La contamination bactérienne des concentrés plaquettaires (CPs) représente le risque le plus fréquent d'infection transmise par transfusion. Malgré les progrès techniques accomplis dans le dépistage des agents infectieux, le risque de transmission de ces agents par transfusion ne peut encore être considéré comme totalement maîtrisé. Nous avons pu développer une méthode de détection par la technique de MALDI-TOF. Notre protocole permet une détection dans un délai de 8 heures à de faibles concentrations bactériennes.Nous avons évalué les performances d’un automate d’hémoculture, le VersaTREK, par rapport au Bactec, qui est fréquemment utilisé par les centres de transfusion. Nos résultats démontrent son intérêt dans la détection de la contamination bactérienne des CPs. Afin de caractériser l’origine de la contamination bactérienne, nous avons comparé, dans une étude rétrospective, les espèces bactériennes identifiées dans les hémocultures des patients de l’hôpital de la Timone, à celles identifiées dans des poches de CPs contaminés. Les espèces responsables de la contamination des CPs sont essentiellement issues de la flore cutanée. Elles sont donc différentes des espèces responsables d’épisodes de bactériémies.Ainsi, ce travail propose de nouvelles méthodes de détection de la contamination bactérienne. La mise en œuvre de ces nouvelles stratégies de diagnostic couplées à une meilleure connaissance de la prévalence et de l’origine de la contamination bactérienne des CPs pourrait permettre d’améliorer davantage la prévention et la réduction de la morbi-mortalité induite par une transfusion de CPs. / Bacterial contamination of platelet concentrates (PCs) is the most common risk of transfusion-transmitted infection. Despite better control of the elements of the technical progress achieved in the detection of infectious agents, the risk of transmission of these agents by transfusion cannot yet be considered as totally mastered. We have developed a method for detecting PCs contamination by MALDI-TOF. To the best of our knowledge, this is the first time that MALDI-TOF is used in the detection of microbial contamination in a complex medium such as PCs. Our protocol allows detection within 8 hours at low bacterial concentrations. In a second step, we evaluated the performance of a blood culture machine, VersaTREK®, compared to Bactec®, frequently used by transfusion centers. Our results demonstrate its interest in the detection of bacterial contamination of PCs. In order to characterize the origin of bacterial contamination of PCs, we compared in a retrospective study, bacterial species identified in patients' blood cultures treated at the Timone hospital, with those identified in infected PC units. The species responsible for the contamination of the PCs are mainly resulting from the cutaneous flora and are therefore different from the species responsible for episodes of bacteremia.Thus, this work proposes new methods for detecting bacterial contamination. The implementation of these new diagnostic strategies coupled with a better knowledge of the prevalence and origin of bacterial contamination of PCs could further improve the prevention and reduction of both morbidity and mortality induced by blood transfusion of PCs.

Las plaquetas (PLTs) son protagonistas en la reparación de las heridas, ya que contienen factores de crecimiento (GFs), los cuales producen quimiotaxis, proliferación y diferenciación celular, neovascularización y deposición de matriz extracelular (ECM). Se ha propuesto la utilización de concentrados de plaquetas (PCs) autólogos para acelerar la reparación de las heridas y estimular la capacidad de regeneración de los tejidos lesionados. En los capitulos III y IV de esta tesis se presentan dos estudios clínicos sobre el efecto de un concentrado plaquetario (PC) autólogo en caballos con patología músculo-esquelética grave. Este PC fue preparado por medio de una nueva técnica de doble centrifugación en tubo. Una caracterización celular y molecular de este PC se presenta en el cápitulo V de ésta tesis. Se evaluó el efecto de la inyección intra-articular del PC en 7 caballos con enfermedad articular grave clasificada así según el grado de cojera (DL) y la efusión sinovial (JE). El PC produjo una mejoría estadísticamente significativa del DL y JE (p<0.05). La mejoría más notable fue observada a los dos meses de finalizado el tratamiento y permaneció hasta 8 meses después (ver cápitulo III). En el capitulo IV se evaluó el efecto del PC en 7 caballos con lesiones tendinosas (tendonitis del tendón flexor digital superficial (SDFT) y ligamentosas (desmitis del ligamento suspensorio (DSL)). Todos los caballos mejoraron significativamente su DL y la respuesta a la prueba de flexión (p<0.05). Los registros ultrasonográficos mejoraron en los caballos con tendinopatías, pero no cambiaron en los que tenían desmopatías. Dos caballos con tendinitis retornaron con