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Background We investigated the presence of interference in a patient who had an elevated CA19-9 concentration using the ADVIA Centaur but results within reference limits with ROCHE Modular Analytics E170 and Brahms KRYPTOR analysers. Methods We performed repeat analyses using the same (ADVIA Centaur) and alternate immunossays (Roche Modular Analytics E170 and Brahms KRYPTOR) on the patient's sample and investigated for known interferences. To determine the nature of the interference, we measured CA19-9 on the ADVIA Centaur after dilution experiments and after incubation with non-immune animal sera and in heterophilic blocking tubes (HBT). We also undertook polyethylene glycol precipitation, lectin inhibition experiments and gel filtration chromatography. Results A curvilinear response to dilution was observed with the ADVIA Centaur. Other known interferences were excluded. Treatment with HBT or non-immune animal sera did not give clinically different results from untreated samples. There was only 0.59% recovery after PEG precipitation in the sample from the case patient. Lectin reduced the assay signal in four patient samples (recovery = 1.9–14.1%) but not in the case patient (recovery = 106.2%). Gel filtration studies suggested the presence of a low molecular weight (~ 100 kDa) interference in the case patient's serum. Conclusions We report a novel mode of interference and show a non-CA19-9, low molecular-weight interference affecting the ADVIA Centaur CA19-9 immunoassay.