Diagnosis and management of keratoconus in the paediatric age group: a review of current evidence.

The diagnosis and management of keratoconus in the paediatric age group presents additional challenges to those encountered in adults. The most significant of these, encountered in some young patients, are delayed presentation of unilateral disease, more advanced disease at diagnosis, difficulty in obtaining reliable corneal imaging, faster rates of disease progression and challenges in contact lens management. The stabilisation effect of corneal cross-linking (CXL), more extensively studied in adults with randomised trials and long-term follow-up, has been much less rigorously examined in children and adolescents. The high heterogeneity of published studies in younger patients, particularly in the choice of tomography parameters designated as primary outcome measures and the definitions of progression, indicates that improved standardisation for future studies on CXL will be necessary. There is no evidence that corneal transplant outcomes in young patients are poorer than those in adults. This review provides a current perspective on the optimal diagnosis and treatment of keratoconus in children and adolescents.

Design and Conduct of Randomized Clinical Trials Evaluating Surgical Innovations in Ophthalmology: A Systematic Review.

PURPOSE: Surgical innovations are necessary to improve patient care. After an initial exploratory phase, novel surgical technique should be compared with alternative options or standard care in randomized controlled trials (RCTs). However, surgical RCTs have unique methodological challenges. Our study sought to investigate key aspects of the design, conduct, and reporting of RCTs of novel surgeries. DESIGN: Systematic review. METHODS: The protocol was prospectively registered in PROSPERO (CRD42021253297). RCTs evaluating novel surgeries for cataract, vitreoretinal, glaucoma, and corneal diseases were included. Medline, EMBASE, Cochrane Library, and Clinicaltrials.gov were searched. The search period was January 1, 2016, to June 16, 2021. RESULTS: A total of 52 ophthalmic surgery RCTs were identified in the fields of glaucoma (n = 12), vitreoretinal surgery (n = 5), cataract (n = 19), and cornea (n = 16). A description defining the surgeon's experience or level of expertise was reported in 30 RCTs (57%) and was presented in both control and intervention groups in 11 (21%). Specification of the number of cases performed in the particular surgical innovation being assessed prior to the trial was reported in 10 RCTs (19%) and an evaluation of quality of the surgical intervention in 7 (13%). Prospective trial registration was recorded in 12 RCTs (23%) and retrospective registration in 13 (25%); and there was no registration record in the remaining 28 (53%) studies. CONCLUSIONS: Important aspects of the study design such as the surgical learning curve, surgeon's previous experience, quality assurance, and trial registration details were often missing in novel ophthalmic surgical procedures. The Idea, Development, Exploration, Assessment, Long-term follow-up (IDEAL) framework aims to improve the quality of study design.

Graphical comparison of surgeon outcomes for the audit of a national corneal transplant registry (OTAG study 32).

PURPOSE: To compare Kaplan-Meier survival curves and funnel plots for the audit of surgeon-specific corneal transplantation outcomes. METHODS: We obtained data on all patients with Fuchs endothelial dystrophy (FED) receiving a first corneal transplant in one eye between January 2012 and December 2017. We produced 2-year Kaplan-Meier graft survival curves to compare a simulated individual surgeon's graft survival rate to national pooled data. We used funnel plots to compare all surgeon outcomes to the national graft survival rate with superimposed 95 and 99.8% confidence limits. We defined an outlier as a surgeon who performed ≥10 transplants and had graft survival below the 99.8% national lower limit. To assess the effect of the surgeon case mix, we also compared unadjusted and risk-adjusted graft survival rates. RESULTS: There were 3616 first corneal transplants for FED patients with complete data, performed or overseen by 196 surgeons. The 2-year national graft survival rate was 88%. The median change from the unadjusted to the risk-adjusted graft survival rate for individual surgeons was 0% (IQR: 0%- -2%). Of the 108 surgeons who had performed ≥10 transplants, we identified two outliers based on the unadjusted graft survival funnel plot, compared to four outliers based on the risk-adjusted graft survival funnel plot. CONCLUSION: Funnel plots provide a visually accessible method for comparing individual graft survival rates to the national rate. Risk-adjustment accounts for clinical factors, and this has advantages for audit and clinical governance.

New Pharmacological Approaches for the Treatment of Neurotrophic Keratitis.

