Effect of Head Trauma-Related Deaths on Corneal Endothelial Cell Loss in Eye Bank Donors.

PURPOSE: The purpose of this study was to examine the effect of head trauma-related deaths on corneal endothelial cell density (ECD) in eye bank donors. METHODS: This is a retrospective study of 287 corneas from donors with causes of death secondary to motor vehicle accident with sustained head trauma (n = 50), gunshot wound to the head (n = 138), fall with sustained head trauma (n = 2), and non-head-related traumatic causes of death (n = 97). Donors older than age 50 years were excluded due to concern for undiagnosed Fuchs endothelial dystrophy as a potential confounder for the cause of endothelial cell loss. Donor characteristics, ECD, and focal endothelial cell loss on specular microscopy were compared between the groups. Donors in the head trauma and nonhead trauma groups were matched by age; there were 42 age-matched donors in both groups. RESULTS: Age and ECD were negatively correlated (Pearson correlation coefficient = -0.57). Death-to-preservation time was not significantly different between the 2 groups ( P value = 0.59). The mean ECD in the head trauma group was 2859 ± 370 cells/mm 2 and 3041 ± 464 cells/mm 2 in the nonhead trauma group. The head trauma group had a lower ECD (178 ± 70 cells/mm 2 , P value = 0.013). After matching for age, the difference in ECD between the 2 groups was -94 ± 82 cells/mm 2 ( P value = 0.26). The adjusted odds of having focal endothelial cell loss was not statistically significant ( P value = 0.50) between the groups. CONCLUSIONS: After statistical adjustments, there were no differences between the head trauma and nonhead trauma groups.

Donor Diabetes Mellitus Severity and Corneal Transplant Suitability in a US Eye Bank Donor Population.

PURPOSE: To determine whether donor diabetes mellitus (DM) severity is associated with differences in endothelial cell density (ECD) and surgically unsuitable tissue. METHODS: Raw data were obtained from Saving Sight Eye Bank (Kansas City, MO) including 10,454 donated eyes from 5346 eligible donors from July 2014 through May 2017. Donors were grouped into 5 categories by their insulin use and the presence of microvascular end-organ complications. The categories were non-DM (NDM), noninsulin-dependent DM without complications (NIDDMnc), noninsulin-dependent DM with complication (NIDDMc), insulin-dependent DM without complications, and insulin-dependent DM with complication. Outcome variables included ECD and tissue transplant suitability. Mixed effects models were used to adjust for the random effect of repeated measures and fixed effects of donor age, race, lens status, and death to refrigeration and death to preservation times. Interaction effects of DM severity group and donor age and DM severity group and lens status were included in the models. RESULTS: One thousand six hundred eighty-four (32.1%) donors had a diagnosis of DM. Six hundred fifty-eight donors were in the NIDDMnc group, 225 in the NIDDMc group, 404 in the insulin-dependent DM without complication group, and 397 in the insulin-dependent DM with complication group. Compared with non-DM, donors with DM were older (P < 0.001) and more likely to be pseudophakic (P < 0.001). DM severity groups did not affect adjusted ECD at mean donor age. There was no statistically significant ECD interaction between DM severity group and lens status. There was a statistically significant ECD crossover interaction with NIDDMnc and donor age (P < 0.001). In phakic eyes, NIDDMc was associated with a statistically significantly lower odds of transplant suitability (odds ratio 0.62, P = 0.006). CONCLUSIONS: DM severity does not affect lowering adjusted ECD at mean donor age. DM severity and pseudophakia were not associated with lower adjusted ECD. NIDDMnc was associated with an attenuation of the age-dependent decrease in ECD. NIDDMc was associated with decreased transplant suitability in phakic eyes. Future studies should include age, lens status, and interaction effects in their models of ECD and transplant suitability.

Effect of Diabetes Mellitus and Metformin on Central Corneal Endothelial Cell Density in Eye Bank Eyes.

