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Erythrocyte concentrates (ECs) are the major labile blood product being transfused worldwide, aiming at curing anemia of diverse origins. In Switzerland, ECs are stored at 4 °C up to 42 days in saline-adenine-glucose-mannitol (SAGM). Such storage induces cellular lesions, altering red blood cells (RBCs) metabolism, protein content and rheological properties. A hot debate exists regarding the impact of the storage lesions, thus the age of ECs on transfusion-related clinical adverse outcomes. Several studies tend to show that poorer outcomes occur in patients receiving older blood products. However, no clear association was demonstrated up to date. While metabolism and early rheological changes are reversible through transfusion of the blood units, oxidized proteins cannot be repaired, and it is likely such irreversible damages would affect the quality of the blood product and the efficiency of the transfusion. In vivo, RBCs are constantly exposed to oxygen fluxes, and are thus well equipped to deal with oxidative challenges. Moreover, functional 20S proteasome complexes allow for recognition and proteolysis of fairly oxidized protein, and some proteins can be eliminated from RBCs by the release of microvesicles. The present PhD thesis is involved in a global research project which goal is to characterize the effect of processing and storage on the quality of ECs. Assessing protein oxidative damages during RBC storage is of major importance to understand the mechanisms of aging of stored RBCs. To this purpose, redox proteomic-based investigations were conducted here. In a first part, cysteine oxidation and protein carbonylation were addressed via 2D-DIGE and derivatization-driven immunodetection approaches, respectively. Then, the oxidized sub- proteomes were characterized through LC-MS/MS identification of proteins in spots of interest (cysteine oxidation) or affinity-purified carbonylated proteins. Gene ontology annotation allowed classifying targets of oxidation according to their molecular functions. In a third part, the P20S activity was evaluated throughout the storage period of ECs, and its susceptibility to highly oxidized environment was investigated. The potential defensive role of microvesiculation was also addressed through the quantification of eliminated carbonylated proteins. We highlighted distinct protein groups differentially affected by cysteine oxidation, either reversibly or irreversibly. In addition, soluble extracts showed a decrease in carbonylation at the beginning of the storage and membrane extracts revealed increasing carbonylation after 4 weeks of storage. Engaged molecular functions revealed that antioxidant (AO) are rather reversibly oxidized at their cysteine residue(s), but are irreversibly oxidized through carbonylation. In the meantime, the 20S proteasome activity is decreased by around 40 % at the end of the storage period. Incubation of fresh RBCs extracts with exogenous oxidized proteins showed a dose-dependent and protein-dependent inhibitory effect. Finally, we proved that the release of microvesicles allows the elimination of increasing quantities of carbonylated proteins. Taken together, these results revealed an oxidative pathway model of RBCs storage, on which further investigation towards improved storage conditions will be based. -- Les concentrés érythrocytaires (CE) sont le produit sanguin le plus délivré au monde, permettant de traiter différentes formes d'anémies. En Suisse, les CE sont stocké à 4 °C pendant 42 jours dans une solution saline d'adénine, glucose et mannitol (SAGM). Une telle conservation induit des lésions de stockage qui altèrent le métabolisme, les protéines et les propriétés rhéologique du globule rouge (GR). Un débat important concerne l'impact du temps de stockage des CE sur les risques de réaction transfusionnelles, certaines études tentant de démontrer que des transfusions de sang vieux réduiraient l'espérance de vie des patients. Cependant, aucune association concrète n'a été prouvée à ce jour. Alors que les modifications du métabolisme et changement précoces des propriétés rhéologiques sont réversibles suite à la transfusion du CE, les protéines oxydées ne peuvent être réparées, et il est probable que de telles lésions affectent la qualité et l'efficacité des produits sanguins. In vivo, les GR sont constamment exposés à l'oxygène, et sont donc bien équipés pour résister aux lésions oxydatives. De plus, les complexes fonctionnels de proteasome 20S reconnaissent et dégradent les protéines modérément oxydées, et certaines protéines peuvent être éliminées par les microparticules. Cette thèse de doctorat est imbriquée dans un projet de recherche global ayant pour objectif la caractérisation des effets de la préparation et du stockage sur la qualité des GR. Evaluer les dommages oxydatifs du GR pendant le stockage est primordial pour comprendre les mécanismes de vieillissement des produits sanguin. Dans ce but, des recherches orientées redoxomique ont été conduites. Dans une première partie, l'oxydation des cystéines et la carbonylation des protéines sont évaluées par électrophorèse bidimensionnelle différentielle et par immunodétection de protéines dérivatisées. Ensuite, les protéines d'intérêt ainsi que les protéines carbonylées, purifiées par affinité, sont identifiées par spectrométrie de