exito a su nivel de competición sin recidivar. Un caballo con tendinosis crónica recidivó. El resto de pacientes con desmopatías volvieron a su nivel de competición pre-lesión. Se obtuvieron un promedio de 250 ± 71.8 x 106 plaquetas, 8.68 ± 3.78 leucocitos x 106 y 12515 ± 2443 pg de TGF- ?1/ml de PC. No se observaron signos clínicos adversos asociados con el tratamiento. En el cápitulo V se realizó un análisis celular y molecular del PC usado clínicamente (PC¬C), sangre entera y 4 fracciones de PCs adicionales (PC-A, PC-B y PC-D), los cuales son obtenidos durante la elaboración del PC-C. El objetivo fue evaluar el método de obtención de este PC, en el que son necesarios dos periodos de centrifugación. Todas las muestras fueron analizadas mediante citometría de flujo y determinación de los niveles de TGF- ?1. Las concentraciones de plaquetas para PC-A, PC-B, PC-C y PC-D fueron un 45%, 44%, 71% y 21%, respectivamente más altas en comparación con cada PC y la sangre entera. Las concentraciones de TGF- ?1 para PC-A, PC-B, PC-C y PC-D fueron un 38%, 44%, 44% and 37%, respectivamente más altas en comparación con cada PC y la sangre entera. Se concluyó que el método empleado para concentrar plaquetas es valido para producir PCs con niveles potencialmente terapéuticos de TGF-?1. Los resultados obtenidos en esta tesis abren un nuevo y prometedor campo de investigación para conocer los efectos clínicos y moleculares de los PCs en caballos con enfermedad crónica músculo-esquelética. Los resultados que se puedan obtener en caballos serán de gran valor para conocer el uso potencial de los PCs autólogos en seres humanos con similares patologías. / Platelets (PLTs) play a central role in wound healing, since they contain growth factors (GFs), which produce chemotaxis, cellular proliferation and differentiation, neovascularization, and extra¬cellular matrix (ECM) deposition. The use of autologous platelet concentrates (PCs) has been proposed to accelerate wound repair and to stimulate the regenerative capacity of injured tissues. Two clinical studies about the effect of an autologous platelet concentrate (PC) in horses with severe musculoskeletal pathology are presented in chapters III and IV of this thesis. The PC was prepared by a novel double centrifugation tube method. A cellular and molecular characterization of this PC is presented in chapter V of this thesis.The effect of the intraarticular injection of this PC in 7 horses with severe joint disease was evaluated on the basis on degree of lameness (DL) and joint effusion (JE). When PCs were injected into the joint a statistically significant improvement in both DL and JE (p<0.05) were observed. The most marked improvement was maximun 2 months after the last injection and persisted up to 8 months later (see chapter III). The clinical effect of PC injection in 7 horses with soft tissue musculoskeletal injuries namely: SDFT tendinopathy and desmopathy of the susensory ligament (DSL) was also evaluated (see the chapter IV). All the horses presented a clinical and a statistical (p<0.05) decrease of the DL and the response to flexion test. Ultrasonic appearance improved in the horses with SDFT lesions, but remained the same in the horses with DSL. Two horses with acute SDFT tendinopathy returned successfully to competition level without reinjury. One horse with chronic SDFT tendinopathy relapsed. The rest of the horses with DSL returned successfully to competition level without reinjury. A mean of 250 ± 71.8 x 106 platelets, 8.68 ± 3.78 leucocytes x 106, and 12515 ± 2443 pg TGF-?1 were obtained per ml of the PC. No adverse reactions resulted from this treatment. A cellular and a molecular study of the PC clinically used in this thesis (PC-C),of whole blood and of three additional PCs (PC-A, PC-B, and PC-D) obtained during the PC-C preparation were performed (see the chapter V) to compare the single and the double centrifugation tube methods for concentrating equine platelets. Whole blood and the 4 PCs were analyzed using flow cytometry for cellular quantification and determination of TGF- ?1 in all the samples. Platelet concentrations for PC-A, PC-B, PC-C and PC-D were 45%, 44%, 71% and 21% higher, respectively, compared to the same values for citrated whole blood samples. TGF- ?1 concentrations for PC-A, PC-B, PC-C and PC-D were 38%, 44%, 44% and 37% higher, respectively, compared to citrated whole blood sample values. In conclusion, the single and double centrifugation tube methods are reliable methods for concentrating equine platelets and obtaining potentially therapeutic TGF- ?1 levels. The results obtained in this thesis open a new encouraging research field on clinical and molecular effects of PCs in equine chronic musculoskeletal pathology. The future potential results obtained in horses can be of key value to determine the potenetial use of autologous PCs in human beings with chronic musculoskeletal pathology.

