A caracterização fenotípica e o isolamento de células-tronco hematopoiéticas (CTH) podem fornecer informações relevantes quanto ao desenvolvimento biológico do sistema hematopoiético. A habilidade em detectar e purificar essas células implica no desenvolvimento de condições para manutenção e expansão dessas células em culturas in vitro. Na medicina veterinária, a purificação de células-tronco hematopoiéticas caninas (CTHc) vem de encontro com interesses em estabelecer métodos para o desenvolvimento de terapias celulares, principalmente em doenças que levam à aplasia medular ou anemia aplástica nesta espécie animal. Neste cenário, a escassez de informação acerca da caracterização fenotípica bem como sobre a capacidade de proliferação e pluripotência celular de CTHc precisa ser superada. O presente projeto visou o isolamento, a caracterização e a expansão, por meio da análise imunofenotípica e de ensaios CFU de células-tronco/progenitoras hematopoiéticas provenientes da fração estromal vascular (FEV) do tecido adiposo de cães. Os resultados demonstraram que o painel de imunofenotipagem CD45-/CD117+/CD34+constitui uma melhor estratégia de isolamento destas células em comparação ao painel CD45-/CD38-/low/CD34+. Além disso, a detecção da atividade da enzima aldeído desidrogenase (ALDH) também pode representar uma grande aliada no enriquecimento desta fração celular. Por meio das técnicas empregadas no presente projeto foi possível verificar que a freqüência de CTH é baixa em tecido adiposo canino. / Phenotypic characterization and isolation of hematopoietic stem cells (HSC) provide relevant information to allow us to elucidate the biological development of the hematopoietic system. The ability to detect and purify these cells involves the development of conditions for maintenance and expansion of these cells in in vitro cultures. In veterinary medicine, purification of canine hematopoietic stem cells (CTHc) is of interesting to provide methods for the development of cell-based therapies, particularly in diseases causing bone marrow aplasia or aplastic anemia in this species. In this scenario, the lack of information about the phenotypic characterization as well as on the ability of cell proliferation and pluripotency of CTHc needs to be overcome. The present study aimed at the isolation, characterization, and expansion, by means of immunophenotyping and CFU assays of stem cells/ progenitor hematopoietic from stromal vascular fraction (SVF) from adipose tissue of dogs. The results showed that immunophenotyping panel CD45-/CD117+/CD34+ is a better strategy for isolation of these cells compared to CD45-/CD38&#X2d/low/CD34+. Furthermore, the detection of the enzyme aldehyde dehydrogenase (ALDH) activity may also represent a great enrichment coupled to this cell fraction. By the techniques employed in this project we found that the frequency of CTH is low in canine adipose tissue.

Os cães são afetados por doenças inflamatórias e neoplásicas que compartilham diversas similaridades com as desordens em humanos, assim seu estudo representa um importante modelo animal para as condições humanas. As células dendríticas (DCs) representam a população mais potente de células apresentadoras de antígenos. As DCs representam também um novo alvo promissor de imunoterapia em cães; no entanto o uso terapêutico de DC caninas é restrito, dentre outros fatores, devido a falta de padronização nas técnicas de isolamento e limitado numero de informações específicas da espécie a esse respeito. Este projeto tem por finalidade avaliar a geração de células dendríticas caninas geradas in vitro e ativadas por diferentes estímulos biológicos na presença e ausência de extrato tumoral de melanoma canino. Os resultados demonstraram que as DCs caninas geradas na presença de extrato tumoral em grandes concentrações apresentavam atividade funcional semelhante as DCs maduras / Dogs are affected by inflammatory and neoplastic diseases that share many similarities with the disorders in humans, so their study is an important animal model for the human condition. Dendritic cells (DCs) are the most potent population of antigen presenting cells. DCs also represent a promising new target for immunotherapy in dogs; However, the therapeutic use of canine DC is restricted among others factors due to lack of standardization in isolation techniques and limited number of species-specific information in this regard. This project aims to assess the generation of canine dendritic cells generated in vitro and activated by different biological stimuli in the presence and absence of tumor extract of canine melanoma. The results showed that the canine DCs generated in the presence of high concentrations tumor extract showed similar functional activity of mature DCs

