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CONGRESSO SOCIEDADE IBÉRICA

DE CITOMETRIA

XIII CONGRESSO SIC13

9-11MAIO2013

Universidade de AveiroPORTUGAL

IPST

®Instituto Portuguésdo Sangue e daTransplantação, IP

XIII CONGRESSO SIC

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XIII CONGRESSO SIC

Junta Directiva De la sociedade ibérica de

citometria (sic)

comitÉ organizaDor

comitÉ científico

Presidente: Jordi Petriz González

vicepresidente: Alfredo Prieto Martín

Secretaria-tesouraria: Julia Almeida Parra

vogais: Alberto Álvarez-Barrientos Jorge Monserrat Sanz

Julia almeida Parraalberto Álvarez-Barrientos rosário DominguesSónia mendoJorge monserrat Sanzalberto Órfão de matosartur Paiva

Julia almeida Parraalberto Álvarez-Barrientosfrederico cerveira rosário Domingues Pilar echániz aizpuruaana espada de Sousa maria Jorge arrozmargarida lima eduardo lópez granados João loureiroPaulo lúcio Sónia mendoJorge monserrat Sanz

marta morado esmeralda neves alberto Órfão artur PaivaJosé antónio Pereira da SilvaJordi Petriz gonzálezalfredo Prieto martínelmano ramallheira maria Soares ana tenreiroHélder trindadeJuan antonio vargas

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BoaS vinDaS

Caros colegas,Em nome da Sociedade Ibérica de Citometria e da Comissão Organizadora, gostaría-mos de vos dar as boas-vindas ao XIII Congresso da Sociedade Ibérica de Citometria, que terá lugar de 9 a 11 de Maio de 2013 em Aveiro, Portugal.O programa científico deste congresso foi esboçado tendo em conta o interesse e a actualidade dos temas, abrangendo estes um leque diversificado de áreas científicas, onde a imunologia, a hematologia, a biotecnologia e a oncologia ocupam um lugar de destaque. A Comissão Organizadora sente-se honrada pela presença dos cinquenta convidados, cujo mérito científico é internacionalmente reconhecido, que irão presti-giar e valorizar o XIII Congresso da Sociedade Ibérica de Citometria.A presença de cerca de duzentos congressistas e a recepção de noventa e nove tra-balhos científicos, é a maior gratificação que poderíamos esperar de todo empenho e dedicação que colocámos na organização e concretização deste Congresso. De igual modo, o apoio recebido por parte das empresas patrocinadoras, deu um impulso definitivo para a realização deste evento. Aveiro é uma bonita cidade litoral do centro de Portugal, situada entre Porto e Coimbra, conhecida como “Veneza de Portugal” e considerada um dos destinos mais encantadores do país. Esta cidade, de grande tradição marítima e com uma longa história de comércio naval, pesca e produção de sal, apresenta uma deslumbrante paisagem, recortada pelos diversos canais da ria de Aveiro, ladeados por belos edi-ficios de estilo Arte Nova do século XIX e por onde se passeiam os coloridos barcos moliceiro.Desejamos que a vossa estadia na cidade de Aveiro seja agradável e que este Con-gresso preencha as vossas expectativas.

A Comissão Organizadora

XIII CONGRESSO SIC

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BienveniDa

Estimados colegas,

En nombre de la Sociedad Ibérica de Citometría (SIC) y de la Comisión Organiza-dora, os damos la más cordial bienvenida al XIII Congreso de la Sociedad Ibérica de Citometría, que se celebra del 9 al 11 de mayo de 2013 en Aveiro, Portugal.Para la elaboración del programa científico se ha tenido en consideración el interés y actualidad de los temas, y se ha procurado que estén representadas las diversas áreas científicas en las que se enmarca la actividad de nuestra Sociedad, teniendo las áreas de inmunología, hematología, biotecnología y oncología un lugar destacado. La Comisión Organizadora se siente muy pri-vilegiada por la presencia de 50 ponentes invitados de reconocido prestigio internacional que, sin duda, van a contribuir a elevar el nivel científico del XIII congreso de la SIC.La asistencia de cerca de 200 profesionales al congreso y la recepción de 99 trabajos científicos son la mejor gratificación que podríamos recibir al empeño y dedicación que ha puesto la Comisión Organizadora para hacer realidad este congreso. De igual modo, queremos reconocer el apoyo recibido por las casas comerciales patrocinadoras, que ha supuesto un impulso definitivo para la rea-lización del evento. Aveiro es una bonita ciudad costera del centro de Portugal, situada entre Opor-to y Coimbra, conocida como la “Venecia de Portugal” y considerada como uno de los destinos con más encanto del país. Es una ciudad de gran tradición marí-tima y tiene una larga historia de comercio naval, pesquero y de producción de sal; cuenta con estupendas playas y con el encanto que le dan los numerosos canales de la ría de Aveiro que la surcan, a cuyos márgenes se pueden con-templar bellos edificios de estilo Art Nouveau del siglo XIX, por donde pasean los coloridos barcos típicos (moliceiros).Deseamos que vuestra estancia en la ciudad de Aveiro sea muy agradable, y que este congreso cumpla todas vuestras expectativas.

La Comisión Organizadora

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cursos pré-congresso / cursos precongreso / Pre-congress courses

09:00-15:00 h Entrega de documentação / Entrega de documentación Registration open

10:30-13:00 h Anfiteatro da Reitoria • Curso/Course 1 Workshop EuroFlow Padronização de equipamentos e técnicas para painéis EuroFlow Estandarización de equipos y técnicas para la aplicación de los paneles EuroFlow Standardization of equipment and techniques for EuroFlow panels Juan Flores. Salamanca (España)

Sala de Atos • Curso/Course 2 Curso de iniciação à citometria: Princípios básicos e aplicações Curso de iniciación a la citometría: principios básicos y aplicaciones Introduction to cytometry: basic principles and applications Alexandre Salvador, Florencio Carretero. Lisboa (Portugal)

Sala da Livraria • Curso/Course 3 Citómica e stress oxidativo Citómica y estrés oxidativo Cytomics and oxidative stress Enrique O’Connor, Angela Gomes, Guadalupe Herrera, Francisco Sala-de Oyanguren. Valencia (España)

Anfiteatro da Livraria • Curso/Course 4 Aplicações da citometria ao estudo da transdução de sinal Aplicaciones de la citometría a los estudios de transducción de señales Applications of cytometry for the study of signal transduction Cedric Favre. Marsella (Francia)

13:00-14:30 h Almoço / Almuerzo / Lunch. Cantina de la Universidade de Aveiro

9 de maio / 9 de mayo / may 9th

Programa científico / Scientific Program

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Anfiteatro da Reitoria15:00-15:15 h Sessão inaugural / Sesión inaugural / Opening Session

15:15-16:30 h Conferência inaugural / Conferencia inaugural Opening Conference Moderador: Manuel Santos Rosa

Novas estratégias de análise de dados de células hematopoiéticas normais e neoplásicas por citometria de fluxo

Nuevas estrategias de análisis de datos de células hematopoyéticas normales y neoplásicas por citometría de flujo New strategies for analyzing data from normal and neoplastic hematopoietic cells by flow cytometry Alberto Órfão. Salamanca (España)

16:30-17:00 h Café / Cofee-break

17:00-19:00 h Anfiteatro da Reitoria Sessão / Sesión / Session A SíNdROmES dE FAlêNCIA mEdulAR SíNdrOmES dE iNSuFiCiENCiA mEduLAr BONE mARROW FAIluRE SyNdROmES Moderadoras: Marta Morado, Margarida Lima

17:00-17:30 h Indicações técnicas e clínicas para o rastreio de células com deficiência da âncora GPI por citometria de fluxo

indicaciones técnicas y clínicas para el rastreo de células GPi deficientes por citometría de flujo Technical and clinical indications for GPI-deficient

cell screening by flow cytometry Martín Pérez-Andrés. Salamanca (España);

en nombre del grupo de trabajo de HPN de la SIC

17:30-18:00 h A citometria no diagnóstico das patologias da membrana eritrocitária / La citometría en el diagnóstico de las hemopatías

eritrocitarias de membrana Cytometry in the diagnosis of erythrocyte membrane disorders Tabita Magalhães Maia. Coimbra (Portugal)


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18:00-18:30 h índice de proliferação dos compartimentos celulares da medula óssea no diagnóstico, prognóstico e seguimento de doentes com mielodisplasia

índice proliferativo de los compartimentos celulares de la médula ósea en pacientes con Smd y su utilidad en el diagnóstico, pronóstico y seguimiento de los pacientes

The proliferation index of bone marrow cell compartments in the diagnosis, prognosis and follow-up of myelodysplastic syndromes

Sergio Matarraz. Salamanca (España)

18:30-19:00 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communications

HEM C0-01 Estudo da maturação eritróide com base no CD44 e no CD35 em medula óssea normal e de síndromes mielodisplásicasRaquel Rodrigues1,3, Paula Laranjeira4, Tiago Carvalheiro4, Margarida Farinha1, Paula Rocha2, Isabel Silva4, Maria Jesus Inácio4, Maria Reis Andrade2, João Ribeiro1, Helena Vitória2, Artur Paiva3,4

1Serviço de Patologia Clínica. Centro Hospitalar Tondela. Viseu. 2Serviço de Hematologia. Centro Hospitalar Tondela. Viseu. 3Escola Superior de Tecnologia da Saúde de Coimbra. 4Instituto Português de Sangue e Transplantação (Portugal).

HEM C0-02 Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesisCarlos Fernández-Giménez1, Maria Claudia Santos-Silva2, Antonio López1, Sergio Matarraz1, María Jara-Acevedo1, Laura Gutiérrez1, María Luz Sánchez1, Carlos Cervero3, Oliver Gutiérrez4, Nicolás González5, Carlos Salvador-Osuna6, Alberto Orfao1

1Department of Medicine and Service of Cytometry. IBSAL and Centro de Investigación del Cáncer (IBMCC USALCSIC). University Hospital of Salamanca and University of Salamanca. Salamanca (Spain). 2Laboratório de Oncologia Experimental e Hemopatias do Departamento de Análises Clínicas. Universidade Federal de Santa Catarina. Santa Catarina (Brazil). 3Hematology Department. Hospital General Virgen de la Luz. Cuenca (Spain). 4Hematology Department. Hospital Universitario Río Hortega. Valladolid (Spain). 5Hematology Department. Hospital Obispo Polanco. Teruel (Spain). 6Hematology Department. Hospital Universitario Miguel Servet. Zaragoza (Spain).

HEM C0-03 Utilidad de eosín-5-maleimida en el diagnóstico de anemias hemolíticas con presencia de esferocitos en sangre periféricaBeatriz Álvarez, F. Ataulfo González, Diego Velasco, Luz Conejo, María Medrano, Juan M. Alonso, A. Vlagea, Marta Jiménez, Jesús Villarrubia, Raquel GuillénLaboratorio Central de la Comunidad de Madrid BRsalud. Hospital Infanta Sofía. Madrid (Spain).

XIII CONGRESSO SIC

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17:00-19:00 h Sala de Atos Sessão / Sesión / Session B ESTudOS dAS AlERGIAS POR CITOmETRIA EStudiO dE LA ALErGiA POr CitOmEtríA FlOW CyTOmETRy IN AllERGy Moderadoras: Esmeralda Neves, Pilar Echániz

17:00-17:30 h utilidade do teste de activacão de basófilos no estudo da alergia a proteínas do leite de vaca

utilidad del test de activación de basófilos en el estudio de la alergia a las proteínas de la leche de vaca

Benefit of the basophil activation test in the evaluation of cow’s milk proteins allergy

Pilar Echániz. San Sebastián (España)

17:30-18:00 h Alterações induzidas pela imunoterapia específica subcutânea nos diferentes compartimentos das células B em rinite alérgica

Alteraciones de los diferentes compartimentos de células B inducidas por la inmunoterapia subcutánea específica en la rinitis alérgica

Alterations on blood B-cell subsets induced by specific subcutaneous immunotherapy in allergic rhinitis

Ana Henriques. Coimbra (Portugal)

18:00-18:30 h Alergia em mastocitoses Alergia en la mastocitosis mastocytosis and allergy Luis Escribano. Toledo (España)

18:30-19:00 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communicationsINM C0-01 A case of delayed hypersensitivy to anisakis with clinical digestive

manifestations diagnosed by lymphocyte activation test (LAT)C. Cámara, A. Rodríguez Trabado*, S. Romero, J.A. García Trujillo, S. Larios, I. Tovar, L. Fernández PereiraImmunology Unit. Complejo Hospitalario de Cáceres. *Allergology Unit. Hospital Campo Arañuero. Navalmoral de la Mata. Cáceres (Spain).


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INM C0-02 Monitorización de inmunoterapia con venenos de himenópteros mediante test de activación de basófilos seriadosC. Cámara, A. Rodríguez Trabado*, S. Romero, J.A. García Trujillo, S. Larios, Immunology Unit. Complejo Hospitalario de Cáceres. *Allergology Unit. Hospital Campo Arañuero. Navalmoral de la Mata. Cáceres (Spain).

17:00-19:00 h Sala de Traduções Sessão / Sesión / Session C BIOTECNOlOGIA / BiOtECNOLOGíA / BIOTECHNOlOGy Moderadores: João Loureiro, Alberto Álvarez-Barrientos

17:00-17:30 h Rmax: um método simples para a avaliação da performance da separação

rmax: un método fácil para evaluar el rendimiento de la separación celular Sorting out recovery with Rmax: A simple methodto evaluate

sorter performance Rui Gardner. Lisboa (Portugal)

17:30-18:00 h Aplicação da citometria de fluxo na produção de plantas e horticultura

Aplicaciones de la citometría de flujo en horticultura Applications of flow cytometry in plant breeding and horticulture João Loureiro. Coimbra (Portugal)

18:00-18:30 h Toolbox de biomarcadores para avaliar os efeitos citostáticos de fitocompostos em células cancerígenas: uma abordagem in vitro

Biomarcadores para evaluar los efectos citostáticos de fitocomponentes sobre células cancerosas: una aproximación in vitro

Toolbox of biomarkers to assess the cytostatic effects of phytocompounds on cancer cells: an in vitro approach

Helena Oliveira. Aveiro (Portugal)

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18:30-19:00 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communicationsBIOTEC C0-01 Utilização da citometria de fluxo multiparamétrica na monitorização

da produção de biocombustíveisTeresa Lopes da Silva, Alberto ReisLaboratório Nacional de Energia e Geologia. Lisboa (Portugal).

BIOTEC C0-02 Integrating cytomic assays for assessing metabolic effects of candidate drugs: proof of concept with tideglusibGuadalupe Herrera, Laura Díaz, José-Enrique O’ConnorLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain).

BIOTEC C0-03 An assay for autophagy and apoptosis using multispectral imaging flow cytometry and SW872 adipocytic cells expressing GFP-LC3, an autophagosome markerFrancisco Sala de Oyanguren1, Sara de Biasi1, Lara Gibellini2, Marcello Pinti3, Massimo Riccio2, Milena Nasi1, Anto de Pol1, Andrea Cossarizza, José-Enrique O’Connor1

1Laboratory of Cytomics. Mix Unit CIPF-UVEG. Valencia (Spain). 2Department of Surgery, Medicine, Dentistry and Morphological Sciences. University of Modena and Reggio Emilia. Modena (Italy). 3Department of Life Sciences Sciences. University of Modena and Reggio Emilia. Modena (Italy).

