Evaluation of the interference of platelets in reticulocyte counting by flow cytometry using thiazole orange / Avaliação da interferência de plaquetas na contagem de reticulócitos por citometria de fluxo, utilizando laranja de tiazol

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.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.

The crude latex of Euphorbia tirucalli modulates the cytokine response of leukocytes, especially CD4+ T lymphocytes

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.

Clinical and laboratory status of patients with chronic Chagas disease living in a vector-controlled area in Minas Gerais, Brazil, before and nine years after aetiological treatment

Twenty-eight Chagas disease patients (CD), 22 with the indeterminate clinical form (IND) and six with the cardiac or digestive form (CARD/DIG), were treated with benznidazole and underwent clinical and laboratorial analysis before (IND and CARD/DIG) and nine years after [patients after treatment (CDt), patients with the indeterminate clinical form at treatment onset (INDt) and with the cardiac or digestive form at treatment onset (CARD/DIGt)] treatment. The data demonstrate that 82.1 percent of CDt patients (23/28) remained clinically stable and 95.4 percent of the INDt (21/22) and 33.3 percent of the CARD/DIGt (2/6) patients showed unaltered physical and laboratorial examinations. The clinical evolution rate was 2 percent/year and was especially low in INDt patients (0.5 percent/year) relative to CARD/DIGt patients (7.4 percent/year). Positive haemoculture in treated patients was observed in 7.1 percent of the cases. None of the INDt (0/21) and 33.3 percent of the CARD/DIGt (2/6) patients displayed positive cultures. The PCR presented a positive rate significantly higher (85.2 percent, 23/27) than haemoculture and two samples from the same patient revealed the same result 57.7 percent of the patients. Conventional serology-ELISA on 16 paired samples remained positive in all individuals. Semi-quantitative ELISA highlighted significant decreases in reactivity, particularly in INDt relative to IND. Non-conventional serology-FC-ALTA-IgG, after treatment, showed positive results in all sera and 22 paired samples examined at seven and nine years after treatment, demonstrated significantly lower reactivity, particularly in INDt patients. This study was retrospective in nature, had a low number of samples and lacked an intrinsic control group, but the data corroborate other results found in the literature. The data also demonstrate that, even though a cure has not been detected in the none-treated patients, the benefits for clinical evolution ...

Otimização da técnica de citometria de fluxo para análise do fator tissular em monócitos de sangue periférico / Flow cytometric assay optimization for peripheral blood monocyte tissue factor analysis

A citometria de fluxo permite a análise individual de células quando há expressão de moléculas de membrana, produtos citoplasmáticos e nucleares. Quando se utiliza a marcação prévia das células com anticorpos monoclonais é possível estudar constituintes de membrana característicos de uma determinada linhagem celular. Assim, esta metodologia tem sido largamente utilizada, principalmente na área de onco-hematologia e dos transplantes de medula óssea. A possibilidade de se utilizarem dois ou mais anticorpos monoclonais marcados com fluorocromos diferentes permite o estudo em uma determinada população celular de substâncias pró-coagulantes produzidas por células envolvidas nos mecanismos da hemostasia, abrindo-se a possibilidade de aplicação desta metodologia em áreas de estudo da fisiopatologia relacionada aos estados de hipercoagulabilidade. Os monócitos estão altamente envolvidos nos processos fisiopatológicos envolvendo a formação de trombo e placas ateromatosas, e o Fator Tissular (FT) consiste no principal ativador fisiológico do sistema de coagulação sangüínea. Monócitos estimulados ou não por lipopolissacárides de Escherichia coli expressam FT em condições fisiológicas normais. Neste trabalho buscou-se otimizar as condições de determinação da expressão de FT em monócitos por citometria de fluxo, a partir de metodologia já descrita, em duas populações de indivíduos sadios, com faixas etárias diferentes, no sentido de se estabelecerem os níveis de expressão de FT. Para a análise dos resultados foram utilizadas duas metodologias que definem o percentual de células expressando FT. Dos resultados obtidos, pode-se concluir que não há diferença significativa na expressão de FT de monócitos em função da idade e que os dois métodos de análise utilizados não diferem entre si

The development of the flow cytometric assay usingmonoclonal antibodies labeled with differentfluorescent substances enables the identification of aparticular cell population even if it is present inheterogeneous cell samples. This technique has beenapplied to oncohematology and bone marrowtransplantation studies. Two combined fluorescentmonoclonal antibodies enable the study of a particularcell population in the expression of procoagulantsubstances produced by cells involved in homeostaticmechanisms. The application of this methodologycreates the perspective of pathophysiologic studiesrelated to hypercoagulability states. Considering thatmonocytes are highly involved in pathophysiologicmechanisms contributing to thrombus formation andatheromatous plaques and that Tissue Factor representsthe principal physiologic activator of the clotting system,this study constitutes a potential tool for obtainingnew insights of the role of monocytes in diseasesassociated to hypercoagulability states. The present work aimed to establish the optimization of conditionsfor measuring the tissue factor expression in monocytesstimulated or not by lipopolysaccharides fromEscherichia coli and analyzed by flow cytometric assaybased on a previous methodology. Blood samples werecollected from healthy subjects and divided in two ageranges. Studies on monocytes were carried outcomparing two methods of analysis, which define thepercentage of cells expressing tissue factor on theirsurface. From the results, it was concluded that thereis no difference between the two age ranges related tothe tissue factor expression in monocytes. In addition,there were no significant differences between the twoassessed methods of analysis. Rev. bras. hematol.

Infecçäo e doença pelos vírus linfotrópicos humanos de células T (HTLV-I/II) no Brasil / Infection and disease caused by the human T cell lymphotropic viruses type I and II in Brazil

A infecçäo pelos vírus HTLV-I/II encontra-se presente em todas as regiöes brasileiras, mas as prevalências variam de um estado para outro, sendo mais elevadas na Bahia, Pernambuco e Pará. As estimativas indicam que o Brasil possui o maior número absoluto de indivíduos infectados no mundo. Testes de triagem de doadores e estudos conduzidos em grupos especiais (populaçöes indígenas, usuários de drogas intravenosas e gestantes) constituem as principais fontes de informaçäo sobre essas viroses em nosso país. O HTLV-I causa a leucemia/linfoma de células T do adulto (LLTA), a paraparesia espástica tropical/mielopatia associada ao HTLV (TSP/HAM), uveíte associada ao HTLV (HAU) e anormalidades dermatológicas e imunológicas. O HTLV-II näo se mostrou associado a nenhuma doença até o momento. O diagnóstico é feito com testes de triagem (ELISA, aglutinaçäo) e confirmatórios (Western Blot, PCR). Estes vírus säo transmitidos pelo sangue e agulhas contaminadas, através de relaçöes sexuais e de mäe para filho, especialmente através do aleitamento materno. Medidas de prevençäo devem focalizar a orientaçäo de doadores soropositivos, mäes infectadas e usuários de drogas intravenosas