## 1 Introduction  
Red blood cell (RBC) transfusion is a life-saving procedure whose primary objective is to sustain tissue and organ oxygenation in patients with massive bleeding or acute anemia. Packed red blood cell (PRBC) donations for transfusion are routinely stored for up to 35 to 42 days, depending on the preservation solution [1]. During cold storage, PRBCs undergo slow detrimental changes that are collectively termed storage lesion [1; 2; 3]. Aging-related processes (that are at least partially due to the sensitivity of cells to oxidative stress [4]) lead to significant metabolic and structural changes in red blood cells [5; 6; 7; 8], and involve global biochemical and biophysical alteration, remodeling of the cell membrane, and cytoplasm composition [3; 9]. The most studied changes include: adenosine triphosphate (ATP) and 2,3-bisphosphoglycer (2.3-DPG) depletion [9], loss of cellular antioxidant capability [10; 11], changes in K+ and Na+ concentration [12; 13], loss of membrane and skeleton proteins [14; 15], loss of membrane lipids, and changes in their in/out distribution, vesicle generation [16; 17], oxidation and remodeling of skeleton proteins [18], clustering of band 3 proteins [19; 20], alteration of nitric oxide signaling [21; 22], decrease in antioxidant activity [23; 24; 25], etc. A number of these changes are interrelated and initiate a cascade of biochemical and structural changes, which in turn lead to impairment in RBC functionality, specifically--alteration in the biophysical/mechanical properties of cells [5; 7]. Each of the reported storage-induced alterations in PRBC properties occurs on its specific time-scale [26; 27; 28; 29; 30; 31; 32; 33; 34]. Some of these changes take place at an early stage of storage (during the first seven days), while others occur later. Changes in the composition and property of the cell membrane begin at the very early stages of PRBCs storage [35; 36; 37; 38]. [PERSON] et al. summarized this cascade of changes as follows [36]: oxidative damage-induced, high-affinity binding of hemoglobin to the cytoplasmic domain of band 3, activation of Ca\({}^{2+}\)-permeable channels, phosphorylation-controlled alterations in morphology and metabolism affecting ATP production and redox status, degradation of band 3 and/or aggregation of band-3 fragments, binding of immunoglobulin-G, and micro vesicles generation. Obviously, such changes in the composition of the cell membrane, its shape, and properties must inevitably lead to a change in its hydrophobicity [39]. Furthermore, bio-active substances accumulate in storage medium [40]. The accumulation of immunomodulatory factors in PRBC concentrates [41] has been implicated as a potential cause of transfusion reactions associated with the use of PRBCs [42; 43]. These include lipids (that prime recipient neutrophils and have been implicated in transfusion-related acute lung injury [44]), cytokines [26; 41], arachidonic acid [45], and malondialdehydedehyde (MDA, a marker of lipid peroxidation) [2]. In addition, PRBC lesion causes the alteration of sodium/potassium balance [46], accumulation of lactate [41; 46], lactate dehydrogenase [2], iron [26; 47; 46], and free hemoglobin [2; 41; 46] in the supernatant in parallel with decreasing of glucose concentration [46] and reducing of medium pH [26; 40]. Our recent Microwave Dielectric Spectroscopy (MDS) studies have shown that it is possible to evaluate the dielectric response of cytoplasmic water by measuring the suspension of stored red blood cells (RBC) [48]. From the analysis of the dielectric relaxation as a function of storage time (ex vivo aging), it was assumed that the behavior was rooted in the delicate interplay between bound and bulk water in the cellular interior. Based on these results, we hypothesized that the state of intracellular water might be used as a marker of the functionality of stored cells. Furthermore, the RBC aging is associated with a specific pattern of changes in intracellular water.