Circulating macroprolactin exhibits molecular heterogeneity and is not exclusively an antibody complex

•Macroprolactin is heterogenous and represents both antibody-prolactin complex and aggregates.•Samples positive for macroprolactin contained variable proportions of prolactin variants.•Samples containing macroprolactin exhibited positive bias in at least two immunoassays. Macroprolactin (macPRL) is considered to be solely a prolactin antibody complex. We examined macPRL heterogeneity in samples from thirteen patients suspected of macroprolactinemia. Polyethylene glycol (PEG) precipitation, gel permeation (GPC), protein-G affinity, and Lectin affinity chromatography were used to investigate the nature of macPRL. Using PEG, 8, 3, and 2 samples were macPRL positive, negative, and indeterminate respectively. Using GPC, prolactin appeared at high (H) (≥150 kDa), mid (M) (≥30