19:15 h Cocktail de boas vindas / Cóctel de bienvenida / Welcome cocktail Restaurante Olá Ria (Cais da Fonte Nova. 3800-200 Aveiro)


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08:30-10:30 h Anfiteatro da Reitoria Sessão / Sesión / Session A APlICAçõES dA CITOmETRIA dE FluxO Em TumORES SólIdOS APLiCACiONES dE LA CitOmEtríA dE FLujO EN LOS

tumOrES SóLidOS / FlOW CyTOmETRy IN SOlId TumORS Moderadores: F. Pimentel, HélderTrindade

08:30-09:00 h diagnóstico e classificação de tumores pediátricos diagnóstico y clasificación de los tumores pediátricos diagnosis and classification of pediatric tumors Elaine Sobral da Costa. Rio de Janeiro (Brasil)

09:00-09:30 h Importância prognóstica do infiltrado leucocitário nos tumores do sistema nervoso central

Significado pronóstico del infiltrado leucocitario en los tumores del sistema nervioso central Prognostic relevance of leukocyte infiltration

in central nervous system tumors Patrícia Henriques Domingues. Salamanca (España)

09:30-10:00 h As células-tronco malignas e desdiferenciação: o estroma match-point! Células madre malignas y desdiferenciación: ¡el match-point estromal! Cancer stem cells and dedifferentiation: the stromal match-point! Maria Carmen Alpoim. Coimbra (Portugal)

10:00-10:30 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communicationsTUM C0-01 Screening of carcinoma metastasis by flow cytometry

Maria Acosta1, José Pereira1, Lylliane Luz1, Ana Costa1, Sílvia Amaro1, Martinha Chorão2, Maria José Carneiro3, Esmeraldina Júnior1, Maria Arroz1

1CHLO. Serviço de Patologia Clínica. Hospital S. Francisco Xavier. 2CHLO. Serviço de Anatomia Patológica. Hospital S. Francisco Xavier. 3Serviço de Anatomia Patológica. Hospital Garcia de Orta (Portugal).


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TUM C0-02 Expressão de queratinas nas células tumoraias de carcinoma gástricoCélia Nogueira1, Paulo Figueiredo2, Maria Augusta Cipriano3, Rui Gradiz1,3, Maximino Leitão3, Fernando Oliveira3, Francisco Castro e Sousa3, Fernando Martinho3, João M. Pereira2, Francisco Caramelo1, A. Meliço-Silvestre1, Tiago Carvalheiro4, Artur Paiva4

1Faculdade de Medicina. Universidade de Coimbra. 2IPO de Coimbra. 3Hospitais da Universidade de Coimbra. 4Centro de Histocompatibilidade do Centro (Portugal).

TUM CO-03 Significado clínico de la detección de células no-hemopoyéticas por citometría de flujo

R. Martínez1, F. Barriopedro1, E. García Fernández2, S. Alonso2, S. Láinez3, J.M. Machín3, G. Hernando3, B. Pinedo1, D. Subirá1

1Servicios de Hematología, 2Anatomía Patológica y 3Medicina Interna. Hospital Universitario de Guadalajara (Spain).

08:30-10:30 h Sala de Atos Sessão / Sesión / Session B ImuNOdEFICIêNCIAS PRImáRIAS E SECuNdáRIAS iNmuNOdEFiCiENCiAS PrimAriAS y SECuNdAriAS PRImARy ANd SECONdARy ImmuNOdEFICIENCIES Moderadores: Eduardo López Granados, Ana Espada de Sousa

08:30-09:00 h Imunopatogénese do HIV em 2013: o que estamos a perder? inmunopatogenia del ViH en 2013: ¿qué nos falta? HIV immunopathogenesis in 2013: what are we missing? Ana Espada de Sousa. Lisboa (Portugal)

09:00-09:30 h Aplicações recentes da citometria de fluxo como ferramenta básica para o diagnóstico de imunodeficiências primárias

recientes aplicaciones de la citometría de flujo como herramienta básica del diagnóstico de inmunodeficiencias primarias

Recent applications of flow cytometry in the diagnosis of primary immunodeficiency’s

Eduardo López-Granados. Madrid (España)

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10:00-10:30 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communications

INM C0-01 Candida albicans stimulates in vivo differentiation of hematopoietic stem and progenitor cells toward macrophages by a TLR2 dependent signalingJosé Enrique O’Connor, Javier Megías, Victoria Maneu, Pedro Salvador, Daniel Gozalbo, M. Luisa GilLaboratorio de Citómica. Unidad Mixta CIPF-UVEG. Universitat de València. Valencia. Departamento de Microbiología y Ecología. Universitat de València. Burjassot (Valencia). Departamento de Óptica, Farmacología y Anatomía. Universidad de Alicante. Alicante (Spain).

INM C0-02 Immune response during treatment of melanoma with high doses of interferon-alphaMaría Carmen Algueró, Jelena Vucetic, José Enrique O’Connor, Eduardo Nagore, Víctor Traves, Virtudes Soriano, Carlos Guillén, Rafael Botella-EstradaLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia. Dermatology Service. Valencian Institute for Oncology-IVO. Valencia. Dermatology Service. University Hospital La Fe. Valencia (Spain).

INM C0-03 The invariant natural killer T cells are differently affected in Fabry and Gaucher diseasesCatia S. Pereira1, M. Luz Maia1, Ana F. Dias1, Filippo Vairo2,3, Olga Azevedo4, Esmeralda Rodrigues5, Teresa Cardoso6, Fatima Ferreira7, Esmeralda Martins8, Ida V.D. Schwartz2,3, Elisa L. Teles5, Clara Sa-Miranda1, M. Fatima Macedo1,9

1Lysosome and Peroxisome Biology Unit (UniLiPe). IBMC. Instituto de Biologia Molecular e Celular. Universidade do Porto (Portugal). 2Servico de Genética Medica. Hospital de Clinicas de Porto Alegre (Brasil). 3Programa de Pós-Graduação em Genética e Biologia Molecular. Universidade Federal do Rio Grande do Sul (UFRGS). Brasil. 4Serviço de Cardiologia. Centro Hospitalar do Alto Ave. Guimarães (Portugal). 5Pediatria. 6Medicina Interna. Unidade de Doenças Metabólicas. 7Hematologia. Centro Hospitalar São João EPE. Porto (Portugal). 8Serviço de Pediatria. Centro Hospitalar do Porto (Portugal). 9SACS. Universidade de Aveiro (Portugal).

INM C0-04 Development of a flow cytometry-based potency assay for the immunomodulatory properties of mesenchymal stromal cells

Andreia Ribeiro, Matthew Griffin, Thomas Ritter, Rhodri CeredigRegenerative Medicine Institute (REMEDI). National Centre for Biomedical Engineering Science (NCBES). National University of Ireland. Galway (Ireland).

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1o:30-11:30 h Anfiteatro da Reitoria Simpósios Empresas / Simposios empresas / Symposium companies Moderador: Artur Paiva. Coimbra (Portugal)

Harmonia no diagnóstico das hemopatias malignas La armonía en el diagnóstico de las neoplasias hematológicas Harmony in the diagnosis of hematological malignancies Antoine Pacheco. Beckman Coulter

Cytognos, novos produtos 2013 Cytognos, nuevos productos 2013 Cytognos, new products 2013 Marta González Salinas. Cytognos

11:30-11:45 h Café / Cofee-break / Posters

11:45-13:15 h Anfiteatro da Reitoria Actualização das recomendações para o screening

diagnóstico de HPN / Actualización de las recomendaciones para el cribado diagnóstico de HPN / update of recommendations for the diagnostic screening of PNH

Alberto Órfao. Alexion

Sony Cell Sorter SH800, simplificando a separação celular Sony Cell Sorter SH800, la selección simplificada Sony Cell Sorter, sorting made simple

Greg Veltri. Izasa

um Prémio Nobel aplicado à citometria / un Premio Nobel aplicado a la citometría / A Nobel Prize aplied to cytometry

Gemma Coma, Marco A. Fernández. BD Biosciences

13:15-13:45 h Sala de Atos Assembleia Geral SIC/Asamblea General de la SiC SIC General meeting

13:45-14:30 h Almoço / Almuerzo / Lunch. Cantina de la Universidade de Aveiro


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14:30-15:45 h Anfiteatro da Reitoria Sessão plenária / Sesión plenaria / Plenary session Moderadora: Rosário Domingues dissecando as vias de sinalização com microesferas

fluorescentes por citometria de fluxo Evaluación de las vías de señalización celular

mediante análisis masivos con arrays de microesferas por citometría de flujo

dissecting signaling pathways by FC bead array Raquel Bartolomé. Salamanca (España)

15:45-17:15 h Anfiteatro da Reitoria Sessão / Sesión / Session A mICROBIOlOGIA / miCrOBiOLOGíA / miCrOBiOLOGy Moderadoras: Ana Tenreiro, Sónia Mendo

15:45-16:15 h utilidade da citometria de fluxo na malária: análise de dNA, hemozoína, GFP, entre outras

utilidad de la citometría de flujo en el paludismo: análisis de AdN, hemozoína, proteína verde fluorescente y otros

usefulness of flow cytometry in malaria: dNA stains, hemozoin, GFP and more

Thomas Hanscheid. Lisboa (Portugal)

16:15-16:45 h monitorização da fermentação da levedura do vinho por citometria de fluxo multiparamétrica: avaliação do estado fisiológico das células

Evaluación de la fermentación del vino inducida por levaduras mediante citometría de flujo

Following wine yeast fermentations by multiparameter flow cytometry: assessment of cell physiological state

Ana Tenreiro. Lisboa (Portugal)

16:45-17:00 h Avaliação da resistência a drogas anti-malária por citometria de fluxo

un nuevo método de citometría de flujo para evaluar la resistencia al tratamiento frente al paludismo

A novel flow cytometric approach to assess antimalarial drug resistance

Maria Rebelo. Lisboa (Portugal)

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17:00-17:15 h dispersão lateral da luz despolarizada no estudo das respostas imunes de monócitos com hemozoína em doentes com malária

depolarized side scatter en el estudio de la respuesta inmune de los monocitos con hemozoína de pacientes con paludismo depolarized side scatter to study immune responses

of hemozoin-containing monocytes from malaria patients Ana Góis. Lisboa (Portugal)

15:45-17:15 h Sala de Atos Sessão / Sesión / Session B SymPOSIum EuROFlOW dIAGNóSTICO E ClASSIFICAçãO dAS HEmOPATIAS mAlIGNAS diAGNóStiCO y CLASiFiCACióN dE LAS HEmOPAtíAS mALiGNAS dIAGNOSIS ANd ClASSIFICATION OF HEmATOlOGICAl mAlIGNANCIES Moderadores: Paulo Lúcio, Julia Almeida

15:45-16:15 h Inovação da citometria de fluxo pelo Consórcio do EuroFlow: novas estratégias para o diagnóstico e classificação das doenças hematológicas

innovación en citometría de flujo por el Consorcio EuroFlow: nuevas estrategias para el diagnóstico y clasificación de las hemopatías malignas

Innovation of flow cytometry by the EuroFlow Consortium: new strategies for diagnosis and classification of hematological malignancies

Jacques van Dongen. Rotterdam (Holland)

16:15-16:45 h Novas estratégias EuroFlow para o estudo das vias de diferenciação mielóide: aplicação no estudo de doenças mielóides

Nuevas estrategias de EuroFlow para el estudio de la maduración mieloide: aplicación en el diagnóstico de las hemopatías mieloides

Novel EuroFlow strategies for the study of myeloid differentiation pathways: application to the study of myeloid malignancies

Sergio Matarraz. Salamanca (España)


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16:45-17:15 h Construção de painéis EuroFlow para o diagnóstico e classificação de doenças linfoproliferativas crónicas de célula B

Construcción de paneles EuroFlow para el diagnóstico y clasificación de los síndromes linfoproliferativos crónicos B

Construction of the EuroFlow panels for the diagnosis and classificationof B-ClPd

Paulo Lúcio. Lisboa (Portugal)


17:30 h Sala 2 Reuniões dos grupos de trabalho da SIC reuniones de los grupos de trabajo de la SiC meetings of the working groups of the SIC



17:30 h Anfiteatro da Reitoria Seminário: Construção de painéis multicor para protocolos

por citometria de fluxo / Seminario: Construcción de paneles multicolor en ensayos de citometría de flujo / Seminary: Construction of panels in multicolor flow cytometry assays

Gemma Coma. BD Biosciences

18:30 h Passeio / Paseo / Boat trip moliceiro

20:30 h Jantar do congresso / Cena de clausura / Closing dinner. Hotel Meliá Ria (Cais da Fonte Nova Lote 5. 3810-200 Aveiro)

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09:00-11:00 h Anfiteatro da Reitoria Sessão / Sesión / Session A dOENçA RESIduAl míNImA Em HEmOPATIAS mAlIGNAS ENFErmEdAd míNimA rESiduAL EN HEmOPAtíAS mALiGNAS mINImAl RESIduAl dISEASE IN HEmATOlOGICAl mAlIGNANCIES Moderadores: Alberto Órfão, Maria Jorge Arroz

09:00-09:30 h Presente e futuro dos estudos da doença residual mínima Presente y futuro de los estudios de enfermedad mínima residual Present and future of minimal residual disease studies Jacques van Dongen. Rotterdam (Holland)

09:30-10:00 h Podemos aumentar a sensibilidade das técnicas imunofenotípicas para detecção de dRm por citometria de fluxo?

¿Podemos aumentar la sensibilidad de las técnicas inmunofenotípicas para detectar enfermedad mínima residual por citometría de flujo?

Can we increase the sensitivity of immunophenotypic techniques to detect minimal residual disease by flow cytometry?

Juan Flores. Salamanca (España)

10:00-10:30 h utilidade dos estudos de doença residual mínima em mieloma múltiplo

utilidad de los estudios de enfermedad mínima residual en el mieloma múltiple

utility of minimal residual disease detection in multiple myeloma

Bruno Paiva. Salamanca (España)


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10:30-11:00 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communicationsHEM C0-01 Can immunophenotypic CR be also achieved in relapsed multiple myeloma

patients?Bruno Paiva, Mauricio Chandia, M.ª Belén Vidriales, José J. Pérez, Noemi Puig, Lucía López-Corral, Enrique M. Ocio, Ramón García-Sanz, Norma C. Gutiérrez, María Victoria Mateos, Jesús F. San MiguelHospital Universitario de Salamanca. Salamanca (Spain)

HEM C0-02 Aproximación a la ontogenia de la leucemia linfática crónica y linfocitosis B monoclonal: identificación de marcadores serológicos de exposición a virus ubicuosJulia Almeida1, Santiago Muñoz-Criado2, Belén Espinosa1, Ignacio Criado1, Wendy G. Nieto1, Cristina Teodosio1, Alfonso Romero3, Paulino Fernández-Navarro4, Arancha Rodríguez-Caballero1, Marcos González Díaz5, Alberto Orfao1, and the Primary Health Care Group of Salamanca for the Study of MBL1Instituto de Biología Molecular y Celular del Cáncer. Centro de Investigación del Cáncer/IBMCC (CSIC-USAL). IBSAL. Departamento de Medicina y Servicio de Citometría. Universidad de Salamanca. Salamanca. 2Servicio de Microbiología, Hospital Universitario de Salamanca. Salamanca. 3Centro de Atención Primaria de Salud Miguel Armijo Salamanca. Sanidad de Castilla y León (SACYL). Castilla y León. 4Centro de Atención Primaria de Salud de Ledesma. Salamanca. Sanidad de Castilla y León. 5Servicio de Hematología. Hospital Universitario de Salamanca. IBMCC. IBSAL. Departamento de Medicina. Universidad de Salamanca (Spain).

HEM C0-03 Value of complete remission by flow cytometry in auto trasplanted patients diagnosed with multiple myelomaA. Lemes, B. Sevillano, D. Fiallo, T. Molero, M. Torres, C. Rodríguez, S. de la Iglesia, M. García1

Servicio de Hematología y 1Unidad de Investigación. Hospital Universitario de Gran Canaria Dr. Negrín. Las Palmas de Gran Canaria (Spain).

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09:00-11:00 h Sala de Atos Sessão / Sesión / Session B mICROPARTíCulAS POR CITOmETRIA miCrOPArtíCuLAS POr CitOmEtríA mICROPARTIClES By CyTOmETRy Moderadores: Frederico Cerveira, Elmano Ramalheira

09:00-09:30 h detecção de micropartículas e a sua utilidade na prática clínica detección de micropartículas y su utilidad

en la práctica clínica detection of microparticles and its clinical relevance Isabel Crespo. Barcelona (España)

09:30-10:00 h Contagem de micropartículas de plaquetas por citometria de fluxo Cuantificación de plaquetas por citometría de flujo Enumeration of platelet microparticules by flow cytometry Romaric Lacroix. Marseille (France)

10:00-10:30 h monitorização do dano tecidual em monócitos/macrófagos circulantes monitorización del daño tisular en los monocitos/macrófagos circulantes monitoring of tissue damage in circulating monocytes/macrophages Jacques van Dongen. Rotterdam (Holland)

10:30-11:00 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communicationsMICRO C0-01 Glycated and glycoxidized phosphatidylethanolamines on the stimulation

of monocytes and dendritic cellsCláudia Simões1, Ana C. Silva1,2, Pedro Domingues1, Paula Laranjeira2, Artur Paiva2, Rosário Domingues1

1Mass Spectrometry Center. UI-QOPNA. Department of Chemistry. University of Aveiro. Aveiro. 2Cytometry Service of the Blood and Transplantation Center of Coimbra. Coimbra (Portugal).

