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Thesis (doctoral)--Freie Universität Berlin, 1979.

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M.Cur. / Awareness of the growing list of potential and inherent risks and hazards associated with receiving donor blood has created a mushrooming interest in alternatives to blood transfusion. Despite the fact that there are programmes, protocols and guidelines in place in the private health sector, blood conservation has not got off the ground. Therefore the aim of this study was to explore and describe the perceptions of health-care professionals regarding blood conservation in the private health sector. An exploratory descriptive contextual design was employed. Data was collected through the use of semi-structured focus group and individual interviews. Conceptualisation as well as data from the interviews served as the basis for the formulation of guidelines for health-care professionals to improve blood conservation. The results of this research show that the interaction among health-care professionals are negatively influenced by the lack of communication, feedback, support and uncertainty, a lack of trust, education, planning, implementation, involvement, commitment and co-ordination. Therefore the outcome of blood conservation cannot be achieved. Effective communication, education and participatory management have to improve in order for these negative factors to be overcome. It is recommended that these guidelines be implemented to improve blood conservation in the private health sector. Conclusions, limitations and further recommendations were made based on the results of this study.

Multicomponent transfusion, or the transfusion of two or more different blood products, has been poorly studied to date, as most of the existing literature has focused on the use of individual blood products. This is of concern as multicomponent transfusion recipients likely differ with respect to characteristics and health outcomes from patients transfused with only one type of blood component (e.g. greater illness severity). Consequently, available data on individual blood product use and outcomes may not be applicable to multicomponent transfused patients. This thesis project identified and synthesized existing literature on the epidemiology of multicomponent transfusion in hospital inpatients, as well as the characteristics and outcomes of its recipients. Based on 37 observational studies, we found that the prevalence of multicomponent transfusion varied greatly by patient population, transfusion timeframe, and type of multicomponent transfusion being studied. The most common types of multicomponent transfusion across the 37 studies were co-transfusions of red blood cells (RBCs) and platelets, and co-transfusions of RBCs and plasma. Multicomponent transfusion was found to be associated with several negative health outcomes, however this was based on low quality evidence due to lack of control for confounding by indication. Our systematic review on multicomponent transfusion identified several knowledge gaps, including the need for studies focusing on patients with hematological malignancies, and studies identifying patient characteristics predictive of multicomponent transfusion. To address areas of knowledge deficiency, and to characterize multicomponent transfusion locally at our own center, we designed and conducted a retrospective cohort study of adult, transfused hospital inpatients. Based on 55,719 transfused inpatient admissions at the Ottawa Hospital between 2007 and 2017, we calculated the overall prevalence of multicomponent transfusion to be 25.1% (95% CI: 24.7%, 25.5%). Similar to the findings of our systematic review, the prevalence varied greatly by patient type, transfusion timeframe, and type of multicomponent transfusion. In particular, in hematology patients, the prevalence of multicomponent transfusion was 51%. Other patient groups frequently receiving multicomponent transfusions at our institution were cardiac surgery, critical care, cardiology, vascular surgery, trauma, surgery, and internal medicine patients. Using multivariable regression analysis, we found that patient sex, age, and type were predictive of multicomponent transfusion requirement. Additionally, controlling for illness severity and burden, multicomponent transfusion was associated with increased odds of in-hospital mortality, institutional discharge compared to discharge home, and greater length of hospital stay compared to patients transfused with only RBCs. Given our findings that multicomponent transfusion recipients make up a large proportion of transfused hospital patients, and that they have poorer outcomes, it is of importance to continue characterizing these patients – and not only focus on patients receiving a single type of blood component – and to evaluate and monitor the appropriateness of multicomponent transfusion. Additionally, as transfusion practice and guidelines are known to vary from region to region, it is important to study multicomponent transfusion locally, as generalizing results from other studies and centers may not be appropriate. Obtaining robust information on multicomponent transfusion – including prevalence, predictors, and potential health consequences – can aid clinicians in their decision-making for patient blood management, potentially minimizing unnecessary patient exposure to blood products, and maximizing the use of transfusion alternatives and blood conservation methods.

