Effects of storage and leukocyte reduction on the concentration and procoagulant activity of extracellular vesicles in canine packed red cells

Abstract

Objectives To characterize the size and procoagulant activity of extracellular vesicles (EV) that accumulate in canine packed red blood cells (pRBCs) over time and the effect of leukocyte reduction on these characteristics. Design Prospective cohort study. Setting Private small animal specialty referral hospital and university research laboratories. Animals Ten healthy blood donor dogs. Interventions Five pRBCs units were obtained according to standard protocols, and 5 were leukocyte‐reduced prior to processing. Platelet‐free supernatant from the pRBC units was collected on days 0, 10, 20, 32, and 42. Measurements and main results Nanoparticle tracking analysis was performed to determine the size and concentration of EVs. Thrombin generation associated with phosphatidylserine‐positive EVs was determined using a capture assay. Factor Xa generation associated with phosphatidylserine‐positive EVs and tissue factor‐positive EVs was measured in a subset of EVs isolated by centrifugation of the supernatant at 20,000 × g. R package nparLD and the Mann–Whitney U‐test were used to determine the effect of duration of storage and the effect of leukocyte reduction, respectively. Small (mean < 125 nm) procoagulant EVs accumulated over time, with significant increases occurring on or after day 20 in both non‐leukocyte reduced and leukocyte‐reduced units. The procoagulant activity of the EVs was due to phosphatidylserine, not tissue factor. Increases in EV concentration and procoagulant activity occurred earlier in non‐leukocyte reduced units. Extracellular vesicle accumulation and procoagulant activity were not decreased at any individual time point by leukocyte reduction. Conclusions Further studies characterizing and determining the clinical relevance of small procoagulant EVs in pRBCs are warranted.

Effect of Leukoreduction by Pre-Storage Filtration on Coagulation Activity of Canine Plasma Collected for Transfusion

“…Owing to the relatively small sample size, the probability of a type II error is increased, and there might be some significant effects of filtration that we could not detect. However, the number of units analyzed in our study reflects what has previously been reported in veterinary medicine, in which published studies have analyzed numbers of plasma units ranging between 8 and 15 units [18][19][20][21][22][23][24][26][27][28][29][30]. This limited number of units analyzed in veterinary medicine is due to ethical and practical constraints when dealing with a precious resource such as blood and blood components.…”

“…In recent decades, leukoreduction has become more common in veterinary transfusion medicine and more studies are being published [ 20 , 21 , 22 , 24 , 26 , 27 ]. Successful pre-storage leukoreduction of canine blood has also been reported [ 18 , 22 ].…”

“…While leukoreduction of blood products is extensively used in human medicine, as demonstrated by the numerous papers published on leukoreduction [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ], it is still rarely used or reported in veterinary medicine, where leukoreduction is usually achieved by filtration of whole blood (WB) before blood components are separated [ 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. Based on evidence in human studies that leukoreduction decreases the rate of febrile non-hemolytic transfusion reactions, a recent Transfusion Reaction Small Animal Consensus Statement suggested that leukoreduction should also be considered in veterinary medicine [ 25 ], even though there is currently insufficient evidence to show that the use of leukoreduction prevents or reduces any type of transfusion reaction in veterinary medicine.…”

“…Most published veterinary reports have studied the efficacy of blood filters for reducing the leukocyte count in final blood components [ 22 , 24 , 26 ], and their effect on RBC viability and on reduction of red cell storage lesions [ 19 , 20 , 21 , 22 , 23 , 24 , 27 , 28 , 29 , 30 ]. However, little is known about the effect of leukoreduction on the hemostatic activity of plasma obtained for transfusion purposes and, to the author’s knowledge, only two veterinary studies have evaluated this using human and canine blood collection systems [ 24 , 26 ].…”

Red Blood Cell-Derived Extracellular Vesicles: An Overview of Current Research Progress, Challenges, and Opportunities

“…However, further research is needed to fully elucidate the underlying mechanisms. Using healthy blood donor dogs, Avenick et al reported that the procoagulant activity of the RBC EVs was due to their exposure to PS [48,49]. Kim et al found that RBC EVs only induce a transient (few hours) hypercoagulable state in C57BL/6 mice [50].…”