Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Transl Med. Aug 6, 2022; 10(2): 14-28
Published online Aug 6, 2022. doi: 10.5528/wjtm.v10.i2.14
Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status
Nusrath Fathima, Sandhya Manorenj, Sandeep Kumar Vishwakarma, Aleem Ahmed Khan
Nusrath Fathima, Sandeep Kumar Vishwakarma, Aleem Ahmed Khan, Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
Sandhya Manorenj, Department of Neurology, Princess Esra Hospital, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
Author contributions: Fathima N performed lab experiments, collected the data, performed the statistical analysis, and wrote and formatted the manuscript; Manorenj S provided samples and clinical inputs for the study; Khan AA and Vishwakarma SK provided inputs, designed the study, helped in data analysis and presentation, and wrote and edited the manuscript.
Institutional review board statement: All study procedures were carried out with the approval of the Institutional Review Board of Deccan College of Medical Sciences, Hyderabad, Telangana, India.
Institutional animal care and use committee statement: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals.
Conflict-of-interest statement: All authors declare that they have no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: All authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Aleem Ahmed Khan, PhD, Senior Scientist, Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, Telangana, India.
Received: April 2, 2022
Peer-review started: April 2, 2022
First decision: May 31, 2022
Revised: June 14, 2022
Accepted: July 16, 2022
Article in press: July 16, 2022
Published online: August 6, 2022
Research background

Role of circulating cell-free mitochondrial DNA (cf-mtDNA) in assessing disease status and treatment response of acute ischemic stroke (AIS) patients. Quantitative discrimination of AIS patients from the general population using cf-mtDNA. Compared sensitivity and specificity of nanodrop reading and real-time quantitative polymerase chain reaction (RT-qPCR) tools for quantifying cf-mtDNA.

Research motivation

AIS results in a continuously increasing rate of morbidity and mortality, and reduced quality of life worldwide. Cellular apoptosis and necrosis are major events during AIS. The amount of DNA present in circulation is directly proportional to the host cell’s death and response.

Research objectives

To validate the quantitative role of cf-mtDNA in discriminating AIS patients from the general population and identifying the treatment response while comparing the sensitivity and specificity of nanodrop reading and RT-qPCR tools.

Research methods

Nanodrop reading and RT-qPCR were used to quantify cf-mtDNA in circulation. The sensitivity and specificity of both the assays were measured using relative operator characteristic (ROC) curve analysis. Correlation analysis of cf-mtDNA was performed with NIHSS score.

Research results

The findings of our study revealed significantly higher values of cf-mtDNA concentration as well as differences in relative fold expression of ND1 gene in AIS patients at the disease onset compared to healthy control participants. ROC analysis showed the higher diagnostic significance of cf-mtDNA concentration estimated through nanodrop reading than RT-qPCR. Intergroup analysis of patients at onset and at treatment showed significantly reduced levels of both cf-mtDNA measured by nanodrop reading, and ND1 relative expression assays at 72 h of treatment. During the technical comparison, we observed that ND1 expression also provides a significant difference at 24 h, and at 72 h of treatment; however, nanodrop quantification of cf-mtDNA didn't reveal any such difference.

Research conclusions

Quantification of cf-mtDNA in circulation using nanodrop reading or RT-qPCR-based assays may provide a simple, highly sensitive and specific, non-invasive, and affordable approach for real-time monitoring and prognostication of AIS patients at stroke onset and during treatment.

Research perspectives

This approach may provide a widely acceptable and applicable platform at a relatively lower cost and time for different clinical conditions other than AIS with further exploration.