34. The effects of biochemical rejuvenation on red blood cell microvesiculation, phosphatidylserine and CD47 expression during hypothermic storage

Abstract

Red blood cells (RBCs) under blood bank storage conditions undergo biophysical and biochemical alterations, including biochemical depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), as well as cholesterol and phospholipid loss through microvesiculation. Microvesiculation is the process by which microparticles (MPs), phospholipid vesicles that are less than 1 μm in diameter, are released in response to different stimuli and conditions. RBC microparticles (RMPs) have been shown to be harmful effectors of inflammation and coagulation post transfusion, as well as in vitro indicators of RBC function and viability throughout ex vivo storage. Recent studies suggested that microvesiculation during storage results from biochemical depletion and/or cell senescence (Chernitskii˘ et al. 38 (8) (1993)18–21). Rejuvenation solutions have been shown to be able to restore ATP and 2,3-DPG levels in hypothermically stored RBCs. The purpose of this study was to examine the effects of the biochemical rejuvenation on red blood cell microvesiculation, as well as phosphatidylserine (PS) and CD47 expression during hypothermic storage. Leukoreduced packed RBC units (n = 14) were produced using the standard buffy coat method from whole blood in citrate–phosphate–dextrose (CPD) and stored in saline–adenine–glucose–mannitol (SAGM) at 1–6 °C for up to 49 days. Units were divided into three different experimental groups: untreated control, sham control and rejuvenated group. On days 28, 35, and 42 of hypothermic storage the rejuvenated group was incubated for one hour with 50 mL of rejuvenation solution containing pyruvate, inosine, phosphate and adenine (PIPA), while the sham control group was incubated for 1 h with 50 mL of 1× phosphate buffered saline. RBC units were tested weekly for ATP concentration, quantity of RMPs, and PS and CD47 expression. Microvesiculation was assessed using multicolor flow cytometric (FC) analysis and a spectrophotometric assay was used to determine ATP concentration. The results indicated that ATP significantly decreased throughout the hypothermic storage in untreated and sham RBC units (p