Survival of red blood cells after transfusion: processes and consequences

Lifespan

The maximal lifespan of erythrocytes is 120 days, with a rather small variation of approximately 10 percent. This variation may be due to variations in methodology, such as the analysis of appearance and disappearance of metabolic labels from the circulation, and of the disappearance of erythrocytes labeled with various markers after autologous transfusion, or after transfusion of erythrocytes with differences in minor blood groups (Werre et al., 2004; Bosman et al., 2012a). Alternatively, the variability in maximal lifespan may also be due to inter-individual variations in erythrocyte homeostasis, as has become apparent especially in recent blood bank donor research (Wenk et al., 2011; Dinkla et al., 2013). The relatively small variation in lifespan observed in all studies, however, suggests a gradual, multi-step mechanism rather than a random, disastrous insult, as well as a very efficient removal process.

Volume and density

With increasing time in the circulation, erythrocytes become smaller and more dense. A detailed analysis of these changes shows that, with age, erythrocytes loose 30% of their volume and 15–20% of their hemoglobin, whereas the hemoglobin concentration increases by 14%. This implies that, with age, erythrocytes lose proportionally more water than hemoglobin. Because the decrease in volume is larger than the decrease in surface area, the surface to volume ratio increases. This theoretically positive effect on deformability is abolished by the increase in the hemoglobin concentration, and probably by a decrease in the membrane elasticity (Bosch et al., 1994). Using the percentage of glycated hemoglobin, HbA1c, as a marker of cell age in combination with cohort labeling, survival studies and hemocytometry, Werre and coworkers (Van der Vegt et al., 1985; Bosch et al., 1992, 1994) established that age-related purification of cell fractions on the basis of density alone has inherent restrictions. A similar conclusion can be drawn using another cell age marker, the 4.1a:4.1b ratio, which increases as the result of non-enzymatic deamidation (Mueller et al., 1987; Lutz et al., 1992; Ciana et al., 2004). The lighter fractions are strongly enriched for reticulocytes and young erythrocytes, but the dense fractions are much more heterogeneous with respect to cell volume and cell age. When counterflow centrifugation is followed by density centrifugation, the mean corpuscular volume decreases from 101 fl in the fraction containing the lightest and largest erythrocytes to 72 fl in the fraction with the most dense and smallest cells (Bosch et al., 1992). Combining the two separation techniques results in a considerable reduction in the cell volume-based distribution curves, an almost complete absence of overlap in the erythrocytograms of the lighter-larger and the denser-smaller fractions, and the largest difference in the percentage HbA1c between these fractions (Bosch et al., 1992). Thus, a combination of separation techniques based on volume and density yields erythrocyte fractions with a greater difference in mean cell age than does separation on the basis of density or volume alone.

Vesiculation

Based on observations in splenectomized individuals and on the analysis of subcellular blood fractions, it has been postulated that, under normal conditions, vesiculation is responsible for the aging-associated loss of hemoglobin. The striking resemblance between the hemoglobin composition of blood-borne vesicles and that of old erythrocytes, supports the conclusion that there is a continuous loss of hemoglobin in vesicles, which accelerates during the second half of the erythrocyte lifespan (Willekens et al., 2003). In the oldest erythrocytes of asplenic individuals, the decrease in hemoglobin is absent, concomitant with an increase in the absolute amounts of glycated and otherwise modified hemoglobin species (Willekens et al., 2003). Together with the previous observations that erythrocytes of patients without a functional spleen have an increased number of hemoglobin-containing vacuoles (Reinhart and Chien, 1988), and that there is a positive relation between the vacuole-containing erythrocytes and the percentage HbA1c (De Haan et al., 1988), these data suggest that hemoglobin-containing vesicles within old erythrocytes are removed from the erythrocytes in the spleen. However, it is not likely that vesiculation and vesicle removal occur only in the spleen. A rough calculation based on the vesicle concentrations measured in the blood (Willekens et al., 2008), combined with the kinetics of vesicle disappearance as measured in a rat model (Willekens et al., 2005), indicates that erythrocyte-derived vesicles are phagocytized almost directly after they are generated, even before they can reach the venous circulation. Immunological, biochemical and proteomic analysis of the vesicles generated in vivo suggests that their origin and removal are intimately interwoven with the erythrocyte aging process, and especially with breakdown of band 3 (Willekens et al., 2008; Bosman et al., 2008a, 2012b). The increase in erythrocyte-derived vesicles described in various pathological conditions, most of which are directly related to erythrocyte-specific abnormalities in hemoglobin or membrane proteins, suggests a disturbed aging process in these diseases (Kozuma et al., 2011; Mahfoudhi et al., 2012). However, it has become clear that vesicles may be generated by various mechanisms, emphasizing the need for extensive analysis of these vesicles and comparison with those generated in healthy individuals (Willekens et al., 2005, 2008; Bosman et al., 2010a, 2012b; Kozuma et al., 2011; Xiong et al., 2011; Mahfoudhi et al., 2012; Nantokomol et al., 2012).

