Dissertation committee:
Μαριάννα Αντωνέλου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Ιωάννης Τρουγκάκος, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Παναγούλα Κόλλια, Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Ουρανία Τσιτσιλώνη, Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Βασιλική Οικονομίδου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Διαμάντης Σίδερης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Αναστάσιος Κριεμπάρδης, Αναπληρωτής Καθηγητής, Τμήμα Βιοϊατρικών Επιστημών, ΠΑΔΑ
Summary:
Erythrocytes are the most frequently transfused blood product. However, during their storage in blood bank conditions, a series of time dependent or independent, reversible, or irreversible, physiological, structural and biochemical changes occur, which are attributed to the term "Erythrocyte Storage Lesion". Defects during ex vivo storage can be distinguished into: a) Biochemical changes (ATP depletion, susceptibility to oxidative stress, etc.), b) Mechanical defects (deformability, morphology changes, etc.) and c) Membrane lesions (externalization of removal markers, changes in the membrane proteome, etc.). In the last years, a huge variability in storage capacity and post-transfusion recovery has been observed after storing blood from different donors. For unknown reasons the erythrocytes of distinct donors, categorized by genetically determined characteristics (e.g., sex) or environmental factors (e.g., smoking), show divergent storage capacity. Indeed, many physiological properties of stored erythrocytes can now be considered "heritable characters" or dependent on intrinsic characteristics of the blood donor. Among them are in-bag hemolysis, 24-hour in vivo recovery, ATP levels, oxygen transport efficiency, accumulation of oxidative stress markers, etc.
In the current thesis, the distinct blood donor group that was studied were eligible donors with heterozygosity for beta-thalassemia (bThal), a frequent genetic trait in our country. It is known that bThal heterozygotes who show intracellular hemoglobin above the threshold for blood donation are considered eligible. At the same time, their red blood cells (RBCs) differ compared to the average blood donor in classic hematological parameters, redox balance, and ion exchange, among other. However, neither the storability profile nor recovery and other characteristics of the post-transfusion state are known for stored erythrocytes from these blood donors. Based on the above, the aim of this thesis was the extensive and thorough examination of blood, and mainly of RBCs, from heterozygous bThal blood donors, in the three links of the transfusion chain: in vivo (namely, before storage), in the blood unit during the storage period, and post-storage or after transfusion by using in vitro or in vivo (animal) models of transfusion, respectively. Physiological parameters (hemolysis, redox balance, morphology, proteostasis) as well as metabolomic analysis of RBCs were performed in fresh blood and stored erythrocytes of blood donors with or without bThal trait. In addition, proteomic analysis of erythrocyte membranes and extracellular vesicles (which accumulate in the supernatant of the transfusion unit) was performed during storage. For the in vitro transfusion model, stored RBCs of the two cohorts of blood donors were incubated in plasma from potential recipients (with bThal major or healthy) for 24 h at body temperature, prior the analyses. Finally, in the animal model, erythrocytes from peripheral blood and from the blood units of the two categories were labeled with fluorescent dyes and administered to immunocompetent and immunodeficient mice to comparatively calculate their 24-hour recovery.
According to the results, blood donors with heterozygosity for bThal mutations constitute a non-negligible percentage of the blood donation population of our country. Their erythrocytes cope with storage stress, as they were found resistant to lysis regardless of the stimulus and exhibited an improved redox balance compared to those of the average blood donor. In fact, resistance to cell lysis appeared to be inextricably linked to the distinct geometry of the heterozygotes’ cells and the stability of structural proteins during the progression of storage time. Furthermore, the results in total, whether they concern physiology and metabolism or protein composition and proteostasis, point to a cell well-adapted to steady but mild levels of oxidative stress, ready to face further oxidative challenges. For example, the levels of antioxidant and proteostatic enzymes were found to be more advantageous in RBCs from bThal heterozygotes, while the same cells also showed reduced oxidative damage and lower oxidative load, especially towards the end of the storage period. Given the key role of oxidative stress in the progression of storage damage, it seems that bThal-trait erythrocytes are able, thanks to their "training" to a slightly increased oxidative load, to deal with it more effectively.
The "superior" storability of bThal units (strongly reflected in the reduced levels of storage hemolysis, namely, the gold quality parameter of blood storage according to the World Health Organization) was also accompanied by an improved pattern of post-transfusion stress response. Results from both in vitro and animal models of transfusion converge and (a) highlight the maintenance of the more advantageous cellular physiology post transfusion, and (b) imply an increased 24-hour in vivo recovery of transfused RBCs (the gold quality measure of successful transfusion according to World Health Organization) in the case of bThal heterozygotes. Through correlation analysis, distinguishing characteristics of heterozygotes, such as low RBC fragility indices and cell size, as well as strong extracellular antioxidant capacity were shown to be associated with low post-transfusion hemolysis and better survival of their erythrocytes in the recipient's circulation. It therefore appears that some parameters that differentiate heterozygotes in vivo and/or during storage are not neutral with respect to the post-transfusion physiology/adequacy of their erythrocytes.
