Stability

The stability of GFAP in plasma exposed to various temperatures and freeze-thaw cycles was determined. Sets of 19 aliquots were prepared from low, intermediate, and high GFAP concentration plasma pools and treated as outlined by Andreasson et al. [23]. Briefly, aliquot 1 was immediately frozen and stored at − 80 °C. Aliquots 2 to 6 were exposed to 1, 2, 3, 5, or 7 freeze-thaw cycles where specimens were thawed and stored at room temperature for 2 h then stored again at − 80 °C. Aliquots 7 to 12 were stored at room temperature for 1, 2, 4, 24, 72, or 168 h before freezing at − 80 °C. Aliquots 13 to 18 were stored at 4 °C for 1, 2, 4, 24, 72, or 168 h before freezing at − 80 °C. Aliquot 19 was stored at − 20 °C for 30 days before freezing at − 80 °C. On the day of the assay, all aliquots were thawed together and assayed in duplicate.

Assay selectivity was evaluated by comparing assay measurements between serum and plasma from the same animal and investigating assay performance in the presence of hemolysis. Male and female C57Bl/6 mice, aged 2.5 to 4 months old, and APOE3 mice, aged 5 months old, were exposed to a 2.5 J head impact with the CHIMERA device or sham procedures as described in the “Proof-of-concept studies in TBI and AD mice” section. Blood from each animal was collected 6 h after TBI by cardiac puncture, as described in the “Animals and tissue collection” section, into tubes with or without EDTA, and then processed into plasma or serum, respectively, as described in the “Animals and tissue collection” section. Plasma and serum were frozen and stored at − 80 °C then assayed together in duplicate. Thawed plasma specimens were also used to test the effect of hemolysis on assay performance. Red blood cells were pooled from the blood collected into EDTA-containing tubes from 3 sham animals. Plasma specimens were spiked with pooled red blood cells at 0%, 5%, 25%, or 50% volume. Assay diluent was added to the specimens spiked at 0%, 5%, and 25% such that an equal total volume of spike and diluent was added to each specimen. Spiked and neat specimens were frozen at − 80 °C for 1 h then thawed and assayed in duplicate.

The stability of GFAP in blood collected and processed by various methods was also determined. Male C57Bl/6 mice were exposed to a 2.5 J head impact with the CHIMERA device or sham procedures as described in the “Proof-of-concept studies in TBI and AD mice” section. Blood was collected 6 h after TBI, first by the saphenous vein and then by cardiac puncture as described in the “Animals and tissue collection” section. Blood collected by saphenous vein was stored on ice then centrifuged at 3000g for 10 min at room temperature within 1 h of collection. Blood collected by cardiac puncture was divided into 2 equal aliquots. Aliquot 1 was centrifuged as above within 1 h of collection. Aliquot 2 was incubated on ice for 4 h then centrifuged as above. Plasma specimens were assayed in duplicate except in the case of most saphenous blood samples where volume limitations only allowed a single measurement.