Introdução: A doação de plaquetas por aférese é um método de coleta que vem aumentando em relevância. Sabe-se que esta técnica apresenta inúmeras vantagens em comparação à doação de sangue total. Observamos que há uma preocupação na qualidade dos hemocomponentes enviados ao paciente, no entanto, não se observam muitas pesquisas em busca do cuidado com o doador. Órgãos como o Food and Drug Administration (FDA) já publicaram normas mais restritivas em relação à doação de plaquetas por aférese, pois pesquisas apontaram uma diminuição de algumas células e proteínas do sistema imunológico em doadores de repetição. Objetivos: Analisar doadores de plaquetas de repetição quanto a parâmetros hematimétricos e quantificação de subpopulações linfocitárias comparando-os com um grupo controle composto por doadores de sangue total que não doam há no mínimo um ano ou doando pela primeira vez e, ainda avaliar se a frequência de doações, o tempo de procedimento e o número de plaquetas doadas influenciam na contagem de leucócitos totais e nas subpopulações de linfócitos. Metodologia: Foram analisados 88 indivíduos em um estudo caso-controle, sendo que o grupo controle (CO) incluído foi de doadores de sangue total que haviam doado pela primeira vez ou haviam doado sangue total há mais de um ano. Os casos (CA) incluídos foram os doadores de repetição de plaquetaférese (quatro ou mais doações no último ano). O pareamento foi feito por sexo e idade. As amostras de sangue periférico foram coletadas em tubos contendo EDTA e analisadas em até 6 horas por citometria de fluxo, através da utilização de anticorpos monoclonais anti-CD3, CD4, CD8, HLADR, CD19 e CD56. Resultados: Foram avaliados 44 pares de doadores (caso vs controle). Destes, 81,8% eram homens, a média de idade dos grupos foi de 46 ±13 anos nos casos e 47 ±11 nos controles. Comparando os dois grupos, observou-se diferença estatisticamente significativa (p<0,05) na média de quantificação de leucócitos absolutos CA= 6476,6/μL vs CO=7115,4/μL (p=0,017), na média de linfócitos absolutos CA= 1862,6/μL vs CO= 2239,2/μL (p=0,007) e nos marcadores: CD3+/CD8+ (absoluto) CA= 437/μL vs CO= 597/μL (p=0,01), CD3+/CD4+(%) CA= 47,3/μL vs CO= 42,77/μL (p=0,007). Conclusões: Neste estudo foi possível observar que há uma diminuição em algumas células linfoides dos doadores de repetição em relação aos doadores convencionais, no entanto essa diferença não tem relevância clínica, demonstrando que os intervalos de doações que estes doadores estão sendo submetidos é adequado. A contagem de plaquetas dos doadores de repetição se mantiveram no decorrer do ano, este dado nos auxilia para mantermos um banco de dados de doadores de repetição com uma quantificação de plaquetas adequada, podendo ser convocado sem risco de ser bloqueado por contagem inferior ao preconizado. / Introduction: The donation of platelets by apheresis as a collection method has lately grown in relevance. This technique presents several advantages when compared to total blood donation. We understand there is a concern about the quality of the hemocomponents that are administered to the patients; however, there are not many researches concerned with caring for the donor. Entities such as the Food and Drug Administration (FDA) have published more restricting regulations regarding the donation of platelets by apheresis, since researches indicate a decrease in some cells and proteins present in the immunological systems of repeat donors. Objectives: To analyze repeat donors of platelets with regards to hematimetric parameters and quantification of lymphocyte sub-populations by comparing them with a control group consisting of total blood donors that have not donated blood