Figure 1

Experimental design flow diagram. (A) In the first experiment, to assess the effect of the type of anticoagulant system and storage-temperature on BDNF stability, peripheral blood was collected from the cubital vein by venipuncture into five plasma collection tubes (K2EDTA, Li-Hep, Li-Hep (gel), Na-Hep, and Na-Hep (glass). For each participant (14 female and 10 males), approximately 200 µl of plasma from each of the five plasma collection tubes was aliquoted into 1.8 mL cryovials. One 200 µl aliquot of plasma from each of the five plasma collection tubes was refrigerated at 4 °C overnight, and used to quantify BDNF the following day (Time 0; never frozen). Four aliquots per collection tube were frozen at − 20 and four were frozen at − 80 °C until required for analysis at 1, 3, 6, and 12 months post-collection. (B) To evaluate the effect of repeated freeze–thaw cycles (1–3 cycles) on BDNF stability, plasma samples (n = 16) collected in K2EDTA tubes from women recruited to a previous study were used for subsequent BDNF analysis. The first freeze–thaw to quantify BDNF by ELISA occurred within 6 months of freezing and subsequent freeze-thaws (#2 and 3) occurred randomly within the next 6 months. K2EDTA = potassium EDTA, Li-Hep = lithium heparin, and Na-Hep = sodium heparin.