Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status

BACKGROUND Acute ischemic stroke(AIS)is one of the major causes of the continuous increasing rate of global mortality due to the lack of timely diagnosis,prognosis,and management.This study provides a primitive platform for non-invasive and cost-effective diagnosis and prognosis of patients with AIS using circulating cellfree mitochondrial DNA(cf-mtDNA)quantification and validation.AIM To evaluate the role of cf-mtDNA as s non-invasive,and affordable tool for realtime monitoring and prognosticating AIS patients at disease onset and during treatment.METHODS This study enrolled 88 participants including 44 patients with AIS and 44 healthy controls with almost similar mean age group at stroke onset,and at 24 h and 72 h of treatment.Peripheral blood samples were collected from each study participant and plasma was separated using centrifugation.The cf-mtDNA concentration was quantified using nanodrop reading and validated through real-time quantitative polymerase chain reaction(RT-qPCR)of NADH-ubiquinone oxidoreductase chain 1(ND1)relative transcript expression levels.RESULTS Comparative analysis of cf-mtDNA concentration in patients at disease onset showed significantly increased levels compared to control individuals for both nanodrop reading,as well as ND1 relative expression levels(P<0.0001).Intergroup analysis of cf-mtDNA concentration using nanodrop showed significantly reduced levels in patients at 72 h of treatment compared to onset(P<0.01).However,RT-qPCR analysis showed a significant reduction at 24 h and 72 h of treatment compared to the disease onset(P<0.001).The sensitivity and specificity were relatively higher for RT-qPCR than nanodrop-based cfmtDNA quantification.Correlation analysis of both cf-mtDNA concentration as well as ND1 relative expression with National Institute of Health Stroke Scale score at baseline showed a positive trend.CONCLUSION In summary,quantitative estimation of highly pure cf-mtDNA provides a simple,highly sensitive and specific,non-invasive,and affordable approach for real-time monitoring and prognosticating AIS patients at onset and during treatment.

Role of cell-free DNA for predicting incidence and outcome of patients with ischemic stroke

Early diagnosis and prognosis of ischemic stroke remains a critical challenge in clinical settings.A blood biomarker can be a promising quantitative tool to represent the clinical manifestations in ischemic stroke.Cell-free DNA(cfDNA)has recently turned out to be a popular circulating biomarker due to its potential relevance for diagnostic applications in a variety of disorders.Despite bright outlook of cfDNA in clinical applications,very less is known about its origin,composition,or function.Several recent studies have identified cell-derived mitochondrial components including mitochondrial DNA(mtDNA)in the extracellular spaces including blood and cerebrospinal fluid.However,the time course of alterations in plasma mtDNA concentrations in patients after an ischemic stroke is poorly understood.DNA is thought to be freed into the plasma shortly after the commencement of an ischemic stroke and then gradually decreased.However,the importance of cell-free mtDNA(cf-mtDNA)in ischemic stroke is still unknown.This review summarizes about the utility of biomarkers which has been standardized in clinical settings and role of cfDNA including cfmtDNA as a non-invasive potential biomarker of ischemic stroke.