Removal signals

Alterations in band 3, as indicated by the presence of breakdown products and/or band 3-containing high-molecular-weight complexes, constitute the most consistent finding in the membranes of old erythrocytes (Kay, 2005; Pantaleo et al., 2008; Willekens et al., 2008; Bosman et al., 2010a,b). Together with the specificity of the senescent cell-specific IgG, this implies a band 3-derived antigen as the main factor responsible for the removal of old erythrocytes from the circulation. There is no convincing evidence for the involvement of phosphatidylserine (PS) in physiological removal of healthy, aged erythrocytes (Willekens et al., 2008; Franco et al., 2013). Recently, however, data have been presented indicating the involvement of the “self” antigen CD47 in phagocytosis of old erythrocytes (Burger et al., 2012). The observations that vesicles are enriched in IgG and band 3 breakdown products, and that most of them expose PS at their surface, have led to the theory that vesiculation serves to dispose damaged membrane patches (Willekens et al., 2008; Tissot et al., 2010). This mechanism would postpone the elimination of functional erythrocytes, and the fast removal of vesicles may prevent uncontrolled coagulation and inflammation (Xiong et al., 2011; Mahfoudhi et al., 2012). The current state of knowledge as described here is schematically depicted in Figure 1.

Lifespan

Erythrocyte survival and lifespan measurements obtained with transfused erythrocytes are in good accordance with those obtained using metabolic labeling studies, indicating an overall good survival with a maximum of 135 days after transfusion (Mollison et al., 1987; Luten et al., 2004). However, almost all studies show the disappearance of 5–10% within the first 24 h after transfusion, which is followed by a linear disappearance curve. This percentage of rapidly disappearing erythrocytes increases with storage time to 25% or more (Luten et al., 2008a). These data suggest that, during their stay in the blood bank, erythrocytes become increasingly vulnerable to as yet unknown stressful conditions they encounter after transfusion. We have postulated that the fraction of quickly disappearing erythrocytes is the main cause of the adverse events of transfusions, especially in transfusion-dependent patients. An overload of the reticulo-endothelial system could lead to hemolysis and the accumulation of neoantigens, triggering iron acccumulation, inflammation and the formation of alloantibodies.

Volume and density

Blood bank storage affects volume regulation as indicated, among other things, by the increase in erythrocyte volume already in the first week of storage (Luten et al., 2008b). This is most likely due to the far from physiological composition of most storage solutions, and seems to be readily reversible upon incubation in buffers with a pH and ion concentrations similar to those of the blood. As a complicating factor, storage may affect cation transport and volume regulation differently in young and old erythrocytes (Minetti et al., 2001). There is a striking change in erythrocyte shape with storage, apparently progressing from echinocytes and stomatocytes to an irreversible spherocyte morphology (Blasi et al., 2012). Density separation results in cell fractions in which the increase in density is accompanied by an increase in HbA1c, suggesting that this method is, in principle, also suitable for the study of aging in vitro (D'Alessandro et al., 2013b). It is tempting to speculate that the more severely and apparently irreversibly deformed erythrocytes comprise at least a fraction of the removal-prone erythrocytes. Cell volume and density are important factors in deformability. Most data indicate that deformability may decrease with storage time, but to an extent that depends heavily on the methods to measure this parameter. The outcomes of most ektacytometry measurements, for example, are likely to be determined by a decrease in the surface/volume ratio, resulting from vesiculation, and a possibly compensatory decrease in the cellular hemoglobin concentration (Cluitmans et al., 2012).

Vesiculation

The availability of an easily accessible source of relatively pure preparations in the form of erythrocyte concentrates in the blood bank has enabled the generation of many data on storage vesicles (Greenwalt, 2006). However, there is no comprehensive theory on the mechanism of their generation, or on their putative contribution to the adverse side effects of transfusion. Blood bank vesicles are enriched in modified hemoglobin species, as are the erythrocyte-derived vesicles in the plasma (Willekens et al., 2003; Bosman et al., 2008a). Their membrane protein composition, on the other hand, is different from that of erythrocyte-derived vesicles from the plasma, suggesting different vesiculation mechanisms in vitro and in vivo (Greenwalt, 2006; Bosman et al., 2008b; Kriebardis et al., 2008; Salzer et al., 2008). Also, blood bank vesicles contain more and different plasma-derived proteins, especially immunoglobulins and complement proteins (Bosman et al., 2008b; Kriebardis et al., 2008). The comparison of blood bank vesicles with plasma vesicles is complicated by their accumulation over time in the blood bag, as this results in a heterogeneity in vesicle age that is likely to be much higher than those of freshly isolated vesicles. Also, there is a distinct possibility that the mechanism of vesiculation changes with storage time and storage medium (Greenwalt and Dumaswala, 1988; Bosman et al., 2010a; Sparrow et al., 2013). A more extensive study especially of the biological activity of blood bank vesicles is warranted by their biological activity, and likely effect on the circulation and immune system of the patient (Gould et al., 2007; Donadee et al., 2011; Kozuma et al., 2011; Xiong et al., 2011).

