Release of Microparticles During Blood Storage Is Influenced by Residual Platelets, Leukocytes and Oxygen Levels
Authors: Jy Wenche , Bidot Carlos J , Yoshida Tatsuro , Vassallo Ralph , Johansen Max E , Horstman Lawrence L , Morgan John , Gomez-Marin Orlando , Shariatmadar Sherry , Ahn Yeon S 
Published: October 2019

Abstract
Abstract 3435

Introduction
Packed red blood cells in blood bank undergo a series of changes and this so-called “storage lesions” increases with time. It is believed that longer storage is associated with adverse transfusion-related complications, but the reason for this is not clear. Many bioactive substances are generated during blood storage and one or more of them may be responsible for adverse events. Among them, red cell microparticles (RMP) are a leading candidate for adverse effects, but conditions influencing their release are not well identified. Elucidation of these conditions is an important step toward minimizing storage lesions.

Methods
(I) MP generation: Non-leukoreduced and leukoreduced PC of known blood types (A+, B+, AB+, O+) were obtained from the blood bank within 2–4 days of collection, then stored at 4°C. Time of receipt was defined as day 0. At days 0, 10, 20, 30, and 40, forty mL samples were centrifuged at 1000×g for 20 min to remove cells. The supernatants were then assayed for subtypes of MP by flow cytometry comprising (a) RMP assessed by CD235a, (b) leukocyte MP (LMP) by CD45, (c) platelet MP (PMP) by CD41, and (d) generic MP by Ulex Europaeus (Ulex) or annexin V (AnV); MP-mediated procoagulant and proinflammatory activities were determined by TEG and CD11b expression, respectively. (II) Storage under anaerobic conditions: Anaerobic test units were processed by an OCDD (Oxygen Carbon dioxide Depletion Device) to deplete O2 and CO2, then stored anaerobically in an Anaerobic Storage Bag (ASB) [Transfusion 2011:51S, SP89]. After 42 days, MP were assayed as above.

Results (a)
Time of storage: In non-leukoreduced RBC, multiple MP types (PMP, LMP, RMP) were detected and seen to increase with time, but at distinctive rates. RMP increased little until day 10 when they rose exponentially; PMP counts rose steadily from day 0 and peaked at day 20; LMP showed little change until day 20 when they began to increase, then rose sharply after day 30. Levels of PMP (days 0 to 20) and RMP (days 20 to 40) correlated with increasing MP-mediated procoagulant and inflammatory markers. (b) Leukoreduction: Pre-storage leukoreduction decreased RMP generation by 20–40% and completely suppressed PMP and LMP generation. Leukoreduction decreased total MP-mediated procoagulant and inflammatory markers by 40–60%. This suggests that residual leukocytes and/or platelets potentiate RMP generation. (c) Residual platelet concentrations: We added increasing numbers of platelets to leukoreduced RBC, and then evaluated RMP generation during storage. The levels of RMP released were proportional to platelet counts in the storage bags. (d) Residual oxygen: We observed that storage of leukoreduced RBC under anaerobic conditions resulted in 40 – 60% reductions in RMP and annexin V+ MP generation measured at 42 days.

Conclusions
Multiple MP species (RMP, PMP, LMP) are released during storage of non-leukoreduced PC and increased with time. Procoagulant and proinflammatory activities increased in parallel. Leukoreduction eliminates LMP and PMP generation and reduces RMP generation by 40–50%. This was accompanied by reduction of procoagulant and proinflammatory activities by 60%. We have identified residual platelets, leukocytes, and oxygen levels as important factors governing MP release in stored blood. Reduction or elimination of factors influencing MP generation such as residual platelets, leukocytes and oxygen levels would improve future blood storage condition.

Disclosures:
No relevant conflicts of interest to declare.