Imaging and Quantification of Endothelial Cell Loss in Eye Bank Prepared DMEK Grafts Using Trainable Segmentation Software

Abstract

Establishing an accurate, efficient and reproducible means of quantifying ECL in graft preparation and surgical techniques can provide insight to the safety, long-term potential of the graft tissues as well as provide a quality control measure for eye banks and surgeons. Trainable segmentation in Fiji software using CAM is a novel approach to measuring ECL that captured a statistically significantly higher percentage of ECL comparable to Adobe and was more accurate in standardized testing. Interestingly, ECL as determined using both methods in eye bank-prepared DMEK grafts exceeded 18% on average.

Risk Factors for Eye Bank Preparation Failure of Descemet Membrane Endothelial Keratoplasty Tissue

“…13,14 However, risk factors for failure in DMEK graft preparation and, consequently, for a higher endothelial cell loss or tissue wastage, have not been addressed in a large-volume, multitechnician eye bank setting and have been limited to surgeon preparation of donor tissue. In this latter context, difficulty in graft peeling for the first eye has been shown to be the most important factor for failure in the fellow eye.…”

Quantitative Analysis of Endothelial Cell Loss in Preloaded Descemet Membrane Endothelial Keratoplasty Grafts

“…Endothelial cell viability staining was performed using Calcein AM dye (CAM; Catalog number C3100MP, Molecular Probes, Eugene, OR) as previously described by Jardine et al 17 Briefly CAM was thawed, and 50 ul was added to 100 ul of dimethyl sulfoxide (DMSO; Molecular Probes, Eugene, OR). The solution was manually mixed, then pipetted into 20 ml of BSS (Alcon, Ft. Worth, TX, USA).…”

“…17 Briefly, the image contained 500 × 500 pixels (250,000). Ten squares of multiple sizes and differing shades of grey were placed within the image; 150039 pixels, 60.02% area.…”

Eye Bank–Prepared Femtosecond Laser–Assisted Automated Descemet Membrane Endothelial Grafts

“…Nonmasked analysis could introduce biases into our outcomes, although the segmentation algorithm in FIJI is partially automated. 20 An additional limitation is that analysis of central versus peripheral endothelial loss was not undertaken because total cell loss between graft techniques was a more straightforward comparison. Differentials in central versus peripheral cell loss would have multiple contributions, including trephination, laser energy, central bubbling, and striae induced by manual graft manipulation.…”

“…20 Briefly, 50 mg of calcein AM stain (Invitrogen, Carlsbad, CA) was dissolved in 100 mL of dimethyl sulfoxide (Fisher, Fairlawn, NJ) and further diluted into 20 mL of buffered saline solution (Alcon). Several drops were applied to the corneal trephined tissue on a glass slide and incubated at room temperature for 15 minutes to allow the stain to be metabolized.…”