PURPOSE: To determine whether metformin use and diabetes mellitus (DM) affect central corneal endothelial cell density (ECD) by examining an eye bank corneal donor database. METHODS: The Lions Eye Institute corneal donor database, which consists of 38,318 corneal samples, was examined. Associations of ECD with metformin use and DM were tested by mixed effects linear models that account for correlations of outcomes between eyes within subjects adjusting for age, intraocular lens status, and glaucoma. Subjects (N = 17,056) with observed ECD counts for both eyes are included for analysis. RESULTS: Average donor age was 56.3 (SD = 15.0). ECD was not associated with metformin use (mean ± SE = 2592 ± 11.9 (N = 1014) versus nonuse [2592 ± 3.0 (N = 16,042), P = 0.302]; further analysis showed that ECD was not significantly associated with metformin use in patients with diabetes. However, metformin use was significantly associated with lower ECD among patients with glaucoma: [2658 ± 50.7 (N = 27) for use versus 2789 ± 19.0 (N = 164) for nonuse, P = 0.018]. The presence of DM was significantly associated with lower ECD 2581 ± 5.6 (N = 4766) for DM versus 2595 ± 3.4 (N = 12,290) for non-DM, P = 0.031). CONCLUSIONS: Lower ECD was associated with DM. Lower ECD was not associated with metformin use except in a subgroup of patients with glaucoma, in which subgroup analysis showed lower ECD. The differences in ECD observed were small and unlikely to affect the suitability for transplantation of donor corneas.

Technician Consistency in Specular Microscopy Measurements: A "Real-World" Retrospective Analysis of a United States Eye Bank.

PURPOSE: To quantify consistency of endothelial cell density (ECD) measurements among technicians in a single US eye bank operating under typical operating conditions. METHODS: In this retrospective analysis of 51 microscopy technicians using a semiautomated counting method on 35,067 eyes from July 2007 to May 2015, technician- and date-related marginal ECD effects were calculated using linear regression models. ECD variance was correlated with the number of specular microscopy technicians. RESULTS: Technician mean ECDs ranged from 2386 ± 431 to 3005 ± 560 cells/mm. Nine technicians had statistically and clinically significant marginal effects. Annual mean ECDs adjusted for changes in technicians ranged from 2422 ± 433 to 2644 ± 430 cells/mm. The period of 2007 to 2009 had statistically and clinically significant marginal effects. There was a nonstatistically significant association between the number of technicians and ECD standard deviation. CONCLUSIONS: There was significant ECD variability associated with specular microscopy technicians and with the date of measurement. We recommend that eye banks collect data related to laboratory factors that have been shown to influence ECD variability.

Age-Stratified Analysis of Diabetes and Pseudophakia Effects on Corneal Endothelial Cell Density: A Retrospective Eye Bank Study.

PURPOSE: To perform an age-stratified analysis of the effect of diabetes and pseudophakia on corneal endothelial cell density (ECD). METHODS: This is a comparative analysis of donor characteristics from data supplied by the Lions Eye Institute for Transplant and Research on tissue harvested from July 1, 2007, through May 23, 2014. The mixed-effects model was used to compare age-adjusted mean corneal ECD between donors with and without diabetes. RESULTS: A total of 20,026 nondiabetic donor eyes and 13,617 diabetic donor eyes were included in this study. ECD was 2604 cells per square millimeter in nondiabetic corneas and 2576 cells per square millimeter in diabetic corneas (P < 0.001). Among phakic patients, diabetic ECD was significantly less in the middle-age subgroups: -33 cells per square millimeter in the 21-to-40-year-old subgroup (P = 0.048) and -25 cells per square millimeter in the 41-to-60-year-old subgroup (P = 0.009). Among pseudophakic patients, diabetic ECD was significantly less only in the subgroup 61 years or older: -56 cells per square millimeter (P = 0.026). The magnitude of difference in ECD between phakic and pseudophakic donors was greater in patients with diabetes in the subgroup 61 years or older (P < 0.001). CONCLUSIONS: Donor eyes with a history of diabetes had a slightly lower ECD (-29 cells/mm) than eyes without a history of diabetes. Although this statistical relationship is consistent with our pathophysiologic understanding of diabetes and the corneal endothelium, such a minor difference in ECD would be expected to have minimal clinical impact on overall corneal endothelial function.

