Abstract
Cell Biology/Immunology and Biochemistry (Basic and Preclinical Research) - Red Cells
Philip Norris
VP Research
Vitalant Research Institute
Mill Valley, California
Disclosure information not submitted.
Methods: From a previous study of hemolysis in 13,403 blood donors, a second blood unit was drawn from 651 donors 2-12 months after the first, was stored at 4°C, and was sampled at days 10, 21, and 42. mtDNA levels were determined by qPCR using primers that target a region in mitochondrial cytochrome oxidase III subunit c. To determine the proportion of mtDNA that was protected from degradation (likely in extracellular vesicles [EVs]), supernatant samples were treated with Triton, DNase I, or Triton plus DNase I. DNase alone should only degrade mtDNA not protected by EVs, while triton plus DNase should degrade free and EV-protected mtDNA.
Background: Mitochondrial DNA (mtDNA) shares characteristics with bacterial DNA and activates immune cells via Toll like receptor (TLR)-9. Furthermore, RBCs have been shown to express TLR9 that can associate with mtDNA that can activate immune cells. Blood product processing method has been shown to influence RBC unit mtDNA levels. We characterized the evolution of mtDNA in stored blood products.
Study
Design/Methods:
Results/Findings:
Results: Levels of mtDNA showed a 3-log variation among donors, and while levels rose modestly through storage time, the changes over time were smaller than the variation from donor to donor. mtDNA levels were higher in RBC units processed in AS-3 compared to AS-1 and units from females of post-menopausal age. mtDNA levels were highly correlated within donors for 98 donations obtained 2-12 months apart (r=0.74, p< 0.0001). This led us to explore a genetic underpinning for the stability from donation to donation. Quantitative trait locus analysis revealed several genetic associations, most notably linking mtDNA levels with polymorphisms in ANKLE1, which encodes an erythroid-specific protein that preferentially cleaves mtDNA. The lead single nucleotide polymorphism (SNP), rs61494113 encodes for a G to A variant, with the minor allele A present in approximately 30% of the population and 28% in the current study. The rs61494113 SNP was significantly associated with mtDNA level at each of the RBC storage time points studied (p=8.4x10-22 at day 42, Figure A). There was a dose-dependent decrease in mtDNA levels with each copy of the A allele at rs61494113, with mtDNA levels correlating as follows: GG >GA >AA. The mean signal for mtDNA fell by 1.7 Cq or 3.2-fold with addition of DNase I (p=0.36) and by 6.0 Cq, or 64-fold with addition of Triton plus DNase I (p< 0.001), consistent with 85% of mtDNA being protected from DNase I.
Conclusions:
Conclusion: These data suggest that donor-intrinsic factors may influence mtDNA levels found in RBC units. This finding lends impetus to determining if genetic or environmental factors control levels of these immune mediators in blood donors, and whether mtDNA levels in donors correlate with clinical outcomes in transfusion recipients.