Background: In-vitro diagnostic (IVD) bilirubin reagents based on oxidation with bilirubin oxidase (BOX) or vanadic acid (VA) for total and direct-reacting bilirubin (TB and DB) are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin (UCB and CB) and delta bilirubin (DLB) has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four IVD bilirubin reagents.

Methods: Porcine UCB solution, chemically synthesized ditaurobilirubin (DTB) solution, and chemically synthesized DLB solution were used as surrogates of naturally occurring UCB, CB, and DLB, respectively. The TB and DB concentrations were measured by three BOX methods and one VA method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure (RMP).

Results: The UCB and DLB concentrations were similar when any of the four IVD bilirubin reagents were used during TB measurement. This was consistent with RMP and exhibited a converged inter-method variation. Compared with RMP, significantly low DTB concentrations were observed by the IVD bilirubin reagents despite the converged inter-method variation. In DB measurement, some reagents reacted doubtfully with UCB, while showed lower DTB concentrations than its corresponding TB concentration. Reactivity with DLB was different for each method including RMP. Some reagents were developed to react less with DLB and others to strongly react with DLB.

Conclusions: We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with RMP. The DB data results vary depending on the reagents used.

背景：基于胆红素氧化酶（BOX）或钒酸（VA）氧化的体外诊断（IVD）胆红素试剂用于总胆红素和直接反应胆红素（TB和DB）在日本被广泛使用；然而，制造商尚未完全披露它们对非结合和结合胆红素（UCB 和 CB）和 δ 胆红素（DLB）的反应性。我们使用人工制备的胆红素材料来研究与四种体外诊断胆红素试剂的反应性。

方法：猪UCB溶液、化学合成二牛胆红素（DTB）溶液和化学合成DLB溶液分别作为天然UCB、CB和DLB的替代物。通过三种 BOX 方法和一种 VA 方法测量 TB 和 DB 浓度，并将观察到的浓度与通过重氮基参考测量程序 (RMP) 获得的浓度进行比较。

结果：当在 TB 测量期间使用四种 IVD 胆红素试剂中的任何一种时，UCB 和 DLB 浓度相似。这与 RMP 一致，并表现出收敛的方法间变化。与 RMP 相比，IVD 胆红素试剂观察到显着低的 DTB 浓度，尽管方法间有收敛变化。在 DB 测量中，一些试剂可能与 UCB 发生反应，而 DTB 浓度低于其相应的 TB 浓度。对于包括 RMP 在内的每种方法，与 DLB 的反应性都不同。一些试剂被开发为与 DLB 反应较少，而另一些试剂与 DLB 反应强烈。

结论：我们揭示了 IVD-TB 和 IVD-DB 试剂对人工制备的胆红素材料的反应性，以及它们与 RMP 的一致性。 DB 数据结果因使用的试剂而异。