一例游离T4偏高问题的分析

A 60-year old male was referred to the Endocrinology Department due to confusing results of thyroid tests during a routine medical check-up. The asymptomatic patient presented with increased free thyroxine (FT4) concentrations, and thyroid-stimulating hormone (TSH) and total thyroxine (T4) concentrations within the reference range, with no tachycardia, distaltremor, visual impairment, or any other symptoms of thyrotoxicosis.

一般验证干扰最简单的办法就是稀释，但是正如作者所说，游离激素没法稀释，因为这样会破坏游离和结合激素的平衡。

FT4 is quantified in our laboratory by electrochemiluminescence using an Elecsys module (Cobas 8000 autoanalyzer, Roche Diagnostics) with a 1-step competitive configuration, including biotin-bound T4 as the marker and a ruthenium-labeled anti-T4 antibody. Two possible types of interference—biotin or heterophilic antibodies—were considered. Endogenous increased biotin concentrations could compete with the biotin-coupled T4 for the streptavidin coated particles, rendering falsely increased measured FT4 concentrations. However, the patient denied having taken biotin supplements.

To check potential interferences related to heterophilic antibodies, the sample was reanalyzed after pretreatment in heterophilic blocking tubes (ScantibodiesLaboratory). The results remained unchanged with the exception of total T4, which was slightly increased, similar to the Lab 2 results (Table 1). Although unchanged FT4 concentrations after pretreatment do not completely rule out the presence of heterophilic antibodies, the probability of an interference of this type is reduced.

从检测原理来分析，作者用的是罗氏Cobas 8000系统，采用的是一步竞争法，使用了生物素标记的T4和钌标记抗体。两种常见的干扰要考虑：生物素和嗜异性抗体。患者否认使用了生物素成分补品。使用嗜异性抗体阻断剂后的结果类似于Lab2结果，降低了这种干扰可能性。

The sample was analyzed by a third laboratory, also using a Siemens Centaur analyzer (Table 2, Lab 3). Similar to the Roche assay, the Centaur assay uses a competitive format, differing mainly in the origin of anti-T4 antibody and the molecule that originates the luminescent signal. Both free and total T4 concentrations were increased (Table 1, Lab 3). This eliminated both antiruthenium and antisheep antibodies as the cause of the interference in the Elecsys method.

标本又送到第三家实验室分析，采用西门子Centaur分析系统，结果仍然类似FT4和T4偏高。这样就排除了不同系统标记物的原因。

An increased FT4 concentration was more noticeable than that of total T4. A T4 binding alteration can be due to T4 displacement from its binding sites by drugs and endogenous molecules or by mutations in binding proteins. The pharmacological history of the patient failed to reveal any drug capable of causing such a displacement. Fatty acids, which can also displace hormones from their binding to proteins, were considered not interfering since triglyceride concentrations had been consistently within the reference range for years.

血清FT4升高可以是因为药物或者内源性物质将T4从结合位点置换下来，但是这个病人没有服用相关药物。游离脂肪酸同样能将激素从结合位置置换下来，但患者甘油三酯结果一直都是正常的。

Consequently, potential mutations of the albumin gene (ALB) were investigated by next-generation sequencing, with an R218H missense mutation being detected in heterozygosity. The R218H is the most common mutation in familial dysalbuminemic hyperthyroxinemia (FDH), a benign autosomal dominant condition where this amino acid change provokes a higher albumin affinity for thyroid hormones. This can increase the percentage of T4 transported by albumin by up to 30%, which results in increased total T4 concentrations with TSH values within the reference interval.

作者进一步检测了ALB基因变异情况，发现了患者存在R218H错义突变。R218H是家族性异常性甲状腺功能亢进症（FDH）中最常见的突变，是一种良性常染色体显性疾病，这种氨基酸变化引起对甲状腺激素的白蛋白亲和力变高。这可以使白蛋白转运的T4的百分比增加高达30%，从而导致总T4浓度增加而TSH正常。然而这个不能解释患者为何FT4升高。

The gold standard for free thyroid hormone measurement is equilibrium dialysis by which free and protein-bound hormones are physically separated.However, given that the process is complex and time consuming, automated immunoassays are the most widely used techniques in clinical laboratories. These immunoassays do not involve physical separation, and antibodies sequester only a minimum part of free hormone to preserve the equilibrium between free and bound hormones. Nevertheless, they can be affected by issues specifically affecting thyroid hormone–binding proteins or general interferences common to immunoassays. The higher affinity with albumin in patients with FDH could thus affect FT4 concentrations measured using certain immunoassays, provoking substantial differences in their results depending on assay design.

游离甲状腺激素测量的标准方法是通过平衡透析方法将结合和游离激素分离。然而这种方法过程复杂且耗时，所以目前还是广泛使用自动化化仪器。但是目前的方法没有物理分离，抗体只隔离最小部分的游离激素，以保持游离激素和结合激素之间的平衡。然而，它们可能会受到甲状腺激素结合蛋白或常见的一般干扰的影响。因此，作者分析到FDH患者与白蛋白的较高亲和力可能会影响使用某些免疫分析法测定的FT4浓度，导致不同检测系统的结果产生显著差异。

In competitive assays, endogenous hormone and marked analogue compete for a limited quantity of antibody. Mutant albumin in 1-step competitive assays can bind more FT4 and labeled analogue than usual, due to its increased affinity, reducing the amount of analogue available to bind to the antibody. This leads to a weaker signal and higher measured results. In 2-step competitive designs, albumin is washed away prior to analogue addition, and therefore results should be less affected by this analytical interference. Our laboratory, and both Labs 2 and 3, used 1-step FT4 assays and obtained similar results, which was consistent with the concordance observed in patient’s results. Nevertheless, one group demonstrated that the Vitros (Ortho Diagnostics) 1-step competitive FT4 assay generated much lower concentrations of FT4 in patients with FDH who had the R218H mutation, suggesting that other factors may be involved in the differences.

在竞争性分析中，内源性激素和标记的类似物争夺有限数量的抗体。一步法中的突变白蛋白可以比通常结合更多的FT4和标记类似物，因为它增加了亲和力，减少了可与抗体结合的类似物的数量。这导致了一个较弱的信号和更高的测量结果。在两步法中，白蛋白在添加类似物之前被洗掉，因此结果应该较少受到这种分析干扰的影响。作者实验室以及Lab2和3都使用了一步法测，获得了类似的结果。然而，使用Vitros (Ortho Diagnostics)系统，虽然也是一步法，但FT4浓度要低得多，这表明可能还有其他原因。

One of these factors seems to be the buffer chloride content. Chloride concentrations have been reported to increase FT4 measurement by equilibrium dialysis, especially in patients with FDH, due to the inhibition of T4 binding to albumin.

其中一个因素是缓冲液中氯的含量。据报道，由于抑制T4与白蛋白的结合，氯离子浓度可以增加平衡透析的FT4测量，特别是在FDH患者中。而Vitros (Ortho Diagnostics)系统没有使用这样的缓冲液。

However, buffer influence must not be limited to chloride content, since other components such as pH or detergent composition and concentration may also alter T4 binding to albumin, or other parameters involved in T4 measurement. 

然而，缓冲液的影响不能限于氯含量，因为其他成分如pH或洗涤剂组成和浓度也可能改变T4与白蛋白的结合。