Bacterial contamination of platelet concentrates (PCs) represents the highest post-transfusion infectious risk. The skin flora bacterium Staphylococcus epidermidis has been reported to be the predominant aerobic contaminant of PCs. The Ramirez' group has shown that S. epidermidis can form surface-attached bacterial aggregates known as biofilms, and can outcompete other coagulase-negative staphylococci, such as Staphylococcus capitis, in PCs. The ability of S. epidermidis to form biofilms has been linked to increased pathogenicity and missed detection during PC screening with an automated culture system (BacT/ALERT). This thesis aimed at investigating the proliferative advantage and resistance mechanisms displayed by S. epidermidis in the PC milieu. Furthermore, in an effort to enhance PC safety for transfusion patients, I studied the anti-biofilm properties of essential oils and antimicrobial peptides (AMPs). My studies aimed at improving PC safety by focussing on both the point of introduction of bacterial contaminants (blood collection), and the stage at which bacterial contaminants can form biofilms and proliferate (PC storage). S. epidermidis can be found in the skin of blood donors as biofilms, which are resistant to the blood donor skin disinfectant currently used by Canadian Blood Services, chlorhexidine-gluconate and isopropyl alcohol (CHG-IPA). Here, several plant-extracted essential oils were evaluated for their ability to enhance the anti-biofilm activity of CHG-IPA. Data revealed that the Lavandula multifida oil and its main component (linalool) greatly enhanced the activity of CHG-IPA against S. epidermidis biofilms. Furthermore, the ability of a combination of three synthetic AMPs to inhibit S. epidermidis biofilm formation during PC storage was assessed These results showed that the combination of AMPs could inhibit biofilm formation but was ineffective against pre-formed S. epidermidis biofilms. The accumulation associated protein (Aap) encoded by the aap gene, found in most S. epidermidis strains and absent in S. capitis, plays a role in biofilm formation. When S. epidermidis aap is transformed into S. capitis, this bacterium displayed increased biofilm formation and proliferated to higher concentrations compared to untransformed S. capitis and to a S. epidermidis aap deletion mutant. Based on these results, aap appears to play a role in providing S. epidermidis a proliferative advantage in PCs by enhancing biofilm formation. Lastly, the GraRS system and SepA were studied for their role in S. epidermidis resistance to platelet-derived AMPs using the synthetic AMP PD4 as a model molecule. Results indicate that the GraS mechanism is involved in resistance towards PD4. The work presented in my thesis provides further insights into why S. epidermidis has a proliferative advantage in the PC storage environment and allows for the proposal of alternative methods to enhance PC safety for transfusion patients.

Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics) / In recent years, the development and use of autologous platelet rich concentrates (PC's) has gained traction within the rapidly progressive, multidisciplinary field of regenerative medicine. A PC subtype, marketed as advanced platelet rich fibrin (A- PRF), is a recent advancement of the original PRF protocol and promoted as a "blood concentrate" containing platelets, leukocytes, circulating stem cells and endothelial cells. A-PRF in the form of membranes, plugs, or even shredded particulates are increasingly being used as surgical adjuncts in areas of previous infection or left exposed within the microbial rich oral environment. Although recent literature has noted the biologic benefits of this material within the context of wound healing and regeneration, the antimicrobial potential of APRF has remained unexplored. The aim of this investigation is to determine if A-PRF displays antimicrobial activity against microbes of the oral cavity with a null hypothesis that its activity is no different to a clot of unprocessed venous blood. Methodology: A-PRF and whole blood samples were obtained from consenting individuals and utilised to conduct an in-vitro agar disk diffusion investigation to determine their antimicrobial activity. Standardised samples of A-PRF, unprocessed clotted blood and 0.2% chlorhexidine gluconate (CHX) were tested against organisms cultured from fresh oral rinse samples and pure cultures of candida albicans, streptococcus mutans, staphylococcus aureus and enterococcus faecalis. The antimicrobial activity was assessed in accordance to the established principles of the agar disk diffusion method and measurement of inhibition