Removal signals and mechanisms

Band 3 is very sensitive to proteolytic breakdown, as becomes apparent not only upon immunoblot analysis of purified erythrocytes kept in physiological buffer solutions, but also when stored as whole blood or during storage in blood bank conditions. This sensitivity is most apparent using antibodies against parts of the cytoplasmic domain, suggesting that this domain is especially vulnerable (Bosman et al., 2008a). The changes in this domain are likely to affect the connection between lipid bilayer and the cytoskeleton, and the binding of key enzymes of the glycolysis. Thus, early changes in the cytoplasmic domain may very well be responsible for the storage-associated changes in erythrocyte morphology and metabolism. On the other hand, new metabolomic data support the hypothesis that storage exacerbates the effect of naturally occurring oxidative stress by disturbing the physiological balance between glycolysis, pentose phosphate pathway, and glutathione homeostasis (Gevi et al., 2012; Rinalducci et al., 2012). The apparent reversibility of these changes, especially within the first weeks of storage, may be explained by the redundancy of available binding sites. There are much more band 3 than ankyrin molecules, and the dynamic equilibrium between band 3 monomers, dimers and tetramers may compensate for a small decrease in ankyrin-binding (or enzyme-binding) sites.

The number of IgG molecules that is bound per erythrocyte only slightly increases with storage time. Even after the maximal storage period, the percentage of erythrocytes with an amount of IgG that is sufficient for recognition by macrophages is still very small (Kay, 2005). It is noteworthy that the binding of IgG increases within the first weeks of storage, and then decreases again (Luten et al., 2004; Dinkla et al., 2012a). We speculate that this may be caused by the lysis of the very old erythrocytes early in the storage period, leading to association of spectrin or actin to other, intact cells. This could trigger the binding of low-affinity antibodies, that are normally present in the blood (Garratty, 2005). The number of IgG-containing erythrocytes at the end of the maximal storage time, even after incubation with autologous plasma, is much lower than predicted if the aging process in vitro would proceed in an identical manner and with the same speed as it does in vivo. If the latter scenario, the percentage of senescent cell antigen-exposing (and thus IgG-containing) erythrocytes would be much higher than observed, even when corrected for the difference in temperature. Therefore, we must conclude that storage in blood bank conditions may have some molecular features of aging in vivo (see also Figure 1), but that the processes underlying the fast removal of up to 30 percent of the erythrocytes after transfusion cannot be ascribed to a physiological aging process. The recently described, storage-associated increase in the binding of IgG from patients with autoimmune hemolytic anemia to blood bank erythrocytes, shows that aging in vitro may, in certain conditions, assume a pathological form (Dinkla et al., 2012a).

Similarly, the very small increase in the number of PS-exposing erythrocytes during storage is far from sufficient to explain the fast removal of up to 30 percent of the stored erythrocytes after transfusion (Verhoeven et al., 2006; Bosman et al., 2011). However, with storage the erythrocytes become very sensitive to stress-induced PS exposure, especially to the near-physiological stress consisting of incubation in a hyperosmotic buffer (Bosman et al., 2011). Together with a concomitant storage-associated increase in susceptibility to generate vesicles (Burger et al., 2013), this observation emphasizes the predisposing, “sublethal” nature of the events that occur in the blood bank.

In a probably analogous manner, there is a detectable change in CD47 conformation during storage only after transfusion in vitro, i.e., after incubation of the stored erythrocytes with whole blood (Burger et al., 2012). Also, storage is associated with an increased sensitivity to a band 3 ligand-induced binding of autologous IgG (Bosman et al., 2010b). The storage-associated increase in sensitivity to lipase-induced morphological and biochemical alterations points toward a hitherto little-studied involvement of lipids and lipid organization in erythrocyte vesicle formation and antigen presentation (Salzer et al., 2008; Dinkla et al., 2012b). In general, parameters for a “sublethal injury” such as these are likely to carry more physiologically and clinically relevant weight than osmotic or mechanical fragility (Raval et al., 2010, 2013; Cluitmans et al., 2012).

Conflict of interest statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.