Introduction:
Packed red blood cells (PRBCs) are transfused to treat anaemia and improve the oxygen carrying capacity of blood. They have a shelf-life of 42 days during which a storage lesion develops, including an increase in extracellular vesicle (EV) concentration. Methods to deplete EVs from PRBC supernatant would be helpful to discern the potential effects of these EVs relative to other factors related to the storage lesion.
Aim:
To develop an EV-depletion protocol for PRBC supernatants.
Methods:
PRBCs (n=3) were sampled at day 42, processed (dual centrifugation at 3,000 g for 15 minutes), and supernatants stored (-80°C). Thawed supernatants were filtered sequentially through 800nm, 450nm and 220nm filters. This initially filtered supernatant (S1) was then filtered through either a 100nm, 50nm or 20nm filter. Alternatively, S1 was either filtered sequentially though the same set of filters, or through the same size filter up to 5 times. Filtered samples underwent nanoparticle tracking analysis (NTA; Nanosight NS300).
Results:
Filtration of S1 supernatant through any single filter reduced mean/mode EV size but not EV levels. Sequential filtration reduced mean/mode EV size and EV levels. Multiple pass throughs of each filter size improved EV depletion; however, it remained incomplete. Notably, for all filter sizes there was incomplete depletion of EVs of the size associated with that filter, even with sequential filtrations or with multiple pass throughs of a single filter size.
Conclusion:
The filters assessed here only partially depleted EVs from PRBC supernatant. Further experiments using alternative depletion methods prior to filtration are on-going.