Priya  M Miranda,Viswanathan  Mohan,Sekhar  Ganthimathy,Meera  Govindarajan,Ranjit  M Anjana,S  Gunasekaran,Venkatachalam  Thiagarajan,Michael Alexander,Jonathan Lakey,
Designing and Validating a New Method the TUNEL Microwave (TUNEL-MW) for Rapid Quantification of Apoptosis in Islets Following Isolation and Post-Thaw
International Journal of Regenerative Medicine
2020
2613-5914
http://dx.doi.org/10.31487/j.RGM.2020.02.05
https://www.sciencerepository.org/designing-and-validating-a-new-method-the-tunel-microwave_RGM-2020-2-105 
Abstract: 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.Keywords:  Islet transplantation, cryopreservation, post-thaw culture, islet isolation, TUNEL