Designing and Validating a New Method the TUNEL Microwave (TUNEL-MW) for Rapid Quantification of Apoptosis in Islets Following Isolation and Post-Thaw

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Author Info

Priya M Miranda Viswanathan Mohan Sekhar Ganthimathy Meera Govindarajan Ranjit M Anjana S Gunasekaran Venkatachalam Thiagarajan Michael Alexander Jonathan Lakey

Corresponding Author

Jonathan Lakey
University of California, Irvine, California, USA

A B S T R A C T

Background: Among the current quality control assays used in islet transplantation, there is an urgent need for more appropriate assays that measure cell damage via apoptosis that are accurate and rapid. Although the Terminal Uridine Nucleotide End Labeling (TUNEL) is a popular marker for apoptosis, the protocol takes 4 hours to complete. In this regard, microwave assisted histoprocessing, which shortens the time taken for processing, holds promise. Keeping this in mind, a new TUNEL Microwave (TUNEL-MW) method, for rapid quantification of apoptosis, was designed, developed and validated. Method: Two lots of post-thaw isolated human islets cultured for 24 hours, 3 days, 5 days and 7 days i.e. 8 samples, were used for the study. Dewaxed and rehydrated tissues were processed for routine histology, stained with haematoxylin and eosin (H&E) and the conventional TUNEL was carried out as per manufacturer’s instructions. For the TUNEL-MW, kit instructions were modified and microwave-assisted histoprocessing was done. The assessment of apoptotic index (AI%) by light microscopy (LM) was carried out by a pathologist who was completely blinded to the study. Results: The new TUNEL-Microwave (TUNEL-MW) developed by us reduced processing time from 4 hours to 30 minutes (saving 3½ hours). Results were validated by univariate linear regression (r2>0.990), coefficient of variation (<5% between all three methods) and the Bland Altman plot comparing AI% determined by the new TUNEL-MW with the conventional TUNEL and with LM (gold standard). Conclusion: TUNEL Microwave appears to be an ideal method. It is simple and takes just 30 minutes to perform and can therefore be used along with existing quality control measures to rule out or measure apoptosis prior to islet release for islet transplantation.

Article Info

Article Type

Research Article

Publication history

Received: Mon 06, Jul 2020
Accepted: Mon 20, Jul 2020
Published: Fri 31, Jul 2020

Copyright

© 2023 Jonathan Lakey. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Hosting by Science Repository.
DOI: 10.31487/j.RGM.2020.02.05