A centrifugação é o principal método utilizado para fracionar o Sangue Total (ST) nos componentes sanguíneos: Concentrados de Hemácias (CH), Concentrados Plaquetas (CP), Plasma e Crioprecipitado, (também chamados de Hemocomponentes). Com intuito de prevenir ou controlar hemorragias, os CP obtidos por centrifugação tornaram-se o padrão no atendimento inicial aos pacientes com baixas contagens de plaquetas. Porém, para obter uma unidade terapêutica (UT), no Hemocentro RP, é necessário agregar seis CP, formando o Pool de CPST (PCP), o que leva a exposição do receptor a vários doadores diferentes. O CP obtido por aférese (CPAF), advindo de equipamento que permite a seleção já na coleta, fornece a dose necessária em apenas uma doação. Baseado no levantamento de dados internos do Hemocentro de RP o presente estudo analisou, através da metodologia de Custeio Baseado em Atividade (ABC), os custos atribuídos às distintas metodologias para a produção dos CP, sabendo que no Hemocentro RP, os CP são dispensados como UT independente da forma de obtenção. Os resultados obtidos foram utilizados na Análise de Custo-Efetividade, evidenciando que a coleta por Centrifugação de ST com a produção de PCP foi mais custo efetiva em 2015 do que a coleta de aférese, porém, por uma diferença relativamente pequena, devido ao grande percentual de obtenção de dupla Unidade Terapêutica (CPAF2). / Centrifugation is the main method used for fractionating Total Blood (TB) to blood components: Red Cells Concentrate (CR), Platelet Concentrates (PC), Plasma and Cryoprecipitate (also called Hemocomponents). In order to prevent or control bleeding, CPs obtained by centrifugation became the standard treatment of patients with low platelet counts. However, to obtain a therapeutic unit (TU), at Hemocentro RP, it is necessary to get together six PCs, forming a Pool of TBPC (PCP), exposing the receptor to several different donors. PC obtained by apheresis (CPAF), which is provided by a equipment that allows selective collection, achieve the dose necessary for the selection in just one donation. Based on the collection of internal data from the Hemocentro RP, the present study analyzed, through the methodology of ActivityBased Costing (ABC), the costs attributed to the different methodologies for the production of PCs knowing that in the Hemocenter RP PCs are dispensed as TU independent of the way of obtaining. The results obtained were used in the CostEffectiveness Analysis, showing collecting by TB Centrifugation producing PC is more cost-effective, however CPAF was almost as effective as in 2015, a great achievement of Dual Therapeutic Unit in a single donation (CPAF2).

Les plaquettes sanguines sont des cellules qui ont un rôle majeur au cours des processus de l’hémostase primaire et jouent un rôle primordial dans l’immunité innée mais aussi adaptative. Ces cellules anucléées ont une capacité sécrétoire très importante de facteurs solubles notamment de cytokines, de chimiokines (CK/CH) et de facteurs immunomodulateurs. L’émergence du rôle inflammatoire des plaquettes sanguines dans la communauté scientifique a soulevé de nombreuses questions auxquelles nous essayons de répondre dans ce manuscrit. La majorité de ces questions repose sur la capacité de ces cellules anucléées à répondre de manière régulée à des stimuli complexes. Nos investigations pour répondre à ces questions ont été réalisées dans un contexte transfusion sanguine. Au cours de nos travaux, nous avons mis en évidence la corrélation des profils de sécrétion plaquettaire avec les récepteurs membranaires et les voies de signalisations intraplaquettaires engagées. Les plaquettes expriment plusieurs récepteurs immunitaires sur leur surface notamment les « Pattern recognition receptors » (PRR) et des récepteurs aux CK/CH. Nous avons démontré et caractérisé la fonction d’un nouveau récepteur plaquettaire, le Siglec-7. Ce récepteur est localisé dans les granules a ; son expression sur la membrane est corrélée avec l’état d’activation plaquettaire. Le Siglec-7 a une avidité élevée avec les molécules composées d’α2,8-disialyl (NeuAcα2,8NeuAcα2,3Gal) et de α2,6-sialyl (Gal-b1,3[NeuAcα2,6]HexNAc) (comme les gangliosides GD2, GD3 et GT1b). L’engagement de ce récepteur peut induire l’apoptose plaquettaire par la voie intrinsèque et extramitochondriale. Ce processus nécessite l’engagement du récepteur GPIIbIIIa et P2Y1 et la signalisation de la voie de PI3k. Nous avons également étudié et mis en évidence une composante inflammatoire multifactorielle dans les effets indésirables des receveurs (EIR) et trouvé dans les concentrés plaquettaires (CP), plusieurs facteurs solubles ayant une valeur prédictive élevée pour la survenue des EIR, notamment le sCD40L et l’IL-13. Nous avons confirmé que la concentration de ces facteurs augmente au cours de temps de stockage des CP, étant, en partie, responsable du taux élevé de l’EIR des CP âgés. Enfin, en plus de la conservation, les processus