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MICRO-C002 Evaluation of reactive oxygen species production and mitochondrial membrane potential on adherent cell lines using flow cytometryTiago Pedrosa1, Helena Oliveira1, Sónia Pinho1, Cristina Monteiro1, Francisco Pinho1, Pedro Pinto1, Catarina Remédios1, Maria Costa1, Susana Barros1,4, Miguel de Oliveira1, Andreia Ascenso3, Sofia Varanda2, Manuel Santos2, Conceição Santos1

1Laboratory of Biotechnology and Cytomics. Department of Biology & CESAM. University of Aveiro (Portugal). 2Laboratory of Biology of the RNA and DNA microarrays. Department of Biology & CESAM. University of Aveiro (Portugal). 3Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL). Faculdade de Farmácia. University of Lisboa (Portugal). 4Department of Biochemistry and Molecular Biology. School of Pharmacy. University of Barcelona. Barcelona (Spain).

MICRO C0-03 Alkaline phosphatase live stain for stem cell researchM.D. García-Godoy, J. Petriz Vall d’Hebron Institut de Recerca. Barcelona (Spain).


11:30-13:30 h Anfiteatro da Reitoria Sessão / Sesión / Session A APlICAçõES dA SEPARAçãO CElulAR APLiCACiONES dE LA SEPArACióN CELuLAr CEll SORTING APlICATIONS Moderadores: Jorge Monserrat, Maria Soares

11:30-12:10 h Separação celular em hemopatias malignas Separación celular en hemopatías malignas Cell sorting in hematological malignancies Alberto Órfão. Salamanca (España)

12:10-12:35 h Novos avanços na separação celular magnética Nuevos avances en separación inmunomagnética New advances in magnetic cell sorting Alex Adán. Madrid (España)

12:35-13:00 h Separação e caracterização de subpopulações de espermatozóides: relevância clínica Separación y caracterización de poblaciones

de espermatozoides: relevancia clínica Isolation and characterization of spermatozoid subsets: Clinical relevance Ana Paula Sousa. Coimbra (Portugal)

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13:00-13:30 h Relevância do estudo de citogenética por FISH em células plasmáticas purificadas no diagnóstico e prognóstico de gamapatias monoclonais relevancia del estudio citogenético por FiSH realizado sobre

células plasmáticas purificadas en el diagnóstico y pronóstico de gammapatías monoclonales

Relevance of the cytogenetic study by FISH performed on purified plasma cells in the diagnosis and prognosis of monoclonal gammopathies

Alfredo Minguela Puras. Murcia (España)

11:30-13:30 h Sala de Atos Sessão / Sesión / Session B dOENçAS AuTOImuNES: CONTRIBuTOS dA CITOmETRIA dE FluxO ENFErmEdAdES AutOiNmuNES: CONtriBuCiONES dE LA CitOmEtríA dE FLujO AuTOImmuNE dISEASES: CONTRIBuTIONS OF FlOW CyTOmETRy Moderadores: António Pereira da Silva, Juan Antonio Vargas

11:30-12:10 h Recentes progressos no conhecimento da autoimunidade Avances recientes en el conocimiento de la autoinmunidad Recent progress in the understanding of autoimmunity Timothy Radstake. Nijmegen (Holland)

12:10-12:35 h O papel das células T Cd8+ em autoimunidade. Foco na artrite Papel de las células t Cd8+ en autoinmunidad, con especial atención en la artritis The role of Cd8+ T cells in autoimmunity. Focus in arthritis Helena Carvalheiro. Coimbra (Portugal)

12:35-13:00 h Aterosclerose em lúpus eritematoso sistémico Arterioesclerosis en el lupus eritematoso sistémico Atherosclerosis in systemic lupus erythematosus Raquel Castejón. Madrid (España)


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13:00-13:30 h Comunicações orais selecionadas Comunicaciones orales seleccionadas Select oral communicationsINM C0-01 Study of TLR expression on peripheral blood monocyte subsets

in rheumatoid arthritis patientsMercedes Martín-Manzanares1, Carolina García-Torrijos1, Esther San Antonio-Sánchez1, Patricia Ramírez-Guijarro1, Ana Gómez-Lahoz1, Beatriz Salvador-Barbero1, Darío Antolín Amérigo1,2, José Barbarroja-Escudero1,2, Jorge Monserrat-Sanz1, Melchor Álvarez-Mon Soto1,2

1Departamento de Medicina y Especialidades Médicas. Facultad de Medicina. UAH. Madrid. 2Servicio de Enfermedades del Sistema Inmune. Hospital Príncipe de Asturias. Alcalá de Henares, Madrid (Spain).

INM C0-02 NK cells dysfunction in systemic sclerosisTiago Carvalheiro1, Magda Lemos2, Cláudia Silva1,3, Mariana Raposo1,3, Mariana Santiago4, Maria João Salvador4, José António P. Silva4, Artur Paiva1,2

1Blood and Transplantation Center of Coimbra. Portuguese Institute of Blood and Transplantation. Coimbra. 2College of Health Technology of Coimbra. Coimbra. 3University of Aveiro. Aveiro. 4Rheumatology Department. University Hospital Center of Coimbra. Coimbra (Portugal).

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Posters

Biotecnologia

BIOTECH P0-01 Cytomic screening of candidate drugs on bacterial biosensors of oxidative stressGuadalupe Herrera, Laura Díaz, Susana Balaguer, José Enrique O’ConnorLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain)

citometria funcional

CIT PO-01 Microesferas fluorescentes: un nuevo método para estudios de ingesta y selectividad alimentaria en larvas de pescado por citometría de flujoJavier Gómez-Arbonés, Montserrat Teixidó, Miquel Angel Gallart, Roser Valles, Kruno Bonacic, Alicia Estévez, Sofia MoraisInstitut de Recerca Biomédica. Laboratorio clínico ICS Lleida. Hospital Universitario Arnau de Vilanova. Lleida (Spain). Universitat de Lleida. Instituto de Investigación y Tecnología Agroalimentaria. Unidad de Cultivos Acuícolas. Sant Carles de la Ràpita. Tarragona (Spain).

CIT PO-02 A cytomic in vitro assay to assess oxidative versus non-oxidative cytotoxicity using acoustic flow cytometry and violet laserGuadalupe Herrera, José Enrique O’Connor, Laura Díaz, Manuel BlancoLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain).

CIT PO-03 Prediction of human in vivo chemical toxicity by in vitro screening of oxidative stress with HepG2 and HepaRG cellsAngela Gomes, Luke Noon, Deborah J. Burks, José Enrique O’ConnorLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain). Laboratory of Molecular Endocrinology. Príncipe Felipe Research Center. Valencia (Spain).

CIT PO-04 Phenotypic and functional characterization of efflux pumps in cell lines used for in vitro toxicological studiesAngela Gomes, Alicia Martínez-Romero, José Enrique O’ConnorLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain). Cytomics Service. Príncipe Felipe Research Center. Valencia (Spain).

CIT PO-05 Analysis by multispectral image-in-flow cytometry of oxidative stress and antioxidant protection on mitochondrial compartmentFrancisco Sala de Oyanguren1, Guadalupe Herrera1, Lara Gibellini2, Marcello Pinti3, Sara De Biasi2, Andrea Cossarizza2, José-Enrique O’Connor1

¹Laboratory of Cytomics. Mix Unit CIPF-UVEG. Valencia (Spain). ²Department of Surgery, Medicine, Dentistry and Morphological Sciences. University of Modena and Reggio Emilia. Modena (Italy). 3Department of Life Sciences. University of Modena and Reggio Emilia. Modena (Italy)

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CIT PO-06 Study of Plasmodium falciparum blood life cycle by flow cytometry in a murine model of infectionVanessa Gómez1, Helen Garuti1, Noemí Magán-Marchal1, Lorena Cortés-Gil1, Sara Viera1, Leonard D. Shultz2, David M. Wilson1, Iñigo Angulo-Barturen1, María Belén Jiménez-Díaz1

1GlaxoSmithKline. Diseases of the Developing World Medicines Development Campus. Tres Cantos, Madrid (Spain). 2The Jackson Laboratory. Bar Harbor. Maine, ME (USA)

CIT PO-07 Utility of autoanalyser Sysmex XE-5000TM (SXE) in the detection of neoplastic cells in cerebrospinal fluid (CSF)A. Lemes1, D. Fiallo1, B. Sevillano1, T. Molero1, C. Rodríguez1, S. de la Iglesia1, M. García2

1Servicio de Hematología y 2Unidad de Investigación. Hospital Universitario de Gran Canaria Dr. Negrín. Las Palmas de Gran Canaria (Spain).

CIT PO-08 Oxidative stress during the time-course of erythroid differentiation in vivo: role of erythroid piruvate kinaseRebeca Sánchez-Domínguez, María García-Gómez, Miguel Ángel Martín Rey, José C. SegoviaUnidad de Diferenciación y Citometría. División de Terapias Innovadoras del Sistema Hematopoyético. CIEMAT. Madrid (Spain).

CIT PO-09 Expression of cytokines in monocytes and dendritic cells exposed to oxidized phosphatidylserinesTânia Melo1, Deolinda Santinha1, Raquel Nunes da Silva1, Ana Cristina Silva1,2, Elisabete Maciel1, Cláudia Simões1, Sara Horta1,2, Paula Laranjeira2, Artur Paiva2, Pedro Domingues1, M. Rosário M. Domingues1

1Mass Spectrometry Centre. QOPNA. Department of Chemistry. University of Aveiro. Aveiro (Portugal). 2Instituto Português do Sangue e da Transplantação, IP. Coimbra (Portugal). University of Aveiro. Aveiro (Portugal).

CIT PO-10 Vybrant® DyeCycle™ Violet stain (DCV) for side population assaysM.ª Dolores García-Godoy, Jordi PetrizVall d’Hebron Institut de Recerca. Barcelona (Spain).

Hematologia

HEM PO-01 Expression of the chemokine receptors CXCR3 and CCR5 in T lymphocytes and natural killer cells from umbilical cord bloodMarika Bini Antunes1,2, Magdalena Leander1, Rita Rebelo2, Patricia Benevides2, Ana Helena Santos1, João Rodrigues1, Lurdes Oliveira1, Maria Luis Queiros1, Marlene Santos1, Marta Gonçalves1, Sonia Fonseca1, Catarina Lau1, Maria Anjos Teixeira1, Margarida Lima1

1Haematology. Centro Hospitalar Porto. Porto (Portugal). 2Laboratory Department. Bebevida-Ciências para a vida. Porto (Portugal).

HEM PO-02 Anemia neonatal grave secundaria a hemorragia fetomaternaCarmen Buesa García, Enrique Colado Varela, Marta Costa Romero, Ana Escudero Gomís, Marina Navarro López, Regina Llorente de JesúsHospital Universitario Central de Asturias. Oviedo (Spain).

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HEM PO-03 Estudio de concordancia entre la citometría de flujo y otras técnicas para la valoración de infiltración de médula ósea por células de linfoma no Hodgkin BAida Bermejo1, Lizbeth Arriarán1, M.D. de Juan1, Álvaro Prada1, M.T. Iglesias2, M. Carmen Cipitria1, Pilar García1, Pilar Echániz1, Mercedes Rey1

1Laboratorio de Inmunología. 2Clinical Epidemiology Unit. CIBER-ESP. Hospital Universitario Donostia. San Sebastián (Spain).

HEM PO-04 Quantitation of residual leukocytes using acoustic-focusing flow cytometry and a permeant DNA dyeAlicia Martínez-Romero1, Guadalupe Herrera1, Laura Díaz1, Amparo Pinilla2, José Enrique O’Connor1

1Laboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain). 2Hematology Service. University Hospital, Doctor Peset. Valencia (Spain).

HEM PO-05 Histiocitosis de células de Langerhans. Diagnóstico por citometría de flujoFrancisco Sala, Guillermina Hurtado, Amaya Zabalza, Eva Vicente, Intza AoizServicio de Hematología A. Complejo Hospitalario de Navarra. Pamplona (Spain).

HEM PO-06 Pesquisa de clones de hemoglobinúria paroxística noturna em medula óssea de síndromes mielodisplásicasDiana Santos1, Ana Henriques2, Isabel Silva2, Maria Jesus Inácio2, Tiago Carvalheiro2, Artur Paiva1,2

1Escola Superior de Tecnologia da Saúde. Coimbra. 2Centro do Sangue e da Transplantação. Coimbra. Instituto Português do Sangue e da Transplantação. Coimbra (Portugal).

HEM PO-07 Trombopenia y neutropenia leve: ¿caso de HPN? Discusión sobre la aportación de CD16 y Flaer en la detección de clona HPN en sangre periférica y médula óseaBeatriz Álvarez1, F. Ataulfo González1, Elena Ruiz2, María Medrano1, Luz Conejo1, Diego Velasco1, Juan M. Alonso1, Alexandru Vlagea1, Jesús Villarrubia1, Raquel Guillén1

1Laboratorio Central de la Comunidad de Madrid BRSalud. Hospital Infanta Sofía. Madrid (Spain). 2Servicio de Hematología. Hospital del Tajo. Aranjuez (Spain).

HEM PO-08 Tumor burden influences the number of circulating normal cell populations in advanced-stage B-cell chronic lymphoid leukemiaGeorgiana Emilia Grigore1,2, Susana Barrena1, Martín Pérez-Andrés1, Miriam Fierro1, Julia Almeida1, Alberto Orfao1

1Instituto de Biología Molecular y Celular del Cáncer. Centro de Investigación del Cáncer/IBMCC (CSIC-USAL), IBSAL. Departamento de Medicina y Servicio de Citometría. Universidad de Salamanca. Salamanca (Spain). 2Gr. T. Popa University of Medicine and Pharmacy. Iasi (Romania).

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HEM PO-09 Immunomodulatory effects of novel therapies in B-cell chronic lymphoid leukemia patientsGeorgiana Emilia Grigore1,2, Susana Barrena1, Martín Pérez-Andrés1, Rosa Ana Rivas1, Julia Almeida1, Alberto Orfao1

1Instituto de Biología Molecular y Celular del Cáncer. Centro de Investigación del Cáncer/IBMCC (CSIC-USAL), IBSAL. Departamento de Medicina y Servicio de Citometría. Universidad de Salamanca. Salamanca (Spain). 2Gr. T. Popa University of Medicine and Pharmacy. Iasi (Romania).

HEM PO-10 Assessment of mature lymphocyte recovery after ammonium chloride red blood cell lysis for flow cytometry analysisJ. Flores-Montero1, J. Almeida1, S. Bötcher2, J. Caetano3, J.J. Pérez4, G. Gregori1,5, J. Te Marvelde6, M Pérez-Andrés1, J.J.M. van Dongen6, A. Orfao1, on behalf of the EuroFlow Consortium1Instituto de Biología Molecular y Celular del Cáncer. Centro de Investigación del Cáncer/IBMCC (CSIC-USAL), IBSAL. Departamento de Medicina y Servicio de Citometría. Universidad de Salamanca. Salamanca (Spain). 2Second Department of Medicine. University Hospital of Schleswig Holstein. Campus Kiel. Kiel (Germany). 3Department of Hematology. Portuguese Institute of Oncology (IPOLFG). Lisbon (Portugal). 4Department of Hematology. University Hospital Salamanca (HUS) and IBSAL. Salamanca (Spain). 5Gr. T. Popa University of Medicine and Pharmacy and Regional Institute of Oncology. Iasi (Romania). 6Department of Immunology. Erasmus MC. University Medical Center Rotterdam. Rotterdam (The Netherlands).

HEM PO-11 Estudio mediante citometría de flujo multiparamétrica de la recuperación de las poblaciones linfocitarias en pacientes trasplantados con células hematopoyéticas de sangre de cordón umbilicalLourdes Cordón1, Lineth López1,2, Amparo Sempere1, Jaime Sanz1, Pau Montesinos1, Guillermo Sanz1, Miguel A. Sanz1

1Servicio de Hematología y Hemoterapia. Hospital Universitario La Fe. Valencia (Spain). 2Servicio de Hematología. Complejo Hospitalario Metropolitano Dr. Arnulfo Arias Madrid. Panamá.