for the past year at least or that are donating for the first time. Additionally, to evaluate if the frequency of donations, the duration of the procedure, and the donated platelet counts influence in the total leukocyte counts and in the sub-populations of lymphocytes. Methodology: We analyzed 88 individuals in a control case study. The control group (CG) consisted of total blood donors in their first donation or that had donated for the last time more than a year before. The cases (CA) included were the repeat donors by platelet apheresis (four or more donations in the past year). We matched the individuals by gender and age. Peripheral blood samples were collected in tubes containing EDTA and analyzed up until 6 hours later by flow cytometry, through monoclonal antibodies anti-CD3, CD4, CD8, HLADR, CD19, and CD56. Results: 44 pairs of donor were evaluated (case vs control). Among them, 81.8% were men, the average age of the groups was 46 (±13) years in the cases and 47 (±11) in the controls. When comparing the two groups, we observed a statistically significant difference (p<0,05) in the average of the quantification of absolute leukocytes CA= 6476.6/μL vs CG=7115.4/μL (p=0.017), in the average of absolute lymphocytes CA= 1862.6/μL vs CG= 2239.2/μL (p=0.007), and in the markers: CD3+/CD8+ (absolute) CA= 437/μL vs CG= 597/μL (p=0,01), CD3+/CD4+(%) CA= 47.3/μL vs CG= 42.77/μL (p=0.007). Conclusions: We were able to note in this study that there is a significant decrease in some lymphoid cells of repeat donors when compared to conventional donors. This difference, however, is not clinically relevant, which demonstrates that the donation intervals to which the donors are subject are appropriate. Platelet numbers of repeat donors remained the same throughout the year. This piece of data helps us keep a database of repeat donors with an adequate platelet number. These donors can be called for without risking of their being blocked in the screening for a number lower than the recommended.

Os cães são afetados por doenças inflamatórias e neoplásicas que compartilham diversas similaridades com as desordens em humanos, assim seu estudo representa um importante modelo animal para as condições humanas. As células dendríticas (DCs) representam a população mais potente de células apresentadoras de antígenos. As DCs representam também um novo alvo promissor de imunoterapia em cães; no entanto o uso terapêutico de DC caninas é restrito, dentre outros fatores, devido a falta de padronização nas técnicas de isolamento e limitado numero de informações específicas da espécie a esse respeito. Este projeto tem por finalidade avaliar a geração de células dendríticas caninas geradas in vitro e ativadas por diferentes estímulos biológicos na presença e ausência de extrato tumoral de melanoma canino. Os resultados demonstraram que as DCs caninas geradas na presença de extrato tumoral em grandes concentrações apresentavam atividade funcional semelhante as DCs maduras / Dogs are affected by inflammatory and neoplastic diseases that share many similarities with the disorders in humans, so their study is an important animal model for the human condition. Dendritic cells (DCs) are the most potent population of antigen presenting cells. DCs also represent a promising new target for immunotherapy in dogs; However, the therapeutic use of canine DC is restricted among others factors due to lack of standardization in isolation techniques and limited number of species-specific information in this regard. This project aims to assess the generation of canine dendritic cells generated in vitro and activated by different biological stimuli in the presence and absence of tumor extract of canine melanoma. The results showed that the canine DCs generated in the presence of high concentrations tumor extract showed similar functional activity of mature DCs

