Comparison of two techniques for evaluation of pancreatic islet viability: flow cytometry and FDA/PI staining

Abstract

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus is dependent, in part, on the number of isolated islets as well as their quality, which is assessed by functional and viability tests. The currently employed test to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release clinical islet product for transplantation, is the FDA/PI staining. However, the efficacy of this method is dependent on the researcher experience. In this context, a quantitative method may be useful. Therefore, the aim of this study was to compare islet viability assessed by flow cytometry and FDA/PI assay. Methods: Viability was evaluated in islets isolated from 10 male Wistar rats. Upon FDA and PI staining, 50 islets from each animal were analyzed under fluorescence microscope by two well-trained researchers. For flow cytometry, islets were dispersed, and then 100,000 single cells were incubated with 7AAD fluorophore (dyes necrotic and late apoptotic cells) and Annexin V-APC antibody (marker of early apoptotic cells). Results: A moderate correlation was found between the two techniques (r = 0.6; P= 0.047). The mean islet viability measured by flow cytometry was higher than the viability estimated using FDA/PI staining (95.5 ± 1.4% vs. 89.5 ± 5.0%; P= 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique, not dependent of the researcher experience. Thus, flow cytometry appears to be the technique of choice when aiming a more precise determination of islet viability.

Keywords

• islet transplantation
• islet isolation
• viability
• flow cytometry.