Neurotrophic keratitis (NK) is a rare degenerative condition that is caused by damage to the trigeminal nerve, with partial or complete loss of corneal sensory innervation. The loss of innervation leads to impaired healing of corneal epithelium, which subsequently results in punctate epithelial erosions, persistent epithelial defects, corneal ulcers and corneal perforation. Management of NK is often supportive and aims to promote epithelial healing and prevent progression of disease. Multiple novel pharmacological approaches have been proposed to address the underlying pathophysiology of NK, which are discussed in this paper.

Survey of Corneal Surgeons' Attitudes Regarding Keratoplasty Rejection Risk Associated With Vaccinations.

PURPOSE: To investigate the attitudes and practice of corneal specialists if patients with keratoplasty sought advice regarding common vaccinations and risk for potential graft rejection. METHODS: An online questionnaire was posted on the Kera-net listserv and the EuCornea Web site in early 2020. Attitudes toward vaccinations and keratoplasty were obtained. Decision making for common keratoplasty (endothelial keratoplasty, deep anterior lamellar keratoplasty, and penetrating keratoplasty) scenarios at early and late time points was explored regarding the herpes zoster and influenza vaccines. RESULTS: There were 142 respondents: 51.1% (70/137) specifically advise their patients with keratoplasty to get all vaccinations; 19.7% (27/137) stated clinical experience of a vaccine-associated rejection episode; 42.2% (57/135) were unaware of any such cases; and 64% (27/42) of those concerned would recommend delay if within 3 months of transplant surgery, recent corneal infection, or a recent rejection episode. The 2245 total responses to 18 clinical scenarios demonstrated wide variability in management of grafts in the setting of vaccination. Generally, 45.9% would not alter management, 26.2% would increase frequency of topical steroids, and 22.2% would recommend delay to vaccinations. Increased concern was expressed with recent surgery, live zoster vaccine and higher-risk penetrating keratoplasty scenarios. CONCLUSIONS: Nearly half of the respondents do not alter management in the setting of keratoplasty and zoster and/or influenza vaccinations. Anecdotal rejection episodes possibly associated with vaccinations were reported by some. Vaccine-related rejection has not been shown in higher-level research, but that has not eliminated clinical concerns. Prospective research into the true vaccine-related risks in keratoplasty is necessary if evidence-based management guidelines are to be developed or definitive reassurance provided.

Effect of Corneal Cross-linking versus Standard Care on Keratoconus Progression in Young Patients: The KERALINK Randomized Controlled Trial.

PURPOSE: To examine the efficacy and safety of corneal cross-linking (CXL) for stabilization of progressive keratoconus. DESIGN: Observer-masked, randomized, controlled, parallel-group superiority trial. PARTICIPANTS: Sixty participants 10 to 16 years of age with progressive keratoconus, one eye of each deemed the study eye. METHODS: The study eye was randomized to either CXL plus standard care or standard care alone, with spectacle or contact lens correction as necessary for vision. MAIN OUTCOME MEASURES: The primary outcome was steep keratometry (K2) in the study eye as a measure of the steepness of the cornea at 18 months. Secondary outcomes included keratoconus progression defined as a 1.5-diopter (D) increase in K2, visual acuity, keratoconus apex corneal thickness, and quality of life. RESULTS: Of 60 participants, 30 were randomized to CXL and standard care groups. Of these, 30 patients in the CXL group and 28 patients in the standard care group were analyzed. Mean K2 in the study eye 18 months after randomization was 49.7 D (standard deviation [SD], 3.8 D) in the CXL group and 53.4 D (SD, 5.8 D) in the standard care group. The adjusted mean difference in K2 in the study eye was -3.0 D (95% confidence interval [CI], -4.9 to -1.1 D; P = 0.002), favoring CXL. Adjusted differences between groups in uncorrected and corrected vision favored eyes receiving CXL: -0.31 logarithm of the minimum angle of resolution (logMAR; 95% CI, -0.50 to -0.11 logMAR; P = 0.002) and -0.51 logMAR (95% CI, -1.37 to 0.35 logMAR; P = 0.002). Keratoconus progression in the study eye occurred in 2 patients (7%) randomized to CXL compared with 12 patients (43%) randomized to standard care. The unadjusted odds ratio suggests that on average, patients in the CXL arm had 90% (odds ratio, 0.1; 95% CI, 0.02-0.48; P = 0.004) lower odds of experiencing progression compared with those receiving standard care. CONCLUSIONS: CXL arrests progression of keratoconus in the majority of young patients. CXL should be considered as a first-line treatment in progressive disease. If the arrest of keratoconus progression induced by CXL is sustained in longer follow-up, particular benefit may be derived from avoiding a later requirement for contact lens wear or corneal transplantation.