Les plaquettes, fragments de cellules, sont très abondantes dans le sang où elles favorisent l'hémostase. Activées, elles forment des microparticules (MPs) et libèrent des mitochondries libres ou encapsulées. Comme la membrane mitochondriale est un substrat de la phospholipase A2 sécrétée qui libère des signaux inflammatoires, nos résultats permettent d'identifier les mitochondries extracellulaires au centre d'un mécanisme inflammatoire puissant qui fournit une explication possible de l'augmentation des niveaux sanguins d'ADNmt rapporté dans de multiples pathologies telles que les réactions transfusionnelles où l’ADNmt est présent à des niveaux plus élevés dans les PCs. Ici, nous avons étudié les MPs et les mitochondries extracellulaires dans différents types de PCs Les concentrations des MPs et des mitochondries extracellulaires étaient significativement plus élevées dans le PRP que les autres PC et sont stables dans l’entreposage. Le mode de préparation, plutôt que de la durée d’entreposage a un impact sur leur libération dans les PCs. / Platelets, anucleate cell fragments that contain mitochondria, are highly abundant in blood where they promote hemostasis. Activated platelets shed microparticles (MPs) and release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. As the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), which yields inflammatory mediators, our findings identify extracellular mitochondria at the midpoint of a potent mechanism leading to inflammatory responses. This mechanism provides a potential explanation for the increased levels of extracellular mtDNA reported in blood in multiple pathologies, such as adverse transfusion reactions where mtDNA is present at higher levels in PCs. Herein, we aimed to quantify MPs and extracellular mitochondria in different types of PCs through storage. Concentrations of MPs and extracellular mitochondria were significantly higher in PRP than other PCs and were stable over storage. The mode of preparation, rather than storage duration, impacts release of MPs and mitochondria in PCs.

Bartonella bacilliformis is a pathogen that is endemic in some areas of the Andean region of Peru, southern Ecuador and southern Colombia. This pathogen causes so-called Carrion's disease, a biphasic disease with acute and chronic phases (called Oroya fever and "Peruvian wart" respectively1-3). In the absence or delay of antibiotic treatment, the mortality rate in the acute phase is up to 88%1. The acute phase is characterised by fever and severe anaemia and may be followed, several weeks or months later, by the chronic eruptive phase due to endothelial cell proliferation2. No animal reservoir has been identified to date and it is considered that healthy carriers act as a pathogen reservoir in endemic areas

La transfusion sanguine est un traitement essentiel à la survie de millions de patients. Son principal risque immunologique est l’allo-immunisation post-transfusionnelle. Elle se traduit par la production d’allo-anticorps contre des antigènes de globules rouges (GR) conduisant à des hémolyses post-transfusionnelles. Les mécanismes à l’origine de la tolérance des GR ou de son inhibition lors de l’allo-immunisation sont mal connus. Ainsi, mes travaux de thèse, portant sur la compréhension de ces effets, se sont articulés en trois parties avec 1/ l’étude des conditions optimales aux réponses allo-immunes, 2/ l’étude des effets d’une stratégie thérapeutique utilisant un anticorps monoclonal et 3/ l’étude des effets immunomodulateurs, incluant la tolérance, médiée par des composants présents dans les concentrés de globules rouges (CGR).Afin d’étudier l’allo-immunisation, nous avons utilisé le modèle murin. Nous montrons qu’une variation du délai entre la transfusion et la stimulation du TLR3 impacte la réponse immune dans la rate. Une activation importante des lymphocytes T CD4+ (LT CD4+) allo-réactifs accompagnée d’une production accrue d’allo-anticorps ont été montrées à 7 jours de délai. Afin de limiter l’allo-immunisation, l’utilisation d’un anticorps anti-CD20 déplétant les lymphocytes B (LB) montre une altération des LB mais surtout des LT CD4+ impliqués dans le processus d’induction de l’allo-immunisation. Enfin, la modification du phénotype des cellules dendritiques CD11c+ de la rate des souris transfusées, observée hors contexte inflammatoire, suggère une maturation incomplète à l’origine d’une tolérance antigénique. Pour finir, l’analyse de différents composants présents dans les CGR confirme l’existence de microparticules (MPs) lymphocytaires. Ces MPs présentent des molécules inhibitrices et pourraient donc être impliquées dans la tolérance des antigènes transfusés.En conclusion, mes travaux montrent la coopération des DCs avec les LT CD4+ permettant celle des LT CD4+ avec les LB pour induire une réponse immune. Comme toute réponse humorale, nous confirmons que l’allo-immunisation fait intervenir des DCs, des LT CD4+ et des LB. Ces résultats ouvrent de nouvelles voies de recherche pour mieux caractériser l’allo-immunisation en particulier chez les patients drépanocytaires qui sont les plus touchés. / Red blood cell (RBC) transfusion is a life-saving treatment for millions of patients. However, its main immunological risk is RBC alloimmunization resulting in antibody production against RBC antigen. Alloimmunization can lead to severe complications threatening the life of the patient. The mechanisms explaining RBC alloimmunization are poorly understood. Therefore, my doctoral work aiming at understanding transfusion effects, was subdivided into three parts with 1/ the study of optimal conditions for alloimmune responses, 2/ the impact of a therapeutic strategy using a monoclonal antibody to inhibit alloimmunization and 3/ the study of immunomodulatory effects of transfusion, including tolerance, through components present in the RBC concentrates.We also used HOD murine model for the study of alloimmunization to show an impact of the delay between the TLR3 agonist injection and the transfusion on immune responses against RBCs. At 7 days of delay, we have demonstrated an important alloreactive CD4+ T-cell activation and a wider alloantibody production. Furthermore, B-cell depletion, using a monoclonal anti-CD20 antibody, revealed potential changes on LB implicated in alloimmunization induction and mostly on alloreactive CD4+ T cells. We finally observed a modification of splenic CD11c+ DC phenotype from transfused mice out of a TLR context. This suggest an incomplete maturation that could explain antigen-specific tolerance. The investigation of several components in RBC concentrates confirmed the presence of microparticules (MPs) issued from T lymphocytes. These MPs carry inhibitory markers and could thus inhibit DC activation to induce antigen-specific tolerance.Therefore, my doctoral work highlights the implication and the cooperation of DCs with CD4+ T cells to allow cellular cooperation between CD4+ T cells and B cells for immune response induction. As in any humoral response, we confirmed that RBC alloimmunization involves DCs, CD4+ T cells and B cells. In addition, these results are opening up new areas of investigation for a better characterization of alloimmunization in particular in the most affected patients, the SCD patients.

