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Human milk and occasional serum samples contain high concentrations of unsaturated haptocorrin, which influence accurate measurement of cobalamins.Cobalamins in serum samples spiked with increasing amounts of unsaturated haptocorrin were measured employing the Centaur, Cobas and Architect analysers. Cobinamide-coated EAH sepharose was employed for pretreatment of the samples. Human milk samples were collected from 24 healthy mothers. Haptocorrin was measured by ELISA.The measured concentration of cobalamins either increased (Centaur analyser) or decreased (Architect, Cobas analysers) significantly for haptocorrin >10 nM, and was 220%, 52% or 45% of the expected values in a serum sample containing 50 nM haptocorrin. Following pretreatment with cobina-mide-sepharose, the expected cobalamin concentration was obtained (Centaur). The milk samples contained 4.5–180 nM haptocorrin. In samples containing >10 nM haptocorrin (n = 19), the median concentration of cobalamins decreased from 1.3 nM to 0.67 nM after pretreatment with cobinamide-sepharose.Haptocorrin in concentrations above 10 nM influences measurement of cobalamins giving rise to falsely elevated or decreased results. Removal of unsaturated haptocorrin by pretreatment with co-binamide-sepharose solves the problem.