Plasma vitellogenin (VTG) concentration is one of the primary apical endpoints of regulatory test methods using fish models where estrogen/androgen/steroidogenesis modalities are evaluated (e.g., OECD 229/230/234 and OCSPP 890.1350). As VTG is relied upon as a biomarker of exposure to endocrine active compounds, there has been a focus on the high variation observed in reported VTG levels, in the impacts this variation may have regarding assay robustness/animal usage/repeat testing, and how the methodology utilized by different labs to assess VTG concentrations may be a factor in the observed variation. One potential source of variation is sample storage.
It is currently unclear how storage methodology and storage duration influence the stability and subsequent analysis of fathead minnow plasma VTG. While relevant guidelines outline a storage procedure for plasma samples (i.e., store with a protease inhibitor at -80 °C), not all laboratories are using the described procedure. In addition, these guidelines provide no clear indication of the stability of samples under this storage regime. The paucity of information regarding the effects of storage method and duration makes it difficult to determine if these factors may be influencing the previously noted variation in VTG concentrations. Therefore, the objective of this project was to evaluate the impacts of storage method, storage duration, and freeze-thaw cycles on plasma vitellogenin concentrations.
To achieve this objective, composite plasma samples from male and female fathead minnows were stored under multiple storage scenarios (i.e., with/without a protease inhibitor at -20°C or -80°C) and sample VTG concentrations were measured via ELISA at 0.14 (1 day), 2, 6, and 15 weeks after collection for VTG concentration. The results of this project indicate that storage time and method have an impact on measured plasma VTG concentrations and may be a contributing factor to the high inter- and intralaboratory variation in control VTG concentrations. In addition, this dataset confirms that freeze-thaw cycles should be avoided if possible; however, if freeze-thaw cycles are anticipated in advance samples should be stored neat.
Dr. Julie Krzykwa, Senior Scientist, presented this research during the SETAC Europe 33rd Annual Meeting
, 30 April – 4 May 2023, in Dublin, Ireland.