HPA1a är ett antigen på trombocytytan som kan orsaka alloimmunisering, såsom neonatal alloimmun trombocytopeni och post-transfusion purpura, vilket kan ge svåra blödningssymptom. I fall med antikroppar mot HPA1a måste kompatibla trombocyter finnas tillgängliga. Syftet med studien var att etablera en flödescytometrisk screeningmetod för fenotypning av HPA1a antigen på trombocyter samt att finna HPA1a negativa blodgivare. Före flödescytometrisk analys poolades två till fem blodprover samman till ett prov och vid förekomst av HPA1a negativa trombocyter i poolen analyserades proverna individuellt. Fluorokrommärkta anti-humana antikroppar mot CD42a och CD61 användes för att särskilja HPA1a negativa trombocyter från HPA1a positiva. Totalt fenotypades 177 blodprover varav sju (4%) typades som HPA1a negativa. Av de sju fynden genotypades fyra vid ett externt laboratorium vilket bekräftade att de var HPA1a negativa. Flödescytometrisk screening av HPA1a är snabb, pålitlig och lämplig för storskalig screening. För att fastställa prevalensen av HPA1a negativa individer behöver mer omfattande studier utföras där en större population ingår. Att ha flera HPA1a negativa blodgivare registrerade ger möjligheten att hjälpa patienter i andra regioner. / HPA1a is an antigen on the platelet surface that can cause alloimmunization, such as neonatal alloimmune thrombocytopenia and post transfusion purpura, which can cause severe bleeding symptoms. In case of antibodies against HPA1a, compatible platelets must be available. The purpose of the study was to establish a flow cytometric screening method for phenotyping HPA1a antigen on platelets and to find HPA1a negative donors. Before the flow cytometric analysis, two to five blood samples were pooled into one sample and in the presence of HPA1a negative platelets in the pool, the samples were analyzed individually. Fluorochrome –labeled anti-human antibodies to CD42a and CD61 were used to distinguish HPA1a negative platelets from HPA1a positive. A total of 177 blood samples were phenotyped, of which 7 (4%) were HPA1a negative. Of the seven findings, four samples were genotyped at an external laboratory confirming that they were HPA1a negative. Flow cytometric screening of HPA1a is fast, reliable and suitable for large scale screening. In order to determine the prevalence of HPA1a negative individuals, more extensive studies need to be performed involving a larger population. By having many registered HPA1a negative donors, it can provide opportunities to help patients in other regions.

(v AJ) The aim of this diploma thesis was namely to find out whether the saline solution, intended for the continuous flushing of arterial catheters, is as effective in preventing arterial blockage as compared to saline solution with heparin. In this diploma thesis I also deal with the comparison of material consumption by using both methods of flushing arterial sets and the financial costs associated with them. Patients with diagnosis of sepsis or septic shock were included in the study. Pseudorandomization was used to group 52 patients into either experimental and control group. The testing was performed using a non-parametric Wilcoxon two-assay test and Fisher precision test. Based on the results of my study, regard to the occurrence of complications (catheter closure) an arterial catheter flushing by saline solution is as effective as flushing by a saline solution with heparin. My research also confirmed that the use of saline solution as a flushing solution is less costly (regarding material) and therefore it is advisable to use saline solution for flushing arterial catheters. Based on my study, saline solution is now used as a flushing solution of arterial catheters at an anesthesiology-resuscitation department, where only a saline solution with heparin was used for the flushing. Using saline...

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Thrombotic microangiopathies (TMA) are disorders characterized by microangiopathic hemolytic anemia, thrombocytopenia, and microthrombi leading to organ dysfunction. Atypical hemolytic uremic syndrome (aHUS) is a rare subtype of TMA mediated by complement dysregulation. We present a case of a 59-year-old female who presented with acute kidney injury and mild thrombocytopenia but with normal hemoglobin. We highlight the importance of prompt diagnosis of aHUS and initiating appropriate treatment with eculizumab.