zones. Results: A-PRF displayed antimicrobial activity against all of the individual organisms tested within this study following a 24 hour incubation period. However, no significant differences were noted between A-PRF and a natural clot of blood when tested against cultures of the oral rinse sample. Finally, the antimicrobial activity of A-PRF is significantly inferior to an equal volume of the CHX preparation. Conclusion: Although A-PRF displays antimicrobial activity; its strength, spectrum and biologic activity within a polymicrobial environment requires further investigation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Blood transfusions have always been great support for monitoring the treatment of cancer, especially patients in the sectors of hematology-oncology, but still stand out as a major source of transmission of infectious diseases, today the bacteria. Platelet concentrates (PCs) are the blood components with higher frequency of bacterial contamination and are responsible for the vast majority of septic transfusion reactions. Bacterial infection is a major cause of morbidity and mortality due to platelet transfusions. Gram-positive bacteria, particularly Staphylococcus epidermidis, are mainly responsible for the contamination of PCs. Therefore, this study aimed to determine the prevalence of bacterial contamination in PCs, the isolation and identification of microorganisms found. In addition, we sought to identify septic transfusion reactions and to evaluate conventional techniques for bacterial detection. A total of 691 samples of PCs (665 whole blood-derivaded platelets and 26 apheresis platelets) was analyzed. These samples were from the Blood Center of the State of Rio Grande do Sul (HEMORGS), located in Santa Maria, Rio Grande do Sul. Were employed culture techniques qualitative, quantitative, daily growth and also metabolic markers for the detection of bacteria. Tests for identification of microorganisms were made by conventional techniques and the phenotypic and genotypic screening of septic reactions was performed by passive haemovigilance. The prevalence of bacterial contamination found in this study was 1.47% and S. epidermidis bacteria was responsible for all contamination. This prevalence is considered high when compared to recent studies conducted in other countries, in Brazil we have very few studies in this area. All samples were contaminated platelet random. The test metabolic markers were nonspecific for the detection of bacteria. There was a great difficulty in performing the daily growth of the technical methodology make it impossible to be proposed to HEMORGS. Through haemovigilance passive transfusion reactions were characterized and it was possible to confirm the occurrence of a septic transfusion reaction. Therefore, we suggest the combination of methodologies for the detection of bacterial contamination screening of PCs, since it is a health problem. The combination of culture systems can reduce the risks of transfusions contaminated CPs. We believe that the septic transfusion reactions can be minimized with early recognition by the clinical team. / As transfusões sanguíneas sempre foram o grande suporte para o seguimento do tratamento de câncer, principalmente em pacientes dos setores de hematologia-oncologia, mas ainda destacam-se como uma das principais fontes de transmissão de doenças infecciosas, atualmente as bacterianas. Os concentrados plaquetários (CPs) são os hemocomponentes com maior frequência de contaminação bacteriana e são os responsáveis pela grande maioria das reações sépticas transfusionais. A infecção bacteriana é uma das principais causas de morbidade e mortalidade decorrentes de transfusões plaquetárias. Bactérias gram-positivas, em especial Staphylococcus epidermidis, são majoritariamente os responsáveis pela contaminação de CPs. Sendo assim, este estudo objetivou determinar a prevalência da contaminação bacteriana em CPs, o isolamento e a identificação dos microrganismos encontrados. Além disso, buscou identificar reações sépticas transfusionais bem como avaliar técnicas convencionais de detecção bacteriana. Um total de 691 amostras de CPs (665 plaquetas randômicas e 26 plaquetaféreses) foi analisado. Estas amostras foram provenientes do Hemocentro do Estado do Rio Grande do Sul (HEMORGS), localizado na cidade de Santa Maria, Rio Grande do Sul. Empregaram-se técnicas de cultura qualitativa, quantitativa, de crescimento diário e também marcadores metabólicos para a detecção das bactérias. Os testes de identificação dos microrganismos isolados foram feitos por técnicas convencionais fenotípicas e genotípicas e o rastreio das reações sépticas foi