Association Between Corneal Endothelial Cell Density and Topical Glaucoma Medication Use in an Eye Bank Donor Population.

PURPOSE: To evaluate the association between corneal endothelial cell density (ECD) and donor topical glaucoma medication use in an eye bank database. METHODS: Raw eye bank data included 19,159 donors over the period July 2007 to May 2015. Free-text, donor medication lists were retrospectively searched for glaucoma medication. Exclusion criteria were age less than 40 years, history of eye surgery, endothelial trauma, guttae, and cell densities <1000 or >3300/mm. Analysis of covariance was used to test differences in cell density between groups while adjusting for age. Linear regression was used to test the correlation of independent interval variables while adjusting for age. RESULTS: Twelve thousand one hundred fifty-seven donors were included in the final analysis; 134 were on topical glaucoma medication. The mean ECD for donors not on glaucoma medication and pooled donors on glaucoma medication was 2561 ± 348 and 2516 ± 320 cells/mm, respectively (P = 0.42). Subgroup analysis by medication class resulted in nonstatistically significant differences between ECDs of nonmedicated donors and donors on alpha agonists (P = 0.76), beta blockers (P = 0.90), carbonic anhydrase inhibitors (P = 0.13), cholinergics (P = 0.37), and prostaglandin analogs (P = 0.62). The number of glaucoma medication classes used by donors was not a statistically significant predictor of endothelial density (P = 0.298). CONCLUSIONS: Donors on topical glaucoma medication do not have ECDs statistically significantly lower than donors not on medication.

Analyses of Factors Affecting Endothelial Cell Density in an Eye Bank Corneal Donor Database.

PURPOSE: To analyze the factors affecting central corneal endothelial cell density (ECD) in an eye bank corneal donor database. METHODS: The Lion's Eye Institute corneal donor database consisting of 18,665 donors (34,234 corneas) aged 20 years or older was analyzed. In particular, differences in the ECD based on age, sex, race, prior ocular surgery, a history of systemic diseases, and smoking were investigated. Furthermore, risk factors for donor cell count inadequacy (defined here as ECD less than 2000/mm) were identified. RESULTS: ECD decreased with age. Regarding race, the average ECD of African American donors was higher than those of white or Hispanic donors. A history of diabetes mellitus (DM) and ocular surgery were associated with a lower ECD. Donor medical history of hypertension, glaucoma, depression, dementia, Parkinson disease, hyper- or hypothyroidism, or smoking did not seem to affect the ECD. The risk factors for donor cell count inadequacy, based on binary logistic regression analyses were advanced age [65-74 years yielded an odds ratio of 17.8; confidence interval (CI), 10.6-29.8; P < 0.001; and 75-99 years yielded an odds ratio of 24.6 (CI, 14.5-41.61; P < 0.001) when compared with 20-34 years], cataract surgery (odds ratio, 4.3; CI, 4.0-4.8; P < 0.001), and DM (odds ratio, 1.2; CI, 1.1-1.3; P = 0.001). CONCLUSIONS: Age, race, ocular surgery (cataract and refractive), and DM seem to significantly affect donor corneal ECD. Of these variables, age, a history of cataract surgery, and DM were found to be the greatest risk factors for inadequate donor cell density (less than 2000/mm).

Keratoplasty in the United States: A 10-Year Review from 2005 through 2014.