Immune thrombocytopenia (ITP) is the consequence of a complex, still incompletely understood immunological dysregulation. Proposed mechanisms include autoantibody-induced platelet destruction, impaired platelet production as well as abnormalities in T-cell immunity, such as T helper cells (Th1) polarization, a high proportion of Th17 cells, and a reduced number of regulatory T cells. Although the etiology of ITP is incompletely understood and considered multifactorial in most cases, genetic variants are thought to play a key role in susceptibility to ITP, especially in persistent or chronic ITP. Efforts are currently underway to uncover possible predisposing genetic factors for the development of ITP. Single-nucleotide polymorphisms and copy number variations have been identified in several immune-related genes, such as cytokine genes, Fcγ receptor genes or T-cell costimulation genes, and have been associated with patients’ susceptibility to ITP. However, because of the clinical heterogeneity and low incidence of ITP it remains challenging to perform genetic analyses with sufficiently large sample size within informative patient populations, highlighting the need for collection of well-annotated biomaterials in clinical trials or registry projects. Another significant challenge is to go beyond performing association studies alone and to establish genotype-phenotype associations, thus proving causality between a genetic alteration and ITP pathogenesis. This review summarizes our current knowledge on genetic alterations identified as potential predisposing factors for the development of ITP in adults, thereby addressing signaling pathways considered critical for ITP pathogenesis.

Les Thrombopénies Induites par l’Héparine (TIH) sont une complication sévère des traitements par l’héparine dues à des IgG qui ciblent le facteur plaquettaire 4 modifié par l’héparine (FP4/H) et induisent une activation cellulaire via FcγRIIA, conduisant à des complications thrombotiques. Nous avons caractérisé 5B9, IgG1 monoclonale chimérique anti-FP4/H mimant parfaitement les anticorps de TIH et qui est donc un excellent outil pour étudier la physiopathologie des TIH. La pathogénicité des anticorps (Ac) de TIH implique leur fixation aux FcγR. Nous avons montré que le clivage de la région charnière des IgG de TIH par IdeS inhibe ces interactions IgG-FcγR et supprime la pathogénicité des Ac. Nous avons aussi construit un Antibody-Drug Conjugate (ADC) antithrombotique, en bioconjuguant le tirofiban (inhibiteur de l’agrégation plaquettaire) et 5B9 déglycosylé grâce à un linker clivable par la thrombine, protéase générée en excès lors d’une TIH. / Heparin Induced Thrombocytopenia (HIT) is a rare but severe complication of heparin treatments. HIT is due to IgG antibodies specific to platelet factor 4 modified by heparin (PF4/H), which activate blood cells, (especially platelets) after binding to FcγRIIA, this process explaining frequent thrombotic complications. We characterized 5B9, a chimeric IgG1 targeting PF4/H and which fully mimics human HIT antibodies. Therefore, 5B9 is a perfect tool for studying the physiopathology of HIT. IgG antibodies to PF4/H are pathogenic by interacting with FcγR. In this regard, we showed that cleavage by IdeS, a bacterial protease, of the hinge of anti-PF4/H IgG, fully suppressed their pathogenicity. Furthermore, we designed an antithrombotic Antibody-Drug Conjugate that combined tirofiban, a GPIIbIIIa inhibitor with deglycosylated 5B9 using a thrombin cleavable linker.

