The majority of participants gave a blood sample at baseline, which was aliquoted as blood spots on Guthrie cards and stored at room temperature. In addition, 1 mL samples of buffy coats and plasma were stored in liquid nitrogen. For the HealthIron study, the DNA samples from a subsample of participants were extracted from Guthrie cards (n = 23,484) using Chelex reagent or from frozen buffy coats (CorProtocol 14102; Corbett, Sydney, Australia) (n = 7708) and genotyped for the nucleotide changes that correspond to the amino acid substitutions C282Y and H63D in the HFE protein,

using TaqMan (Applied Biosystems, Carlsbad, CA) real-time polymerase chain reaction (PCR) probes as previously described.7 Only samples from participants actively participating Selleckchem PF 01367338 in the cohort who reported being born in Australia, the LY294002 United Kingdom, Ireland, or New Zealand were processed. Participants born in southern Europe (Italy, Greece, or Malta) were excluded due to the lower prevalence of the HFE C282Y mutation in populations from that region. A comprehensive active follow-up of MCCS participants began in 2003 and was completed in June 2007. Letters of invitation to participate in the HealthIron study were sent to a sample of 1438 participants that included all C282Y homozygotes

identified in the MCCS (n = 203) and a stratified random sample of approximately equal numbers of participants from each of the other five HFE genotype groups. All participants gave written,

informed consent to participate in both MCCS and the HealthIron study. Both study protocols were approved by the Human Research Ethics Committee of the Cancer Council of Victoria. Participants attending a study center completed a computer-assisted personal interview (that included questions on medical history, blood donation history, and venesection), provided a cheekbrush DNA sample for confirmatory HFE genotyping using PD184352 (CI-1040) real-time PCR assay with TaqMan probes (Applied Biosystems), and underwent a clinical examination of the abdomen and metacarpophalangeal (MCP) joints by study physicians blinded to HFE genotype. Blood samples were collected for measurement of iron indices, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations using Roche automated assays (Roche Diagnostics, Indianapolis, IN) and were paired for analysis with stored baseline plasma samples for each participant. Blood samples were usually collected in the morning at both baseline and follow-up, and participants were requested to fast. We defined sex-specific and menopause-specific SF upper limit of normal thresholds to be >300 μg/L for men and postmenopausal women and >200 μg/L for premenopausal women. We categorized participants according to their baseline SF concentration.