients should be interviewed. What were the limitations?

Table 1 is nonreadable, please correct. In addition, in the table, the total of research interviews is 126 but number written in results line 154 is 127. Please review and correct.

Line 212 “medical contraindications that allowed selection in the research group” can you please clarify the French rules about research on tissues with medical contraindications for transplant?

End of comments

**********

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9 Mar 2020

PONE-D-19-35417

Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital

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First, we would like to thank the Academic Editor, reviewer #1 and #2 for her/his reviewing and advises. We have chosen to respond to the comments and to resubmit this manuscript. We deeply apologize for grammar or language mistakes.

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No problem, we followed guidelines

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Consent was informed. In France we have opt out system. The type of the consent depended of each eligible deceased : written or oral refusal during lifetime for deceased, or oral consent or refusal (by phone or face to face) from relatives contacted.

We precised it in Methods section.

3. Thank you for including your ethics statement: The Agence de la Biomedecine (ABM) specifically authorized corneas procurement for research (PFS15-008 & PFS16-010) and study was approved by local Institutional Review Board (IORG0007394, N°IRBN272016/CHUSTE).

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We have amended the full name of our institutional review board that approved our specific study Ethics Committee of the CHU de Saint-Etienne, Research Commission of Terre d’Ethique

We precised it in the Methods Section and in the “Ethics statement” field of the submission form.

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The patent does not alter our adherence to PLOS ONE policies on sharing data and materials.

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"P GAIN, S ACQUART and G THURET are inventors on “patent US 20160029618A1” submitted by University Jean Monnet that covers “Medical device intended for long-term storage of a cornea, or for ex vivo experimentation on a human or animal cornea”.

P GAIN and G THURET are consultant for Thea laboratories and Quantel Medical."

We note that one or more of the authors are employed by a commercial company: Thea laboratories and Quantel Medical.

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We apologize for that, we precised this point in reviewed manuscript.

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study is technically sound with a valid scientific question. The results were interesting and surprising. We note that the paper did not mention whether the research cornea donors were tested for relevant communicable diseases and what the disposition of the corneas would be should the results come back positive. Was there any concern that some of the cognitive disorders were due to prion disease?

Thank you for your comment, we have added the following sentences in the discussion section (lines 291 to 307)

“only rabies (constantly fatal), herpes simplex virus, bacteria, fungi and retinoblastoma have been formally demonstrated to be transmissible via the cornea. So, targeting these contraindications allows researchers to benefit from them. Some sporadic cases of prion disease were reported in recipients, and circumstantial evidence has implicated corneal transplantation as a mechanism of transmission of iatrogenic prion disease : transplantation is presumed (but not formal) to be the source of prion disease in recipients [31-34]. Although we could be concern about disease transmission or research bias induced by these contraindications, it clearly depends on the research aim(s). In this study, every cornea was procured by in situ excision with single-use sterile instrument, without any contact with retina or optic nerve, which are be considered as specific risk factors to possible iatrogenic spread of sporadic and variant prion disease [35, 36].”

Our other query would be the consent taking process for biomedical research, perhaps this can be mentioned in the paper? Will this be different from the consent for clinical transplant purposes? Or can we assume that ABM has already provided the necessary authorization and therefore no further consent is required?

Thank you for your comment. We clarified this point lines 122 to 139.

The manuscript is easily understood but there is room for improvement with regard to overall sentence construction, grammatical flow and word choices. We deeply apologize for grammar or language mistakes. We updated this point.

Agree with the author’s view that the high consent rates may be a single centre effect and may not represent the situation in most other institutions around the world. However, it echoes the need for a consistent and sustained messaging to the general public about cornea donation, whether it be for therapy, research or education.

Reviewer #2: In this prospective study, the authors compared the donation acceptance rate for cornea transplantation (deceased patients with no medical contraindication for corneal transplantation) and for research (deceased patients with contraindication for corneal transplantation) and evaluated if the acceptance rate for transplantation donation remained stable over a year.

General comments

Obtaining fresh corneas for research is highly challenging and a main concern for worldwide researchers. While it is highly interesting to see that acceptance rates for research and for transplant were comparable, this paper opens several questions regarding research regulations of corneas. Indeed, in France, historically, ineligible corneas for transplant were being used for research, in consequence, research corneas came from patients without medical contraindication for transplant.