O presente estudo visou avaliar os efeitos da administração do resíduo aquoso final (RAF) da Ipomoea carnea e da suainsonina, sobre o sistema imune de ratos jovens em idade pré-pubere (21 dias) e adultos (70 dias). Para isso, o RAF foi administrado nas doses de 1,0; 3,0 e 7,0 g/kg e a suainsonina na dose de 5,0 mg/kg, por gavage, durante 14 dias para avaliação histopatológica de diferentes órgãos, peso relativo de baço e timo, celularidade de medula óssea, fenotipagem de linfócitos T e B presentes no sangue, timo e baço, e atividade proliferativa de linfócitos T de animais tratados apenas com suainsonina. Além disto, foi realizada toxicocinética da suainsonina de animais jovens e adultos. Dentre os resultados obtidos com o tratamento com RAF, foi verificado que todos os animais apresentaram lesões, tais como: congestão hepática e esplênica e vacuolização renal. Em ratos adultos observou-se ainda diminuição do ganho de peso e do consumo de ração, além de involução tímica e diminuição da celularidade da medula óssea. Já nos jovens, as principais alterações observadas foram quanto à alteração fenotípica das populações de linfócitos T CD8+ no timo, sangue e baço e de linfócitos B no sangue e baço. Entretanto, estas alterações não podem ser atribuídas exclusivamente ao RAF de I. carnea, pois os animais do grupo pear-feeding (grupo sob restrição alimentar para mimetizar a diminuição da ingesta de alimentos daqueles ratos tratados com a maior dose do RAF) apresentaram resultados semelhantes àqueles tratados com a planta. Em relação aos resultados obtidos em ratos jovens tratados com suainsonina, verificou-se menor porcentagem de linfócitos B no baço e no sangue e maior expansão clonal de linfócitos T CD4+ de ratos imunizados in vivo e desafiados ex vivo com anatoxina tetânica. Por fim, o estudo toxicocinético revelou que este alcalóide possui biodisponibilidade de apenas 9 horas tanto nos animais jovens quanto nos adultos. Assim, é possível concluir que a suainsonina, principal princípio ativo da I. carnea, tem atividade imunomodulatória, mas esta atividade difere do efeito imunomodulador causado pelo RAF provavelmente devido a presença de outros alcalóides também encontrados na planta / This study aimed to evaluate the effects of administration of the final aqueous residue (RAF) of Ipomoea carnea and swainsonine on the immune system of young (21 days) and adult rats (70 days). For this, the RAF was administered at doses of 1.0, 3.0 and 7.0 g/kg and swainsonine at dose of 5.0 mg/kg by gavage for 14 days for histopathological evaluation of different organs, relative weight of spleen and thymus, bone marrow cellularity, phenotyping of T and B lymphocytes in the blood, thymus and spleen, and proliferative activity of T lymphocytes from animals treated with swainsonine. Moreover, we performed toxicokinetics of swainsonine in young and adult rats. Among the results obtained with treatment with the RAF, it was observed that all animals had lesions such as hepatic and splenic congestion and kidney vacuolization. In adult rats there was also reduction in weight gain and feed intake, thymic involution and decreased bone marrow cellularity. Already in the young rats, the main changes observed were in the populations of T CD8+ lymphocytes in the thymus, spleen and blood and B lymphocytes in the blood and spleen. However, these changes cannot be attributed solely to the RAF of I. carnea, because the animals of the pear-feeding group (group under food restriction to mimic the decrease in food intake of those rats treated with the higher dose of the RAF) had similar results to those treated with the plant. Regarding the results obtained in young rats treated with swainsonine, there was a lower percentage of B lymphocytes in the spleen and blood and increased clonal expansion of T CD4+ lymphocytes from rats immunized in vivo and challenged ex vivo with tetanus toxoid. Finally, the toxicokinetic study has revealed that bioavailability of this alkaloid is only 9 hours in the both animals young and adult. Thus, we conclude that swainsonine, the main active principle of I. carnea, has immunomodulatory activity, but this activity differs from the immunomodulatory effect caused by the RAF probably due to the presence of other alkaloids also found in the plant

