Development of an easy non-destructive particle isolation protocol for quality control of red blood cell concentrates

The extracellular vesicle release in red blood cell concentrates reflects progressive accumulation of storage lesions and could represent a new measure to be implemented routinely in blood centres in addition to haemolysis. Nevertheless, there is currently no standardized isolation protocol. In a previous publication, we developed a reproducible ultracentrifugation-based protocol (20,000 × g protocol) that allows to classify red blood cell concentrates into three cohorts according to their vesiculation level. Since this protocol was not adapted to meet routine requirements, the goal of this study was to develop an easier method based on low-speed centrifugation (2,000 × g protocol) and limited red blood cell concentrate volumes to match with a non-destructive sampling from the quality control sampling tubing. Despite the presence of contaminants, mainly in the form of albumin and lipoproteins, the material isolated with the 2,000 × g protocol contained red blood cell-derived vesicular structures. It was reproducible, could predict the number of extracellular vesicles obtained with the 20,000 × g protocol and better discriminated between the three vesiculation cohorts than haemolysis at the legal expiry date of 6 weeks. However, by decreasing red blood cell concentrate volumes to fit with the volume in the quality control tubing, particle yield was highly reduced. Therefore, centrifugation time and relative centrifugal force were adapted (1,000 × g protocol), allowing for the recovery of a similar particle number and composition between small and large volumes sampled from the main unit, in different vesiculation cohorts over time. A similar observation was made with the 1,000 × g protocol between small volumes sampled from the quality control tubing and the mother-bag. In conclusion, our study paves the way for the use of the 2,000 × g protocol (adapted to a 1,000 × g protocol with the quality control sampling tubing) for particle measurement in blood centres.