Proteomic analysis of red blood cells from donors exhibiting high hemolysis demonstrates a reduction in membrane-associated proteins involved in the oxidative response.

Abstract

The development of hemolysis during ex vivo hypothermic storage is multifaceted. Standardization of collection and production processes is used to minimize variability in biologics manufacturing and to maximize product quality. However, the influence of various donor characteristics on product quality is often difficult to evaluate and to control. Using a proteomic approach, we aimed to decipher relevant donor characteristics that may predict red blood cell (RBC) quality during storage. Ten healthy volunteer donors exhibiting repeated high hemolysis at outdate (>0.8%; RBCHH ) and 10 age- and sex-matched control donors (RBCCtrl ) were studied. Common quality variables were measured on Days 5, 14, 21, 28, and 42 of storage. Protein profiles of hemoglobin-depleted membrane fractions from RBCHH and RBCCtrl donors were analyzed using a quantitative proteomics approach based on iTRAQ (isobaric tags for relative and absolute quantitation). Time-dependent lesion development was apparent in both donor populations. RBCHH exhibited reduced 2,3-bisphosphoglycerate levels (p < 0.001) and morphologic score (p < 0.001), but displayed elevated hemolysis level (p < 0.001), RBC-derived microvesicle formation (p < 0.001), and mean corpuscular fragility (p < 0.001) compared to RBCCtrl , indicating notable differences at the membrane between the two donor populations. Proteomic findings revealed a significant reduction in the level of proteins involved in oxidative response pathways at early time points in RBCHH compared to that of RBCCtrl . The recruitment of these candidate proteins might be part of a response mechanism altered in RBCHH donors and therefore may be useful as a donor screening tool.