Figure 1. ACD changes in DMEK grafts before and after loading and ejecting. Fluorescence images of CAM-stained DMEK grafts “before” (A) and “after” (B) loading an ejection from glass cannulas. A, “before” images were obtained after partial peel and trephination of the graft on the corneoscleral disk. B, “after” images were obtained after loading, ejection and flattening of the graft in viscoelastic. Hypo-fluorescent areas indicate endothelial cell damage. Arrows indicate the location of artifacts introduced by forceps gripping, which was subtracted out in the final ACD analysis.
Figure 2. Change in ACD from loading and ejection of DMEK grafts through glass cannulas. A, absolute ACD values of the DMEK grafts “before” and “after” loading and ejection from standard Straiko (n=6, P=0.0003, paired t test). B, Absolute ACD values “before” and “after” loading and ejection from LWVI micro cannulas (n = 6, p=0.0007, paired test). C, Comparison of mean increase in ACD from loading and ejection between Strike and LWVI cannulas (p=0.42, student t test).
Table 1: Endo-out vs Endo-In tissue processing-related cell damage
| Processing (Inserter) | Added %ACD | 2 Sample T-Test |
| Endo-in (EndoGlide) | 8.6 +/- 2.9% | N = 6 |
| Endo-out (Straiko) | 4.9 +/- 1.3%* | p=0.055, n=6 |
| Endo-out (LWM) | 4.2 +/- 1.4%* | p=0.032, n=6 |
| Endo-out (Combined) | 4.6 +/- 1.3%` | p=0.044, n=12 |
*Endo-out injector group means are from previously published data from Lee et al., using the same methods
`Endo-out (combined) compiled from both sets of Endo-in injector data.
AlamarBlue assay
Research Scientist Sung Lee was awarded an Eye Bank Association of America Pilot Research Grant for his study, “Evaluation of alamarBlue as an ‘in-situ’ Endothelial Cell Viability Assay in Preloaded DMEK grafts.”
In addition to the current microscopic approach, Lions Gift of Sight research staff have developed a new way to evaluate transplant DMEK grafts. Using a non-toxic, highly sensitive cell viability dye, alamarBlue (Fig 1) and a perfusion system, we are able to measure the total biochemical output of viable endothelial cells of the entire DMEK grafts while they are kept inside the DMEK storage/injector device (Fig 2 video).
Utilizing this method in conjunction with existing methods of endothelial cell analysis (spectral microscopy and slit-lamp evaluation) can offer a further comprehensive assessment of transplant corneal grafts. The video visualizes the alamarBlue signal, representing the biochemical readout from viable endothelial cells, flushed out of the modified Jones tube.
Direct In-Situ Viability Assessment of DMEK Endothelial Cells in a Modified Jones Tube
Fig 2. A DMEK graft inside a modified Jones tube was perfused to collect the fluorescent alamarBlue assay product under a blue light illumination.
Fig. 3. Endothelial cell viability in DMEK grafts decreases during storage. DMEK grafts were perfused with an alamarBlue solution after being loaded into glass cannulas, both before and after storage at 4oC for 48 hrs. Using the alamarBlue perfusion assay developed by the LGS R&D team, we observed that grafts stored in capped glass injectors (right, "w/ cap decrease") displayed a larger decrease in alamarBlue fluorescence (~17%) compared to the 6% decrease in uncapped DMEK injectors (left, "No cap decrease"). (Silicon end cap indicated by the arrow in the mock setup shown)