HEM PO-12 Hypoxia alters the DNA damage response of mouse mesenchymal stromal cells to γ-radiation treatmentTara Sugrue1, Noel F. Lowndes2, Rhodri Ceredig1

1Regenerative Medicine Institute. Department of Physiology. School of Medicine. Nursing and Health Sciences. 2Genome Stability Laboratory. Centre for Chromosome Biology. Department of Biochemistry. School of Natural Sciences. National University of Ireland. Galway (Ireland).

HEM PO-13 Estudio genómico de una leucemia con inmunofenotipo de linfoblástica aguda y “recaída” con inmunofenotipo de leucemia mieloide agudaJ. Melero Ruiz1, I. Vallcorba1, M.A. Mori Álvarez2, E. Doblaré Castellano1, M. Fernández-Cavada1, R. Vázquez1, E. Vallespín García2

1Servicio de Inmunología y Genética. Complejo Hospitalario Universitario de Badajoz. 2INGEMM. Hospital Universitario La Paz. Madrid (Spain).

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HEM PO-14 Expressão de CD27 nos plasmócitos de mieloma múltiplo e sua relação com as alterações genéticas mais frequentes nesta entidadeAna Sofia Gonçalves1, Tiago Carvalheiro1, Catarina Geraldes2, Ana Lopes1, Isabel Silva1, Maria de Jesus Inácio1, Susana Pedreiro1, Adriana Teixeira2, Artur Paiva1

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. 2Serviço de Hematologia Clínica. Centro Hospitalar e Universitário de Coimbra (HUC). Coimbra (Portugal).

HEM PO-15 Síndromes linfoproliferativos CD5+ no LLC: caracterización inmunofenotípica en relación con la presencia de t(11;14)L. López-Anglada1, M.B. Vidriales1, E. de Cabo2, M.C. Montes-Fernández3, R. Martos4, F.J. Díaz-Gálvez5, S. Fernández-Ferrero6, S. Redondo-Blasco7, J.M. Alonso8, G. Martín-Núñez9, R.H. Cantalejo10, C. Aguilar11,N. Puig1, A. Martín1, M. Díez-Campelo1, J.F. San Miguel1

1Hospital Clínico Universitario de Salamanca. Salamanca (Spain). 2Hospital del Bierzo. Ponferrada (Spain). 3Hospital Virgen de la Concha. Zamora (Spain). 4Hospital General de Segovia (Spain). 5Hospital Clínico de Valladolid (Spain). 6Hospital Universitario de León. León (Spain). 7Hospital Nuestra Señora de Sonsoles. Ávila (Spain). 8Hospital Río Carrión. Palencia (Spain). 9Hospital Virgen del Puerto. Plasencia (Spain). 10Hospital Santos Reyes. Aranda de Duero (Spain). 11Hospital de Santa Bárbara. Soria (Spain).

HEM PO-16 Multiparameter flow cytometry detection of clonal plasma cells in the bone marrow of patients with solitary plasmacytoma determines the risk of progression to active myelomaBruno Paiva, Mauricio Chandia, María Belén Vidriales, José J. Pérez, Noemí Puig, Lucía López-Corral, Enrique M. Ocio, Ramón García-Sanz, Norma C. Gutiérrez, María Victoria Mateos, Jesús F. San MiguelHospital Universitario Salamanca. Salamanca (Spain).

HEM PO-17 Alterações citogenéticas de alto risco e fenótipo das células plasmáticas em gamopatias monoclonais induzem alterações significativas nos compartimentos das células B na medula ósseaSofia Ramos, Tiago Carvalheiro, Catarina Geraldes, Adriana Teixeira, Artur PaivaInstituto Português do Sangue e Transplantação. Centro de Histocompatibilidade do Centro. Coimbra (Portugal)

HEM PO-18 Multidimensional flow cytometry immunophenotypic quantification and characterization of in vivo mesenchymal stem cells in MGUS and multiple myelomaRui Leite1, Bruno Paiva2, Tiago Carvalheiro3, Paula Rocha4, Helena Vitória4, Maria Amélia Pereira5, Catarina Geraldes6, Sofia Ramos6, Adriana Teixeira6, Hélder Trindade3, Artur Paiva1,3

1College of Health Technology of Coimbra (Portugal). 2University of Salamanca (Spain). 3Flow Cytometry Lab. Blood and Transplantation Center of Coimbra. Portuguese Institute of Blood and Transplantation, IP (Portugal). 4Clinical Hematology Department. Hospital Center of Tondela-Viseu (Portugal). 5Medicine Department. Distrital Hospital of Figueira da Foz (Portugal). 6Clinical Hematology. University Hospital Center of Coimbra (Portugal).

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HEM PO-19 Alterações nos diferentes compartimentos das células B normais da medula óssea em leucemia linfocítica crónica de células BTiago Carvalheiro1, Liliana Costa2, José Pedro Carda3, Sofia Ramos3, Ana Lopes1, Isabel Silva1, Maria de Jesus Inácio1, Susana Pedreiro1, Adriana Teixeira3, Artur Paiva1,2

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. Coimbra (Portugal). 2Escola Superior de Tecnologia da Saúde de Coimbra. Coimbra (Portugal). 3Serviço de Hematologia Clínica. Centro Hospitalar e Universitário de Coimbra (HUC). Coimbra (Portugal).

HEM PO-20 Respuesta de expresión de P-selectina en plaquetas activadas con ADP y trombina en pacientes con síndrome mielodisplásico.María José Cejalvo, Pilar Léon, Amparo Pinilla, Amparo López, Guadalupe Herrera, Secundino Ferrer, José Enrique O’ConnorServicio de Hematología. Hospital Universitario Doctor Peset. Laboratorio de Citómica. Unidad Mixta CIPF-UVEG. Valencia (Spain).

HEM PO-21 A new protocol for alkaline phosphatase live staining using unlysed whole bloodM.ª Dolores García-Godoy, Jordi PetrizVall d’Hebron Institut de Recerca. Barcelona (Spain).

HEM PO-22 Vybrant® DyeCycle™ Violet stain (DCV) reveals the heterogeneity of the CD34+ compartmentC. García-Espinosa, M.D. García-Godoy, G. Viscor, J. PetrizVall d’Hebron Institut de Recerca. University of Barcelona. Barcelona (Spain).

HEM PO-23 Avaliação das gamapatias monoclonais seguidas no Centro Hospitalar Cova da Beira por citometria de fluxo: introdução de novos marcadoresAndreia Monteiro1,2, Daniela Coelho1, Patrícia Amantegui1,2, Conceição Faria1,2, Mafalda Fonseca1

1CICS-UBI, Centro de Investigação em Ciências da Saúde. Faculdade Ciências da Saúde. Universidade da Beira Interior. Covilhã (Portugal). 2Serviço de Patologia Clínica. Centro Hospitalar Cova da Beira. Covilhã (Portugal).

inmunologia

INM PO-01 La respuesta inflamatoria en el líquido cefalorraquídeo de pacientes con infiltración leptomeníngea difiere en tumores epiteliales y linfomasJulia Illán1, Cristina Serrano2, Marta Simo3, Susana Castañón2, Raquel Gonzalo2, Lidia Gómez4, Javier Pardo5, Miguel Navarro6, María Martínez-García7, Javier Pérez8, Jordi Bruna3, Dolores Subirá9

1Unilabs Diagnósticos S.L.U. Madrid (Spain). 2Fundación Jiménez Díaz. Madrid (Spain). 3Hospital de Bellvitge-ICO Duran i Reynals. Hospitalet de LLobregat (Spain). 4Hospital Quirón. Madrid (Spain). 5Hospital Rey Juan Carlos. Móstoles (Spain). 6Hospital Universitario de Salamanca (Spain). 7Hospital del Mar. Barcelona (Spain). 8Hospital General de Elche (Spain). 9Hospital Universitario de Guadalajara (Spain).

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INM PO-02 BAL immune cell patterns identified by flow cytometry1Melina Noelia Nava González, 1Ainara Etcheverría, 2Manuel Menéndez, 3Miguel Arias, 1Lourdes Tricas1Servicio de Inmunología. 2Servicio de Análisis Clínicos. 3Servicio de Neumología. Hospital Universitario Central de Asturias. Oviedo (Spain).

INM PO-03 Altered functional activity of Th17 and Th1 cells in systemic sclerosisMariana Raposo1,2, Mariana Galante Santiago3, Tiago Carvalheiro1, Maria João Salvador3, M. Rosário Domingues2, José A.P. Silva3, Artur Paiva1

1Centro do Sangue e Transplantação de Coimbra. Coimbra (Portugal). 2Universidade de Aveiro. Aveiro (Portugal). 3Serviço de Reumatologia. Centro Hospitalar e Universitário de Coimbra. Hospitais da Universidade de Coimbra. Coimbra (Portugal).

INM PO-04 Caso clínico: “Primary effusion lymphoma”. Diagnóstico por citometría de fluxoCristina Marques1, Ana Teresa Nunes2, Manuela Bustorff 2, Maria José Teles1, Cláudio Reis1, Conceição Magalhães1, Sónia Silva1, Tiago Guimarães1,3

1Serviço de Patologia Clínica. Centro Hospitalar São João. EPE. 2Serviço de Nefrologia. Centro Hospitalar São João. EPE. 3Departamento de Bioquímica. FMUP (Portugal).

INM PO-05 Quantificação e caracterização fenotípica de diferentes subtipos de monócitos e de células dendríticas CD16+ em esclerose sistémicaLetícia Nunes1,2, Sara Horta3, Mariana Santiago3, Maria João Salvador4, José António Pereira da Silva4, Artur Paiva2

1Crioestaminal. Saúde e Tecnologia, SA. Biocant Park. Cantanhede. 2Centro de Sangue e da Transplantação do Centro. Coimbra (Portugal). 3Departamento Química. Universidade de Aveiro. Aveiro (Portugal). 4Serviço de Reumatologia do Centro Hospitalar e Universitário de Coimbra (Portugal).

INM PO-06 Study of bronchoalveolar lavage flow cytometry analysis in the diagnosis of patients with interstitial lung diseasesJ.L. García de Veas Silva, P. Fernandez Riejos, F. Fabiani, V. Sánchez-MargaletVirgen Macarena University Hospital. Seville (Spain).

INM PO-07 Development of an assay for in vitro immunotoxicity based on quantitative analysis by multispectral image-in-flow cytometry of NF-kB translocation in a monocytic cell lineFrancisco Sala de Oyanguren, Teresa Palau, Guadalupe Herrera, José Enrique O’ConnorLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain).

INM PO-08 Frequency and cytotoxic activity of different subtypes of CD4 and CD8 T cells in systemic lupus erythematosusAna Micaela Simões1, Tiago Carvalheiro1, Ana Vieira2, Maria de Jesus Inácio1,2, Ana Henriques1, Luís Inês3, José A.P. da Silva3, Artur Paiva1,2

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. Coimbra (Portugal). 2Escola Superior de Tecnologia da Saúde de Coimbra. Coimbra (Portugal). 3Serviço de Reumatologia. Centro Hospitalar e Universitário de Coimbra. Coimbra (Portugal).

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INM PO-09 Alterações das subpopulações de células B do sangue periférico em lúpus eritematoso sistémico: relevância na monitorização da atividade da doença e da terapêutica.Isabel Silva1, Ana Henriques1, Ana Lopes1, Luís Inês2, José A. Pereira da Silva2, Artur Paiva1

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. 2Serviço de Reumatologia. Centro Hospitalar e Universitário de Coimbra (Portugal).

INM PO-10 Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, NK and T cellsPaula Laranjeira1, Andreia Ribeiro1, Sandrine Mendes1, Isabel Velada1, Cristiana Leite2, Pedro Andrade3, Francisco Santos3, Ana Henriques1, Mário Grãos2, Carla Cardoso4, António Martinho1, M. Luísa Pais1, Cláudia Lobato da Silva3, Joaquim Cabral3, Hélder Trindade1, Artur Paiva1

1Blood and Transplantation Center of Coimbra, Portuguese Institute of the Blood and Transplantation. Coimbra (Portugal). 2Biocant. Technology Transfer Association. Cantanhede (Portugal). 3Department of Bioengineering and IBB. Institute for Biotechnology and Bioengineering. Technical University of Lisbon. Lisbon (Portugal). 4Crioestaminal. Saúde e Tecnologia, S.A. Cantanhede (Portugal).

INM PO-11 Increased immunological response by peripheral blood monocyte and dendritic cell subsets in hypogonadal men under testosterone replacement therapyMaría Almeida, Juan José Corrales, Mar Cordero, Lourdes Martín-Martín, Cristina Méndez, José Manuel Miralles, Alberto OrfaoServicio General de Citometría. Universidad de Salamanca. Servicio de Endocrinología. Hospital Universitario de Salamanca (Spain).

INM PO-12 Quantificação e caracterização fenotípica e funcional das diferentes subpopulações das células T γδ em lúpus eritematoso sistémicoMaria Inácio1, Ana Vieira2, Luís Inês3, Ana Lopes1, Tiago Carvalheiro1, José P. Silva3, Artur Paiva1

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. 2Escola Superior de Tecnologia da Saúde de Coimbra. 3Serviço de Reumatologia. Centro Hospitalar e Universitário de Coimbra (Portugal).

INM PO-13 Polychromatic 10 colours study of cytokine production by CD4 T lymphocytes in RA patients untreated and treated with MTXCarolina García-Torrijos1, Mercedes Martín-Manzanares1, Esther Sánchez-San Antonio1, Darío Antolín-Amérigo1,2, José Barbarroja-Escudero1,2, Ana María Gómez-Lahoz1, Patricia Ramírez-Guijarro1, Beatriz Salvador Barbero1, Jorge Monserrat-Sanz1, Melchor Álvarez-Mon Soto1,2

1Departamento de Medicina y Especialidades Médicas. Facultad de Medicina. Universidad de Alcalá. Alcalá de Henares (Spain). 2Servicio de Enfermedades del Sistema Inmune. Hospital Príncipe de Asturias. Alcalá de Henares (Spain).

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INM PO-14 Alterações no perfil de produção de quimiocinas pelos neutrófilos do sangue periférico em doentes com esclerose sistémicaCristiana Fonseca1, Tiago Carvalheiro2, Sara Horta2,3, Mariana Galante Santiago4, Maria João Salvador4, José A. P. Silva4, Artur Paiva1,2

1Escola Superior de Tecnologia da Saúde de Coimbra. Coimbra(Portugal). 2Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. Coimbra (Portugal). 3Univerisidade de Aveiro. Aveiro (Portugal). 4Serviço de Reumatologia. Centro Hospitalar e Universitário de Coimbra. Coimbra (Portugal).

INM PO-15 Avaliação fenotípica e funcional de diferentes subpopulações de linfócitos T na resposta à terapêutica com ribavarina e IFN-γ peguilado na infecção pelo vírus da hepatite CSusana Pedreiro1, Sílvia Andrade2, Andreia Ribeiro1, J. Eduardo Serra3, Ana Lebre3, Saraiva da Cunha3, Beatriz Pais4, Cristina Valente4, Ana Matos5, Paula Cristina Luxo5, António Martinho Teixeira1, Artur Paiva1

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. 2Faculdade de Ciências e Tecnologias da Universidade de Coimbra. 3Serviço de Infecciosas CHUC-HUC. 4Serviço de Infecciosas CHUC-HG. 5Laboratório de Microbiologia. Faculdade de Farmácia da Universidade de Coimbra (Portugal).

INM PO-16 Subpopulações de linfócito T, B e célula dendrítica na esclerose múltiplaAndreia Monteiro1,2, Mafalda Fonseca2, Luiza Rosado1,2, Pedro Rosado1,2, Catarina Cruto1,2, Tiago Carvalheiro3, Artur Paiva3

1Centro Hospitalar Cova da Beira. Covilhã (Portugal). 2Centro de Investigação em Ciências da Saúde. Faculdade de Ciências da Saúde. Universidade da Beira Interior. Covilhã (Portugal). 3Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. Coimbra (Portugal).