de préparation des CP peuvent aussi avoir des impacts sur les propriétés inflammatoires des plaquettes. Ces travaux montrent que la réponse inflammatoire plaquettaire est régulée en fonction du stimulus, permettant d’argumenter sur le rôle présumé de sentinelle des plaquettes sanguines humaines. Ainsi, mes travaux s’inscrivent dans la ré-exploration de la fonction inflammatoire des plaquettes sanguines et l’étude du rôle des plaquettes comme cellules de l’immunité à composante inflammatoire / Blood platelets are non-nucleated cells and play a major role in primary hemostasis and a key role in inflammation, innate and adaptive immunity. They secrete a large variety of soluble factors including cytokines/chemokines (CK/CH) and immunomodulator factors. The emergence of their inflammatory role has raised numerous questions based on the ability of platelets to respond to complex stimuli. Our investigations to answer these questions were realized in the context of platelet component transfusion. In our study, we demonstrated the correlation between the platelet secretion of soluble factors with their membrane receptors and the signaling pathways involved. Platelets express many immune receptors on their surface, including "Pattern recognition receptors" (PRRs) and receptor for CK/CH. We discovered and characterized the function of a new platelet receptor, the Siglec-7. This receptor is located in the granules a and its expression is correlated to the platelet activation level. The Siglec -7 has a high avidity with the molecules composed of α2,8-disialyl (NeuAcα2,8NeuAcα2,3Gal) and of α2,6-sialyl (Gal-b1,3[NeuAcα2,6]HexNAc) (ganglioside GD2 , GD3 and GT1b). Stimulation of this platelet receptor may induce platelet apoptosis by the intrinsic and extramitochondrial pathway. This process requires the engagement of GPIIbIIIa and P2Y1 receptor and the PI3K pathway. We also demonstrated a multifactorial inflammatory component in adverse effects issuing from platelets transfusion, and identified many soluble factors which have a high predictive value of Acute Transfusion Reactions (ATR) occurrence, such as sCD40L and IL- 13. We confirmed that the concentration of these factors increases during storage time of platelet component (PC), being partly responsible for the high rate of ATR by old PC. Finally, in addition to the PC conservation, the process of PC preparation may also have impacts on the inflammatory properties of platelets. These studies showed that the platelet inflammatory response is regulated by the stimulus, explaining the sentinel role of human blood platelets. Therefore, my work contributes to the re-exploration of inflammatory function of these cells and studies their role as an immune cell with an inflammatory component

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor b1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In the development of this research, 15 New Zealand rabbits were used, with the aim of storing platelet-rich plasma (PRP) during 30 days refrigerated at 4-6oC. There was a preparation of 30 samples of PRP which were sorted in three equal groups. Every three days a sample would be removed for evaluation, before and after storage, for the number of platelets, mean platelet volume (MPV), pH of the plasma, aggregation post addition of calcic thromboplastin and for the presence of bacterial and fungal contamination. In the variables of number of platelets, there was no linear relationship in function of time, when comparing the number of platelets before x after storage, a statistical difference was observed. The hemogram MPV variables, before and after storage also did not relate with time, however there was a statistical difference in the comparisons of MPV on blood count x MPV before storage and of MPV before storage x MPV post storage. On the evaluation of the pH there was no influence of time on the variables, but statistical differences were found in the samples after storage in between the periods of 30 and 6; 30 and 24; 30 and 27 days. Platelet aggregation occurred within twenty seconds in all samples independent of storage time. There was no growth of bacteria or yeast in any sample, however mold growth occurred in the samples 21 days of storage on group one and three. It can be concluded that platelet-rich plasma of rabbits can be stored under refrigeration of 4 to 6 °C for maintaining the number of platelets, without significant pH alteration and absence of bacterial or fungal contamination during 18 days. / Utilizou-se 15 coelhos da raça Nova Zelândia para o desenvolvimento da pesquisa, com o objetivo de armazenar plasma rico em plaquetas (PRP) durante 30 dias sob refrigeração de 4-6ºC. Preparou-se 30 amostras de PRP que foram divididas em três grupos iguais. A cada três dias uma amostra foi retirada para avaliação antes e após o armazenamento quanto ao número de plaquetas, volume plaquetário médio (VPM), pH do plasma, agregação pós adição de tromboplastina