The Composition of Kunkel Reagent Using the Hill Climbing Approach

Abstract

The standard procedure of the zinc sulfate turbidity test (ZTT, Kunkel test), which uses 2.5mmol/l barbiturate buffer as the Kunkel reagent, is limited by the fact that pH change of the reagent on standing causes considerable between-assay variations; the reagent has insufficient buffering capacities to reduce the effect of CO₂ absorption from air. To solve this problem, Tokuda and Hamada investigated several different buffers for the Kunkel reagent and recommended a Bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer instead of the barbiturate buffer (Jpn J Clin Chem 23: 316-322, 1994). Although the Bis-Tris buffer improved reagent stability, we noticed that their reagent conditions did not maintain results that correlated with the standard procedure of the correlation test represented in their paper. Therefore, it was necessary to optimize the reagent conditions for clinical management. We focused on maintaining results that correlated with the standard procedure while determining the best conditions for the Kunkel reagent with Bis-Tris buffer. The appropriateness of each condition was evaluated by Sy·x. in a correlation test with a routine method in which results that correlated with the standard procedure had been verified. The hill climbing approach, which is a heuristic method, was used to identify conditions to minimize Sy·x. The optimal condition determined by this Sy·x-minimization method maintained results that correlated with the standard procedure. Control charts also showed that the reagent was stable for over two weeks. The hill climbing approach is an excellent method to optimize the conditions of an assay consisting of several co-influencing reagents.