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INTRODUÇÃO: A HPN é uma patologia na qual a atividade do sistema complemento, sem oposição, leva a complicações sistêmicas. Ela é caracterizada por anemia hemolítica adquirida com hemoglobinúria intermitente, falência medular e fenômenos tromboembólicos (TE). A trombose venosa é a sua principal causa de mortalidade, entretanto o seu mecanismo fisiopatológico é apenas parcialmente elucidado. O grande número de pacientes com trombocitopenia dificulta o manejo da profilaxia antitrombótica secundária e primária. Optou-se por evidenciar o desequilíbrio hemostático associado ao clone HPN através de um teste de avaliação global da coagulação. MÉTODOS: Para a detecção do potencial hemostático de cada indivíduo foi utilizado um ensaio fluorogênico de geração de trombina, em amostra de plasma pobre em plaquetas na presença e na ausência de trombomodulina (TM). A eficiência na redução do potencial de trombina endógeno (ETP) e da concentração máxima de trombina (pico) pela TM foi utilizada para a identificação do estado de hipercoagulabilidade. O tempo para o início da geração de trombina (tempo de latência) e para a concentração máxima de trombina foram utilizados para a detecção do fenótipo hemorrágico. Os indivíduos foram categorizados em três grupos: HPN, se clone HPN >= 10%; anemia aplástica idiopática adquirida ou associada a clone < 10%, e normais. Os pacientes e controles foram submetidos a avaliação laboratorial que incluiu pesquisa de trombofilia (TB) e do clone HPN, hemograma, testes habituais de avaliação da hemostasia, e no grupo de pacientes análise bioquímica. Os participantes foram avaliados para a identificação da presença de fatores de risco para TE através de questionário. A análise dos resultados foi realizada em duas fases: a primeira incluiu apenas indivíduos com pesquisa de TB negativa e sem fatores de risco para TE; a segunda, realizada apenas no grupo de pacientes, também incluiu indivíduos em uso de contraceptivo hormonal, diagnóstico de infecção assintomática, evento de TE associado a fator de risco temporário, em período superior a 1 ano da inclusão no estudo, e com pesquisa de TB positiva. Esta última fase da análise teve como objetivo incluir um maior número de pacientes com o diagnóstico destas patologias, de baixa prevalência populacional. RESULTADOS: A presença do clone >= 10% foi associada à ineficiência da ação da TM em reduzir o ETP e o pico. O primeiro, de maior relevância científica e clínica, apresentou correlação positiva e negativa, respectivamente, com a atividade do fator von Willebrand (FvW:RCo) e com níveis plasmáticos de proteína C (PC). O grupo HPN apresentou menor tempo para atingir o pico. Na segunda fase, o tempo de latência apresentou correlação negativa com o número de plaquetas no grupo HPN, e houve correlação positiva do clone com a ineficiência da ação da TM na redução do ETP. CONCLUSÕES: o teste de geração de trombina é eficaz na detecção do fenótipo protrombótico associado ao clone HPN. As correlações encontradas com o FvW:RCo e a PC sugerem que a ativação endotelial e o sistema da PC, respectivamente, podem estar comprometidos. A inflamação secundária à ativação do sistema complemento pode levar à redução de expressão endotelial da TM e do receptor da PC. Entretanto os nossos achados, associados à descrição recente da redução de expressão e de atividade da TM, secundária à depleção de óxido nítrico (em estudos com estatinas), podem justificar a característica agressiva da trombofilia na HPN, e o desenvolvimento de TE mesmo nos pacientes em anticoagulação oral. Os parâmetros que avaliam o \'tempo\' no trombograma (tempo de latência e tempo para o pico) podem auxiliar na identificação do risco hemorrágico eventualmente associado à HPN / INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. The pathology is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic event (TE). Venous thrombosis is the main cause of death, however, its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We chose to demonstrate the hemostatic unbalance associated with PNH clone through a global evaluation of the coagulation test. METHODS: To detect the hemostatic potential, we used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without throbomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential (ETP) and of the upper limit of thrombin concentration (peak) was done to identify the hypercoagulable state. Times to initiate thrombin generation (latency time-LT) and for reaching the peak were used to identify hemorrhagic phenotypes. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%), and controls. Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood test, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TE through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TE; the second step, done only in patients, included individuals using hormonal contraceptive, diagnosis of any asymptomatic infection, TE associated to temporary risk factors, and which have occurred in a period longer than one year since inclusion in the study, and positive TB. The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. RESULTS: The presence of the clone >= 10% was associated with TM inefficiency in reducing the ETP (ETP+TM) and peak. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP+TM and the activity of the von Willebrand factor (FvW:RCo), whereas a negative correlation was observed with the levels of protein C (PC). The PNH group presented the shortest time to reach the peak. In the second step of the analysis, the LT showed negative correlation with platelet counts in the PNH group, whereas a positive correlation between the clone and ETP+TM was observed. CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The correlation found with both FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Secondary inflammation to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor. However, our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statin) may explain the aggressive nature of thrombophilia in PNH and the development of TE in these patients, even in those taking oral anticoagulants. The parameters that measure the time of thrombin generation may help identify the hemorrhagic risk that might be associated with PNH

L’objectif de cette thèse est d’étudier, à l’aide de modèles mathématiques, le mécanisme de régulation qui permet au corps de maintenir une quantité optimale de plaquettes sanguines. Le premier chapitre présente le contexte biologique et mathématique. Dans un second chapitre, un modèle pour la mégacaryopoïèse est introduit qui suppose une régulation ponctuelle par le nombre de plaquettes du taux de différentiation des cellules souches vers la lignée mégacaryocytaire et du nombre de plaquettes produites par mégacaryocyte. Nous montrons que la dynamique de ce modèle est régie par une équation différentielle à retard x'(t) = -?x(t)+f(x(t))g(x(t-t)), et nous obtenons ensuite de nouvelles conditions suffisantes pour la stabilité et l’oscillation des solutions de cette équation. Dans le troisième chapitre, nous analysons un second modèle pour la mégacaryopoïèse qui considère cette fois-ci une régulation opérée en continu uniquement via la vitesse de maturation des mégacaryoblastes. L’analyse de stabilité nécessite d’adapter un cadre pré-existant aux cas où le paramètre de bifurcation n’est pas le retard, et permet de montrer que l’augmentation du taux de mort des mégacaryoblastes conduit à l’apparition de solutions périodiques, en accord avec les observations cliniques de la thrombopénie cyclique amégacaryocytaire. Le dernier chapitre est consacré l’analyse de stabilité d’une équation différentielle à deux retards qui apparait notamment dans le cadre de la mégacaryopoïèse lorsque l’on considère que les plaquettes ont une durée de vie limitée / The object of this thesis is the study, using mathematical models, of the regulation mechanism maintaining an optimal quantity of blood platelets. The first chapter presents the biological and mathematical context of the thesis. In a second chapter, we introduce a model for megakaryopoiesis assuming a regulation by the platelet quantity of both the differentiation rate of stem cells to the platelet cell line and the amount of platelets produced by each megakaryocyte. We show that the dynamic of this model corresponds to a delay differential equation x'(t) = -?x(t) + f(x(t))g(x(t - t)), and we obtain for this equation new sufficient conditions for stability and for the oscillation of solutions. In a third chapter, we analyze a second model for megakaryopoiesis in which the regulation is continuous through the maturation speed of megakaryocyte progenitors. The stability analysis requires to adapt a pre-existing framework to problems where the bifurcation parameter is not the delay, and allows to show that increasing the death rate of megakaryocyte progenitors leads to the onset of periodic solutions, in agreement with clinical observation of amegakaryocytic cyclical thrombocytopenia. The last chapter covers a differential equation with two delays that appears among others in a model of platelet production which considers that platelet death can both age-independent and age-dependent