INM PO-17 Alterations in γδ T cells homeostasis in systemic sclerosisCláudia Silva1,2, Tiago Carvalheiro1, Mariana Santiago3, Maria João Salvador3, José António P. Silva3, Artur Paiva1

1Flow Cytometry Lab. Blood and Transplantation Center of Coimbra. Portuguese Institute of Blood and Transplantation. Coimbra (Portugal). 2University of Aveiro. Aveiro (Portugal). 3Rheumatology Department. University Hospital Center of Coimbra (Portugal).

INM PO-18 Avaliação das células T reguladoras por citometria de fluxoEmília Cardoso1, Judite Guimarães2, Esmeralda Neves2

1LabMED Saúde. Clínica Laboratorial Dr. Mário Moreira Lda. 2Serviço de Imunologia do Centro Hospitalar do Porto (Portugal).

INM PO-19 Impacto da imunoterapia específica subcutânea com dermatophagoides pteronyssinus nos basófilos do sangue periférico na rinite alérgicaAna Lopes1, Patrícia Azenha2, Celso Pereira3, Maria Inácio1, Isabel Silva1, Graça Loureiro3, António Martinho1, António S. Luís3, Artur Paiva1,2

1Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. 2Escola Superior de Tecnologia da Saúde de Coimbra. 3Serviço de Imunoalergologia. Centro Hospitalar e Universitário de Coimbra (Portugal).

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INM PO-20 Sera from systemic lupus erythematosus patients have the ability to in vitro activate monocytes and dendritic cellsAna Lopes1, Diane Gomes2, Tiago Carvalheiro1, Susana Pedreiro1, Luis Inês3, António Martinho2, José P. Silva3, Artur Paiva1,2

1Blood and Transplantation Center of Coimbra. Portuguese Institute of Blood and Transplantation. 2College of Health Technology of Coimbra. 3Rheumatology Department. University Hospital Center of Coimbra (Portugal).

INM PO-21 Caracterização fenotípica e funcional de monócitos e células dendríticas do sangue periférico em doentes com hepatite C crónica durante o tratamento anti-viralVanessa Ribeiro1, Andreia Ribeiro2, Susana Pedreiro2, Tiago Carvalheiro2, J. Eduardo Serra3, Ana Lebre3, Saraiva da Cunha3, Beatriz Pais4, Cristina Valente4, Ana Matos1, Artur Paiva2, Cristina Luxo1

1Laboratório de Microbiologia. Faculdade de Farmácia da Universidade de Coimbra. 2Centro do Sangue e da Transplantação de Coimbra. Instituto Português do Sangue e da Transplantação. 3Serviço de Infeciosas Centro Hospitalar e Universitário de Coimbra - HUC. 4Serviço de Infeciosas Centro Hospitalar e Universitário de Coimbra. Coimbra (Portugal).

INM PO-22 A case of intolerant patient for several NSAIDs specific COX-2 inhibitors diagnosed by basophil activation test (BAT)C. Cámara1, A. Rodríguez Trabado2, S. Romero1, J.A. García Trujillo1, S. Larios1, I. Tovar1, C. Muñoz Reja1, J. Manzano1, L. Fernández Pereira1

1Immunology Unit. Complejo Hospitalario de Cáceres (Spain) 2Allergology Unit. Hospital Campo Arañuero. Navalmoral de la Mata, Cáceres (Spain).

INM PO-23 Recuperação do número de linfócitos T CD4 circulantes em doentes com SIDA após vários anos de terapêutica antiretroviralManuela Rebordão1, Teresa Cabral2, Manuel Silva1

1Serviço Patologia Clínica. 2Unidade de Infecciologia. Hospital Forças Armadas. Lisboa (Portugal).

INM PO-24 Study of phagocytosis capacity and reactive oxygen species production of monocyte subsets and neutrophils in rheumatoid arthritis patientsAna María Gómez Lahoz1, Patricia Ramírez Guijarro1, Carolina García Torrijos1, Mercedes Martín Manzanares1, Beatriz Salvador Barbero1, Marga Lario Martínez1, Antolín Amérigo1,2, Jorge Monserrat Sanz1, Melchor Álvarez-Mon1,2

1Departamento de Medicina y Especialidades Médicas. Facultad de Medicina. Universidad de Alcalá. 2Servicio de Enfermedades del Sistema Inmune. Hospital Universitario Príncipe de Asturias. Alcalá de Henares (Spain).

INM PO-25 Construction of an 8-color combination-based panel for the identification of the main functional CD4+ T-cell populationsNicole Marques, Martín Pérez-Andrés, Julia Almeida, Alberto OrfaoInstituto de Biología Molecular y Celular del Cáncer. Centro de Investigación del Cáncer/IBMCC (CSIC-USAL), IBSAL. Departamento de Medicina y Servicio de Citometría. Universidad de Salamanca. Salamanca (Spain).

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INM PO-26 Profiling changes triggered by allergic contact dermatitis in dendritic cells and keratinocytes: a lipidomic approachDeolinda R. Santinha1,2, Tânia Melo1, M. Luísa Doria1, Elisabete A. Maciel1, Cláudia S.O. Simões1, Susana Rosa2, Bruno M. Neves2, Bárbara Macedo3, M. Teresa Cruz2, Pedro Domingues1, M. Rosário1, M. Domingues1

1Mass Spectrometry Centre. Chemistry Department. University of Aveiro. Aveiro (Portugal). 2Faculty of Pharmacy and Center for Neuroscience and Cell Biology (CNC). University of Coimbra. Coimbra (Portugal). 3Metabolomics Core Service. IBMC. Institute for Molecular and Cell Biology. Porto (Portugal).

INM PO-27 A imunidade inata na insuficiência cardíaca: papel dos monócitosCláudio Reis1, Patricia Lourenço2,3, Cristina Marques1, Nuno Silva1, Paulo Bettencourt2,3, Tiago Guimarães1,3

1Serviço de Patologia Clínica. 2Serviço de Medicina Interna. Centro Hospitalar São João. EPE. 3Faculdade de Medicina da Universidade do Porto (Portugal).

INM PO-28 Identification of phosphatidylserine oxidized/glycated/glyco-oxidized products by LC-MS and the study of their anti-inflammatory propertiesElisabete Maciel, Bruno M. Neves, Teresa Cruz, Pedro Domingues, M. Rosário M. DominguesMass Spectrometry Centre. UI-QOPNA. Department of Chemistry. University of Aveiro. Aveiro (Portugal).

INM PO-29 NK cell degranulation in response to human melanoma cell lines correlates with MICA/B, ULBP and CD155 expression: the relevance of multiple receptor-ligand interactions on NK cell activation Sara Morgado1, Beatriz Sánchez-Correa1, Javier G. Casado1,2, Federico Sierra1, Alejandra Pera3, Carmen Campos3, Soledad Sánchez Mateos1, Juan J. Gordillo1, Esther Durán4, Rafael Solana3, Raquel Tarazona1

1Immunology Unit. University of Extremadura. Cáceres (Spain). 2Stem Cell Therapy Unit. Minimally Invasive Surgery Centre. Cáceres (Spain). 3Immunology Unit. Instituto Maimónides para la Investigación Biomédica de Córdoba (IMIBIC). University of Córdoba. Hospital Reina Sofía. Córdoba (Spain). 4Histology and Pathology Unit. University of Extremadura. Cáceres (Spain).

INM PO-30 CMV infection and polyfunctionality of CD8+ T cellsAlejandra Pera1, Carmen Campos1, Corona Alonso1, Beatriz Sánchez-Correa2, Raquel Tarazona2, Rafael Solana1

1Department of Immunology. IMIBIC-Reina Sofia University Hospital. University of Córdoba (Spain). 2Immunology Unit. Department of Physiology. University of Extremadura. Cáceres (Spain).

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microBiologia

MICRO PO-01 Estudio de la sensibilidad a candinas mediante citometría de flujo multiparamétrica: comparación con el método de referenciaLourdes Cordón1, Eva M. González2, Emilia Cantón3, Javier Pemán2, Amparo Sempere1, Isidro Jarque1

1Servicio de Hematología y Hemoterapia, 2Servicio de Microbiología, 3Unidad de Microbiología Experimental. Hospital Universitario y Politécnico La Fe. Valencia (Portugal).

MICRO PO-02 Application of flow cytometry to study the effect of compound combinationsMaría Cándida Monteiro, Nuria de Pedro, Noureddine El Aouad, José R. Tormo, Lorena Rodrigez, Sara Palomo, Ignacio González, Fernando Reyes, Olga Genilloud, Francisca VicenteFundación MEDINA. Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía. Armilla, Granada (Spain).

DiverSoS

DIVER PO-01 Diagnostic value of flow cytometry in adenoid cavum hypertrophyCristina Serrano, Carolina Miranda, Susana Castañón, Raquel Gonzalo, Raquel Mata, Pilar LlamasLaboratorio de Citometría de Flujo. Servicio de Hematología. Fundación Jiménez Díaz-CAPIO. Madrid (Spain).

DIVER PO-02 Evaluation of a novel automatic system to measure confluency and cell count in adherent monolayer culturesLaura Díaz, Alicia Martínez-Romero, Guadalupe Herrera, José Enrique O’ConnorLaboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain).

DIVER PO-03 Applications of cell sorting strategies for biobanks: promoting the advance of biomedical researchM. Almeida, S. Mateos, T. Malvar, M.T. Márquez, M. Pérez-Caro, R. Pinto, M.I. Morante, J. García-Palomo, A. Regalado, E. Chamorro, E. de Álava, A. Orfao, A.C. García-MonteroBanco Nacional de ADN Carlos III (BNADN). Universidad de Salamanca. Salamanca (Spain).

DIVER PO-04 Modulation of macrophage’s lipid profile by Leishmania-infantum promastigotesVanessa Augusto, Deolinda Santinha, Bruno Neves, Anabela da Silva, Rosário DominguesDepartamento de Química. Universidade de Aveiro. Aveiro (Portugal).

DIVER PO-05 Variação do perfil lipídico e resposta imune no enfarte agudo do miocárdioAna Cristina Silva1,2, Cláudia Simões2, Tiago Carvalheiro1, Artur Paiva1, Rosário Domingues2

1Instituto Português do Sangue e da Transplantação. Centro de Sangue e da Transplantação de Coimbra. Coimbra (Portugal). 2Departamento de Química da Universidade de Aveiro. Aveiro (Portugal).

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DIVER PO-06 Mitochondrial dysfunction in cancer-related cachexia: are phospholipids target?Diana Antunes, Ana Isabel Padrão, Deolinda Santinha, Catarina Teixeira, Paula A. Oliveira, Rita Ferreira, Rosário DominguesDepartamento de Química. Universidade de Aveiro. Aveiro (Portugal).

tumoreS SÓliDoS

TUMS PO-01 Urinary protein profiling in urothelial carcinomaAna Isabel Padrão1, Rui Vitorino1, Sandra Magalhães1, Telma Martins1, Rita Nogueira-Ferreira1, Catarina Oliveira2, Paula Oliveira2, Francisco Amado1, Rita Ferreira1

1QOPNA. Department of Chemistry. University of Aveiro. Aveiro (Portugal). 2CECAV. Department of Veterinary Sciences. University of Trás-os-Montes e Alto Douro. Vila Real (Portugal).

TUMS PO-02 Caracterização do infiltrado leucocitário em biópsias de carcinoma gástrico e seu impacto prognósticoCélia Nogueira1, Paulo Figueiredo2, Maria Augusta Cipriano3, Rui Gradiz1,3, Maximino Leitão3, Fernando Oliveira3, Francisco Castro e Sousa3, Fernando Martinho3, João M. Pereira2, Francisco Caramelo1, A. Meliço-Silvestre1, Susana Pedreiro4, Artur Paiva4

1Faculdade de Medicina. Universidade de Coimbra. 2IPO de Coimbra. 3Hospitais da Universidade de Coimbra. 4Centro de Histocompatibilidade do Centro. Coimbra (Portugal)

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informação De intereSSe / informaciÓn De interÉS uSeful information

Sede do congresoUniversidade de AveiroEdifício da Reitoria. Campus Santiago. 3810-193 Aveiro (Portugal)

Secretaria técnicaGrupo Acción Médicac/ Fernández de la Hoz, 61. 28003 MadridTel.: 91 536 08 14 • Fax: 91 536 06 [email protected] Agência de viagensGabo Travelc/ Balcells, 21-25. 08024 BarcelonaTfno.: 93 285 75 55 • Fax: 93 285 75 56 e-mail: [email protected]

Página webwww.congresosic.com

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SPonSorS

maYor SPonSor

Secretaría técnica:

c/ Fernández de la Hoz, 61, entreplanta. 28003 Madrid Tfno.: 91 536 08 14 • Fax: 91 536 06 07

Correo electrónico: [email protected]

www.congresosic.com

Síndromes de falência medular / Síndromes de insuficiencia medular Bone marrow failure syndromes HEM C0-01 Estudo da maturação eritróide com base no CD44 e no CD35 em medula óssea normal e de síndromes mielodisplásicas

Raquel Rodrigues1,3, Paula Laranjeira4, Tiago Carvalheiro4, Margarida Farinha1, Paula Rocha2, Isabel Silva4, Maria Jesus Inácio4, Maria Reis Andrade2, João Ribeiro1, Helena Vitória2, Artur Paiva3,4

1Serviço de Patologia Clínica. Centro Hospitalar Tondela. Viseu. 2Serviço de Hematologia. Centro Hospitalar Tondela. Viseu. 3Escola Superior de Tecnologia da Saúde de Coimbra. 4Instituto Português de Sangue e Transplantação (Portugal).

Introdução: As SMD representam um grupo de doenças clonais da célula stem hematopoiética caracterizadas por hematopoiese ineficaz e risco de progressão para LMA. Considerando que a linha eritróide está muitas vezes afectada em SMD é importante a introdução de novos marcadores para melhor conhecimento da maturação eritróide normal e dessa forma para melhor detecção de aberrações fenotípicas em SMD. Objectivos: Avaliar a expressão de CD44 e CD35 na diferenciação eritróide normal e a forma como essa expressão está alterada em SMD. Material e Métodos: Estudaram-se 16 amostras de MO normais/reactivas e 48 amostras de SMD. De acordo com a OMS, os doentes foram classificados em CRDM (20), AREB-1 (15) e AREB-2 (13) e, de acordo com o IPSS, em Baixo risco (5), Intermédio-1 (8) e Intermédio-2 /Alto risco (14). Identificaram-se 4 estádios maturativos: I:CD34+/HLA-DR+/CD117+/CD35++/CD44++/CD123-/CD133-/CD45+/-; II:CD34-/HLA-DR-/CD117+/CD35+/CD44++/CD123-/CD133-/CD45débil; III:CD34-/HLA-DR-/CD117-/CD35+/CD44+intermédio/CD123-/CD133-/CD45-; IV:CD34-/HLA-DR-/CD117-/CD35+/CD44+débil/CD123-/CD133-/CD45-/SSCbaixo/FSCbaixo. Resultados: Na diferenciação normal verificou-se que a expressão de CD44 aumenta ligeiramente do estádio I para o II, diminuindo nos restantes estádios, ao passo que o CD35 é mais expresso no estádio I, diminui para o II, aumenta ligeiramente no III, tornando a diminuir no estádio IV. Em SMD observou-se aumento da expressão de CD44, mais acentuado em AREB-1 e 2 e na categoria de risco Intermédio-2/Alto risco. Este aumento da expressão de CD44 é independente da presença de anemia e/ou displasia morfológica. Quanto à expressão de CD35, observou-se aumento no estádio I da maturação em AREB-1 e 2 e diminuição nos estádios III e IV em AREB-2.

Conclusão: Os resultados obtidos revelaram o CD35 como um dos primeiros antigénios expressos pelas células CD34+ comprometidas à linha eritróide e a expressão de CD44 aumentada em SMD, parecendo ser anterior a displasia morfológica. Estudo da maturação eritróide com base no CD44 e no CD35 em medula óssea normal e de Síndromes Mielodisplásicas.docxEstudo da maturação eritróide com base no CD44 e no CD35 em medula óssea normal e de Síndromes Mielodisplásicas anónimo.

