Abstract Background Primary aldosteronism (PA) is a condition characterized by hypertension resulting from excess secretion of aldosterone. PA caused by a unilateral aldosterone-producing adenoma can be treated with adrenalectomy but requires diagnosis by adrenal vein sampling (AVS). In this procedure, intravenous cosyntropin is administered to stimulate adrenal cortical secretion, followed by obtaining samples from the right and left adrenal veins and IVC. Laterization index is calculated by obtaining the aldosterone:cortisol ratio for each side, with a ratio cutoff ≥3 is used for confirmation. Iodinated contrast media facilitates visualization of catheter positioning during AVS and sometimes results in contamination of samples, evidenced by abnormal gel separator flotation (i.e. gel above plasma). This study had two aims: 1) to determine at what concentration contrast dye causes gel flotation and 2) discern whether contrast media interferes with our cortisol or aldosterone assays. Methods Gel flotation studies were performed by spiking pooled plasma samples with iohexol contrast media (Omnipaque 240, GE Healthcare) at concentrations ranging from 3%-10% v/v and aliquoting into fresh lithium heparin plasma separator tubes (BD, PSTs). Samples were then centrifuged and visually inspected for abnormal gel flotation. Contrast interference studies were performed using three pools of low (5 µg/dL), medium, (30 µg/dL), and high (55 µg/dL) cortisol concentrations. Sample pools were diluted with 20% contrast media or saline (control) and subsequently mixed at different ratios to create a range of dye concentrations (0.5%-18% v/v). Each mixture was assayed in triplicate for cortisol and aldosterone on a cobas e602 (Roche Diagnostics) and LIAISON XL (DiaSorin) immunoanalyzer, respectively. The total allowable error (TEa) was ±0.4 μg/dL or ±20% for cortisol and ±2 ng/dL or ±20% for aldosterone. Results Flotation of the gel separator was observed at a dye concentration of 4.5% v/v and greater. For the cortisol interference study, the bias was <5% for the three pools at all contrast media concentrations. Aldosterone was also measured in these pools, as bias could affect ratios calculated for PA. Aldosterone interference occurred at contrast dye concentrations ranging from 12%-20% v/v. An average positive bias of 34% was observed in aldosterone samples spiked with contrast media relative to the saline control. Compared to sample pool controls without added saline or dye, measurements for cortisol and aldosterone were 19%-22% and 16-25% lower in media- and saline-spiked samples (20% v/v), respectively, indicating a logical dilution effect. Conclusions Contrast dye contamination of AVS specimens can cause gel flotation in lithium heparin PSTs, preventing their analysis. The presence of contrast media at certain concentrations interfered with the DiaSorin aldosterone assay, leading to falsely elevated results. No cortisol assay interference occurred in the presence of contrast media but results were falsely decreased due to sample dilution. Samples collected during AVS may be contaminated with contrast dye causing gel flotation to occur. This is indicative of a minimum contrast media concentration of 4.5% v/v. Aldosterone:cortisol ratios may be inaccurate due to the positive aldosterone immunoassay bias with contrast media contamination. Care should be taken when reporting and interpreting AVS results with suspected contamination.