In the present study, corneas from deceased patients with medical contraindication for transplant (including cognitive disorders, blood cancers, neurologic disorder, uncontrolled infection, tumor of CNS, severe influenza and hemodilution) were used for research to increase the number of fresh corneas needed for research purpose.

The main question here might be “should we use tissues from cognitive disorders, blood cancers, neurologic disorder, tumor of CNS, uncontrolled infections… for our research?” Indeed, the authors explained that one of the main focus of their lab was to develop an active storage machine for corneal graft. We could be concern about both disease transmission and induced research bias which is not at all discussed in the paper. Thanks for this comment. We added new comment concerning this point. (cf. response to comment of reviewer #1 about prion disease => line 291 to 307)

Thus, the discussion should address those points which can’t be only resumed in one sentence (Line 279-281) “The medical contraindications to corneal donation for transplantation are numerous, based mainly on precautionary principle solely and deprive recipients of potentially great intrinsic quality corneas”. We added new comment concerning this point (line 291 to 307).

Was any risk analysis performed before the use of those corneas? Yes, We added new comment concerning this point (line 303 to 307).

“We did risk analysis before the use of those corneas to develop our active storage machine to make evolve eyebanking, with assessment of their baseline intrinsic quality (endothelial cell density, transparency, presence of scar or not, presence of previous refractive surgery or not) and safety (no infection), following tests used in daily routine in eyebanks.”

Point by point comments

Some additional points need to be clarified and are listed below.

Results

Line 153 the authors reported 1442 intra-hospital deaths but only 371 deceased eligible. Can the authors explain the reasons of the gap since cornea for research are based on medical contraindications meaning that families of all deceased patients should be interviewed. What were the limitations? , We added new comment concerning this point (line 371 to 380).

Gap can be explained by several causes :

1/ legislative framework with death-to-procurement time >24h, which discards deceased from eligibility to for corneal donation for research or transplantation (n= 413)

2/ fast transport of the deceased body from hospital mortuary to private funeral homes or conservative care, which prevent from interviewing families for research or transplantation (n=411)

3/ Only 3 nurses of the coordination team are present to do all the work about corneas but also for other tissues (vessels, bone) and organs (mainly kidney). So, sometimes they do not have enough time to treat every dossier of eligible deceased for research or for transplantation in their daily tasks (between corneas, other tissues, organs) (n=243) ; so increase the resources allocated to the coordinators is crucial.

4/ medico-legal impediment with prosecutor objection (n=4).

Table 1 is nonreadable, please correct. In addition, in the table, the total of research interviews is 126 but number written in results line 154 is 127. Please review and correct. We apologize for that ; we reviewed and corrected on revised manuscript.

Line 212 “medical contraindications that allowed selection in the research group” can you please clarify the French rules about research on tissues with medical contraindications for transplant?

The Rules are edited and updated regularly by the Health Authorities, and this is The Agence de la biomédecine provides the rules : for each tissue medical contraindications exist for transplantation. Ineligibility for transplantation makes tissue discarded and so destructed. But some of this discarded tissue which does not represent infectious risks, may be used by specific, certified and authorized laboratories. For ocular tissue, distribution of tissue are free of charge in France.

End of comments

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Howard Cajucom -Uy

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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Submitted filename: Response to reviewers.docx

Click here for additional data file.

30 Apr 2020

PONE-D-19-35417R1

Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital

PLOS ONE

Dear Dr. GARCIN,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that the manuscript has significantly improved but the reviewers still have some minor concerns. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jun 14 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Please include the following items when submitting your revised manuscript:

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We look forward to receiving your revised manuscript.

Kind regards,

Yu-Chi Liu, M.D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Discussion

Line 268 misspelling of “iin” please replace by “in”

The authors may want to add some additional information regarding the CoVid current situation which will probably change their future practice. In their study, all deceased patients were eligible for research donation, will they now consider testing all deceased patients before harvesting corneas? Are French rules currently changing regarding cornea donation for research?

**********

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Reviewer #1: Yes: Howard Cajucom-Uy

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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1 May 2020

PONE-D-19-35417R1

Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital

PLOS ONE

Saint-Etienne, May 1st 2020

Dear Professor Yu-Chi Liu,

Please find attached a revised version of our article titled “Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital”.

We have carefully considered and responded to all the points addressed by the reviewers.

Per your instructions, all substantive amendments in the revised version are stated in our point-by-point response, and are marked in red in the article.

We greatly hope that this new version will meet the reviewers' expectations and comply with your editorial policy.