HEM C0-02 Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesis

Carlos Fernández-Giménez1, Maria Claudia Santos-Silva2, Antonio López1, Sergio Matarraz1, María Jara-Acevedo1, Laura Gutiérrez1, María Luz Sánchez1, Carlos Cervero3, Oliver Gutiérrez4, Nicolás González5, Carlos Salvador-Osuna6, Alberto Orfao1 1Department of Medicine and Service of Cytometry. IBSAL and Centro de Investigación del Cáncer (IBMCC USALCSIC). University Hospital of Salamanca and University of Salamanca. Salamanca (Spain). 2Laboratório de Oncologia Experimental e Hemopatias do Departamento de Análises Clínicas. Universidade Federal de Santa Catarina. Santa Catarina (Brazil). 3Hematology Department. Hospital General Virgen de la Luz. Cuenca (Spain). 4Hematology Department. Hospital Universitario Río Hortega. Valladolid (Spain). 5Hematology Department. Hospital Obispo Polanco. Teruel (Spain). 6Hematology Department. Hospital Universitario Miguel Servet. Zaragoza (Spain).

INTRODUCTION. Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. MATERIAL AND METHODS. Here, we investigated the clonal vs. reactive nature of residual maturing BM cells in 59 newly-diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome (MDS)-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. RESULTS. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly-diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. CONCLUSION. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly-diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

HEM C0-03 Utilidad de eosín-5-maleimida (EMA) en el diagnóstico de anemias hemolíticas con presencia de esferocitos en sangre periférica

Beatriz Álvarez, F. Ataulfo González, Diego Velasco, Luz Conejo, María Medrano, Juan M. Alonso, A. Vlagea, Marta Jiménez, Jesús Villarrubia, Raquel Guillén Laboratorio Central de la Comunidad de Madrid BRsalud. Hospital Infanta Sofía. Madrid (Spain).

INTRODUCCIÓN: El test de unión de membrana de los hematíes Eosín-5-Maleimida (EMA), está teniendo un uso creciente para el diagnóstico de Esferocitosis Hereditaria (EH) por su especificidad de unión a proteína estructural Banda-3 del hematíe. Objetivo: Mostrar la utilidad de la técnica como prueba de “screening” en pacientes con anemia hemolítica y con presencia de esferocitos en sangre periférica. MATERIAL Y MÉTODO: Se marcaron los hematíes con EMA según King et al (con leves modificaciones) en 95 controles y en 45 casos de anemia hemolítica con presencia de esferocitos y elevación de hematíes con hipercromía. RESULTADOS: En la tabla 1 se describen las diferencias encontradas de EMA, EMAcv, Hb, HIPER, MCV, MCH, MCHC, RDWCV y RETIC entre los casos estudiados. Según la expresión de EMA se encuentran 3 patrones característicos: 1) De Esferocitosis Hereditaria (EH), 2) De Anemia Hemolítica Autoinmune (AHAI) y 3) Patrón sugestivo de EH silente. El patrón de EH se caracteriza por mostrar menor intensidad de EMA que el resto de los grupos estudiados mientras que el patrón característico de AHAI es de mayor intensidad de expresión. El tercero corresponde a los casos de posible EH silente y se caracteriza por una menor expresión de EMA pero de carácter mucho más leve que el de la EH. De los 45 pacientes estudiados, 21 fueron diagnosticados de EH, 12 de AHAI (confirmados por Combs directo) y 12 resultaron sugestivos de EH silente (de los cuales, la mitad correspondían a estudios familiares). CONCLUSIÓN: El estudio de expresión de EMA en los hematíes de pacientes con anemia hemolítica, presencia de esferocitos y elevación de hematíes con hipercromía, resulta de gran utilidad para el diagnóstico diferencial de EH y de AHAI, ya que muestran patrones claramente diferenciados. Se observa un tercer patrón sugestivo de EH Silente, con menor expresión de EMA pero más sutil que el de la EH, pudiendo ser debido al menor porcentaje de esferocitos en esos pacientes, los cuales requerirán de estudios adicionales para confirmar EH.

Estudos das alergias por citometria Estudio de la alergia por citometría Flow cytometry in allergy INM C0-01 A case of delayed hypersensitivy to anisakis with clinical digestive manifestations diagnosed by lymphocyte activation test (LAT)

C. Cámara, A. Rodríguez Trabado*, S. Romero, J.A. García Trujillo, S. Larios, I. Tovar, L. Fernández Pereira Immunology Unit. Complejo Hospitalario de Cáceres. *Allergology Unit. Hospital Campo Arañuero. Navalmoral de la Mata. Cáceres (Spain).

Background: The most common clinical manifestations of anisakis allergy are urticaria-angioedema and anaphylaxis (type I). Nevertheless, cell-mediated delayed hypersensitivity reactions have been involved in few cases of work-related allergic contact dermatitis. We report the case of a 33 year old woman with an episode of hospitalization after acute abdominal pain and vomits 24 hours after ingestion of salmon. She was diagnosed too of asthma due to grass, mites and mold allergy. She worked as a cooker in a restaurant referring hand coetaneous itching while handling raw fish, without eczema. The day before colic she ate undercooked salmon not previously frozen Material and Methods: Prick tests and IgE specific (IgEs) measurement were done as usual. Patch tests were performed in triplicated with frozen raw anisakis larvae. For LAT we used a total peripheral blood method. Briefly, triplicates were cultured for 48 hours in the presence of allergenic anisakis extract for diagnosis (0.1, 1 and 10 mcg/ml). PBS and PHA were negative and positive controls. CD 69 expression on T cells was measured by a triple binding protocol and analyzed on a Navios Flow Cytometer. Positivity criteria was almost 3% of CD4 cells expressing CD 69, being almost the double of the expressed by the negative controls. Three healthy controls were studied. Results: Prick tests result was negative, but a delayed positive reaction 24 after was seen for anisakis (4x4 mm). Total IgE 23mUI/L. IgEs was negative for anisakis and fishes. On patch tests a delayed positive reaction was seen after 48 and 96 hours. TAL was positive for the two highest concentration tested on patients and negative in controls. Conclusions: The application of an additional test to study this kind of reactions can increase the global sensitivity of the study. In the best of our knowledge, no other cases of delayed hypersensitivity to anisakis have been studied by way of T cell CD69 expression.

INM C0-02 Monitorización de inmunoterapia con venenos de himenópteros (ITV) mediante test de activación de basófilos (TAB) seriados

C. Cámara, A. Rodríguez Trabado*, S. Romero, J.A. García Trujillo, S. Larios, Immunology Unit. Complejo Hospitalario de Cáceres. *Allergology Unit. Hospital Campo Arañuero. Navalmoral de la Mata. Cáceres (Spain).

Introducción: El TAB se ha demostrado como una herramienta diagnóstica de utilidad en el diagnóstico de alergia a venenos de himenópteros (S 83.5% y E 100%). Sin embargo, su utilidad para monitorización de la ITV está más discutida, con publicaciones contradictorias. Esa fue la razón de diseñar un estudio prospectivo, valorando tanto reactividad (máxima respuesta) como sensibilidad (cambios en la dosis mínima para lograr un TAB positivo). Material y métodos: 20 pacientes diagnosticados de alergia a venenos de himenópteros con indicación de ITV se siguieron desde el momento basal preITV, desde el año 2008 hasta hoy. Los tiempos de estudio fueron 1, 3, 6 y 12 meses, con posteriores controles anuales. Se realizó en sangre completa y con preincubación con buffer de IL-3). Se estudió un amplio rango de concentraciones del antígeno responsable en cada paciente (2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2500 y 5000 ng/ml). Protocolo de doble marcaje CD63-FITC/CD203c-PE, con lisis y lavado posterior. Adquisición y análisis en un citómetro FC500 con software MXP. Resultados: Al mes se ve un claro efecto, tanto en reactividad (disminuye hasta un 50%) como en sensibilidad (aumento del umbral de concentración mínima entre 10 y 50 veces). Hay un empeoramiento de ambos parámetros a los 6 meses, coincidente con el aumento de IgG4 e IgE específicas descrito. La respuesta se negativiza entre los 12-18 meses hasta concentraciones de 1000 ng/ml, permaneciendo así en los años posteriores. Se observan positivizaciones puntuales en relación tanto con picaduras espontáneas toleradas, como con espaciamiento de dosis. Conclusiones: La monitorización por TAB permite cuantificar de forma objetiva de los cambios que induce la ITV correlacionándose con la historia natural de la enfermedad, por lo que permitiría identificar pacientes con insufciente respuesta terapéutica.

Biotecnologia / BIOTECNOLOGÍA / BIOTECHNOLOGY BIOTEC C0-01 Utilização da citometria de fluxo multiparamétrica na monitorização da produção de biocombustíveis

Teresa Lopes da Silva, Alberto Reis Laboratório Nacional de Energia e Geologia. Lisboa (Portugal).

A actual escassez dos combustíveis fósseis, e os problemas que se a sua utilização acarreta para o ambiente tem levado à procura de fontes renováveis de energia. Alguns microorganismos são produtores de óleo que pode ser transformado em biodiesel. Outros têm a capacidade de produzir bioetanol, biogás, biohidrogénio e bioelectricidade. Estes processos devem ser monitorizados, de forma a obter-se informação, em tempo real, sobre o estado metabólico das células microbianas. Essa informação permite alterar a estratégia de controlo do processo, de forma a atingir-se as máximas produtividades. Nesta comunicação serão exemplificados dois processos de produção de biocombustíveis monitorizados por citometria de fluxo .No primeiro caso, será apresentada a produção de lípidos (óleo) pela levedura Rhodosporidium toruloides e, no segundo, a produção de bioetanol pela levedura Saccharomyces calrsbergensis em presença de inibidores resultantes da hidrólise de matreriais lenhecelulósicos. Discutir-se-á às vantagens da utilização da citometria de fluxo na monitorização destes processos, em relação às técnicas tradicionais de monitorização de bioprocessos atualmente utilizadas.

BIOTEC C0-02 Integrating cytomic assays for assessing metabolic effects of candidate drugs: proof of concept with tideglusib

Guadalupe Herrera, Laura Díaz, José-Enrique O’Connor Laboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia (Spain).

Introduction: Tideglusib, also known as NP031112, is a potent inhibitor of glycogen synthase kinase-3, neuroprotective and anti-inflammatory in animal models. Because of this, it is currently in clinical trials for Alzheimer disease and Progressive Supranuclear Palsy. However, no studies on Tideglusib metabolic effects are available, although some thiazolidinediones (TZDs) are associated with hepatic toxicity. We have designed and applied a strategy of high-content cytomic assays to quantifiy cellular markers and to define possible pathways of in vitro toxicity of Tideglusib, its main metabolite NP04113, other related TZDs and different positive control compounds. Materials and Methods: More than 10 different cellular endpoints of acute or sustained cell dysfunction, protein binding and oxidative biotransformation were integrated by multiparametric flow cytometry (FCM), automated fluorescence bioimaging (HCS), fluorimetry or oxymetry. We designed sensitive cellular in vitro models based on human and animal established cell lines and primary cultures. Results: Tideglusib was cytotoxic only at relatively high concentrations (IC50 > 60 mM) for renal and neural cells but, but not to liver cells. At lower concentrations Tideglusib induced oxidative stress in live cells, likely deriving directly from its biotransformation, as suggested by cell-free experiments. Tideglusib may bind to NH2- groups in cellular- and plasmatic proteins, which may be relevant to the pharmacokinetic and toxicological features of the test compounds. Conclusions: Cytomic strategy shows that the implications of the evaluated biomarkers of dysfunction in the mechanism of the cell death caused by this compound might be not evident in vivo and in all tissues, as the oxidative stress is not accompanied by significant cytotoxicity in HepG2 cells and human hepatocytes, thus classifying this compound as safer than other TZDs. Funding: Ministerio de Economia y Competitividad, Spain (BIO2010-19870)

BIOTEC C0-03 An assay for autophagy and apoptosis using multispectral imaging flow cytometry and SW872 adipocytic cells expressing GFP-LC3, an autophagosome marker

Francisco Sala de Oyanguren1, Sara de Biasi1, Lara Gibellini2, Marcello Pinti3, Massimo Riccio2, Milena Nasi1, Anto de Pol1, Andrea Cossarizza, José-Enrique O’Connor1 1Laboratory of Cytomics. Mix Unit CIPF-UVEG. Valencia (Spain). 2Department of Surgery, Medicine, Dentistry and Morphological Sciences. University of Modena and Reggio Emilia. Modena (Italy). 3Department of Life Sciences Sciences. University of Modena and Reggio Emilia. Modena (Italy).

Introduction: Autophagy is a physiological survival process involving lysosomal degradation of intracellular components. Autophagy dysregulation is associated to cancer, inflammation, degenerative diseases and drug toxicity. During autophagy, cytoplasmic LC3 protein is recruited to autophagosomal membranes and immunofluorescent localization of LC3 puncta by microscopy is mostly used for autophagy studies. We have set up and validated a novel assay by multispectral imaging flow cytometry (MsIFC) using SW872, an adipocytic cell line expressing constitutively GFP-LC3. Material and Methods: SW872 cells were stably transfected with pMSCVGFP-LC3 plasmid. Autophagy was induced by nutrient deprivation and by treatment with Atazanavir (ATV), an inhibitor of HIV protease recently shown to induce autophagy. MsIFC data were obtained for at least 20,000 events per sample using an ImageStream100 system (Amnis, Merck Millipore). Distribution of GFP-LC3 and co-localization of mitochondria, lysosomes and autophagosomes were determined with anti-hMit, anti-LAMP2 and DAPI. Lysosomes were assessed with Lysotracker Green. Apoptosis was revealed by Annexin V/7-AAD. Results: The distribution of LC3 was evaluated in cells starving for 24 hours or treated with ATV up to 200 mM. Cells undergoing autophagy showed LC3-specific puncta, thus indicating the formation of autophagosome. Autophagosomes were present after 6 and 16 hours of treatment only occasionally. Mitochondria, autophagosomes and lysosomes co-localized in cells treated with 50 mM ATV for 16h. Early, and, specially, late apoptotic cells increased in a dose dependent manner with ATV. Conclusions: Here we show that SW872, an adipocytic cell line which expresses constitutively GFP-LC3 is a suitable cell model to investigate autophagy, using MsIFC, a technique allowing to collect imagery of large numbers of cells in quantitative and statistically robust manner. Funding: Ministerio de Economia y Competitividad, Spain (BIO2010-19870).

Aplicações da citometria de fluxo em tumores sólidos Aplicaciones de la citometría de flujo En los tumores sólidos / Flow cytometry in solid tumors TUM C0-01 Screening of carcinoma metastasis by flow cytometry

Maria Acosta1, José Pereira1, Lylliane Luz1, Ana Costa1, Sílvia Amaro1, Martinha Chorão2, Maria José Carneiro3, Esmeraldina Júnior1, Maria Arroz1 1CHLO. Serviço de Patologia Clínica. Hospital S. Francisco Xavier. 2CHLO. Serviço de Anatomia Patológica. Hospital S. Francisco Xavier. 3Serviço de Anatomia Patológica. Hospital Garcia de Orta (Portugal).

Aim: To evaluate the efficiency of flow cytometry to detect malignant cells in different types of specimens as a complementary method to classical cytology and histology. Material and methods: A total of 119 fresh samples of pleural effusions (46), ascitic fluids (15), pericardial effusions (4), FNAs and lymph node biopsies (30), bone marrow aspirates (5), CSF (3) and other tissues (16) were analysed by flow cytometry, using monoclonal antibodies against Ber-EP4, an epithelial antigen, and CD45, a pan-leucocyte antigen. Results were compared with smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks. Results: Cytologic and histologic results were as follows: carcinoma cells, 52 specimens; benign and non-epithelial neoplasia, 67 specimens. The sensitivity of immunophenotyping was 96% and specificity was 99%. Conclusion: Flow cytometry is a rapid and highly effective method for the evaluation of several types of specimens. The detection of Ber-EP4 positive, CD45 negative cells, is strongly indicative of the presence of carcinoma cells, independently of the samples analysed. In our experience flow cytometry is a powerful tool to detect carcinoma cells and should be performed in parallel with classical morphologic and immunocytochemical studies to increase overall diagnostic accuracy.

TUM C0-02 Expressão de queratinas nas células tumoraias de carcinoma gástrico

Célia Nogueira1, Paulo Figueiredo2, Maria Augusta Cipriano3, Rui Gradiz1,3, Maximino Leitão3, Fernando Oliveira3, Francisco Castro e Sousa3, Fernando Martinho3, João M. Pereira2, Francisco Caramelo1, A. Meliço-Silvestre1, Tiago Carvalheiro4, Artur Paiva4 1Faculdade de Medicina. Universidade de Coimbra. 2IPO de Coimbra. 3Hospitais da Universidade de Coimbra. 4Centro de Histocompatibilidade do Centro (Portugal).