masse en tandem. Les protéines cibles de l'oxydation sont classées selon leur fonction moléculaire. Dans une troisième partie, l'activité protéolytique du protéasome 20S est suivie durant la période de stockage. L'impact du stress oxydant sur cette activité a été évalué en utilisant des protéines exogènes oxydées in vitro. Le potentiel rôle défensif de la microvesiculation a également été étudié par la quantification des protéines carbonylées éliminées. Dans ce travail, nous avons observé que différents groupes de protéines sont affectés par l'oxydation réversible ou irréversible de leurs cystéines. De plus, une diminution de la carbonylation en début de stockage dans les extraits solubles et une augmentation de la carbonylation après 4 semaines dans les extraits membranaires ont été montrées. Les fonctions moléculaires engagées par les protéines altérées montrent que les défenses antioxydantes sont oxydées de façon réversible sur leurs résidus cystéines, mais sont également irréversiblement carbonylées. Pendant ce temps, l'activité protéolytique du protéasome 20S décroit de 40 % en fin de stockage. L'incubation d'extraits de GR en début de stockage avec des protéines oxydées exogènes montre un effet inhibiteur « dose-dépendant » et « protéine-dépendant ». Enfin, les microvésicules s'avèrent éliminer des quantités croissantes de protéines carbonylées. La synthèse de ces résultats permet de modéliser une voie oxydative du stockage des GRs, à partir de laquelle de futures recherches seront menées avec pour but l'amélioration des conditions de stockage.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

Two hundred and four untreated patients with Hodgkin's disease were given 6 monthly courses of combined therapy with nitrogen mustard, vincristine, procarbazine, and prednisone (MOPP), followed by monthly vinblastine (protocol H2 65). Fifty of these patients were non-randomly selected to receive complementary or prophylactic irradiation to large fields, either supradiaphragmatic (28 cases), subdiaphragmatic (7 cases) or both of these (15 cases). Similarly, 50 other patients were chosen to receive reinductions with MOPP every three months during the first year, then every 6 months. All treatment was stopped after four years. Splenectomy was not performed routinely but 19 patients underwent splenectomy to define precisely the stage of their disease. Review of lymphography led to reclassification of 37 patients to that of stage II; 91% of whom entered remission, 48% being complete. Of the 109 stage III patients, 52% had a complete remission and 38% a partial remission (P.R.). Of stage IV patients, 40% obtained a C.R. and 26% a P.R. Remission curves reached a plateau at the 42nd month: 74 ± 6% for those who entered into C.R. and 56 ± 8% for those with a P.R. (P < 0·03). These patients have been at risk for more than 42 months and 14 have been followed for periods between 5 and 8 years. Other factors may have influenced these results: increased age (more than 50 years) greatly decreased the rate of remission (P < 0·001); the more advanced the anatomical stage the worse the prognosis: but paradoxically, histological lymphocytic depletion (L.D.) had a better prognosis than other types (P < 0·02). The clinical signs (A or B) did not influence the duration of the remission, but abnormalities in the parameters of biological activity (b) worsened the prognosis (P < 0·01). Patients who were given complementary radiotherapy had a longer remission (P < 0·001). A relapse occurred in 38 patients, but never after the 42nd month. In 21 cases, the relapse occurred in the lymphatic areas which had been involved initially, and in the other 17, there was visceral involvement. In addition, there was a correlation between histological cell type and mode of recurrence: local recurrences occurring predominantly in lymphocytic predominance (L.P.), nodular sclerosis (N.S.) and widespread relapses occurring in mixed cellularity (M.C.) and (L.D.). The overall survival reached a plateau at the 48th month of 72 ± 10%, this being influenced by the same factors as the remission. However, the beneficial effects of complementary irradiation (P < 0·05) and reinductions (P < 0·05) were certain. Thus the value of polychemotherapy is confirmed. Prognosis in Hodgkin's disease may be related directly to the administration of polychemotherapy before the radiotherapy. These two modalities must be used in flexible association dependent on the staging which must include all the parameters.

The filterability of total blood and erythrocytes has been investigated in 36 children from 2 to 14 years old by the filtration method developed by Reid. Red blood cells being obtained by centrifugation, were washed three times and resuspended in saline. Filterability was recorded as the filtration time of 1 ml of total blood or red cells suspension. The filtration time of total blood was: 49.10 +/- 14.5 s/ml; that of resuspended cells was: 18.19 +/- 6.16 s/ml. These results have been correlated with age, fibrinogen, white blood cells, glycaemia, haematocrit, and compared with the results obtained in 42 adults whose filtration times were: 33.04 +/- 7.40; and red cells' time: 13.99 +/- 2.95. Statistical analysis shows that the difference is very significant and not correlated with the investigated factors. Erythrocyte filterability may be influenced by still unknown factor.