The invention relates to a method for determination of the frequency of regulatory T-cells in samples obtained from human blood and to methods for the preparation of compositions comprising predetermined amount of regulatory T-cells. The invention is based on the conception that a large fraction of regulatory T-cells present in human peripheral blood do not express detectable amounts of Foxp3, the master transcription factor used for identification of regulatory T-cells, as a result of cytokine deprivation outside of the tissue context.