Yours sincerely,

Dr. Thibaud GARCIN, M.D., Ph.D., FEBO

Saint-Etienne University Hospital

"Corneal Graft Biology, Engineering, and Imaging" Laboratory EA 2521

Faculty of Medicine

Saint-Etienne

France

Dear Dr. GARCIN,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that the manuscript has significantly improved but the reviewers still have some minor concerns. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Jun 14 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

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Kind regards,

Yu-Chi Liu, M.D

Academic Editor

PLOS ONE

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Discussion

Line 268 misspelling of “iin” please replace by “in”

This has been corrected

The authors may want to add some additional information regarding the CoVid current situation which will probably change their future practice. In their study, all deceased patients were eligible for research donation, will they now consider testing all deceased patients before harvesting corneas? Are French rules currently changing regarding cornea donation for research?

Thank you for the suggestion. The continuation of corneal procurement as early as possible is indeed crucial both for patients waiting for transplants and for laboratories like ours that work on these irreplaceable human tissues. The SARS-Cov-2 epidemic has effectively stopped all but multi-organ donation. It appears that we may soon be able to resume therapeutic procurement from symptomatic, non-at-risk individuals, but there are many unanswered questions.

In order to be able to answer the question of the risk of donor-recipient transmission, we have already started a new research work and obtained the authorization from our health authority (Biomedicine Agency, PFS-2020-011) to resume retrieval for scientific purposes for the next 150 donors. Thus, we will systematically collect and test all donors (nasopharynx, conjunctiva and cornea) and carry out serologies. This will provide us with robust data to make good decisions about what new tests to perform or not to perform on donors, for transplantation and for research. We hope that the new recommendations will be based on evidence and not on an unfounded precautionary principle.

Of course, during this new study we are recording the acceptance rate for corneal donation among all potential donors, and possible reasons for refusal. We will know whether the current epidemic is changing behavior and we will submit a letter to report the results, if you wish.

For this article, as suggested, we would like to add the following paragraph as the end of discussion:

“By the time this article is accepted, the global SARS-Cov-2 epidemic has stopped corneal procurement for therapeutic and scientific purposes altogether. Scientific knowledge is sorely lacking to establish the risk of transmission of this virus via ocular tissues and therefore to make new recommendations on which virological tests should be added or not to ensure total safety. Our French health authority has authorized us in emergency (PFS-2020-011) to perform a large series of scientific procurements from any potential donor (COVID+ or -) in order to objectively analyze the risks. During this particular study we will also analyze whether the epidemic has modified the acceptance rate and the reasons for a possible refusal.”

Respectfully

Dr T Garcin, MD, PhD, FEBO

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Reviewer #1: Yes: Howard Cajucom-Uy

Reviewer #2: No

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Submitted filename: R2_Response to reviewers.docx

Click here for additional data file.

5 May 2020

Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital

PONE-D-19-35417R2

Dear Dr. GARCIN,

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Reviewers' comments:

6 May 2020

PONE-D-19-35417R2

Corneal donation for research versus for transplantation: a-year prospective study of acceptance rates in a French University Hospital

Dear Dr. Garcin:

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Table 1

The acceptance rates for the research and the transplantation groups according to interview type and answer delay.

		Research n = 79	Transplantation n = 132	P
Interview type	Telephone	59% (55/93)	50% (86/171)	0.416
	Face-to-face	68% (24/34)	63% (46/73)	0.670
Answer Delay	Immediate	60% (49/82)	51% (90/176)	0.242
	Delayed	67% (30/45)	62% (42/68)	0.953

Table 2

The characteristics of donors and corneas procured in the research and the transplantation groups.

Continuous data are expressed in mean±SD [min-max]; ECD = endothelial cell density.

	Research n = 79	Transplantation n = 132	P
Sex ratio Male/Female	0.84 (36 M/43 F)	1.93 (87 M/45 F)	0.004
Donor age (years)	81±12 [49;100]	71±13 [25;101]	<0.001
Death-to-refrigeration time (hour)	3h36±2 [1h30;11h20]	7h32±1 [1h00;20h39]	<0.001
Death-to-procurement time (hour)	15h05±6 [0h45;23h58]	15h01±6 [3h00;23h50]	0.911
% of eyes with cataract surgery	30% (48/158)	22% (58/264)	0.120
Average ECD (cells/mm2)	2540±479	2641±596	0.071