La transfusion sanguine permet de sauver des vies et réduit la morbidité pour un grand nombre de maladies et d'affections cliniques, mais elle n'est pas exempte de complications. Un incident néfaste lié à une transfusion, également appelé Effet Indésirable Receveur (EIR), est un incident défavorable survenant chez un patient pendant ou après une transfusion sanguine. Parmi eux, le TRALI est considéré comme l’une des réactions inflammatoires les plus critiques. Cette pathologie se développe généralement dans les 6 heures après transfusion. On en reconnaît deux types, les TRALI immunologiques et les TRALI non-immunologiques. En France, les premiers sont presque entièrement prévenus par une politique de sécurité des produits sanguins, tandis que la fréquence des seconds augmente. La physiopathologie du TRALI reste mal connue. Tandis que certains y accordent une place importante aux plaquettes sanguines du patient transfusé, d’autres les considèrent comme pas réellement impliquées. Le but de ce travail de thèse a été, dans un premier temps, d’investiguer le potentiel inflammatoire des plaquettes sanguines conservées dans les concentrés plaquettaires et l’influence de cette inflammation sur l’endothélium vasculaire général. Ensuite, sera évalué le rôle des plaquettes sanguines de l’organisme, notamment par l’intermédiaire de leurs produits de sécrétion, dans la pathogénie de cette complication transfusionnelle. Pour cela, un ALI (mimant un TRALI) a été déclenché, dans un modèle in vivo, par une injection d’anticorps anti-CMH I chez des souris préalablement stimulées avec du LPS. L’ensemble de nos résultats confirme le potentiel inflammatoire des plaquettes sanguines, au sein des concentrés plaquettaires, pouvant probablement assumer l’entière responsabilité du déclenchement d’un TRALI non-immunologique, ainsi qu’un rôle secondaire des plaquettes sanguines de l’organisme, participant activement à l’amplification de la sévérité de la pathologie. Cette thèse s’inscrit dans la continuité logique des études menées, au sein du laboratoire GIMAP-EA3064, investiguant la place des plaquettes sanguines au sein de l’inflammation, ouvrant ainsi de nouvelles perspectives dans la sécurité transfusionnelle. / Blood transfusion saves lives and reduces morbidity for many diseases and clinical conditions, but it is not without complications. A transfusion-related adverse event, also known as the Adverse Reaction (AR), is an incident occurring in a patient during or after a blood transfusion. Among them, TRALI is considered as one of the most critical inflammatory reactions. This pathology usually occurs within 6 hours after transfusion. Two types are recognized: immune TRALI and non-immune TRALI. In France, the first is almost completely prevented by a blood product safety policy, while the frequency of the second increases. The pathophysiology of TRALI remains poorly understood. While some scientists give an important function of patient blood platelets, others consider them dispensable. The aim of this thesis was, first, to investigate the inflammatory potential of blood platelets stored in platelet concentrates and its impact on the general vascular endothelium. Next, the role of patient blood platelets, including their secretory products, in the pathogenesis of this transfusion complication will be evaluated. For it, an ALI (mimicking a TRALI) was triggered, in an in vivo model, by an injection of anti-MHC I antibody in mice previously stimulated with LPS. Our results confirm the inflammatory potential of blood platelets in platelet concentrates, which can probably assume the entire responsibility for triggering a non-immune TRALI, and a secondary role for patient blood platelets in the amplification of the severity of this pathology. This thesis is the continuity of studies conducted in the laboratory GIMAP-EA3064, investigating the function of blood platelets in inflammation, thus opening up new perspectives in transfusion safety.

