Which of the following is NOT a method of bilirubin determination?

• A. Colorimetric
• B. Direct spectrophotometry
• C. Enzymatic
• D. Immunoassay

Answer: (D)

Bilirubin determination relies on methods that quantify the pigment through color reactions or direct light absorption. Colorimetric assays use a diazo reaction to form a colored product that can be measured photometrically, which is a classic and widespread approach for total bilirubin. Direct spectrophotometry, or measuring bilirubin by its absorbance at a specific wavelength, is another common technique that relies on the pigment’s light-absorbing properties without requiring a chemical reaction. Enzymatic methods exist as well, using enzymes like bilirubin oxidase to generate a detectable signal tied to bilirubin concentration.

Immunoassay, on the other hand, is not a standard method for bilirubin determination because bilirubin is a small, lipophilic molecule with limited immunogenicity, making it difficult to develop specific antibodies and reliable immunoassays for routine measurement. As a result, immunoassay is not used for determining bilirubin in typical clinical chemistry workflows.