Comparative Analysis of Dyes Used in the Assessment of Filgrastim Products Specific Activity by Biological in vitro Methods

Assessment of specific activity of Russian and foreign-made filgrastim products by biological in vitro methods is performed using different types of dyes. It is important to choose one cell staining dye in order to align the procedure of filgrastim specific activity assessment using cell culture. The aim of this study was to perform comparative assessment of tetrazolium and resazurin dyes in tests determining filgrastim ability to activate proliferation of sensitive cells. Materials and methods: NFS-60 (mouse myelogenous leukemia) cell line, 2nd International Standard for Granulocyte Colony Stimulating Factor (IS), as well as МТТ, MTS, WST-1, and alamarBlue dyes were used in the study. Proliferative activity of cells was assessed in vitro. The level of cell proliferation was assessed by fluorescence or absorbance intensity. Origin Pro 9.1. and Microsoft Excel applications were used for statistical processing of the obtained results. Results: the paper compares characteristics of the most widely used dyes. It describes the procedure for choosing optimal test conditions for some of the studied dyes. The authors analysed the potential of some factors, such as duration of cell suspension incubation with IS and with a dye, composition of the lysis buffer (for MTT staining), and different readout modes, to influence the final results. Despite the fact that all the studied dyes gave reproducible dose–response curves under the given test conditions, 50% effective concentrations showed no statistically significant differences in tests with only three dyes: МТТ, MTS, and alamarBlue (р > 0.05). Conclusions: better reproducibility of results was obtained in tests using МТТ and alamarBlue. The test procedure using alamarBlue is easier to perform and less time-consuming, it does not include the cell lysis stage and does not require additional reagents, therefore this dye may be recommended for harmonisation of the test procedure to be elaborated for the Russian Pharmacopoeia.

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