Introdução O Cancro gástrico (CG) é um dos tumores mais frequentes em Portugal e, globalmente é a segunda maior causa de morte por cancro. Com o objectivo de identificar novos marcadores prognósticos de evolução tumoral fomos estudar a presença de células tumorais circulantes (CTC) e caraterizar fenotipicamente os tumores gástricos. Material e Métodos Foram estudados 82 doentes com carcinoma gástrico e 33 indivíduos controlos. A identificação de células tumorais circulantes no sangue periférico foi efetuada por citometria de fluxo, em células CD45 negativas, utilizando anticorpos anti-queratinas (K) (K18, K20 e KPan). Os tumores foram tipados de acordo com o perfil de expressão de Ks e pela ploidia do DNA. Resultados CTC foram detetadas em 31,9% dos doentes e, a K18 revelou-se o marcador mais sensível, sendo encontrada em 23,6% dos casos. Todavia, a presença de CTC não manifestou nenhum valor prognóstico. Nos tumores o padrão de expressão de queratinas permitiu diferenciar 5 fenótipos - fenótipo 1 (K18+/k20-/kPan-), fenótipo 2 (K18+/k20+/kPan-), fenótipo 3 (K18+/k20+/kPan+), fenótipo 4 (K18+/k20-/kPan+) e fenótipo 5 (K18-/k20+/kPan+); em que o mais prevalente foi o fenótipo 1 com 43,1% dos casos. O perfil de expressão de queratinas nos tumores teve impacto significativo na sobrevivência dos doentes: o fenótipo 4 apresenta a melhor sobrevivência e o fenótipo 2 a pior. Em nenhum dos indivíduos saudáveis foi observado a presença de queratinas, quer no tecido gástrico quer no sangue periférico. Na ploidia do DNA 54,5% dos tumores foram diplóides e 45,5% aneuplóides, com fenótipo hiperdiplóide. A aneuploidia apresenta uma relação inversa com o estadiamento dos

tumores, sendo mais frequente nos tumores em fase mais inicial; no entanto associa-se a piores sobrevivências. Conclusão A pesquisa de queratinas parece ser um marcador específico para a deteção de tumores em tecidos, apresentando impacto na sobrevivência.

TUM CO-03 SIGNIFICADO CLÍNICO DE LA DETECCIÓN DE CÉLULAS NO-HEMOPOYÉTICAS POR CITOMETRÍA DE FLUJO. R. Martínez1, F. Barriopedro1, E. García Fernández2, S. Alonso2, S. Láinez3, J.M. Machín3, G. Hernando3, B. Pinedo1, D. Subirá1. Servicio de Hematología1, Anatomía Patológica2 y Medicina Interna3. Hospital Universitario Guadalajara, España. La práctica clínica reciente señala una progresiva demanda para estudiar por citometría de flujo (CMF) muestras que contienen células hemopoyéticas y de otras estirpes. OBJETIVO: describir con qué frecuencia la CMF detecta células no-hemopoyéticas y establecer la correlación con los hallazgos morfológicos. PACIENTES: 102 (54 hombres, 48 mujeres), mediana de edad de 58 años (12-96). MATERIAL: en 2012 se reunieron 110 muestras: 2 líquidos pericárdicos, 2 ascíticos, 19 pleurales, 29 lavados broncoalveolares/LBA, 6 punciones transbronquiales/PTB y material de biopsia o punción con aguja fina de 40 ganglios y 12 tumoraciones sólidas. MÉTODOS: estudio simultáneo de las muestras por morfología y CMF (protocolo habitual marcaje-lisis-lavado). El panel inicial de anticuerpos monoclonales siempre incluyó CD45 y sólo se añadió al estudio el antígeno epitelial EpCAM en los casos en los que se detectó una población CD45-. El requisito necesario fue ≥ 20 eventos agrupados. RESULTADOS: la CMF detectó células CD45- (entre 0,04 y 98% de la celularidad global) en 18/110 muestras (16,36%). Todas pertenecían a diferentes pacientes. La CMF hizo el estudio del antígeno EpCAM en 15/18 casos y los 15 fueron positivos. La morfología confirmó el diagnóstico de carcinoma en 7: 1 líquido pericárdico, 1 ascítico, 2 pleurales, 1 PTB, 1 ganglio y 1 tumoración. En las 8 muestras restantes (2 pleurales, 4 LBA, 1 PTB y 1 ganglio) la citología descartó una neoplasia epitelial. La mediana del % de células EpCAM+ por CMF fue 5% (0,04-89%) en los carcinomas y 0,3% (0,1-33%) en las poblaciones reactivas. CONCLUSIÓN: la CMF es una herramienta muy sensible para detectar células no-hemopoyéticas pero poco específica para discriminar entre población neoplásica y reactiva. Es crucial conocer qué muestras pueden contener células CD45-/EpCAM+ de forma fisiológica. En esta serie, la capacidad para detectar neoplasia epitelial fue máxima en líquido ascítico, pericárdico y tumoración sólida y nula en LBA.

Imunodeficiências primárias e secundárias Inmunodeficiencias primarias y secundarias Primary and secondary immunodeficiencies INM C0-01 Candida albicans stimulates in vivo differentiation of hematopoietic stem and progenitor cells toward macrophages by a TLR2 dependent signaling

José Enrique O’Connor, Javier Megías, Victoria Maneu, Pedro Salvador, Daniel Gozalbo, M. Luisa Gil Laboratorio de Citómica. Unidad Mixta CIPF-UVEG. Universitat de València. Valencia. Departamento de Microbiología y Ecología. Universitat de València. Burjassot (Valencia). Departamento de Óptica, Farmacología y Anatomía. Universidad de Alicante. Alicante (Spain).

Introduction: Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), and may play a role in hematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs toward the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation toward macrophages. Materials and Methods: Purified lineage negative cells (Lin-) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Three days after transplantation, bone marrow and spleen cells were enriched for CD45.2 cells and analyzed by multicolour fluorescence and flow cytometry to detect donor derived cells. Results: Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin- cells from TLR2-/- mice did not give rise to macrophages, whereas Lin- cells from TLR4-/- mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin- cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Conclusion: TLR2-mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that

TLR-mediated signaling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen. Funding: SAF2010-18256 (Ministerio de Economia y Competitividad, Spain).

INM C0-02 Immune response during treatment of melanoma with high doses of interferon-alpha

María Carmen Algueró, Jelena Vucetic, José-Enrique O’Connor, Eduardo Nagore, Víctor Traves, Virtudes Soriano, Carlos Guillén, Rafael Botella-Estrada Laboratory of Cytomics. Mix Research Unit CIPF-UVEG. Valencia. Dermatology Service. Valencian Institute for Oncology-IVO. Valencia. Dermatology Service. University Hospital La Fe. Valencia (Spain).

Introduction: High-dose Interferon-a (IFN) is used in adjuvant therapy of high-risk melanoma. We have assessed the immune response in 32 high-risk patients before treatment, after one month with 20x106 IFN units, 5 times per week, and after five months with 10x106 IFN units, three times per week. Material and Methods: Using flow cytometry and peripheral blood samples, we determined absolute number of leukocytes, T cell subpopulations (Th1, Th2, Treg, Tc), B lymphocytes and NK cells. Myeloid (MDC) and plasmocytoid (PDC) dendritic cells (DC) were also quantified and the expression of ILT-3 and CD33 on MDC and of ILT-3, CD123 and CD2 on PDC was determined. Results: Total leukocytes, MDC and PDC decreased after treatment, but the number of CD2high PDC increased. The absolute number of all the studied T-cell populations remained stable in the first month but decreased after six months following treatment. B lymphocytes and NK cells decreased in the first month but increased after 5 months with IFN. Unsupervised cluster analysis defined three groups of patients with different patterns of immune cell distribution. Consistent with our previous findings on the prognostic value of PDC in untreated melanoma, patients with more than 7 PDC/mL showed more intense immunophenotypic changes after IFN treatment. Conclusion: Our data confirm that IFN induces significant changes in the number and functional phenotype of lymphocytes and DC in peripheral blood of melanoma patients. The decrease of Treg cells might be involved in the effectiveness of the IFN-treatment. On the other hand, increased expression of tolerogenic molecule ILT-3 in both MDC and PDC might explain the decreased number of T cells. However, CD2high PDC increased by IFN treatment. These cells are able to lyse tumor cells without previous activation because they express lysozyme and are efficient in activating naïve T cells. Sponsoring: Fondo de Investigación Sanitaria (PI021679 and PS09/01001)and Izasa, SA.

INM C0-03 The invariant natural killer T cells are differently affected in Fabry and Gaucher diseases

Catia S. Pereira1, M. Luz Maia1, Ana F. Dias1, Filippo Vairo2,3, Olga Azevedo4, Esmeralda Rodrigues5, Teresa Cardoso6, Fatima Ferreira7, Esmeralda Martins8, Ida V.D. Schwartz2,3, Elisa L. Teles5, Clara Sa-Miranda1, M. Fatima Macedo1,9 1Lysosome and Peroxisome Biology Unit (UniLiPe). IBMC. Instituto de Biologia Molecular e Celular. Universidade do Porto (Portugal). 2Servico de Genética Medica. Hospital de Clinicas de Porto Alegre (Brasil). 3Programa de Pós-Graduação em Genética e Biologia Molecular. Universidade Federal do Rio Grande do Sul (UFRGS). Brasil. 4Serviço de Cardiologia. Centro Hospitalar do Alto Ave. Guimarães (Portugal). 5Pediatria. 6Medicina Interna. Unidade de Doenças Metabólicas. 7Hematologia. Centro Hospitalar São João EPE. Porto (Portugal). 8Serviço de Pediatria. Centro Hospitalar do Porto (Portugal). 9SACS. Universidade de Aveiro (Portugal).

Fabry and Gaucher diseases are lysosomal storage disorders (LSDs) caused by specific enzymatic defects that lead to the lysosomal accumulation of glycosphingolipids (globotriaosylceramide in Fabry disease and glucocerebroside in Gaucher disease). In several lysosomal storage disorders mouse models, including the Fabry mice, there is a decrease in a lipid-specific subset of T cells, the invariant natural killer T (iNKT) cells. However, this reduction has not been observed in LSDs patients. Human iNKT cells can be classified into three functionally distinct subsets according to CD4 and CD8 expression and these subsets produce different types of cytokines. We analysed the phenotype of iNKT cells in 15 Fabry, 18 Gaucher disease patients and 27 control subjects by flow cytometry. Interestingly, we found that iNKT CD4+ cells are differently affected in these two diseases: Fabry disease patients present a decrease of iNKT CD4+ cell percentage while Gaucher disease patients show an increase of this subset. Since iNKT CD4+ cells produce mainly the anti-inflammatory cytokine IL-4, while the CD4- iNKT cells produce the pro-inflammatory cytokine IFN-γ, we analysed these cytokines production in in vitro expanded iNKT cells from Gaucher and Fabry disease patients by flow cytometry. We found that Gaucher disease patients presented an increased percentage of iNKT cells expressing IL-4 when compared to control subjects while Fabry disease patients’ iNKT cells have a tendency to express less IL-4. There were no differences in IFN-γ production. These alterations suggest a bias towards a pro-inflammatory status in Fabry disease and anti-inflammatory status in Gaucher disease, that might have impact on disease pathology.

INMU C0-04 Development of a flow cytometry-based potency assay for the immunomodulatory properties of mesenchymal stromal cells

Andreia Ribeiro, Matthew Griffin, Thomas Ritter, Rhodri Ceredig Regenerative Medicine Institute (REMEDI). National Centre for Biomedical Engineering Science (NCBES). National University of Ireland. Galway (Ireland).

Introduction: MSC have been described as possessing immunomodulatory properties, having several effects on immune cells. However, there is a commercial need to develop reliable, rapid, quantifiable assays to assess the potency of allogenic human Bone Marrow Mesenchymal Stromal Cells (hBM MSC) on the recipient patient’s hematopoietic cells. For this purpose, we developed a rapid flow cytometry-‐based whole blood screening assay. The aims of this assay were to determine the effects of hBM MSC on the production of TNF-‐α and IL-‐12 by LPS-‐stimulated monocytes. Material and methods: Several parameters of the assay, such as anticoagulant and incubation time were optimized. hBM MSC were grown and used in different passage number and from different donors. A system of co-‐culturing hBM MSC and Monocytes at different rations was set up. We used the IntraPrep Kit from Beckman Coulter for intracellular staining using the protocol recommended by the manufacturers. We analysed the intracellular expression of TNF-‐α and IL-‐12/IL-‐23 p40 using PE-‐labeled monoclonal reagents. After staining, samples were acquired using the Accuri C6 four colour flow cytometer. Results: Heparin was the best anticoagulant in terms of cell activation. This was because anticoagulants containing divalent cation chelating agents removed Ca2+, found necessary for optimal monocyte activation. TNF-‐α production was optimal at 6H while IL-‐12 became optimally detectable by 24H. Our results also show that hBM MSC have the capability of suppressing monocyte activation. Different ratios of Monocyte:MSC were used and after 6H incubation, we detected a dose dependent suppression of TNF-‐α expression by monocytes at the higher concentrations of hBM MSC. Conclusions: In conclusion, hBM MSC have the capacity to immunoregulate monocyte activation in a dose-‐dependent fashion, and we have established a rapid and quantifiable assay to determinate the effects of hBM MSC on monocytes. Using this assay, the effects of MSC on lymphocyte activation can also be studied.

Doença residual mínima em hemopatias malignas Enfermedad mínima residual en hemopatías malignas Minimal residual disease in hematological malignancies HEM C0-01 Can immunophenotypic CR be also achieved in relapsed multiple myeloma patients?

Bruno Paiva, Mauricio Chandia, M.ª Belén Vidriales, José J. Pérez, Noemi Puig, Lucía López-Corral, Enrique M. Ocio, Ramón García-Sanz, Norma C. Gutiérrez, María Victoria Mateos, Jesús F. San Miguel Hospital Universitario de Salamanca. Salamanca (Spain)

Background: Although the introduction of new therapies has significantly improved outcome in relapse multiple myeloma (MM), the management of these patients remains challenging. Aims: Here, we hypothesized that novel therapeutic options and autologous or allogeneic stem cell transplantation (SCT) may induce minimal residual disease (MRD) clearance and that this may translate into extended survival in the relapse setting. Methods: 30 patients achieving CR after rescue therapy were referred for MRD investigation by multiparameter flow cytometry (MFC) and are the focus of the study. Rescue therapy immediately preceding CR was usually based on novel agent combinations (90%), followed by alloSCT in 37% of cases and autoSCT in 23%. The remaining 40% were not transplanted. Patients were defined to be in immunophenotypic CR (iCR) when < 1 phenotypically aberrant plasma cell was detected among 105 cells analyzed. Results: From the 30 patients in CR, 14 (47%) also achieved iCR whereas the remaining 16 (53%) were MRD+. MRD clearance was most likely achieved in patients submitted to SCT vs. those who were not (61% vs. 25%; P=.05). Only 2/14 (14%) MRD- cases experienced subsequent relapse as compared to 94% in MRD+ cases (P Conclusion: We show that achieving iCR is possible in a subset of relapse MM patients particularly after SCT, and identifies a subset of cases with long term relapse free survival.

HEM C0-02 Aproximación a la ontogenia de la leucemia linfática crónica y linfocitosis B monoclonal: identificación de marcadores serológicos de exposición a virus ubicuos

Julia Almeida1, Santiago Muñoz-Criado2, Belén Espinosa1, Ignacio Criado1, Wendy G. Nieto1, Cristina Teodosio1, Alfonso Romero3, Paulino Fernández-Navarro4, Arancha Rodríguez-Caballero1, Marcos González Díaz5, Alberto Orfao1, and the Primary Health Care Group of Salamanca for the Study of MBL 1Instituto de Biología Molecular y Celular del Cáncer. Centro de Investigación del Cáncer/IBMCC (CSIC-USAL). IBSAL. Departamento de Medicina y Servicio de Citometría. Universidad de Salamanca. Salamanca. 2Servicio de Microbiología, Hospital Universitario de Salamanca. Salamanca. 3Centro de Atención Primaria de Salud Miguel Armijo Salamanca. Sanidad de Castilla y León (SACYL). Castilla y León. 4Centro de Atención Primaria de Salud de Ledesma. Salamanca. Sanidad de Castilla y León. 5Servicio de Hematología. Hospital Universitario de Salamanca. IBMCC. IBSAL. Departamento de Medicina. Universidad de Salamanca (Spain).

