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Background:  Studies have correlated nucleoside reverse transcriptase inhibitor (NRTI)-related decreases in mitochondrial DNA (mtDNA) with mitochondrial toxicity, resulting in several HIV, hepatitis, and oncology therapy-related complications. The Retina Mitox DNA assay, a real time duplex NASBA method, quantitates the levels of mtDNA in peripheral blood mononuclear cell (PBMC) samples. Critical to the interpretation of results is the standardization and the selection of optimal test conditions. This study evaluated the performance of the assay including reproducibility of nucleic acid extraction, intra-run, inter-run, tech to tech and instrument variation, effects of specimen processing, storage and stability of mtDNA levels in PBMC over time.

Methods:  Whole bloods from volunteers (n = 8) was collected in multiple (n = 3) heparin CPT tubes over several days (n = 4). PBMC were isolated at time 0, 24 and 48 hours after storage at room temperature in unspun CPT tubes, counted, and viability determined. PBMC were either frozen, then thawed and 50,000 cells added to multiple lysis buffer tubes (LBT) (n = 10), or PBMC were added directly to LBT at the time of isolation. Nucleic acids were extracted (Boom method) and multiple same sample aliquots were tested in duplicate over multiple runs, over several days, by 3 technicians and using 2 analyzers. Comparable results should demonstrate an inter-run coefficient of variation of < 35%.

Results:  The assay results were highly reproducible with coefficient of variation as follows:  isolation replicates 9.06%; intra-assay 7.52%; inter-assay and tech to tech 8.73%; different analyzers 8.38%. Reproducibility was comparable over the range of mtDNA copy numbers. PBMC processed at 0 and 24 hours were comparable for cell viability (90 to 95%) and mtDNA levels when PBMC were added to LBT directly after isolation. Two PBMC samples frozen first, prior to addition to LBT, had significantly different values (coefficient of variation 44%, 55%). Samples processed at 48 hours yielded PBMC with a cell viability of approx 60% and insufficient quantity for nucleic acid extraction. PBMC stored in LBT at –70^°C gave comparable results when tested over time.  

Conclusions:  To ensure accuracy and reproducibility of mtDNA results, PBMC should be processed and lysed within approximately 24 hours of collection and stored at –70^°C until tested. The Retina Mitox DNA assay is highly reproducible which is critical for assessing the significance of changes in mtDNA levels. The Retina Mitox DNA assay should provide an effective tool for predicting and monitoring mitochondrial toxicities in patients receiving NRTI or other replication inhibiting drugs.

Keywords: mtDNA; toxicity; NASBA