realizado por hemovigilância passiva. A prevalência da contaminação bacteriana encontrada neste estudo foi de 1,47% e S. epidermidis foi a bactéria responsável por todas as contaminações. Esta prevalência é considerada alta quando comparada a estudos recentes realizados em outros países. No Brasil contamos com pouquíssimos estudos nesta área. Todas as amostras contaminadas foram de plaquetas randômicas. O ensaio de marcadores metabólicos foi inespecífico para a pesquisa de bactérias. Ocorreu uma grande dificuldade na execução da técnica do crescimento diário inviabilizando essa metodologia para ser proposta ao HEMORGS. Através da hemovigilância passiva as reações transfusionais foram caracterizadas e foi possível confirmar a ocorrência de uma reação séptica transfusional. Sendo assim, sugerimos a associação de metodologias para a detecção da contaminação bacteriana na triagem dos CPs, uma vez que, se trata de um problema de saúde. A associação de sistemas de cultura pode reduzir os riscos de transfusões de CPs contaminados. Acreditamos que as reações sépticas transfusionais podem ser minimizadas com o seu reconhecimento precoce pela equipe clínica. Palavras-chaves: Concentrados plaquetários; reação transfusional; sepse; bactérias

A transfusão de plaquetas pode ser terapêutica quando indicada para interrupção de sangramento ativo ou profilática com o objetivo de prevenir hemorragias. Os concentrados de plaquetas apresentam um volume menor quando comparado ao sangue fresco total e ao plasma rico em plaquetas elevando a contagem plaquetária rapidamente minimizando, assim, a transfusão de componentes desnecessários e o risco de reações transfusionais o que no entanto podem ocorrer. Em cães, a literatura é escassa em relação à ocorrência de reações transfusionais frente ao emprego de concentrados de plaquetas. Sendo assim, este estudo tem como objetivo documentar a ocorrência de efeitos adversos em relação à administração de concentrados de plaquetas em cães em caráter clínico. Realizou-se a transfusão de concentrados de plaquetas em vinte e oito cães trombocitopênicos com manifestações clínicas de diáteses hemorrágicas ou antes de procedimentos cirúrgicos. Estes hemo componentes foram preparados por centrifugação diferencial seriada do sangue total padronizando-se o fracionamento sanguíneo para cães. Determinou-se um controle de qualidade destes concentrados monitorando-se o pH, níveis de glicose, temperatura de armazenamento, rendimento plaquetário, contaminação bacteriana e swirling. Adotou-se uma proporção de 1 concentrado de plaquetas para cada 10 Kg. Dentre os vinte e oito cães transfundidos, presenciou-se a ocorrência de reações adversas em dez pacientes (35,7%) caracterizadas por manifestações gastrintestinais (6/28), seguidas das alérgicas (5/28) e inflamatórias (2/28). A hipertermia, principal reação relatada em seres humanos, foi constatada apenas em um paciente. O incremento plaquetário observado em 1 e 24 horas foi considerado satisfatório na maioria dos cães transfundidos sendo, em média, de 38.000 e 31.000 plaquetas/&micro;L respectivamente. / Platelet transfusion may be either therapeutic or praphylactic when used to interrupt active bleeding or to avoid it, respectively. Plate1et concentrates have a smaller volume when compared to fresh whole blood and to plate1et-rich plasma. Their use quickly increases plate1et counts and, therefore, minimizes unnecessary transfusion of blood components and subsequent transfusion reactions. Literature is scarce concerning transfusion reactions after the administration of platelet concentrates in dogs. Therefore, the objective of this study was to record c1inical adverse effects after its administration in dogs. Platelet concentrates were administered in twenty-eight thrombocytopenic dogs with c1inical signs of hemorrhage or before surgical procedures. These hemocomponents were prepared by serial differential centrifugation of the whole blood standardizing the whole blood fractionation method to dogs. Quality contraI of platelet concentrates was performed by monitoring pH, glucose, storage temperature, platelet count, bacterial contamination and swirling. The dose administrated was one unit per 10 kg. Transfusion reactions occurred in ten dogs (35.7%), characterized by gastrointestinal manifestations (6/28), followed by allergic (5/28) and inflammatory reactions (2/28). Hyperthermia, the most frequent1y reported reaction in humans, was observed in only one dog. The platelet increment observed 1 and 24 hours post-transfusion was considered satisfactory in the majority of the transfused dogs, being, on the average, 38,000 and 31,000 platelets/ &micro;L, respectively.

