Red-Cell Microparticles Released From Stored Packed Cells: Possible Contributing Factor to Adverse Responses to Transfusion
Authors: Jy Wenche , Bidot Carlos , Johansen Max E , Horstman Lawrence , Shariatmadar Sherry , Ricci Marco , Gomez-Marin Orlando , Ahn Yeon-Soong 
Published: October 2019

Abstract
Abstract 342

Background.
Packed red cells (PC) stored in blood bank undergo a series of changes, so-called “storage lesion”, which increase with time. In view of some but not all recent studies, it is widely believed that transfusion with younger blood carries less risk of adverse reactions than older blood. However, there is no agreement on the “safe” age of blood, nor is it clearly understood why older blood may carry increased risks. The purpose of this study was to identify microparticle-related factors in stored PC at different time intervals that might pose risk of adverse effects. We investigated profiles of cell-derived microparticles (MP), particularly RBC-derived MP (RMP), in stored PC and assessed their procoagulant and inflammatory property.

Methods.
Twelve bags of fresh packed red cells (PC) of known blood types (A+, B+, AB+, O+) were obtained from blood bank (2-4 days since drawing). All were non-leuko depleted and were stored at 4°C. Time of receipt was considered day 0. At intervals of 0, 10, 20, and 30 days, 40 mL was withdrawn and centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for (1) quantity of different species of cell-derived MP by flow cyteometry comprising (a) RMP defined by CD235b; (b) LMP by CD45; (c) PMP by CD41; (d) EMP by CD144; (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV), (2) procoagulant activity by MP-mediated thrombin generation assay (TGA); (3) MP-mediated proinflammatory activity by CD 11b expression in neutrophils following incubation with RMP.

Results.
(1) MP Profiles. The time-course of generation of the MP subtypes varied considerably. For RMP, there was little increase before day 10, but then rose rapidly with time, to 180% at 20 days, and to 450% at 30 days. Small amounts of MP derived from leukocytes (LMP), platelets (PMP), and endothelia (EMP) were present in all bags at day 0, generally <10% by number compared to RMP. For LMP, there was no significant change in the first 20 days but was increased significantly at day 30, to 160% of day 0. For PMP, counts rose steadily from day 0 and peaked to 220% of baseline at day 20. For EMP, counts were very low (<1% of RMP) and no change was observed over 30 days. For total MP, defined by Ulex counts and total protein concentrations, the time course was similar to RMP. Results with AnV+ MP showed significant increase from day 10 to day 20. We found no influence of blood type on MP generation. (2) Procoagulant Activity. There was little change in MP-mediated thrombin generation in the first 10 days, but it rose significantly from day 10 to day 20, correlating well with counts of AnV+ MP or RMP. (3) Proinflammatory Activity. Leukocyte CD11b expression induced by MP from the PC bags showed a nearly linear rate of increase from day 0 to day 30, correlating closely with PMP. (4) Exceptions. In 2 of the 12 PC bags, we observed exceptionally high levels of both RMP and PMP (4-8 fold higher than average) from day 0 to day 30. CBC assay showed that all bags contained similar counts of RBC and WBC, with exception of high concentrations of platelets (>200,000/μL) in the two exceptional bags vs. the others (20,000 – 45,000/μL). In the two exceptional bags, coagulant activities and inflammatory potential were also highly elevated compared to the values in the other bags from day 0 to day 30.

Conclusions.
(i) RMP are the predominant MP species in stored RBC, increasing slowly from day 0 to 10 and thereafter rising exponentially to day 30. (ii) PMP were also present in significant amounts. The time course of RMP and PMP release correlated well with procoagulant and proinflammatory indicators in stored RBC. (iii) Two of the 12 bags (17%) exhibited exceptionally high platelet content and RMP, PMP. The significance of this new finding remains to be clarified. These preliminary results indicate that procoagulant, proinflammatory MP levels increased significantly after 10 days of storage, and that contaminating platelets exacerbate RMP generation. The increase in MP in stored PC constitutes one aspect of the storage lesion and may pose prothrombotic and/or proinflammatory risks in blood transfusions. (Supported by NIH Grant 5R01HL098031-02)

Disclosures:
No relevant conflicts of interest to declare.