INTERNATIONAL JOURNAL OF REGENERATIVE MEDICINE | ISSN 2613-5914 Available online at www.sciencerepository.org Science Repository * Correspondence to: Jonathan RT Lakey, Professor, Department of Surgery and Biomedical Engineering, University of California, Irvine, California, USA; E-mail: jlakey@hs.uci.edu © 2020 Jonathan RT 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. All rights reserved. http://dx.doi.org/10.31487/j.RGM.2020.02.05 Research Article Designing and Validating a New Method the TUNEL Microwave (TUNEL-MW) for Rapid Quantification of Apoptosis in Islets Following Isolation and Post-Thaw Priya M Miranda 1,2 , Viswanathan Mohan 2 , Sekhar Ganthimathy 3 , Meera Govindarajan 4 , Ranjit M Anjana 2 , S Gunasekaran 5 , Venkatachalam Thiagarajan 3 , Michael Alexander 1 and Jonathan RT Lakey 1* 1 University of California, Irvine, California, USA 2 Madras Diabetes Research Foundation & Dr. Mohan’s Diabetes Specialties Centre, ICMR Centre for Advanced Research on Diabetes, WHO Collaborating Centre for Noncommunicable Diseases-Prevention and Control, Chennai, Tamil Nadu, India 3 Professor Saveetha Medical College, Thandalam, Chennai, India 4 R & D Histopathology Laboratory, Chennai, India 5 Christian Medical College, Vellore, Tamil Nadu, India A R T I C L E I N F O Article history: Received: 6 July, 2020 Accepted: 20 July, 2020 Published: 31 July, 2020 Keywords: Islet transplantation cryopreservation post-thaw culture islet isolation TUNEL 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 (r 2 >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. © 2020 Jonathan RT Lakey. Hosting by Science Repository. All rights reserved. Introduction The Terminal Uridine Nucleotide End Labeling (TUNEL) assay is a popular method for the detection of apoptosis [1-18]. Apoptosis first described by Kerr et al. is the endpoint of an energy-dependent cascade of molecular events initiated by numerous stimuli [19-24]. It is a vital process and is a natural form of programmed cell death designed to eliminate unwanted cells. It occurs during development, during situations where tissue homeostasis must be restored, as a defense mechanism and following cell injury [19-24]. The relevance of this phenomenon to islet transplantation is that both cold and warm