Introducción: La alta sensibilidad alcanzada por la citometría de flujo (CMF) ha permitido la identificación de la ´´linfocitosis B monoclonal´´ (LBM); hoy sabemos que prácticamente todas las leucemias linfáticas crónicas (LLC) van precedidas de una fase de LBM. Estudios recientes sugieren que la exposición crónica a agentes infecciosos podría estar relacionada con la etiología de la LBM/LLC. Nuestro objetivo ha sido identificar marcadores serológicos de exposición a virus involucrados en linfomagénesis, en sujetos con LBM/LLC. Métodos: se han estudiado: a) 179 sujetos de la población general de Salamanca >40 años, sin linfocitosis, seleccionados a partir de 639 individuos en los que se había realizado un rastreo de la presencia de clones B circulantes (mediante CMF de alta sensibilidad y rastreo de ≥5x106 leucocitos), de los cuales 79 tenían algún clon LBM (LBMlo); b) 23 sujetos con LBM con linfocitosis (LBMhi); y c) 61 pacientes con LLC. En todos ellos se cuantificaron los niveles plasmáticos de IgM e IgG frente al VEB y CMV mediante ELISA quimioluminiscente; además, en sanos y MBLlo se determinaron Ac frente a VHB, VHC y VIH. Resultados: En todos los grupos, más del 90% de los sujetos tenía un patrón de Ac compatible con infección pasada por VEB y CMV, igualmente sin diferencias (p>0.05) en la prevalencia de infección por VHB (5/26 sanos vs. 0/8 MBLlo), VHC (n=1 sano) o VIH (n=0). Por el contrario, sí encontramos diferencias en los niveles de IgG anti-CMV en sujetos con infección pasada: mucho más elevados en las LLC y LBMhi que en la población general con y sin MBLlo (109±44 y 104±38 vs. 14±7 y 12±7 U/ml IgG anti-CMV, respectivamente; p<0,0001), sin diferencias entre las LBMhi, LLC de buen y de mal pronóstico. Conclusión:

CMV podría estar involucrado en la expansión clonal de células B tipo-LLC; se requieren estudios adicionales para determinar el papel concreto que desempeña el virus en la ontogenia y transformación de la enfermedad.

HEM C0-03 Value of complete remission by flow cytometry in auto trasplanted patients diagnosed with multiple mieloma

A. Lemes, B. Sevillano, D. Fiallo, T. Molero, M. Torres, C. Rodríguez, S. de la Iglesia, M. García1 Servicio de Hematología y 1Unidad de Investigación. Hospital Universitario de Gran Canaria Dr. Negrín. Las Palmas de Gran Canaria (Spain)

We reviewed 24 patients diagnosed with MM under (Tx) to assess the importance of CR determined by FC and immunofixation (IF) in overall survival. Patients: Men (n = 16) and women (n = 8) age (m = 59.6) The mean plasma cells (PC) % in bone marrow (BM) at diagnosis was 17.85%.20 patients received as preTx treatment with bortezomib-dexamethasone (x6), 3 received VBAD plus Bortezomib and one received thalidomide plus dexamethasone. All patients were conditioned with melphalan and one also received fludarabine. PC Immunophenotype (IP) at diagnosis: 75% of patients expressed CD56.33% expressed CD117 and out of the 12 patients that had CD28 tested, 25% of them were positive. 1 patient coexpressed CD28 and CD117 (5%) FISH at diagnosis: 4 patients were positive for t(4, 14) 1 of them also had del (17p). 4 were positive for t (11, 14), and one of them also showed del (13q) and IgH reorg. 1 showed chromosome 13 monosomy. One presented trisomies of chromosomes 5, 9 and 15 and another one had hyperdiploid karyotype. PreTx situation: We found no relationship between the % CP or its IP and the course of the disease. Out of the 11 patients with abnormal FISH at diagnosis, 9 were FC+ in the preTx determination. 2 were FC-, also carrying poor prognosis cytogenetics. In these cases the IF was also negative. PreTx situation of patients and their evolution shown in table 1: 100% of the patients with negative FC preTx, remained in RC after three years. Conclusion In our series, FC was slightly more sensitive than IF in detecting minimal residual disease (MRD), it seems quite important to achieve a negative MRD by CF at the time of Tx in MM patients, as well as negative results early post

RC o VGPR (%) (mfuy)

Relapse (%) (mfuy)

Deceased (%) (mfu months)

positive IF (n=14) 64.2 % (3.5) #28.5% (3.5) 7.14% (5) positive FC (n=16) *62,5 % (3.5) #25% (3.5) 12.5% (5) Negative IF (n=4) 50% (3.5) # 25% (1.0) 25% (6) negative FC (n=4) 100% (3) ------- --------- * 12.5% of became negative by FC in one year follow up by and persisted in CR # 7 of the 24 patients relapsed. In 4 patients, we performed a 2nd AutoTx, 2 of them are now in CR and the other 2 relapsed.

Micropartículas por citometria Micropartículas por citometría Microparticles by cytometry MICRO C0-01 Glycated and glycoxidized phosphatidylethanolamines on the stimulation of monocytes and dendritic cells

Cláudia Simões1, Ana C. Silva1,2, Pedro Domingues1, Paula Laranjeira2, Artur Paiva2, M. Rosario Domingues1 1Mass Spectrometry Center. UI-QOPNA. Department of Chemistry. University of Aveiro. Aveiro. 2Cytometry Service of the Blood and Transplantation Center of Coimbra. Coimbra (Portugal).

Introduction: Glycation of phosphatidylethanolamine (PE) is a reaction known to occur in vivo under the chronic hyperglycemic conditions found in diabetes. Glycated phosphatidylethanolamines were found in plasma and atherosclerotic plaques of diabetic patients, and its presence was correlated with increased oxidative stress. Upregulation of cytokines and other inflammatory mediators can be observed not only in diabetes, but also under oxidized phosphatidylcholine stimulation. Material and methods: We evaluated the effect of glycated (GluPE) and glycoxidized (GluOxPE) of three PEs: dipalmitoyl-phosphatidylethanolamine (DPPE), oleolyl-palmitoylphosphatidylethanolamine (POPE) and lineolyl-palmitoylphosphatidylethanolamine (PLPE); on monocyte and myeloid dendritic cell (mDC) stimulation. A total of 10 peripheral blood samples from healthy adult subjects were incubated with GluPE or GluOxPE and the expression of pro-inflammatory cytokines, IL-1β, IL-6, IL-8, MIP-1β and TNF-α, were determined using flow cytometry. Results: Glycated and glycoxidized PEs were able to increase the stimulation levels of monocytes and mDCs related to basal level, mainly notorious in the frequency of cytokine-producing cells. Besides all GluPE and GluOxPE were able to increase the cell stimulation related to the basal level, each PE induced different effect on cells. GluOxDPPE decreased the frequency of monocytes and mDCs expressing cytokines, when compared with GluDPPE. GluOxPOPE decreased the frequency of monocytes but increased the mDCs expressing cytokines. While GluOxPLPE increased the frequency of monocytes and mDCs expressing cytokines, when compared with GluPE.

Conclusion: Our results indicate that GluPE and GluOxPE stimulate the antigen-presenting cells and that the level of oxidation is an important factor in this process. Thus GluPE and GluOxPE could have an important contribution to the low-grade inflammation associated with diabetes and to the development of diabetic complications.abstract citometria- Claudia Simões.docxabstract citometria.

MICRO-C002 Evaluation of reactive oxygen species production and mitochondrial membrane potential on adherent cell lines using flow cytometry

Tiago Pedrosa1, Helena Oliveira1, Sónia Pinho1, Cristina Monteiro1, Francisco Pinho1, Pedro Pinto1, Catarina Remédios1, Maria Costa1, Susana Barros1,4, Miguel de Oliveira1, Andreia Ascenso3, Sofia Varanda2, Manuel Santos2, Conceição Santos1 1Laboratory of Biotechnology and Cytomics. Department of Biology & CESAM. University of Aveiro (Portugal). 2Laboratory of Biology of the RNA and DNA microarrays. Department of Biology & CESAM. University of Aveiro (Portugal). 3Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL). Faculdade de Farmácia. University of Lisboa (Portugal). 4Department of Biochemistry and Molecular Biology. School of Pharmacy. University of Barcelona. Barcelona (Spain)

Flow cytometry allows the analysis of multiple parameters of individual cells within large and heterogeneous populations. Its main applications vary from immunophenotyping, to ploidy analysis. Oxidative stress assessment, as a mechanism for compounds toxicity has become a focus for many investigators. Furthermore, the association between oxidative stress and several human diseases, including cancer and neurodegenerative diseases, has been demonstrated. The objective of our work is to implement efficient protocols for the evaluation of reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) by flow cytometry, in several cell types. The optimizations were performed with MG-63 cells and then applied to other cell lines, namely HaCaT, HEK293 and MCF-7. ROS assessment was performed with 2′ 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), which as high affinity for H2O2. To induce ROS production, cell cultures were exposed to rotenone. Cells auto-fluorescence was adjusted to first two decades and the concentration of rotenone which promotes maximum fluorescence was adjusted. This concentration was used as positive control for all experiments. The MMP was determined by measuring the accumulation of cationic fluorescent dye rhodamine 123 (Rh123). Rh123 binds to the negatively charged mitochondria proportionally to its membrane potential. To induce a decrease in MMP, etoposide was used as a control.

Both protocols were used to assess the effects caused by cadmium, sulforaphane, methotrexate and by the mistranslations of some amino acids. All compounds tested increased DCFH-DA signal of fluorescence, and decreased the fluorescence of Rh123, as expected. On the whole, with both protocols a good and reproductive performance was achieved in the conditions tested and all cell types. In this way, flow cytometry proved to be a useful tool to measure oxidative stress.

MICRO C0-03 Alkaline phosphatase live stain for stem cell research

M.D. García-Godoy, J. Petriz Vall d’Hebron Institut de Recerca. Barcelona (Spain).

BACKGROUND. Alkaline phosphatase (AP) is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and EGCs. While AP is expressed in most cell types, its expression is highly elevated in PSCs. Since most of stem cells express multidrug resistance transporters, we have validated APLS as a marker for stem-like Side Population cells. MATERIAL AND METHODS. In this study we have used human brain LN405 astrocytoma cells, which contain low numbers of cancer stem cells. Data were collected using the Attune® Acoustic Focusing Cytometer (Life Technologies) equipped with two lasers operating at 405 and 488 nm. Filter combination for APLS analysis was: 555 DLP, BP 530/30 (green). DCV signal was measured using linear scale at 50 mW. APLS was measured using logarithmic scale at 20 mW. RESULTS. APLS is not effluxed by multidrug resistance pumps, making feasible this stain for the identification of differentially expression within stem-like Side Population (SP) cells. In addition, alkaline phosphatase is expressed differentially under normoxic and hypoxic conditions, and overexpressed when SP cells were incubated at low O2 concentrations. CONCLUSIONS. Current available alkaline phosphatase substrates are toxic to the cells, which prevent them from propagating once stained. However, APLS can be used to easily distinguish primitive stem cells, since the stain maintains stem cell viability.

Doenças autoimunes: contributos da citometria de fluxo Enfermedades autoinmunes: contribuciones de la citometría de flujo Autoimmune diseases: contributions of flow cYtometry INM C0-01 Study of TLR expression on peripheral blood monocyte subsets in rheumatoid arthritis patients

Mercedes Martín-Manzanares1, Carolina García-Torrijos1, Esther San Antonio-Sánchez1, Patricia Ramírez-Guijarro1, Ana Gómez-Lahoz1, Beatriz Salvador-Barbero1, Darío Antolín Amérigo1,2, José Barbarroja-Escudero1,2, Jorge Monserrat-Sanz1, Melchor Alvarez-Mon Soto1,2 1Departamento de Medicina y Especialidades Médicas. Facultad de Medicina. UAH. Madrid. 2Servicio de Enfermedades del Sistema Inmune. Hospital Príncipe de Asturias. Alcalá de Henares. Madrid (Spain).

Introduction: Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation and disruption of diarthrodial joints. Previous studies have described the presence of different monocytes subsets in peripheral blood: Classic (CD14++CD16-), regulatory (CD14+CD16++) and transitional (CD14++CD16+ and CD14++CD16++). All of these monocyte subsets present Toll like Receptor (TLR). They play a pivotal role in monocyte activation, antigenic presentation and cytokine secretion, recognizing PAMPs and stimulating inflammatory responses.Objective: The aim was to study the expression of TLR2, 4 and 9 in the different monocyte subsets of RA patients untreated or treated with methotrexate (MTX).Material and Methods: Cohorts comprised forty RA patients according to treatment (untreated and treated with MTX) and activity (inactive and active) and thirteen healthy controls. We used CD14 and CD16, TLR2, 4 and 9 surface antigens to classify the monocyte subsets and to study the TLR expression and was assessed by multiparameter flow cytometry.Results: There was a significant increase of the regulatory monocytes associated to a decrease of the classic. We found that MTX treatment normalized the percentage of classic monocytes to control values. Regulatory monocytes showed a significant increase in the expression of TLR2, in both treated and untreated active patients with respect to healthy controls. Transitional CD16+CD14++ monocytes from MTX treated active patients presented an increase on the TLR2 percentage in contrast with the inactive. Classic monocytes expressed a higher TLR4 percentage in RA untreated active patients when compared with healthy controls. RA patients treated with MTX presented a reduced expression of TLR4 percentage in comparison with untreated.Conclusions: We

found that MTX treatment provoked an increase of the regulatory monocyte percentage and caused an alteration of the TLR2 expression percentage in transitional monocytes.Study of TLR expression on peripheral blood monocyte subsets in Rheumatoid Arthritis patients.docxAnomino.Study of TLR expression on peripheral blood monocyte subsets in Rheumatoid Arthritis patients.

INM C0-02 NK cells dysfunction in systemic sclerosis

Tiago Carvalheiro1, Magda Lemos2, Cláudia Silva1,3, Mariana Raposo1,3, Mariana Santiago4, Maria João Salvador4, José António P. Silva4, Artur Paiva1,2 1Blood and Transplantation Center of Coimbra. Portuguese Institute of Blood and Transplantation. Coimbra. 2College of Health Technology of Coimbra. Coimbra. 3University of Aveiro. Aveiro. 4Rheumatology Department. University Hospital Center of Coimbra. Coimbra (Portugal).

Background: Systemic sclerosis (SSc) is an inflammatory and fibrotic disease. There are some evidences of NK cells involvement in SSc by abnormalities in their activation status and cytokine production. Objectives: Evaluate the frequency of NK cells subsets (CD56bright and CD56dim) and their expression of LAIR1, CD49e and CD29, as well as to determine their functional activity by the expression of Granzime B, Perforin, TNF-a and IFN-g in SSc patients. Materials and Methods: The study enrolled 43 SSc patients, 30 with limited cutaneous subtype (lSSc) and 13 with diffuse subtype (dSSc). The healthy control group (HG) included 20 individuals. Immunophenotyping and functional characterization of NK cells subsets was assessed by flow cytometry. Results: It was observed a decrease in the frequency of total NK cells in SSc patients, more pronounced in SSc patients with pulmonary fibrosis or with digital ulcers, when compared to HG. After in vitro stimulation, it was observed an increase in the frequency of CD56dim NK cells producing TNF-a and IFN-g in patients with pulmonary fibrosis, compared to those without this clinical feature. It was also found a higher frequency of CD56bright NK cells expressing Perforin and Granzime B in SSc patients, particularly according to disease onset. It was verified a decrease in the CD49e expression in CD56dim in lSSc, compared to HG, on the other hand in the same group was found an increased LAIR-1 expression. The expression of CD49e in CD56bright NK cells were lower either in lSSc either in dSSc, in opposition, LAIR-1 expression was higher in lSSc and dSSc. No differences were observed to the CD29 expression. Conclusions: Important alterations were observed in the frequency/absolute values of NK cells subsets, in their phenotype, functional ability to produce cytokines and cytotoxic activity of SSc patients, which seem to be related to disease onset and clinical findings.NK cells dysfunction in Systemic sclerosis.docxNK cells dysfunction in Systemic sclerosis-A.

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