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Abstract(s)
A eritropoiese é um processo complexo de diferenciação de progenitores
eritróides em glóbulos vermelhos que se dá, na medula óssea dos humanos, durante a
infância e a vida adulta. Este processo, regulado por diversos factores, permite a
diferenciação de progenitores eritróides, não identificáveis morfologicamente, em
precursores eritróides (proeritroblastos, eritroblastos basófilos, eritroblastos
policromáticos, eritroblastos ortocromáticos), estes em reticulócitos e, por fim, em
glóbulos vermelhos, células pequenas, anucleadas e bicôncavas, altamente
especializadas no transporte de oxigénio. Nos laboratórios clínicos, o estudo da
diferenciação da linha eritróide é tradicionalmente efectuado através da análise da
morfologia, e contagem diferencial das células nos diferentes estadios de diferenciação
reconhecíveis pelas suas características morfológicas. Neste estudo avaliamos a
maturação da linha eritróide por citometria de fluxo, através da marcação de amostras
de medula óssea com uma combinação de oito anticorpos monoclonais conjugados com
fluorocromos (anti-CD36 FITC, anti-CD105 PE, anti-CD34 PerCP-Cy5.5, anti-CD117
PE-Cy7, anti-CD33 APC, anti-CD71 APC-H7 / APC-Alexa Fluor 750, anti-HLA-DR
PB e anti-CD45 PO) e comparamos os resultados com os obtidos no estudo
morfológico. O estudo por citometria de fluxo permitiu identificar quatro estadios de
maturação com perfil fenotípico distinto. No estadio 1, mais imaturo, as células
expressam todos os antigénios analisados, no estadio 2 perdem a expressão de CD45,
CD34, HLA-DR e CD33, no estadio 3 perdem a expressão de CD117 e no estadio 4,
mais maduro, a expressão de CD105. A expressão de CD71 e de CD36 mantém-se ao
longo de todo o processo maturativo, atingindo o máximo nos estadios 3 e 4,
respectivamente. Os estadios fenotípicos 1, 2, 3 e 4 parecem corresponder, grosso
modo, aos estadios morfológicos de proeritroblasto (podendo incluir préproeritroblastos),
eritroblasto basófilo, eritroblasto policromático e eritroblasto
ortocromático, respectivamente. Em resumo, utilizando um painel de imunofenotipagem
com 8 cores, caracterizamos a maturação da linha eritróide na medula óssea por
citometria, identificamos diferentes estadios maturativos com base no perfil fenotípico e
estabelecemos a sua correlação com os estadios identificáveis por critérios
morfológicos.
Erythropoiesis is a complex process of differentiation of erythroid progenitors into red blood cells that occurs in the bone marrow of humans during childhood and adulthood. This process, regulated by several factors, allows the differentiation of erythroid progenitors, not morphologically identifiable, into erythroid precursors (proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts), these into reticulocytes and finally, into red blood cells, that are small, anucleated, uniform and biconcave cells highly specialized in oxygen transport. In clinical laboratories, the morphology analysis and differential counts are of great importance for the study of the erythroid differentiation. In this study we have studied the maturation of the erythroid lineage by flow cytometry, by staining bone marrow samples with a combination of eight monoclonal antibodies with different specificities, conjugated with different fluorochromes (anti-CD36 FITC, anti-CD105 PE, anti-CD34 PerCP-Cy5.5, anti-CD117 PE-Cy7, anti-CD33 APC, anti-CD71 APC-H7 / APC-Alexa Fluor 750, anti-HLA-DR PB and anti-CD45 PO) and compared the results with those obtained by morphology. Flow cytometry allowed the identification of four stages of red cell maturation with different phenotypic profiles. In stage 1, more immature, erythroid cells expressed all the antigens analyzed, in stage 2 they lose CD45, CD34, HLA-DR and CD33, followed by CD117 in stage 3 and CD105 in stage 4. CD71 and CD36 expression persist till the end of the erythroid maturation, reaching the maximum in stages 3 and 4, respectively. The stage 1 probably correspond to proerytroblasts (although some preproerytoblasts may also be included), stage 2 to basophilic erythroblasts, stage 3 to polychromatic erythroblasts and stage 4 to orthochromatic erythroblast. In summary, using an eight-color staining, we characterized the red cell maturation by flow cytometry and established a relationship with the phenotypically identifiable stages with those traditionally identified by morphology.
Erythropoiesis is a complex process of differentiation of erythroid progenitors into red blood cells that occurs in the bone marrow of humans during childhood and adulthood. This process, regulated by several factors, allows the differentiation of erythroid progenitors, not morphologically identifiable, into erythroid precursors (proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts), these into reticulocytes and finally, into red blood cells, that are small, anucleated, uniform and biconcave cells highly specialized in oxygen transport. In clinical laboratories, the morphology analysis and differential counts are of great importance for the study of the erythroid differentiation. In this study we have studied the maturation of the erythroid lineage by flow cytometry, by staining bone marrow samples with a combination of eight monoclonal antibodies with different specificities, conjugated with different fluorochromes (anti-CD36 FITC, anti-CD105 PE, anti-CD34 PerCP-Cy5.5, anti-CD117 PE-Cy7, anti-CD33 APC, anti-CD71 APC-H7 / APC-Alexa Fluor 750, anti-HLA-DR PB and anti-CD45 PO) and compared the results with those obtained by morphology. Flow cytometry allowed the identification of four stages of red cell maturation with different phenotypic profiles. In stage 1, more immature, erythroid cells expressed all the antigens analyzed, in stage 2 they lose CD45, CD34, HLA-DR and CD33, followed by CD117 in stage 3 and CD105 in stage 4. CD71 and CD36 expression persist till the end of the erythroid maturation, reaching the maximum in stages 3 and 4, respectively. The stage 1 probably correspond to proerytroblasts (although some preproerytoblasts may also be included), stage 2 to basophilic erythroblasts, stage 3 to polychromatic erythroblasts and stage 4 to orthochromatic erythroblast. In summary, using an eight-color staining, we characterized the red cell maturation by flow cytometry and established a relationship with the phenotypically identifiable stages with those traditionally identified by morphology.