ABSTRACT
ABSTRACT Introduction: The reticulocyte count by flow cytometry (FC) - an automated counting method - can present errors due to the presence of interfering factors, contributing to a slight increase in results. However, automated methods have large advantages over the manual method, taken as reference, what justifies efforts to improve their quality. Objective: Evaluate platelet interference with the reticulocyte count by FC, using thiazole orange (TO) (FC/TO). Materials and methods: The method of reticulocyte count by FC/TO and a modified automated equivalent method, which excluded CD61-positive cells (platelets) from analysis (FC/TO/MOD), were compared to the manual method. Conclusion: Results were analyzed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) to assess interchangeability between the methods, by linear regression analysis and paired t-test. The exclusion of interfering fragments from result analysis by the modified method produced results in closer proximity to those of the reference method.
RESUMO Introdução: A contagem de reticulócitos por citometria de fluxo (CF) - um método de contagem automatizada - pode apresentar erros devido à presença de interferentes, contribuindo para uma ligeira elevação dos resultados. No entanto, os métodos automatizados possuem grandes vantagens em relação ao manual, tido como referência, o que justifica esforços para a melhoria de sua qualidade. Objetivo: Avaliar a interferência de plaquetas na contagem de reticulócitos por CF, utilizando laranja de tiazol (thiazole orange [TO]) (CF/TO). Materiais e métodos: O método de contagem de reticulócitos por CF/TO e um método equivalente automatizado modificado, no qual se excluíram células CD61-positivas (plaquetas) da análise (CF/TO/MOD), foram comparados com o método manual. Conclusão: Os resultados foram analisados de acordo com as recomendações do Clinical and Laboratory Standards Institute (CLSI) para avaliar a intercambialidade entre os métodos, por meio da análise de regressão linear e do teste t pareado. A exclusão de interferentes da análise dos resultados pelo método modificado demonstrou maior proximidade dos resultados com aqueles do método de referência.
ABSTRACT
The plants of the Euphorbiaceae family, especially those of the genus Euphorbia, are frequently used by Brazilian folk communities to treat a wide variety of infectious, tumoral and inflammatory illnesses. Among the species of this genus, Euphorbia tirucalli L. is widely used in some Brazilian regions, such as the Jequitinhonha River Valley. There is evidence that the latex produced by E. tirucalli has antiviral and antitumor activities, but little is known about the mechanisms involved in these effects. It is likely that the mechanism for such activities involves leukocyte activation and cytokine production. In this work, we aimed to evaluate the production of type 1 (TNF-α and IFN-γ) and type 2 (IL-4 and IL-10) cytokines by circulating leukocyte subsets submitted to brief stimulation with the crude latex of E. tirucalli. Peripheral blood leukocytes of twenty healthy subjects were submitted to 4 h incubation with crude E. tirucalli latex diluted in dimethylsulfoxide. After the incubation period, the cells were stained with FITC-conjugated monoclonal antibodies specific to the cell surface receptors CD4, CD8 and CD14, and to PE-conjugated monoclonal antibodies specific to the cytokines TNF-α, IFN-γ, IL-4 and IL-10. The acquisition and analysis of data were performed by flow cytometry. The results showed a significant increase (p<0.05) in the percentage of CD4+ T lymphocytes positive for the type 1 cytokines TNF-α and IFN-γ. Neutrophils and CD8+ T lymphocytes showed a mixed profile of cytokine production, characterized by an increase in the percentage of cells expressing IFN-γ, TNF-α, and IL-10. The data indicate a predominant type 1 cytokine response. The findings presented suggest that the effect popularly attributed to E. tirucalli usage may be attributed to its effect on the production of TNF-α and IFN-γ. However, the relationship between the in vitro and in vivo effects of E. tirucalli needs to be investigated.