Urine Bilirubin: Multistix

The bilirubin pad on the multireagent dipstick detects bilirubin using a specific diazotization reaction and is sensitive to 0.2-0.4 mg/dL of conjugated bilirubin. The color change indicating a positive reaction, however, is a rather subtle transition among shades of beige, and sometimes is obscured by color inherent in the urine itself (e.g., marked hemoglobinuria). In such instances, confirmation of a suspected positive reaction used to be done using a tablet version of the test, the Ictotest method, which yielded a purple color reaction which was easier to read. When there was a discrepancy between the dipstick bilirubin result and the Ictotest (e.g. small reaction on the dipstick and negative on the Ictotest), the Ictotest is considered the accurate result (i.e. a negative result in this example). Unfortunately, the Ictotest® is no longer available and we now recommend testing for total bilirubin in urine instead. In our experience, false positive reactions for bilirubin with the dipstick were exceedingly uncommon (results of the dipstick and tablet versions of the test usually matched).

The table below illustrates how results from the dipstick (Multistix) correlates to those from the Criterion, the machine used to "read" the dipstick at Cornell University.

Small (1+)
1 mg/dL
Moderate (2+)
3 mg/dL
Large (3+)
6 mg/dL

Positive reactions

  • Physiologic: Dogs have a low renal threshold for bilirubin, therefore highly concentrated urine samples from this species may have a trace to 1+ reaction. This may be clinically insignificant.
  • Cholestatic hepatobiliary disease: Detection of bilirubin in urine is generally an abnormal finding. Bilirubinuria generally results when conjugated bilirubin levels in blood are elevated as a result of cholestatic hepatobiliary disease. In the dog, bilirubinuria may be seen prior to bilirubinemia due to the low renal threshold for bilirubin in this species. In all other species, bilirubinuria indicates cholestasis.
  • Intravascular hemolysis: In some cases of intravascular hemolytic anemia, bilirubinuria may be secondary to the hemolysis without any evidence of cholestasis. The renal tubular epithelium is capable of absorbing free hemoglobin from the glomerular filtrate and converting it to conjugated bilirubin, which is then excreted in the urine. This will only occur with intravascular hemolysis, when free hemoglobin is filtered by the glomerulus.
False negative reactions
  • Bilirubin crystals: In some samples, bilirubin crystals may be identified in the urine (indicating bilirubinuria), however the dipstick reaction for bilirubin is negative. The reason for this is unknown, however the crystals may not solubilize sufficiently to react with the dipstick in these cases, resulting in a false negative reaction.
  • Aged urine samples: Conjugated bilirubin hydrolyzes to unconjugated bilirubin if left at room temperature.
  • Exposure to UV light: UV light converts bilirubin to biliverdin, resulting in false negative reactions.
  • Ascorbic acid: High concentrations of vitamin C inhibit the reaction.


Cornell University