A modification of the acid diazo coupling method (Malloy-Evelyn) for the determination of serum total bilirubin

Abstract

A simple and reliable method for the determination of total bilirubin from human serum is described. In this method, indirect bilirubin is liberated by the tenside in 0.12 mol T⁻¹ HC1 (Rl), and the total bilirubin is coupled with a 2,5-dichlorobenzene diazonium (DBD) salt to obtain the corresponding azobilirubin having a λ_max of about 520–522 nm. The method can easily be applied to the KONE Delta, a fully automated, discrete random access clinical analyser, and also to less modern instruments. A sample volume of 5 μl, Rl volume of 180 μ1, and R2 volume of 36 μl was used on the KONE Delta. After a 5-min incubation at 37°C, measurement at 575 nm was done (main wavelength). The within-run imprecision (CV%) varied from 2.9 to 0.3% within the serum total bilirubin range of 14–290 (imol 1 (n=10). The between-run imprecision was from 2.2 to 1.3% within the range 13–97 μmol I¹ (n=8). The method is linear up to at least 340 μmol T⁻¹ (19.8 mg dl⁻¹), and dilution extends the test limit to 3400 μmol I⁻¹ (198.8 mg dr¹). The linearity of dilution was good over the practical measuring range. The present method had a strong linear correlation with the Boehringer 2,5-dichlorophenyl diazonium (DPD) method on the Hitachi 717 analyser: y(DBD) = 1.018x(DPD)+0.758, r=0.9955 (n=61). The stability of R2 (diazo reagent) in the analyser reagent compartment lasts at least 2 weeks.

Key Words:

• azobilirubin
• bichromatic spectrophotometry
• blood chemical analysis
• clinical chemistry
• diagnosis
• intermethod comparison

Additional information

Notes on contributors

M. T. Parviainen

Joyce Laing works in the Department of Child and Family Psychiatry, Playfield House, Cupar, Fife, and is a Consultant Art Therapist to Psychiatric Hospitals and Prisons and Chairwoman of the Scottish Society of Art and Psychology.