A caracterização fenotípica e o isolamento de células-tronco hematopoiéticas (CTH) podem fornecer informações relevantes quanto ao desenvolvimento biológico do sistema hematopoiético. A habilidade em detectar e purificar essas células implica no desenvolvimento de condições para manutenção e expansão dessas células em culturas in vitro. Na medicina veterinária, a purificação de células-tronco hematopoiéticas caninas (CTHc) vem de encontro com interesses em estabelecer métodos para o desenvolvimento de terapias celulares, principalmente em doenças que levam à aplasia medular ou anemia aplástica nesta espécie animal. Neste cenário, a escassez de informação acerca da caracterização fenotípica bem como sobre a capacidade de proliferação e pluripotência celular de CTHc precisa ser superada. O presente projeto visou o isolamento, a caracterização e a expansão, por meio da análise imunofenotípica e de ensaios CFU de células-tronco/progenitoras hematopoiéticas provenientes da fração estromal vascular (FEV) do tecido adiposo de cães. Os resultados demonstraram que o painel de imunofenotipagem CD45&#x2D;/CD117&#43;/CD34&#43;constitui uma melhor estratégia de isolamento destas células em comparação ao painel CD45&#x2D;/CD38&#x2D;/low/CD34&#43;. Além disso, a detecção da atividade da enzima aldeído desidrogenase (ALDH) também pode representar uma grande aliada no enriquecimento desta fração celular. Por meio das técnicas empregadas no presente projeto foi possível verificar que a freqüência de CTH é baixa em tecido adiposo canino. / Phenotypic characterization and isolation of hematopoietic stem cells (HSC) provide relevant information to allow us to elucidate the biological development of the hematopoietic system. The ability to detect and purify these cells involves the development of conditions for maintenance and expansion of these cells in in vitro cultures. In veterinary medicine, purification of canine hematopoietic stem cells (CTHc) is of interesting to provide methods for the development of cell-based therapies, particularly in diseases causing bone marrow aplasia or aplastic anemia in this species. In this scenario, the lack of information about the phenotypic characterization as well as on the ability of cell proliferation and pluripotency of CTHc needs to be overcome. The present study aimed at the isolation, characterization, and expansion, by means of immunophenotyping and CFU assays of stem cells/ progenitor hematopoietic from stromal vascular fraction (SVF) from adipose tissue of dogs. The results showed that immunophenotyping panel CD45&#x2D;/CD117&#43;/CD34&#43; is a better strategy for isolation of these cells compared to CD45&#x2D;/CD38&#X2d/low/CD34&#43;. Furthermore, the detection of the enzyme aldehyde dehydrogenase (ALDH) activity may also represent a great enrichment coupled to this cell fraction. By the techniques employed in this project we found that the frequency of CTH is low in canine adipose tissue.

O presente estudo visou avaliar os efeitos da administração do resíduo aquoso final (RAF) da Ipomoea carnea e da suainsonina, sobre o sistema imune de ratos jovens em idade pré-pubere (21 dias) e adultos (70 dias). Para isso, o RAF foi administrado nas doses de 1,0; 3,0 e 7,0 g/kg e a suainsonina na dose de 5,0 mg/kg, por gavage, durante 14 dias para avaliação histopatológica de diferentes órgãos, peso relativo de baço e timo, celularidade de medula óssea, fenotipagem de linfócitos T e B presentes no sangue, timo e baço, e atividade proliferativa de linfócitos T de animais tratados apenas com suainsonina. Além disto, foi realizada toxicocinética da suainsonina de animais jovens e adultos. Dentre os resultados obtidos com o tratamento com RAF, foi verificado que todos os animais apresentaram lesões, tais como: congestão hepática e esplênica e vacuolização renal. Em ratos adultos observou-se ainda diminuição do ganho de peso e do consumo de ração, além de involução tímica e diminuição da celularidade da medula óssea. Já nos jovens, as principais alterações observadas foram quanto à alteração fenotípica das populações de linfócitos T CD8+ no timo, sangue e baço e de linfócitos B no sangue e baço. Entretanto, estas alterações não podem ser atribuídas exclusivamente ao RAF de I. carnea, pois os animais do grupo pear-feeding (grupo sob restrição alimentar para mimetizar a diminuição da ingesta de alimentos daqueles ratos tratados com a maior dose do RAF) apresentaram resultados semelhantes àqueles tratados com a planta. Em relação aos resultados obtidos em ratos jovens