Assessment of inflammatory markers

Fasting serum samples were collected between 08.00 and 13.00, stored at −80°C and were not thawed or refrozen during storage. Although serum samples were from three different study phases, blood collection, processing and storage followed the same standard operating procedures.

CRP was measured with a high‐sensitivity immunonephelometric assay in a BN ProSpec nephelometer (Dade Behring, Milton Keynes, UK). IL‐6 was measured with a high‐sensitivity enzyme‐linked immunosorbent assay (ELISA) (R&D Systems, Oxford, UK). Values lower than the detection limit [0.154 mg L⁻¹ for CRP (multiplied by 9524 to express the value in mmol L⁻¹) and 0.08 pg mL⁻¹ for IL‐6] were assigned a value equal to half the detection limit. We excluded samples with CRP concentrations suggestive of acute inflammation and related bacterial infection (>10 mg L⁻¹) 49 (n = 242). To measure short‐term biological variation and laboratory error, a repeat sample was taken from 150 participants for CRP and 241 participants for IL‐6 at phase 3 [with a mean elapsed time between samples of 32 days (SD 10.5)]. Intra‐assay and interassay coefficients of variation were 4.7% and 8.3% for CRP and 7.5% and 8.9% for IL‐6, respectively.

Serum IL‐1 RA was measured in a diabetes case–cohort sample 25, 50 with the Quantikine ELISA kit (R&D Systems, Wiesbaden, Germany). All assays were performed consecutively in the same laboratory (German Diabetes Center), and samples from different study phases from the same participant were always measured using the same ELISA plate to minimize assay imprecision. Mean intra‐assay and interassay coefficients of variation were 2.6% and 7.9%, respectively. The limit of detection was 14 pg mL⁻¹ (all samples were above the limit of detection).