Characteristics of endothelial corneal transplant rejection following immunisation with SARS-CoV-2 messenger RNA vaccine.

AIM: We report two cases of endothelial corneal allograft rejection following immunisation with SARS-CoV-2 messenger RNA (mRNA) vaccine BNT162b2 and describe the implications for management of transplant recipients postvaccination for COVID-19. METHODS: A 66-year-old woman with Fuchs endothelial corneal dystrophy (FECD) and a unilateral Descemet's membrane endothelial keratoplasty (DMEK) transplant received COVID-19 mRNA vaccine BNT162b2 14 days post-transplant. Seven days later, she presented with symptoms and signs of endothelial graft rejection. An 83-year-old woman with bilateral DMEK transplants for FECD 3 and 6 years earlier developed simultaneous acute endothelial rejection in both eyes, 3 weeks post second dose of COVID-19 mRNA vaccine BNT162b2. Rejection in both cases was treated successfully with topical corticosteroids. CONCLUSIONS: We believe this is the first report of temporal association between corneal transplant rejection following immunisation against COVID-19 and the first report of DMEK rejection following any immunisation. We hypothesise that the allogeneic response may have been initiated by the host antibody response following vaccination. Clinicians and patients should be aware of the potential of corneal graft rejection associated with vaccine administration and may wish to consider vaccination in advance of planned non-urgent keratoplasties. Patients should be counselled on the symptoms and signs that require urgent review to allow early treatment of any confirmed rejection episode.

Keratoplasty for Keratoconus in Young Patients: Demographics, Clinical Features, and Post-transplant Outcomes.

PURPOSE: To examine pretransplant findings and outcomes of corneal transplants for keratoconus in children. DESIGN: Retrospective cohort (national registry) study. METHODS: Data on all patients aged 16 or younger (n = 170) who had a first transplant for keratoconus between 2003 and 2018 in all corneal transplant centers in the UK were compared to adult patients aged 17 and older (n = 7,191). The influence of demographic variables, pretransplant corneal findings, and transplant type on 2-year visual, rejection-free, and transplant survival outcomes was examined. RESULTS: Children had poorer pretransplant visual acuity and higher rates of corneal vascularization and ocular surface disease than adults. However, 2-year post-transplant corrected visual acuity reached 20/20 or better in 35% of children compared to 28% of adults (P = .1). Transplant rejection and failure rates were 11% (P = .79) and 3% (P = .31), respectively, for children, which were comparable to rates for adults. Endothelial rejection was reported following penetrating keratoplasty (PK) in 13% of children (10% in adults). Irreversible rejection was not recorded for any transplant in a child. Despite a lack of difference in transplant outcomes, there was a significant age effect in the Cox regression model for transplant rejection, such that for every 5-year increase in age there was a 6% reduction in the hazard of rejection. Transplant survival following anterior lamellar keratoplasty and PK in children was similar. CONCLUSIONS: Young keratoconus patients have excellent transplant outcomes and visual results comparable to adults. Overall, the hazard of rejection was found to decrease with advancing age. However, in this large cohort of young patients with keratoconus and poor vision, there is no evidence of outcome advantage in delaying transplant until adult years.

Clinical and confocal imaging findings in congenital corneal anaesthesia.

