Jendrassik-Grof analysis for total and direct bilirubin in serum with a centrifugal analyzer

Abstract

Total bilirubin is determined here with a multipoint calibration approach and with use of Standard Reference Material No. 916 bilirubin from the National Bureau of Standards. The bilirubin calibration solutions prepared in solutions of Cohn Fraction V human albumin were stable for at least one year when stored at -80°C. Studies for direct bilirubin analysis show that the serum sample should be preincubated with the HCl reagent before the diazo reagent is added to minimize the false reaction with unconjugated bilirubin. With a preincubation of 15 min and a reaction interval of 15 min a 250 mg/L unconjugated bilirubin standard gave only 1 mg of apparent conjugated bilirubin per liter. Day-to-day precision studies gave CVs of 3.3% at 12.4 mg/L, 2.1% at 41.2 mg/L, and 1.0% at 197.4 mg/L for total bilirubin and 1.6% at 61.2 mg/L for direct bilirubin, the latter based on a serum pool stored at -80°C. Lipemic sera caused negligible errors. Hemolysis caused slightly larger errors for total bilirubin but much smaller errors for direct bilirubin when measured at 600 nm rather than 550 nm. Comparison of results with the Rotochem (y) to those with a manual Jendrassik-Grof method (x) for total bilirubin gave y = 0.9009x - 0.04 mg/L, S(y/x) = 1.3 mg/L, r = 0.9987 when different calibrators were used for each method and y = 0.9813x - 2.9 mg/L, S(y/x) = 1.5 mg/L, r = 0.9987 when the same calibrators were used. For direct-bilirubin comparisons, y = 1.2762x + 0.2 mg/L, S(y/x) = 1.3 mg/L, r = 0.9926.