Division of Paediatric Endocrinology and Diabetes, Khoo Teck Puat-National University Children's Medical Institute, National University Health System, Singapore; Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Department of Laboratory Medicine, National University Hospital, Singapore.
Clin Chim Acta. 2018 Jan;476:103-106. doi: 10.1016/j.cca.2017.11.025. Epub 2017 Nov 23.
Two sisters with hirsutism presented with mild hirsutism and isolated, grossly elevated (>34.9nmol/L) serum concentrations of androstenedione measured by competitive, homogeneous immunoassay. The clinically discordant laboratory results prompted us to look for assay interference. In this immunoassay, horseradish peroxidase (HRP)-conjugated androstenedione competes with endogenous androstenedione for binding with the solid-phase polyclonal rabbit IgG antibodies. After a wash step, the amount of signal generated by the bound HRP conjugate is inversely proportional to the androstenedione concentration. Alternative analysis by tandem mass spectrometry (a good first line option for troubleshooting) and repeating the competitive immunoassay after polyethylene glycol treatment returned androstenedione concentrations within reference limits. These findings suggested that the original result was spuriously elevated due to assay interference. Additionally, the patient samples were pre-incubated with heterophile blocking reagents, normal rabbit IgG antibodies and HRP-conjugated normal goat IgG antibodies, followed by repeat measurement using the immunoassay. Only samples pre-incubated with HRP-conjugate returned significantly lower androstenedione (9.5 and 12.5nmol/L, respectively), implying neutralisation of the interfering antibodies. Androstenedione remained grossly elevated in the other experiments. This deductive exercise showed that the interference is due to autoantibodies against the HRP label used in the immunoassay. Another immunoassay using HRP label (5α-dihydrotestosterone) also produced gross elevation that was normal by tandem mass spectrometry analysis. Assay interferences, though not uncommon, are frequently overlooked. Laboratory results discordant with clinical features should prompt consideration of assay interference to avoid unnecessary investigations and treatment. This is the first report of autoantibodies against the HRP label used in immunoassay.
两位患有多毛症的姐妹表现出轻度多毛症和孤立的、显著升高的（>34.9nmol/L）血清雄烯二酮浓度，这些结果是通过竞争性均相免疫测定法测量得出的。临床结果不一致促使我们寻找检测干扰的原因。在这种免疫测定法中，辣根过氧化物酶（HRP）-标记的雄烯二酮与内源性雄烯二酮竞争与固相多克隆兔 IgG 抗体结合。在洗涤步骤之后，与 HRP 结合的信号量与雄烯二酮浓度成反比。通过串联质谱分析（解决问题的首选方法）和聚乙二醇处理后重复竞争性免疫测定法，雄烯二酮浓度均恢复至参考范围。这些发现表明，最初的结果是由于检测干扰而导致的假性升高。此外，患者样本用异嗜性阻断试剂、正常兔 IgG 抗体和 HRP 标记的正常山羊 IgG 抗体预先孵育，然后使用免疫测定法重复测量。只有用 HRP 标记物预先孵育的样本，雄烯二酮显著降低（分别为 9.5 和 12.5nmol/L），这意味着干扰抗体被中和。在其他实验中，雄烯二酮仍然显著升高。这种演绎性的研究表明，这种干扰是由于针对免疫测定法中使用的 HRP 标记物的自身抗体引起的。另一种使用 HRP 标记物（5α-二氢睾酮）的免疫测定法也产生了显著升高的结果，但通过串联质谱分析则显示正常。尽管检测干扰并不罕见，但常常被忽视。与临床特征不一致的检测结果应提示考虑检测干扰，以避免不必要的检查和治疗。这是首次报道针对免疫测定法中 HRP 标记物的自身抗体。