BACKGROUND/AIMS: Congenital corneal anaesthesia (CCA) is an uncommon cause of corneal ulceration in young patients, with a reported poor visual prognosis. We correlated clinical findings in patients with CCA with corneal sub-basal nerve plexus (SBNP) morphology and dendritiform cell density (DCD) on confocal microscopy. METHODS: A prospective, case-control study was conducted at a referral clinic. History includied presenting features in patients with CCA, clinical course and examination findings. Differences in SBNP morphology and DCD on in vivo confocal microscopy (IVCM) were compared in cases and control subjects with healthy corneas. RESULTS: Eight patients with CCA were examined, of which three had a diagnosis of familial dysautonomia. Age at initial diagnosis of corneal disease ranged from infancy to 22 years, the most common presentation being corneal ulceration. All patients with CCA except one with optic neuropathy had corrected visual acuity 6/18 (logMAR 0.35) or better in at least one eye. Measured corneal sensation was minimal in all patients. Major abnormalities were found on confocal microscopy in all patients with CCA, whether or not inherited, including statistically significant reduction in SBNP nerve fibre density, fibre length and branch density. Increased DCD in superficial cornea was found in all patients with CCA. CONCLUSION: Good visual acuity can be maintained in eyes with corneal anaesthesia present from birth. IVCM provides direct evidence of a morphological correlate for measured corneal anaesthesia. Increased DCD may indicate an enhanced role for innate immune cells in superficial cornea in protection of the anaesthetic ocular surface.

Update on Herpes simplex keratitis management.

Herpes simplex keratitis (HSK) is a common, potentially blinding condition characterised by recurrent infections of the cornea, seen by both general ophthalmologists and corneal specialists. Successful treatment of recurrences reduces disease duration, prevents progressive corneal scaring leading to vision loss and reduces risk of further recurrences. In this review we discuss the relative advantages of the diagnostic laboratory investigations including polymerase chain reaction, viral culture and fluorescence-based immunohistochemistry. We review treatment strategies in selected aspects of HSK and discuss the management options in cases not responding to treatment.

Corneal cross-linking versus standard care in children with keratoconus - a randomised, multicentre, observer-masked trial of efficacy and safety (KERALINK): a statistical analysis plan.

BACKGROUND: The KERALINK trial tests the hypothesis that corneal cross-linking (CXL) treatment reduces the progression of keratoconus in comparison to standard care in patients aged 10-16 years. This article describes the statistical analysis plan for this trial as an update to the published protocol. It is written before the end of the patient follow-up, while the outcome of the trial is still unknown. DESIGN AND METHODS: KERALINK is a randomised controlled, observer-masked, multicentre trial in progressive keratoconus comparing epithelium-off CXL with standard care, including spectacles or contact lenses as necessary for best-corrected acuity. Keratoconus is a disorder of the shape of the cornea in which the normally round dome-shaped clear front window of the eye (cornea) thins progressively leading to a cone-like bulge. This impairs the ability of the eye to focus properly, causing reduced vision which requires spectacle or contact lens wear or, in a minority of patients, eventually corneal replacement by a transplant for best vision. The primary outcome measure is the between-group difference in K2 at 18 months adjusted for K2 at baseline examination. K2 is the value of the steepest corneal meridian as measured on Pentacam topography. Secondary outcomes are keratoconus progression, time to keratoconus progression, visual acuity, refraction, apical corneal thickness and adverse events. Patient-reported effects will be explored by questionnaires. We describe in detail the statistical aspects of KERALINK: the outcome measures, the sample size calculation, general analysis principles, the planned descriptive statistics and statistical models, and planned subgroup and sensitivity analyses. DISCUSSION: The KERALINK statistical analysis will provide comprehensive and precise information on the relative effectiveness of the two treatments. The plan will be implemented in May 2020 when follow-up for the trial is completed. TRIAL REGISTRATION: EudraCT, 2016-001460-11. Registered on 19 May 2016.

Differential effects of primary disease and corneal vascularisation on corneal transplant rejection and survival.

AIMS: To investigate the relative risk of pretransplant corneal vascularisation on rate of rejection and graft failure within 5 years of surgery when categorised by indication for transplantation.We analysed all adults recorded in the UK transplant registry who had a first cornea transplant for keratoconus (KC), pseudophakic bullous keratopathy (PBK) or previous infection (viral/bacterial/fungal/protozoan) between 1999 and 2017. We analysed the number of quadrants of the recipient cornea vascularised before transplant and type of vascularisation, the interval post-transplant to rejection, if any, and the outcome at 5 years post-transplant. Risk factors for rejection and transplant failure were modelled by multivariable risk-adjusted Cox regression. RESULTS: Corneal vascularisation was recorded in 10%, 25% and 67% of patients with KC, PBK and infection, respectively. Individuals with PBK had an increased hazard of transplant rejection only when there were more than two quadrants of vascularisation (HR 1.5, p=0.004) when either superficial and/or deep vascularisation was present (HR 1.3 and 1.4, respectively, p=0.004). Individuals who had a transplant for previous infection had an increased hazard of rejection with four quadrants of vascularisation (HR 1.6, p=0.003). There was no risk-adjusted increase in transplant failure associated with vascularisation in any group. There was weak evidence of reduction in risk of rejection and/or failure associated with lamellar compared with penetrating transplantation in KC and PBK in vascularised recipient corneas. CONCLUSION: Vascularisation is a risk factor for corneal allograft rejection within 5 years. The indication for transplantation has a clinically significant effect on the magnitude of this risk.

