Blood Collection and Serum Preparation

The serum samples were collected at four time points. These were a few days after admission to the main stay (T₀), at the halfway point (T₁), upon discharge (T₂), and at the one-week follow-up stay one year after discharge (T₃). The samples were drawn between 8 and 9 am. Sixteen patients on the depression ward submitted their blood samples between noon and 3 pm. The serum samples were collected in 8 mL Vacuette containers. The containers were rotated 8–10 times after the blood collection procedure was completed. They were at rest for 30–60 minutes in order to let the blood clot successfully. The samples were spun in a centrifuge, at room temperature, reaching 1917 g for 10 minutes. Upon completion, the samples were stored at −80°C until assay.

The blood samples were thawed on ice, vortexed, and then spun down a tube with 250 μL serum at 14,000 g for 10 min at 4°C, before dilution (1:5) and further processing. The cytokines were measured with Bio-Plex xMAP Technology (Bio-Rad, Austin, TX, USA) using a Luminex IS 100 instrument (Bio-Rad, Hercules, CA, USA). The Luminex IS 100 was powered by the Bio-Plex Manager (version 6.0.1) software. An additional standard point was added to the manufacturer’s instructions when the assay was performed.

Individual sets of samples from patients were run in the same assay in order to achieve a more reliable result. The samples were assayed in duplicate, and a magnetic plate washer was used during set up. The StatLIA software package (version 3.2; Brendan Scientific, Carlsbad, CA, USA) incorporates a weighted, five-parameter logistic curve-fitting method and was used to calculate serum cytokine levels. For the purpose of this study we analyzed IL-6 and IL-10. These biomarkers were chosen since they have frequently been assessed in previous immunopsychiatric studies, thus allowing for comparisons with the available literature. Longitudinal controls were used to validate inter-assay variation: IL-6 (16%) and IL-10 (5.7%). The mean inter-assay percentage coefficient of variability for all blood sample plates was 10.4%. The unit of measurement was picograms per milliliter (pg/mL).