tratados com suainsonina, verificou-se menor porcentagem de linfócitos B no baço e no sangue e maior expansão clonal de linfócitos T CD4+ de ratos imunizados in vivo e desafiados ex vivo com anatoxina tetânica. Por fim, o estudo toxicocinético revelou que este alcalóide possui biodisponibilidade de apenas 9 horas tanto nos animais jovens quanto nos adultos. Assim, é possível concluir que a suainsonina, principal princípio ativo da I. carnea, tem atividade imunomodulatória, mas esta atividade difere do efeito imunomodulador causado pelo RAF provavelmente devido a presença de outros alcalóides também encontrados na planta / This study aimed to evaluate the effects of administration of the final aqueous residue (RAF) of Ipomoea carnea and swainsonine on the immune system of young (21 days) and adult rats (70 days). For this, the RAF was administered at doses of 1.0, 3.0 and 7.0 g/kg and swainsonine at dose of 5.0 mg/kg by gavage for 14 days for histopathological evaluation of different organs, relative weight of spleen and thymus, bone marrow cellularity, phenotyping of T and B lymphocytes in the blood, thymus and spleen, and proliferative activity of T lymphocytes from animals treated with swainsonine. Moreover, we performed toxicokinetics of swainsonine in young and adult rats. Among the results obtained with treatment with the RAF, it was observed that all animals had lesions such as hepatic and splenic congestion and kidney vacuolization. In adult rats there was also reduction in weight gain and feed intake, thymic involution and decreased bone marrow cellularity. Already in the young rats, the main changes observed were in the populations of T CD8+ lymphocytes in the thymus, spleen and blood and B lymphocytes in the blood and spleen. However, these changes cannot be attributed solely to the RAF of I. carnea, because the animals of the pear-feeding group (group under food restriction to mimic the decrease in food intake of those rats treated with the higher dose of the RAF) had similar results to those treated with the plant. Regarding the results obtained in young rats treated with swainsonine, there was a lower percentage of B lymphocytes in the spleen and blood and increased clonal expansion of T CD4+ lymphocytes from rats immunized in vivo and challenged ex vivo with tetanus toxoid. Finally, the toxicokinetic study has revealed that bioavailability of this alkaloid is only 9 hours in the both animals young and adult. Thus, we conclude that swainsonine, the main active principle of I. carnea, has immunomodulatory activity, but this activity differs from the immunomodulatory effect caused by the RAF probably due to the presence of other alkaloids also found in the plant

Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Introdução: A doação de plaquetas por aférese é um método de coleta que vem aumentando em relevância. Sabe-se que esta técnica apresenta inúmeras vantagens em comparação à doação de sangue total. Observamos que há uma preocupação na qualidade dos hemocomponentes enviados ao paciente, no entanto, não se observam muitas pesquisas em busca do cuidado com o doador. Órgãos como o Food and Drug Administration (FDA) já publicaram normas mais restritivas em relação à doação de plaquetas por aférese, pois pesquisas apontaram uma diminuição de algumas células e proteínas do sistema imunológico em doadores de repetição. Objetivos: Analisar doadores de plaquetas de repetição quanto a parâmetros hematimétricos e quantificação de subpopulações linfocitárias comparando-os com um grupo controle composto por doadores de sangue total que não doam há no mínimo um ano ou doando pela primeira vez e, ainda avaliar se a frequência de doações, o tempo de procedimento e o número de plaquetas doadas influenciam na contagem de leucócitos totais e nas subpopulações de linfócitos. Metodologia: Foram analisados 88 indivíduos em um estudo caso-controle, sendo que o grupo controle (CO) incluído foi de doadores de sangue total que haviam doado pela primeira vez ou haviam doado sangue total há mais de um ano. Os casos (CA) incluídos foram os doadores de repetição de plaquetaférese (quatro ou mais doações no último ano). O pareamento foi feito por sexo e idade. As amostras de sangue periférico