Graft Survival After Penetrating and Endothelial Keratoplasty in Iridocorneal Endothelial Syndrome.

PURPOSE: To compare the survival of a first penetrating keratoplasty (PK) or endothelial keratoplasty (EK) for iridocorneal endothelial (ICE) syndrome with transplant survival in Fuchs endothelial dystrophy (FED) and pseudophakic bullous keratopathy (PBK). METHODS: We compared graft survival of PK and EK for ICE syndrome for 2 time periods. We then compared graft survival in ICE syndrome with graft survival in FED and PBK. Kaplan-Meier estimates of graft survival up to 5 years posttransplant were calculated with 95% confidence intervals (CI), whereas comparisons between the groups were performed using the log-rank test. RESULTS: We included 86 first transplants for ICE syndrome. There was no difference in graft survival between the 58 PKs and the 28 EKs for up to 5 years after surgery (P = 0.717). For the period from 2009 to 2017, the 5-year graft survival rates for ICE syndrome were 64.3% (CI, 21.8%-88.0%) for the 16 PKs and 66.8% (CI, 41.8%-83.0%) for the 26 EKs (P = 0.469). Between 2009 and 2017, the 5-year survival rate for 42 grafts with ICE syndrome was 62.7% (CI, 39.6%-79.0%), which was lower than 75.9% (CI, 74.2%-77.4%) in 7058 transplants for FED but higher than 55.1% (CI, 52.0%-58.0%) in 3320 transplants for PBK, although the numbers of ICE transplants are too small to tell whether this difference was by chance. CONCLUSIONS: The results indicate no difference in graft survival between PK and EK for ICE syndrome. Graft survival in ICE syndrome is intermediate between that of FED and PBK.

A randomised, controlled, observer-masked trial of corneal cross-linking for progressive keratoconus in children: the KERALINK protocol.

INTRODUCTION: The KERALINK trial tests the hypothesis that corneal cross-linking (CXL) treatment reduces the progression of keratoconus in comparison to standard care in patients under 17 years old. KERALINK is a randomised controlled, observer-masked, multicentre trial in progressive keratoconus comparing epithelium-off CXL with standard care, including spectacles or contact lenses as necessary for best-corrected acuity. METHODS AND ANALYSIS: A total of 30 participants will be randomised per group. Eligible participants aged 10-16 years with progressive keratoconus in one or both eyes will be recruited. Following randomisation, participants will be followed up 3-monthly for 18 months. The effect on progression will be determined by K2 on corneal topography. The primary outcome measure is between-group difference in K2 at 18 months adjusted for K2 at baseline examination. Secondary outcomes are the effect of CXL on (1) keratoconus progression, (2) time to keratoconus progression, (3) visual acuity, (4) refraction, (5) apical corneal thickness and (6) adverse events. Patient-reported effects will be explored by questionnaires. ETHICS AND DISSEMINATION: Research Ethics Committee Approval was obtained on 30 June 2016 (ref: 14/LO/1937). Current protocol: V.5.0 (08/11/2017). Study findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: European Union clinial trials register (EudraCT) 2016-001460-11.

Descemet Membrane Endothelial Keratoplasty (DMEK) Graft Dislocation Into the Vitreous Cavity.