PURPOSE: To report evolving indications and preferred techniques of corneal transplantation in the United States. DESIGN: Retrospective review. METHODS: Annual reports from the Eye Bank Association of America on corneal graft distribution in the United States from 2005 through 2014 were reviewed. MAIN OUTCOME MEASURES: Number and percentage of corneal grafts distributed for various types of keratoplasty and their surgical indications in the United States. RESULTS: The total number of corneal transplants increased from 44 277 in 2005 to 46 513 in 2014. In the past decade, penetrating keratoplasty dramatically decreased (from 95% to 42%) and largely has been replaced by various lamellar keratoplasty (LK) techniques (from 5% to 58%). Descemet stripping (automated) endothelial keratoplasty was the most common (50%) type of corneal transplantation performed in the United Stated in 2014. The volume of Descemet membrane endothelial keratoplasty (DMEK) has been doubling every year since 2011 and accounted for 11% of total endothelial keratoplasties in 2014. There was a significant shift in indication for corneal transplantation, with Fuchs' endothelial dystrophy (22%) being the most common, followed by corneal edema occurring after cataract surgery (12%) in 2014. Eye banks supplied precut corneal grafts for 68% of LK techniques in 2014. CONCLUSIONS: In the United States, there has been a major shift in preferred keratoplasty techniques over the past decade, with a wide adoption of new LK techniques.

Protocol for vital dye staining of corneal endothelial cells.

PURPOSE: To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. METHODS: Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. RESULTS: For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. CONCLUSIONS: We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss.

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.

Microkeratome versus femtosecond laser predissection of corneal grafts for anterior and posterior lamellar keratoplasty.

PURPOSE: To compare 2 different techniques for predissection of human anterior and posterior lamellar corneal grafts for eye bank storage. METHODS: A mechanical microkeratome (group 1, N = 5) and a femtosecond laser (group 2, N = 5) were used to dissect intended 350-microm-deep lamellar planes in deepithelialized donor corneas mounted on an artificial anterior chamber. These corneas were replaced in Optisol GS at 4 degrees C postoperatively and examined 2 days later to simulate a clinical scenario. Ultrasonic pachymetry of corneal lamellar sections was measured before and after separation of the lamellar grafts. Group 1 sections were separated by the mechanical microkeratome, whereas group 2 sections were manually separated 2 days after laser dissection. Endothelial cell viability was evaluated in posterior grafts. RESULTS: Total corneal thicknesses immediately before dissection were 559 +/- 61 (group 1) and 578 +/- 79 microm (group 2; P = 0.46). Immediate postdissection anterior and posterior graft thicknesses were 361 +/- 68 and 203 +/- 74 microm (group 1), respectively. Achieved anterior and posterior graft thicknesses 2 days later were 282 +/- 44 and 413 +/- 35 microm (group 1) and 324 +/- 112 and 397 +/- 51 microm (group 2), respectively. Percentage of devitalized endothelial cells were 3.4% +/- 1.6% (group 1) and 1.6% +/- 1.2% (group 2; P = 0.35). CONCLUSIONS: Centralized predissection by both techniques, cold storage, and shipping by airmail results in viable grafts without significant endothelial cell loss 2 days later.

Microkeratome preparation of lamellar corneal grafts.

PURPOSE: To evaluate the thickness and viability of microkeratome-prepared lamellar corneal grafts in cold storage. METHODS: Ten human corneas were sectioned with a mechanical microkeratome with a 350-microm depth head and stored in Optisol GS at 4 degrees C for 2 days to simulate an eye bank scenario. Central corneal thickness before and after mechanical microkeratome sectioning was measured by ultrasonic pachymetry. Endothelial cell viability was evaluated by trypan blue and alizarin red staining. RESULTS: Total corneal thickness immediately before microkeratome dissection was 562 +/- 51 microm. Anterior and posterior graft thicknesses were 296 +/- 111 microm and 270 +/- 74 microm, respectively, immediately after dissection, and 282 +/- 38 microm and 429 +/- 31 microm, respectively, 2 days after storage. There was significant swelling in the posterior (P=0.005) but not the anterior grafts (P=0.386). The percentage of devitalized endothelial cells was 3.0% +/- 1.2%. CONCLUSIONS: Corneal lamellar grafts may possibly be precut in a centralized facility and stored cold before further distribution.