The picture of Dorian Gray: news from the red blood cell storage lesion.

The picture of Dorian Gray is a novel, written by Oscar Wilde, about a young man whose desire to maintain his youth and beauty renders his soul irrevocably damaged. There are allusions in the text indicating that Dorian’ portrait was a representation of his soul. While Dorian remained young, the picture progressively showed signs of age. In the end, when Dorian attempts to destroy the picture, it returns to its original beauty, while he is found lying dead on the ground, old and ugly, as he should have been. The storage lesion confronts us with this paradox of ageing. Red blood cells are produced every day and survive in the circulation for about 120 days. Blood donors are able to give blood from the age of 18 (16 in same countries) to the age of 65 to 70. The parallel between red blood cells and The picture of Dorian Gray arises from the fact that “old” blood donors are able to produce young cells, while the blood of young donors contains heterogeneous populations of old erythrocytes that are condemned to die. Therefore, the question is: “what is aging?” Another interesting paradox is related to the fact that erythrocytes intended for transfusion may be stored for weeks, at 4 °C, a temperature far from the physiological condition of 37 °C. The importance of temperature and the significance of modifications of the cellular membrane that occur with the creation of neoantigens at low temperature and are recognised by liver macrophages (Kupffer’s cells), leading to the elimination of transfused cold-stored platelets1,2, have been well deciphered in the analysis of storage conditions for platelets. In addition to the effect of temperature on stored erythrocytes, various storage solutions are added that impart many other non-physiological stresses to red blood cells. All of these phenomena lead to the so-called storage lesion, and numerous attempts have been made to mitigate this lesion or improve the duration of storage at 4 °C. Paradoxically, although red blood cells are well-equipped to tackle oxidative stress (they actually pass through the lungs approximately every 20 seconds), our attempts to store them in a functional condition trap them in a situation in which, deprived of essential metabolites after 2 weeks of storage, they can no longer fight against the oxidative stress to which they are exposed during storage. What can be done to maintain the youth and beauty of a red blood cell, either during its circulation in the blood of an individual or during its storage? One old-fashioned approach was the transfusion of neocytes collected by aphaeresis. The modern approach will be the transfusion of homogeneously young and beautiful red cells generated in vitro3. These two approaches are difficult to apply in daily life, which is why blood banks have urged (and still urge) manufacturers to develop storage media that allow the conservation of red blood cells at 4 °C for up to 56 days or even longer4. They are also encouraging manufacturers to develop storage solutions that will reduce the deleterious effects of transfusing “old” blood into a recipient, particularly after cardiovascular surgery, as described by Koch et al5. This paper has been the subject of many editorials and comments, including one in Blood Transfusion6, followed by a meta-analysis7, a review8, and even a study suggesting that “old” blood might be better for transfusion than fresh blood9. What is the truth? At the beginning of 2014, no clear answer can be given. Nevertheless, accumulating evidence indicates that “old” blood may be associated with a poorer outcome in transfused patients than fresh blood, as observed in patients undergoin liver transplantation. However, well-controlled clinical studies are still ongoing. Nevertheless, basic science has given us a better understanding of the pathophysiology of the storage lesion, what can be done to decrease it, and how to ameliorate the “wellness” of red blood cells during storage. In this context, the pioneering works of Dr. Tibor Greenwald should be acknowledged10. The biochemical modifications associated with storage at 4 °C can no longer be ignored by clinicians, and provide insights into the potential consequences that are observed in transfused patients. It is well known that hypothermic storage of red cells induces membrane remodelling and many biological modifications. Many research protocols have been proposed to decrease such alterations. Modifications of the additive solutions may improve the quality of the ex vivo-stored erythrocytes11. Rejuvenation leads to numerous metabolic changes, notably an increase in intracellular ATP, regeneration of 2,3-diphosphoglycerate, restoration of several electrolyte gradients such as those of potassium, sodium and calcium, as well as improvement of the morphology of red cells. Rejuvenation solutions are licensed for use in some countries, at any time between a few days after collection up to the expiry of the unit. Tentative efforts are also being made to optimise the biopreservation of red blood cells by changes in the biochemical composition of the additive solutions. “Systems biology”-oriented understanding of the biochemistry of red blood cells has helped to dissect the pathways associated with both in vivo and in vitro ageing of red cells. In this context, the works of Lello Zola’s group should be highlighted, because they provide many new insights into the understanding of the pathophysiology of RBC storage12. An important observation was the effect of oxygen removal during storage, which did not result in lower levels of oxidative stress13. In another interesting study, published in this issue of Blood Transfusion, Zolla’s group have just added a new cornerstone to the role of oxygen as an important player of the storage lesion, by showing the benefit of supplementing additive solutions with anti-oxidants14. In this work, Pallotta et al. use various methodological approaches, known under the term “metabolomics”, to provide evidence that the addition of vitamin C and N-acetylcysteine is able to prevent oxidative stress-related lesions14. These observations are of importance because they show that anti-oxidants are able to maintain the “quality” of red blood cells by controlling the glycolytic metabolism and by modulating the redox poise through the promotion of glutathione homeostasis14. This research further confirms the toxicity of oxygen during the storage of red blood cells. Evidence has accumulated that when oxygen is present in the storage medium, the red blood cell is the subject of oxidative stress15, but that it can mount defences by eliminating oxidized proteins within microvesicles16–18. In addition to the many biochemical modifications detected during the storage of red blood cells, notably an increased concentration of potassium and decreases in both ATP and the expression of CD47 (a “self” protein of red blood cells), other changes that can be observed include accumulation of malondialdehyde and altered size and morphology of the cells, resulting in changes in osmotic resistance and vesiculation19. Classification of membrane vesicles, protocols for their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Microvesicles were identified in the blood circulation many years ago and were first considered as cell fragments. In fact, microvesicles are quite heterogeneous and at least two main distinct types have been identified: exosomes and microparticles17. Both types of vesicles are detected in flowing blood and arise from cells such as erythrocytes, platelets, leucocytes and endothelial cells. In the context of blood banking, accumulation of microvesicles can definitively be seen as a marker of the storage lesion. Very interestingly, it was demonstrated that those microvesicles may have procoagulant activities19 as well as immunosuppressant effects20, both biological functions that can be linked to the clinical complications observed in patients after transfusion of “old” blood. Thus, all efforts to decrease the alterations of ageing should be explored and, more importantly, should be validated in well-designed clinical studies. Taken together, basic science and a small amount of antioxidants, such as vitamin C, may provide better control of the ageing of the picture of a red blood cell (unlike the picture of Dorian Gray, who still represents a complex balance between eternal youth and ageing). Translational studies and controlled clinical trials are of pivotal importance for further assessment of whether the addition of vitamin C and N-acetylcysteine to the storage medium (with or without adsorption of the residual reactive oxygen species) can provide benefit to the recipients of red blood cell transfusions. Nevertheless, there are already evidences that such a treatment may be beneficial, as shown by another study from Zolla’s group, published in this issue of Blood Transfusion21. The authors show pictures of red blood cells showing that, in the particular storage conditions, stored red blood cells look like young. The road is long, but the direction seems to be clear!