Fig 1.
Representative electron micrographs of pelleted material from supernatant of RBC storage units.
A, B, Negative-stain EM images of the unfixed material, showing pleomorphic structures. C,D, Thin-section TEM images, showing membrane encapsulate vesicles. E,F, SEM images. G,H, Negative-stain EM of fixed (G) versus unfixed (H) structures. Vesicles were isolated and imaged as described in Methods section.
Fig 2.
Representative 3D cryo-electron tomograpahy data and visual characterization of RBC-derived microparticles (RMPs).
A, Slices through 3D volumes of RMPs. B, RMPs were measured and were characterized as either round or pleiomorphic, and as either full or empty. The number of each particle type, percent of the total, mean diameter, and standard deviation are presented in the table.
Fig 3.
Analysis of bacterial DNA in pelleted material from RBC storage units and serum.
RMPs were isolated from ~100 mL of RBCs stored under standard conditions for 42 days. A, Genomic DNA was isolated and analyzed by gel electrophoresis. Molecular grade water and DNA from S. Pneumoniae served as negative and positive controls, respectively. Band reflective of genomic DNA was only observed in positive control lane. B, Isolated DNA was also subjected to 16S rDNA PCR analysis, using universal primers. PCR product was observed for all samples assessed. C, Serum from 3 healthy donors was incubated at 30°C for 5 and 10 days, and then analyzed using a 16S rDNA qPCR assay. No significant difference in 16S rDNA was observed between the different time points.
Fig 4.
Vesicles isolated from supernatant of RBC storage units are membrane-bound, intact, and contain RBC surface antigen and RBC-specific miRNA.
A, Unstained vesicles (red, bottom left) or vesicles co-stained with calcein-AM and fluorescent anti-GPA (blue, top right) were analyzed by flow cytometry. >99% of the vesicles were positive for calcein-AM and anti-GPA. B, RNA from vesicles was analyzed by Agilent Bioanalyzer. Peak on electropherogram at 25 nt is internal standard and small peak to the right reflects small RNA. This electropherogram is representative of Bioanlyzer data from six different RBC storage units. C, Levels (Ct values) of miR-451 were assessed by qRT-PCR in stored RBCs, the RMP pellet, and in human aortic endothelial cells (HAECs, negative control). D, Hemoglobin-alpha content of RMP pellet and stored RBCs, as assessed by Western blot. Blot is representative of six different RBC storage units.