Indices of immune function used by ecologists are mostly unaffected by repeated freeze-thaw cycles and methodological deviations

Over the past couple of decades, measuring immunological parameters has become widespread in studies of ecology and evolution. A combination of different immunological indices is useful for quantifying different parts of the immune system and comprehensively assessing immune function. Running multiple immune assays usually requires samples to be repeatedly thawed and re-frozen. There is some evidence that repeated freezing and thawing can affect assay

Our data suggest that repeated freeze-thaw cycles do not impair assay results to the point of influencing ecological or evolutionary conclusions. Plasma samples can be safely stored in one tube and thawed repeatedly for different assays. Nevertheless, we recommend consistent treatment of samples in terms of freeze-thaw cycles or other laboratory treatments to minimize the potential for introducing a systematic bias.

Repeated freezing and thawing of plasma had no effect on lysis titres, haptoglobin concentrations, bacterial killing capacity, or total immunoglobulin levels. Agglutination titres were unaffected by up to five cycles but were lower after ten freeze-thaw cycles. For the haemolysis-haemagglutination assay and haptoglobin concentrations, re-analysing samples 1 year apart yielded highly correlated data. For the haemolysis-haemagglutination assay, the source of rabbit blood did not influence the results, and the untreated vs. blocked plates differed slightly overall, but at the individual level assay results were highly correlated. Using different rabbit blood sources or different types of microtitre plates yielded highly correlated data. Conclusions: Our data suggest that repeated freeze-thaw cycles do not impair assay results to the point of influencing ecological or evolutionary conclusions. Plasma samples can be safely stored in one tube and thawed repeatedly for different assays. Nevertheless, we recommend consistent treatment of samples in terms of freeze-thaw cycles or other laboratory treatments to minimize the potential for introducing a systematic bias.