PURPOSE: To present our experience of Descemet stripping endothelial keratoplasty (DMEK) graft luxation into the vitreous cavity in 2 cases. METHODS: DMEK was performed in 2 patients with aphakic bullous keratopathy. The indications for keratoplasty were endothelial failure caused by chronic intermediate uveitis and glaucoma in 1 case and decompensated previous penetrating keratoplasty in the other. Both cases had enlarged pupils and had previously undergone pars plana vitrectomy. In both cases, the DMEK graft dislocated into the vitreous cavity during unfolding maneuvers and could not be retrieved during the same procedure. RESULTS: No signs of retinal detachment were observed during follow-up (6 months and 1 year). Although visualization of the graft was not possible on examination, B-scan confirmed the presence of the lenticule lying over the retina. One case underwent repeat DMEK, and 1 case underwent repeat penetrating keratoplasty. In 1 case, the graft was retrieved after a month and sent for histopathology. In both cases, corneal transparency and corrected visual acuity improved to full potential after the final procedure. Histopathology of the retrieved graft showed only endothelial cell loss and no fibrocellular proliferation. CONCLUSIONS: The risk of fibrous proliferation and retinal detachment after posterior dislocation of DMEK grafts may be less than in grafts including corneal stroma, but pars plana vitrectomy and retrieval of the dislocated corneal transplant are still indicated after revision corneal transplant surgery where visual symptoms or signs of fibrotic change around the dislocated graft are evident.

Longitudinal changes in corneal leucocyte density in vivo following transplantation.

AIMS: To prospectively evaluate the changes in corneal leucocyte density with in vivo confocal microscopy (IVCM) following transplantation and to determine if leucocyte density post-transplant is an indicator of graft rejection risk. METHODS: IVCM imaging of cornea pre-transplant and post-transplant at 1 week, 1, 3 and 12 months. The changes in leucocyte density associated with diagnosis, vascularisation, type of keratoplasty, topical steroid and immunosuppression treatment, allograft rejection and failure within 4 years post-transplant were analysed. RESULTS: Sub-basal nerve plexus total central leucocyte density (SBNP-TCLD) varied with diagnosis (p<0.001), interval post-transplant (p<0.001), degree of vascularisation (p=0.001) and rejection episodes in eyes off topical steroid (p=0.01). The highest SBNP-TCLD was found in eyes with inflammation pre-transplant. Mean 12-month SBNP-TCLD in eyes which had rejection episodes was almost double that in eyes which did not (79.0 and 39.8 cells/mm2, respectively). SBNP-TCLD >63.5 cells/mm2 was associated with a higher risk of rejection within 1 year (p=0.04) and 4 years (p=0.007). Changes in leucocyte density on the donor endothelium significantly differed between penetrating keratoplasty and deep anterior lamellar keratoplasty grafts (p<0.01) and in those in which rejection episodes were observed (p<0.001). CONCLUSIONS: Leucocyte density varies with corneal diagnosis, extent of vascularisation and interval post-transplant. Topical steroid treatment is associated with reduced leucocyte density and risk of graft rejection. Higher endothelium leucocyte density correlates significantly with previous or subsequent rejection episodes. Leucocyte density measurement by IVCM may be useful in identifying transplants at risk of rejection.

Fungal infection after endothelial keratoplasty: association with hypothermic corneal storage.

PURPOSE: To compare the incidence of fungal infection after endothelial keratoplasty (EK) when donor tissue had been stored in hypothermic medium or organ culture. METHODS: We describe the clinical features of 10 cases of fungal infection (keratitis or endophthalmitis) following EK identified at three European centres. Case definition was the culture of fungus or a positive PCR from the host cornea or anterior chamber after EK. A survey of the incidence of infection after EK was conducted by the European Eye Bank Association. The main outcome measure was the number of cases in which donor tissue had been stored in hypothermic medium compared with organ culture. RESULTS: The 10 cases occurred between 2014 and 2017. All donor corneas had been stored in hypothermic medium sourced from three US eye banks. Three pairs of mate corneas caused infections in six recipients. Candida spp were identified from nine cases, with one isolate of Purpureocillium lilacinum. Data on 16 862 corneas supplied for EK were available from 16 European eye banks for the 5-year period from 2012. There were 17 reported cases of infection, of which 15 (88%) were fungal infections and 14 (82%) were Candida spp. Fungal infection was reported from 3 of 14 476 (0.02%) corneas supplied in organ culture compared with 12 of 2386 (0.50%) corneas supplied in hypothermic medium (p<0.0001). The incidence of infection after hypothermic storage was similar for material sourced from Europe (0.52%) or the USA (0.61%). CONCLUSIONS: Infection after EK is strongly associated with Candida spp. The possible explanations for the higher incidence of infection when tissue is stored in hypothermic medium are discussed.