Au analyser

The main exposure of interest for this study was maternal iron status during pregnancy, which was assessed in terms of SF concentrations, an established biomarker of body iron. Maternal ferritin (µg/L) levels were measured at a single point in the first trimester (mean 9.6 weeks, standard deviation (SD) 2.5) or second trimester (mean 14.6 weeks, SD 2.3) of pregnancy in serum samples collected and stored at − 80 °C until assessment. SF measurements in the first and second trimester as a whole (combined) were defined as the first period of pregnancy and analysed as total SF levels. For the Gipuzkoa and Sabadell cohorts, SF concentrations were quantified by time-resolved fluorescence immunoassay (DELFIA Ferritin kit A069–101) at the Gipuzkoa Public Health Laboratory, and for the Valencia cohort they were quantified by immunoturbidimetry (Beckman Coulter AU analysers) at La Fe hospital. The secondary exposure of interest was Hb. Data on maternal Hb levels (g/dL) were also obtained during the first trimester (mean 9.6 weeks, standard deviation (SD) 2.7) and third trimester (mean 27.2 weeks, SD 4.2) of pregnancy from medical records. Iron deficiency was defined as SF levels < 12.0 µg/L and anaemia as Hb levels < 11.0 g/dL.

Peripheral inflammation was measured as serum levels of hsCRP, which has been demonstrated as a reliable biomarker of inflammation associated with MDD. Participants fasted for 8 h and abstained from strenuous exercise for 72 h prior to their blood draw, which was carried out between 8:00 and 10:00. Blood samples were collected in clotting tubes, allowed to coagulate at room temperature for 30–60 min, then centrifuged at 1600 Relative Centrifugal Force for 15 min. The serum samples were separated and transported to a central laboratory (Q2 solutions) where they were analysed on the day of collection. Samples were exposed to anti-CRP-antibodies on latex particles, and the increase in light absorption due to complex formation was used to quantify hsCRP levels, using Turbidimetry on Beckman Coulter AU analysers. Inter and intra-assay coefficient of variations were <10%. The measure of hsCRP was calculated from one blood draw taken at the time of clinical assessment, for each participant. In line with previous studies, clinically elevated hsCRP levels were defined as hsCRP ⩾3 mg/L (Miller & Raison, 2016 (link); Pearson et al., 2003 (link)).

Laboratory investigations were carried out on overnight fasting samples at two certified laboratories with regular system calibration of validated devices and quality measures in place. High sensitivity CRP was measured via Beckman Coulter AU analyser with detection limits of 0.2–160 mg/L. Per hsCRP latex package insert, CVD relative risk is considered as follows: low < 1mg/L, average 1-3mg/L and high > 3mg/L. Urine microalbumin of <3 mg/mmol is considered normal/mildly increased, 3-29mg/mmol is moderately increased, and >30mg/mmol is severely increased. Fasting glucose levels of < 5.6, ≥ 5.6, 6–6.9, ≥ 7 mmol/L are normal, meets metabolic syndrome criteria, impaired fasting glucose and diagnostic of diabetes mellitus, respectively. Levels in mmol/L of serum total cholesterol (TC) > 5, HDL < 1.2, LDL > 3 and triglyceride > 1.7 are abnormal in adult females.
HIV-1 viral load was measured using COBAS AmpliPrep/COBAS TaqMan HIV-1 test. For the purposes of this study, participants with viral load <200 copies/ml (cp/ml) were considered as virally suppressed. CD4 count was determined by the Becton Dickinson Facscalibur flow cytometer.