A trombocitopenia induzida por heparina (TIH) é uma síndrome imunohematológica mediada por um anticorpo que causa ativação plaquetária na presença de heparina, induz à agregação plaquetária e pode estar associada a graves e paradoxais complicações trombóticas e morte. A freqüência de TIH nos pacientes que recebem heparina por mais de cinco dias é de 1% a 5%, e está relacionada a vários fatores. Este é um estudo pioneiro no Brasil, que objetivou avaliar aqui a freqüência de TIH nos pacientes em uso de heparina, a relação ao gênero, ao tipo de heparina e a associação do genótipo da FcRIIa de receptores plaquetários. Foram selecionados 278 pacientes das Unidades de Terapia Intensiva e Unidades Coronariana do InCor-HCFMUSP, que receberam anticoagulação por heparina não fracionada (HNF) e/ou heparina de baixo peso molecular (HBPM), por pelo menos 5 dias, e excluídas as possíveis causas conhecidas de trombocitopenia. Foi realizada a contagem plaquetária pré e pós terapia com heparina, e o teste de detecção do anticorpo anti-fator 4 plaquetário/heparina (ID-PaGIA, DiaMed; e Asserachrom®-HPIA, Stago). O estudo da genotipagem da FcRIIa de receptores plaquetários foi realizado pelo método de digestão com enzima de restrição alelo específica. A freqüência de TIH encontrada foi de 6 (2,2%), e a freqüência de trombocitopenia com a presença do anticorpo anti-fator 4 plaquetário foi de 24,3%. A análise do gênero do paciente não demonstrou correlação com a TIH, nem com a trombocitopenia e nem com o anticorpo anti-fator 4 plaquetário. As mulheres apresentaram mais trombose do que os homens. A trombocitopenia ocorreu com maior freqüência nos pacientes que utilizaram os dois tipos de heparina (HNF-HBPM) e, com menor freqüência, nos que utilizaram apenas HBPM. O genótipo da FcRIIa de receptores plaquetários não apresentou relação com o gênero, nem com a TIH. Este estudo determinou a freqüência de TIH em uma população brasileira com uso de heparina e auxiliou no diagnostico. O melhor teste para detectar o anticorpo anti-fator 4 plaquetário/heparina, na presença de trombocitopenia e trombose, foi o teste de imunoaglutinação ID-PaGIA (DiaMed). A utilização dos dois tipos de heparina promoveu uma maior freqüência de trombocitopenia. Porém, novos estudos precisam confirmar as relações entre o tipo de heparina, com a trombocitopenia e com a TIH / Heparin induced thrombocytopenia (HIT) is an immune-hematologic syndrome mediated by a heparin dependent antibody that causes platelet activation, platelet aggregation, and can be associated with thrombosis and death. HIT occurs in about 1-5% of patients receiving heparin therapy up to 5 days or more. Many factors influence on the frequency of HIT. This is a pioneer Brazilian study to determine the frequency of HIT on patients under heparin therapy, and the relationship of HIT with gender, heparin type and the FcRIIa platelet receptor genotype. 278 patients from the Intensive Care Unit and Cardiac Care Unit at InCor-HCFMUSP treated with Unfractionated Heparin (UFH) and/or Low Molecular Weight Heparin (LMWH) for 5 or more days were studied. Known causes of thrombocytopenia were excluded. Platelet count was monitored pre and post heparin therapy. All selected patients were tested for detection of anti-heparin/PF4 antibody (ID-PaGIA, DiaMed; and Asserachrom®-HPIA, Stago). HIT frequency found was 6 (2,2%) and the frequency of thrombocytopenia (determined by a decrease in the platelet count below 50%, after the introduction of heparin therapy) and positive anti-heparin/PF4 antibody test was 24,3%. Patients gender was not related to TIH, neither to thrombocytopenia nor to the presence of antiheparin/ PF4 antibody. Thrombosis events were more frequent in women than in men. Thrombocytopenia, related to the type of heparin, was more frequent in patients that had used both types of heparin and less frequent in those that used only LMWH. FcRIIa platelet receptor genotype was associated with neither HIT nor with gender. This study has provided the frequency of HIT in a Brazilian patient population under heparin therapy and auxiliary in the HIT diagnosis. The ID-PaGIA (DiaMed) was shown to be the best test to correlate the presence of anti-heparin/PF4 antibody to thrombocytopenia and thrombosis event. The use of both heparin types promotes more thrombocytopenia. New studies are needed to confirm the relationship between heparin type and thrombocytopenia with HIT