Prevention of hemoglobin interference on the formazan reaction

BackgroundHb interference induces false test results and thus hinders the establishment of further applications that use the formazan method. We attempted to eliminate hemoglobin (Hb) interference on the formazan reaction.MethodsTotal serum bile acid (TBA) reagent was constructed to elucidate the mechanism of Hb interference. We determined the interaction between formazan reagents and Hb using spectral analysis and investigated the optimal condition of the reagent to establish a practical technique for its prevention.ResultsHb caused overestimation during TBA measurement, and its influence was attributed to the errors of both the sample and reagent blank signals. These errors were triggered by the reaction between Hb and formazan reagents. The main finding was that the addition of both imidazole and sodium nitrite in the reagent considerably accelerated the oxidation of Hb, even at neutral pH. The overestimation of TBA concentration was reduced to < 3 μmol/l even with Hb concentrations ≥ 500 mg/dl.ConclusionsWe elucidated the mechanism of Hb interference on the formazan reaction and succeeded in preventing its influence. This will help to solve the technical difficulties associated with utilizing formazan reactions as routine diagnostic tools.