P02-A121†Precut DSAEK stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: to measure endothelial viability of precut DSAEK after 3 or 10 days of storage in our ASM in a preclinical study. METHODS: Human pairs of corneas were included. The endothelial cell density (ECD in cells/mm2), and central corneal thickness (CCT in µm) were measured to ensure their initial intra pair comparability. After deswelling (CorneaJet, Eurobio) grafts preparation was performed by cutting the anterior stroma with a Moria linear microkeratome and keeping the anterior lamellae attached during storage. After randomization, one cornea was kept in the corneajet bottle (CJ) and the other was inserted into the ASM allowing a renewal or storage medium (CorneaMax, Eurobio) at 2.6 µL/min with 21 mmHg of pressure in the endothelial chamber. Both group of corneas were stored for 3 or 10 days at 31°C. The final viable ECD (vECD) was determined using the triple staining with Hoechst-Ethidium-Calcein-AM by an independent experimenter in a masked fashion. RESULTS: Initial ECDs were comparable: 2595±878 in ASM versus 2654±954 cells/mm2 in CJ for the 3-days period (n=5 pairs) and 2416±712 in ASM versus 2492±764 cells/mm2 in CJ for the 10-period (n=5 pairs). CCTs were also comparable. The anterior lamellae stayed attached in either the ASM or CJ. vECD was significantly higher in ASM than in CJ with respectively 2062±695 cells/mm2 versus 1632±633 cells/mm2 after 3 days either a cell loss of 20.5% and 38.5% respectively (p=0.0062) and 1082±649 versus 935±691 cells/mm2 for the 10-day period either a cell loss of 132% and 164% respectively (p=0.005). Grafts thickness did not differ after 3 days 219±25 µm in ASM versus 182±39 µm (p=0.063) or 10 days respectively 221±58 µm versus 189±48 µm (p=0.06). CONCLUSION: The storage of precut DSAEKs into the ASM allows a better preservation of grafts without use on deswelling storage medium. Nevertheless, the cell loss remains high after 10 days, suggesting a significant cell stress.

P01-A120†Pre-dissected dmek stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: To measure the endothelial viability of pre-dissected DMEK after 3 and 10 days of storage in our ASM in a preclinical study. METHODS: Pairs of human corneas were included. The endothelial cell density (ECD in cells/mm2), thickness and transparency of corneas were measured before graft preparation. Descemet's membrane (DM) was peeled using the no-touch technique leaving the graft attached to the center of the cornea (on approx. 1mm2). After randomization, one cornea was kept in organ culture (OC) and the other in the ASM (21 mmHg, 2.6 µL/min) in the same medium (CorneaMax, Eurobio). The final viable ECD was determined using the triple staining with Hoechst-Ethidium-Calcein-AM. In addition, the expression of CD166 and NCAM (lateral membranes), ZO-1 (apical junctions), Na+/K+ ATPase (endothelial pump function) and COX-IV (mitochondrial content) was studied by immunostaining to characterize endothelial cells after the storage. RESULTS: Initial ECDs were comparable: 2185±232 cells/mm2 in the ASM versus 2276±328 in OC for the 3-day period and 2680±416 cells/mm2 in the ASM versus 2644±420 in OC for the 10-day period. The DMs did not fold back in either BR or OC. The viable ECD did not differ significantly between the ASM and OC for either storage period: 2378±501 (ASM) versus 2342±503 (OC) for the 3-day period (n=8 pairs and p=0.624) and 2482±288 (ASM) versus 2579±315 (OC) for the 10-day period (n=5 pairs and p=0.176). Corneas were more transparent and thinner in the ASM than in OC after 3 days (916±86 versus 1193±136µm, p=0.0001) and 10 days (957±128 versus 1220±105µm, p=0.0625). The functional and structural markers studied were expressed in both groups after 3 and 10 days, some better preserved in the ASM. CONCLUSION: The storage of precut DMEKs is possible in ASM and OC for at least 10 days. Interestingly, a pre-dissected endothelium continues to partially exert its pump function into the ASM. In practice, this could allow the stroma to be used for DALK without further deswelling. In addition to improving the storage of whole grafts, the ASM allows the storage of precut DMEKs for up to 10 days with excellent endothelial survival.

P38-A108†Optimization of the triple HEC staining for laboratory assessment of DMEK graft quality.

INTRODUCTION: The quality of the endothelial graft is critical to the success of DMEK and to the survival time of the graft. The peeling technique, preservation method, and skill level of graft preparers need to be evaluated and validated. The most reliable method of evaluation is the viability test based on a triple staining of Hoechst- Ethidium-Calcein AM (H-E-C) which allows the determination of the total number of viable cells on the graft. However, this test has some shortcomings for DMEK grafts: 1) The undesirable fluorescence of the Calcein AM stain prevents accurate viability analysis, especially in cases where the graft is attached to the cornea for preservation; 2) Incompatibility with immunofluorescence (IF) that could provide additional information. The objective of this study is to develop technical tricks to overcome these drawbacks. METHODS: Two strategies were employed to improve Calcein AM staining: 1. Increase the specific fluorescence intensity by changing the diluent and the concentration of Calcein AM; 2. Decrease undesired fluorescence from keratocytes by adding Trypan Blue (BT). In order to combine the IF after the HEC test, an extension wash in PBS was performed. RESULTS: Calcein AM at 4µM diluted in OptiMEM increased fluorescence intensity 3-fold (p=0.0017, n=5) compared with conventional staining at 2µM in PBS. BT decreased the undesired fluorescence of Calcein and thus optimized count variability between different operators by 42% (p=0.0027, n=10) and saved 40% (p=0.0002, n=10) of count time. To perform IF after HEC, prolonged washing in PBS is an effective method to remove residual Calcein fluorescence and allows release of the FITC/Alexa 488 filter. CONCLUSION: This study provides effective technical tips for optimizing the endothelial viability assay using Calcein AM and for performing IF after the viability assay.

Pauci-symptomatic foodborne botulism due to Clostridium botulinum type B with predominant ophthalmologic presentation possibly after consumption of honey.

Foodborne botulism, a toxin-mediated illness caused by Clostridium botulinum, is a public health emergency, and rarely reported in France. We report herein the case of two family members (a father and his son) from Franche-Comté, France, presented with ophthalmological symptoms which occurred after non-specific gastro-intestinal symptoms after a trip to Serbia with a recent consumption of artisanal honey, and suggestive of botulism. The suspected intoxication appeared to be caused by a type B strain of C. botulinum, as demonstrated by toxin-neutralization in the lethal mouse bioassay. Regarding the mild-to-moderate form, the patients were treated symptomatically with monitoring, against antitoxins, with no evidence of relapse afterwards. We want to highlight the importance of recognizing clinical ophthalmologic botulism symptoms as unreactive bilateral mydriasis with lack of accommodation to contribute to earlier diagnosis in case of pauci-symptomatic botulism.