Platelets play an important role in the haemostatic process in order to seal damaged blood vessels. The platelets form a platelet plug at the damaged area and prevent blood loss. Once the damage to the vessel wall has been covered, the platelets retract the coagulum, to allow the blood to flow freely in the vessel. Free oscillation rheometry (FOR) can be used for analysis of coagulation as measured by clotting time and changes in clot elasticity (G'). Clot G' provides information about the fibrin network in the coagulum and the platelets’ ability to retract the coagulum. FOR analysis is performed using the ReoRox® 4 instrument. The blood sample is added to a cylindrical sample cup, which is set into free oscillation. The frequency and damping of the oscillation is recorded over time as the blood coagulates. The change in G' is calculated from the frequency and damping measured. Patients with malignant haematological diseases are often thrombocytopaenic and require platelet transfusions to prevent or stop bleeding. To ensure good haemostatic function in the recipient it is important that the quality of the platelets used for transfusion is well preserved. The aim of this thesis was to determine the quality of platelet concentrates (PCs), during storage, using various in vitro methods, including FOR, and to investigate how various preparation processes affect the quality. We also investigated whether FOR can be used to evaluate the haemostatic status in subjects at risk for thrombosis or bleeding as well as how the haemostatic status was affected by a platelet transfusion. We show that FOR can provide information about the coagulation properties in subjects at risk of thrombosis (pregnant women) or bleeding (thrombocytopaenic patients). We also show that the coagulation as measured by FOR is influenced by red blood cells and the fibrinogen concentration. However, the presence of functional platelets accounted for 90% of the G'. Furthermore we present data that FOR can provide information on the haemostatic effect of platelet transfusions and on the function of the transfused platelets. PCs produced by two different cell separators showed similar quality during storage for 7 days as assessed by FOR analysis. Leukocytes in the PCs can cause transfusion-associated graft-versus-host disease which can be prevented by gamma irradiation of the PCs. Gamma irradiation did not affect the quality of PCs during 7 days of storage as analysed by FOR. The clotting time was unchanged during the storage period. The capacity of platelets to retract the coagulum was reduced from days 1 to 5 of storage as seen by a prolonged time to reach maximum G' and the reduced mean change in G' per minute. However, if sufficient time is allowed for the platelets to regain their function, the clot will be fully retracted (as seen by a well maintained maximum G'). The FOR parameters were similar for 5- and 7-day old PCs, which, combined with other in vitro tests (e.g. hypotonic shock response, changes in pH, swirling, lactate and glucose), support the prolongation of the platelet storage period to 7 days. Intercept™ treatment of PCs can be performed to inhibit replication of contaminating bacteria in PCs. Intercept™ treatment of PCs did not diminish the clot-promoting capacity of the platelets as assessed by FOR clotting time. In conclusion, FOR is a promising method for assessing hyper- and hypocoagulability. It can provide information on the haemostatic effect of platelet transfusions and the function of the transfused platelets. FOR was also shown to be useful for analysing PC quality during different preparation and storage conditions.