cálcica e presença de contaminação bacteriana e fúngica. Nas variáveis número de plaquetas, não houve relação linear em função do tempo, na comparação do número de plaquetas pré x pós armazenamento, observou-se diferença estatística. As variáveis de VPM do hemograma, pré e pós armazenamento também não apresentaram relação com o tempo, porém obteve-se diferença estatística nas comparações VPM no hemograma x VPM pré armazenamento e VPM pré-armazenamento x VPM pós armazenamento. Na avaliação do pH não houve influência do tempo nas variáveis, mas houve diferença estatística nas amostras pós armazenamento entre os períodos 30 e 6; 30 e 24; 30 e 27 dias. A agregação plaquetária ocorreu dentro de vinte segundos em todas as amostras independente do tempo de estocagem. Não houve crescimento bacteriano e de leveduras em nenhuma amostra, porém ocorreu crescimento de bolores nas amostras 21 dias de armazenamento dos grupos um e três. Pode-se concluir que o plasma rico em plaquetas de coelhos pode ser armazenado sob refrigeração de 4 a 6ºC por manter o número de plaquetas, sem alteração significativa do pH e ausência de contaminação bacteriana e fúngica durante18 dias. / Mestre em Ciências Veterinárias

Trombocyttransfusion är en viktig behandlingsmetod för att antingen förebygga eller förhindra blödning. Trombocyter för transfusion (trombocytkoncentrat) framställs med två metoder: antingen genom aferesteknik eller genom att separera helblodet i erytrocytenhet, plasma, leukocytenhet och interim platelet unit (IPU). Sedan placeras IPU-enheter på en agitationsmetod. Efter det framställs trombocytkoncentrat genom att poola 4–6 IPU  tillsammans med platelet additive solution (PAS). Syftet med denna studie var att undersöka en ny agitationsmetod för framställning av trombocytkoncentrat, Trombomixer 307, och jämföra den med nuvarande agitationsmetoden, trombocytvagga. Dessutom att undersöka om införandet av Trombomixer 307 kan minska antal IPU-enheter som har  trombocytaggregat. Studien utfördes genom att placerade 100 IPU-enheter på trombocytvagga och 100 placerades på Trombomixer 307. Dessutom framställdes totalt 56 trombocytkoncentrat (28 från Trombomixer 307 och 28 från trombocytvagga) som sedan undersöktes genom kvalitetskontroll av platelet yield index-summa (PYI-värde), volymen, leukocytpartikalkoncentration (LPK) och trombocytpartikalkoncentration (TPK). Resultaten visade att 31 av de 100 IPU-enheterna på Trombomixer 307 hade trombocytaggregat, medan 47 av de 100 IPU-enheter från trombocytvagga hade trombocytaggregat. IPU-enheter på Trombomixer hade en signifikant färre antal IPU-enheter som hade trombocytaggregat jämfört med IPU-enheter från trombocytvagga (p = 0,029). Dessutom visade resultateten att trombocytkoncentrat från Trombomixer hade lägre LPK- och TPK-värde jämfört med trombocytkoncentrat från trombocytvagga. Trombocytkoncentrat från trombocytvagga visade bättre korrelation mellan PYI-summa och TPK-värde. Slutsatsen är att Trombomixer 307 uppfyller krav som en agitationsmetod för framställning av trombocytkoncentrat och att Trombomixer 307 minskar andelen  IPU-enheter som har bildat trombocytaggregat. Dock leder trombocytvagga till bättre kvalitet av trombocytkoncentrat. / Platelet transfusion is a treatment to prevent or stop bleeding. Platelets for transfusion (platelet-concentrate) are produced by separating the blood into erythrocyte-unit, plasma, leukocyte-unit, and interim platelet-unit (IPU). The IPUs are then placed on an agitation method. Platelet-concentrate is prepared by pooling 4–6 IPUs with platelet additive solution (PAS). The purpose of this study was to examine Trombomixer 307 as new agitation method for producing platelet-concentrate, and compare it with the current agitation method, the platelet-rocker. It is also investigated whether the implantation of the Trombomixer 307 can reduce the number of aggregated IPUs. The study was performed by placing 100 IPUs on the platelet-rocker and 100 IPUs on the Trombomixer 307. Additionally, 56 platelet-concentrates were prepared (28 from the Trombomixer 307 and 28 from the platelet-rocker), which then examined through quality control of the platelet yield index sum (PYI value), volume, leukocyte particle concentration (LPK) and platelet particle concentration (TPK). The results showed that 31 out of the 100 IPUs from Trombomixer and 47 of the 100 IPUs from the platelet-rocker were aggregated. IPUs from the Trombomixer had a significantly lower number of aggreged IPUs compared to IPUs from the platelet rocker (p = 0.029). The results also showed that platelet-concentrate from Trombomixer had lower LPK and TPK, while Platelet-concentrate from platelet-rocker showed better correlation between PYI sum and TPK. In conclusion, Trombomixer 307 fulfills requirements as an agitation method to produce platelet-concentrates and reduces the percentage of aggregated IPUs. However, platelet-rocker leads to better quality of platelet-concentrate.