Active caspase-3 expression levels as bioindicator of individual radiosensitivity

ABSTRACT Several molecules and events involved in cell response to radiation-induced damage have been investigated towards a personalized radiotherapy. Considering the importance of active caspase-3 in the proteolytic cascade that ensures radiation-induced apoptosis execution, this research was designed to evaluate the expression levels of this protein as a bioindicator of individual radiosensitivity. Peripheral blood samples of 10 healthy individuals were gamma-irradiated (cobalt-60 source) with 1, 2 and 4 Gy (control: non-irradiated samples), and active caspase-3 expression levels were measured in lymphocytes, by flow cytometry, ex vivo and after different times of in vitro incubation (24, 48 and 72 hours). Short-term incubation of 24 h was the most adequate condition to evidence correlations between dose radiation and active caspase-3 expression. For each radiation dose, it was observed a significant inter-individual variation in active caspase-3 expression intensity, suggesting that this parameter may be suitable for evidence individual radiosensitivity. The methodology presented and discussed in this work may help to predict healthy tissues response to radiation exposure toward the better patient outcome.

Importância e vantagem da citometria de fluxo frente aos testes de triagem no diagnóstico da hemoglobinúria paroxística noturna / Importance and advantages of flow cytometry in screening tests for paroxysmal nocturnal hemoglobinuria diagnostic

Hemoglobinúria paroxística noturna (HPN) é uma doença clonal adquirida da célula tronco hematopoética em decorrência de uma mutação somática no gene PIG-A, causando inabilidade dos eritrócitos, leucócitos e plaquetas de se protegerem contra lise mediada pelo sistema complemento. Assim, avaliamos a eficiência dos testes de triagem para HPN (teste de Ham e pesquisa dos antígenos CD55 e CD59 em coluna de gel) utilizando a citometria de fluxo (CMF), que é capaz de detectar e quantificar o clone HPN. Inicialmente, selecionamos 63 pacientes que foram testados pelo teste de Ham entre janeiro/2003 e dezembro/2004, na Fundação Hemope. Destes, 15 tiveram seus testes positivos para HPN. O critério de inclusão dos casos para avaliação por CMF foi a obtenção de resultados do teste em gel concordantes com o teste de Ham positivo. Dessa maneira, quatro pacientes foram incluídos no grupo de estudo. Foram adicionados a esse grupo dois casos que exibiam clínica exuberante da doença, mas tiveram os resultados discordantes, explicado pelo fato de que o teste em gel foi realizado após terapia transfusional recente, provocando a suspeita de falso resultado normal. Submetemos esses seis casos à CMF, os quais todos se mostraram verdadeiros portadores da doença através da confirmação da existência do clone HPN em eritrócitos e granulócitos, em expressões variáveis. Os resultados da CMF comprovaram a limitação dos testes de triagem além de demonstrarem a relevância da citometria em identificar variações de intensidade do clone, garantindo inclusive o diagnóstico preciso em pacientes previamente transfundidos.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of the hematopoietic stem cell caused by a somatic mutation in the PIG-A gene, resulting in the inability of erythrocytes, granulocytes and platelets to protect themselves against complement system mediated lysis. Thus, PNH screening tests (Ham's test and CD55 e CD59 proteins investigation through gel column agglutination) were evaluated using flow cytometry, a test useful to detect and measure the PNH clone. Initially, 63 patients evaluated using the Ham's test between January 2003 and December 2004 from the Hemope Foundation were selected. From these, 15 cases were positive for PNH. The inclusion criterion for cytometry evaluation was a positive Ham's test. Thus, four patients were included in the study group. Furthermore, two cases with clinical symptoms of the disease but with negative results for PNH were included in this group. Negative results were explained by the gel test being performed after blood transfusion, giving a suspicion of false negative results. These six cases were submitted to flow cytometry with all cases proving to be positive for the disease as the PNH clone was confirmed, to different degrees, in both erythrocytes and granulocytes. The flow cytometry results proved the limitation of screening tests as well as showing the importance of cytometry in the identification of the intense variations of clone guaranteeing precise diagnosis in previously transfused patients.