foram coletadas em tubos contendo EDTA e analisadas em até 6 horas por citometria de fluxo, através da utilização de anticorpos monoclonais anti-CD3, CD4, CD8, HLADR, CD19 e CD56. Resultados: Foram avaliados 44 pares de doadores (caso vs controle). Destes, 81,8% eram homens, a média de idade dos grupos foi de 46 ±13 anos nos casos e 47 ±11 nos controles. Comparando os dois grupos, observou-se diferença estatisticamente significativa (p<0,05) na média de quantificação de leucócitos absolutos CA= 6476,6/μL vs CO=7115,4/μL (p=0,017), na média de linfócitos absolutos CA= 1862,6/μL vs CO= 2239,2/μL (p=0,007) e nos marcadores: CD3+/CD8+ (absoluto) CA= 437/μL vs CO= 597/μL (p=0,01), CD3+/CD4+(%) CA= 47,3/μL vs CO= 42,77/μL (p=0,007). Conclusões: Neste estudo foi possível observar que há uma diminuição em algumas células linfoides dos doadores de repetição em relação aos doadores convencionais, no entanto essa diferença não tem relevância clínica, demonstrando que os intervalos de doações que estes doadores estão sendo submetidos é adequado. A contagem de plaquetas dos doadores de repetição se mantiveram no decorrer do ano, este dado nos auxilia para mantermos um banco de dados de doadores de repetição com uma quantificação de plaquetas adequada, podendo ser convocado sem risco de ser bloqueado por contagem inferior ao preconizado. / Introduction: The donation of platelets by apheresis as a collection method has lately grown in relevance. This technique presents several advantages when compared to total blood donation. We understand there is a concern about the quality of the hemocomponents that are administered to the patients; however, there are not many researches concerned with caring for the donor. Entities such as the Food and Drug Administration (FDA) have published more restricting regulations regarding the donation of platelets by apheresis, since researches indicate a decrease in some cells and proteins present in the immunological systems of repeat donors. Objectives: To analyze repeat donors of platelets with regards to hematimetric parameters and quantification of lymphocyte sub-populations by comparing them with a control group consisting of total blood donors that have not donated blood for the past year at least or that are donating for the first time. Additionally, to evaluate if the frequency of donations, the duration of the procedure, and the donated platelet counts influence in the total leukocyte counts and in the sub-populations of lymphocytes. Methodology: We analyzed 88 individuals in a control case study. The control group (CG) consisted of total blood donors in their first donation or that had donated for the last time more than a year before. The cases (CA) included were the repeat donors by platelet apheresis (four or more donations in the past year). We matched the individuals by gender and age. Peripheral blood samples were collected in tubes containing EDTA and analyzed up until 6 hours later by flow cytometry, through monoclonal antibodies anti-CD3, CD4, CD8, HLADR, CD19, and CD56. Results: 44 pairs of donor were evaluated (case vs control). Among them, 81.8% were men, the average age of the groups was 46 (±13) years in the cases and 47 (±11) in the controls. When comparing the two groups, we observed a statistically significant difference (p<0,05) in the average of the quantification of absolute leukocytes CA= 6476.6/μL vs CG=7115.4/μL (p=0.017), in the average of absolute lymphocytes CA= 1862.6/μL vs CG= 2239.2/μL (p=0.007), and in the markers: CD3+/CD8+ (absolute) CA= 437/μL vs CG= 597/μL (p=0,01), CD3+/CD4+(%) CA= 47.3/μL vs CG= 42.77/μL (p=0.007). Conclusions: We were able to note in this study that there is a significant decrease in some lymphoid cells of repeat donors when compared to conventional donors. This difference, however, is not clinically relevant, which demonstrates that the donation intervals to which the donors are subject are appropriate. Platelet numbers of repeat donors remained the same throughout the year. This piece of data helps us keep a database of repeat donors with an adequate platelet number. These donors can be called for without risking of their being blocked in the screening for a number lower than the recommended.