New kinetic procedure for the measurement of conjugated bilirubin with a centrifugal analyzer

A procedure has been developed for the automated measurement of conjugated bilirubin in serum with a centrifugal analyzer. The conjugated bilirubin is measured by a fixed time kinetic method which monitors the reaction between conjugated bilirubin and diazotized sulfanilic acid at 550 nm. Results are calculated based on a comparison of the reagent blank-corrected absorbance changes between 15 seconds and 75 seconds for sample vs changes in an empirical standard. The standard used is N-(1-naphthyl) ethylene diamine dihydrochloride (NEDC) which reacts with diazotized sulfanilic acid at a rate comparable to conjugated bilirubin. The standard is calibrated by comparison with a modified Jendrassik and Grof procedure using a serum blank-corrected centrifugal analyzer reference method. The method is linear to 150 mg per 1 with a sensitivity of 3.0 milliabsorbance units per 1.0 mg per 1 of conjugated bilirubin using a 35 mul sample volume. Within-run precision is 1 percent for elevated concentrations of bilirubin. Hemolysis introduces a negative interference, the nature of which is discussed.