The purified vepoloxamer prevents haemolysis in 42-day stored, DEHP/PVC-free red blood cell units

Abstract

Background: Use of the plasticiser di(2-ethylhexyl) phthalate (DEHP) in polyvinyl chloride (PVC) blood bags poses a potential dilemma. The presence of DEHP in blood bags has been shown to be beneficial to red blood cells during storage by diminishing haemolysis. However, DEHP use in PVC may be carcinogenic or estrogenising. Vepoloxamer is a poloxamer with rheological and cytoprotective rheological properties and a favourable toxicity profile in clinical trials. We hypothesised that vepoloxamer may be sufficient to replace the plasticiser DEHP to prevent elevated haemolysis while conserving the biochemical and redox potential++ in RBCs stored for up to 42 days.

Materials and methods: Paired analyses of aliquots from pooled RBC suspensions of ABO identical donors were aseptically split into test storage containers (DEHP/PVC or DEHP-free/ethylene vinyl acetate [EVA]) supplemented with or without vepoloxamer (at concentrations of 0.1, 1, 5 or 7.89 mg/mL) and cold stored for up to 42 days.

Results: Vepoloxamer significantly prevented the increased haemolysis induced by the absence of DEHP in EVA bags in a dose-dependent manner by days 28 and 42 of storage (approx. 50% reduction of the maximum concentration of vepoloxamer; p<0.001). There was an inverse correlation between the concentration of vepoloxamer used and the haemolysis rate (r²=0.27, p<0.001) and a direct correlation between haemolysis and phosphatidylserine (PS) exposure (r²=0.42; p<0.01). Increased osmotic fragility and shear induced deformability of 42-day stored RBC in EVA bags was significantly corrected by the addition of vepoloxamer.

Discussion: Vepoloxamer, in a concentration-dependent fashion, is able to partly rescue the increased haemolysis and PS exposure induced by the absence of the commonly used plasticiser DEHP. These results provide initial but strong evidence to support vepoloxamer use to replace DEHP in long-term storage of RBC.

Figure 1

Chemical formula for vepoloxamer. Vepoloxamer consists of repeated ethylene and propylene oxide groups. With n=80 and m=27, Vepoloxamer has a calculated molecular weight of 8,624 Daltons.

Figure 2

Addition of vepoloxamer results in a dose-dependent reduction in haemolysis and fragility of red blood cells (RBC) stored in ethylene vinyl acetate (EVA) bags. (A and B) Haemolysis. (C and D) Osmotic fragility. (E and F) Deformability index (ektacytometry at 60 Pa). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA test with Bonferroni correction). DEHP: polyvinyl chloride bag with di(2-ethylhexyl) phthalate (DEHP) plasticiser.

Figure 3

Addition of vepoloxamer results in a dose-dependent reduction of eryptosis with no changes in glycolytic flux, potassium leakage or redox potential by day 42 of storage. (A) Percentage of red blood cell (RBC) binding annexin-V. (B) Intracellular adenosine-5′-triphospate (ATP) concentration levels. Extracellular concentration of (C) glucose and (D) lactate. (E) Extracellular potassium concentration. (F) Intracellular level of oxidised PRX2. Intracellular levels of (G) reduced glutathione (GSH) and (H) oxidised glutathione (GSSG). * p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (ANOVA test with Bonferroni correction). DEHP: polyvinyl chloride bag with di(2-ethylhexyl) phthalate (DEHP) plasticiser.