Bacterial contamination of platelet concentrates (PCs) poses the highest transfusion-associated infectious risk with skin flora, such as Staphylococcus epidermidis and Staphylococcus capitis, being the predominant contaminants. These bacteria are able to form surface-attached aggregates or biofilms, which are present in the skin of healthy blood donors and can subsequently be isolated from contaminated PCs. Disinfection of the venipuncture area before donation with a combination of 2% chlorhexidine-gluconate and 70% isopropanol is used at Canadian Blood Services. However, not all bacteria are eliminated during skin disinfection since contaminated PCs are still captured during routine PC screening. In this thesis, the ability of biofilm-forming S. epidermidis and S. capitis to resist the currently used disinfectants was explored. It was demonstrated that although a combination of chlorhexidine and isopropanol has a bactericidal effect, it is unable to completely eradicate skin flora biofilms. Several countries have implemented Pathogen Inactivation Technologies (PITs) as a measure to help control transfusing bacterially-contaminated PCs by exposing PC units to ultra violet light. However, no investigations have been done to evaluate the ability of PITs against bacterial biofilms, which was one of the objectives of this thesis. Data revealed that the efficacy of a currently used PIT, the Mirasol® system, is similar for S. epidermidis present in PCs produced from whole blood inoculated with biofilm or non-biofilm cells. However, treatment effectiveness was strain dependent. In conclusion, further investigation to improve donor skin disinfection and PITs should be considered. Surveillance at Canadian Blood Services shows that contamination rates in single-donor apheresis PCs (Aph-PCs) is generally higher than in four-donor buffy coat platelet pools (BC-PCs). This study investigated whether the BC-PC production method contributes to this observation as BC-PCs are derived from WB that is left to rest overnight while Aph-PCs are collected directly from the donor. Data showed that WB hold during BC-PC production does not have a broad-spectrum bactericidal effect and therefore other factors contribute to low rates of contamination in BC-PCs. The work presented in this thesis provides an insight to bacterial residence and persistence during blood product manufacturing and makes suggestions for PC safety improvements.

A transfusão de plaquetas pode ser terapêutica quando indicada para interrupção de sangramento ativo ou profilática com o objetivo de prevenir hemorragias. Os concentrados de plaquetas apresentam um volume menor quando comparado ao sangue fresco total e ao plasma rico em plaquetas elevando a contagem plaquetária rapidamente minimizando, assim, a transfusão de componentes desnecessários e o risco de reações transfusionais o que no entanto podem ocorrer. Em cães, a literatura é escassa em relação à ocorrência de reações transfusionais frente ao emprego de concentrados de plaquetas. Sendo assim, este estudo tem como objetivo documentar a ocorrência de efeitos adversos em relação à administração de concentrados de plaquetas em cães em caráter clínico. Realizou-se a transfusão de concentrados de plaquetas em vinte e oito cães trombocitopênicos com manifestações clínicas de diáteses hemorrágicas ou antes de procedimentos cirúrgicos. Estes hemo componentes foram preparados por centrifugação diferencial seriada do sangue total padronizando-se o fracionamento sanguíneo para cães. Determinou-se um controle de qualidade destes concentrados monitorando-se o pH, níveis de glicose, temperatura de armazenamento, rendimento plaquetário, contaminação bacteriana e swirling. Adotou-se uma proporção de 1 concentrado de plaquetas para cada 10 Kg. Dentre os vinte e oito cães transfundidos, presenciou-se a ocorrência de reações adversas em dez pacientes (35,7%) caracterizadas por manifestações gastrintestinais (6/28), seguidas das alérgicas (5/28) e inflamatórias (2/28). A hipertermia, principal reação relatada em seres humanos, foi constatada apenas em um paciente. O incremento plaquetário observado em 1 e 24 horas foi considerado satisfatório na maioria dos cães transfundidos sendo, em média, de 38.000 e 31.000 plaquetas/&micro;L respectivamente. / Platelet transfusion may be either therapeutic or praphylactic when used to interrupt active bleeding or to avoid it, respectively. Plate1et concentrates have a smaller volume when compared to fresh whole blood and to plate1et-rich plasma. Their use quickly increases plate1et counts and, therefore, minimizes unnecessary transfusion of blood components and subsequent transfusion reactions. Literature is scarce concerning transfusion reactions after the administration of platelet concentrates in dogs. Therefore, the objective of this study was to record c1inical adverse effects after its administration in dogs. Platelet concentrates were administered in twenty-eight thrombocytopenic dogs with c1inical signs of hemorrhage or before surgical procedures. These hemocomponents were prepared by serial differential centrifugation of the whole blood standardizing the whole blood fractionation method to dogs. Quality contraI of platelet concentrates was performed by monitoring pH, glucose, storage temperature, platelet count, bacterial contamination and swirling. The dose administrated was one unit per 10 kg. Transfusion reactions occurred in ten dogs (35.7%), characterized by gastrointestinal manifestations (6/28), followed by allergic (5/28) and inflammatory reactions (2/28). Hyperthermia, the most frequent1y reported reaction in humans, was observed in only one dog. The platelet increment observed 1 and 24 hours post-transfusion was considered satisfactory in the majority of the transfused dogs, being, on the average, 38,000 and 31,000 platelets/ &micro;L, respectively.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.