Squitti Rosanna, Pal Amit, Ivanova Irena D, Marianetti Massimo, Rongioletti Mauro
Department of Laboratory Science, Research and Development Division, Ospedale Isola Tiberina-Gemelli Isola, 00186 Rome, Italy.
Department of Theoretical and Applied Sciences, eCampus University, Viale Massenzio Masia, 26, 22100 Como, Italy.
Biomolecules. 2025 May 29;15(6):788. doi: 10.3390/biom15060788.
Copper dyshomeostasis has been implicated in a subset of Alzheimer's disease (AD) patients, characterized by elevated non-ceruloplasmin-bound copper (non-Cp Cu). However, traditional methods for estimating non-Cp Cu are indirect and analytically imprecise. This study introduces and validates a direct assay for exchangeable copper (ExcCu) by inductively coupled plasma-mass spectrometry (ICP-MS), compliant with Clinical and Laboratory Standards Institute (CLSI) guidelines.
We performed analytical validation of the ExcCu assay following CLSI protocols (EP5, EP6, EP7, EP9, EP15, and EP28). ExcCu and other copper-related biomarkers were quantified in serum samples from 154 healthy controls, 82 AD patients, and 10 patients with Wilson disease (WD). Diagnostic performance was evaluated via receiver operating characteristic (ROC) curve analysis, and inter-method agreement was assessed using Bland-Altman plots.
The ExcCu assay demonstrated excellent linearity, precision (CV < 6%), and inter-laboratory reproducibility. Among AD patients, ExcCu levels were significantly elevated compared to controls ( < 0.001). ExcCu distinguished AD from controls with an AUC of 0.80 and a specificity of 95%. Compared to non-Cp Cu, ExcCu yielded no negative values and showed reduced bias. The relative exchangeable copper (REC) index was more effective in differentiating AD from WD (AUC = 0.88).
The validated ExcCu assay overcomes the limitations of the traditional non-Cp Cu calculation, offering a reliable biomarker for copper-related AD subtypes. Its high specificity supports its use in patient stratification, potentially contributing to personalized approaches in AD diagnosis and therapy.
铜稳态失衡与一部分阿尔茨海默病（AD）患者有关，其特征是未与铜蓝蛋白结合的铜（非Cp Cu）升高。然而，传统的估算非Cp Cu的方法是间接的，分析上也不精确。本研究引入并验证了一种通过电感耦合等离子体质谱法（ICP-MS）直接测定可交换铜（ExcCu）的方法，该方法符合临床和实验室标准协会（CLSI）的指南。
我们按照CLSI方案（EP5、EP6、EP7、EP9、EP15和EP28）对ExcCu测定法进行了分析验证。对154名健康对照者、82名AD患者和10名威尔逊病（WD）患者的血清样本中的ExcCu和其他铜相关生物标志物进行了定量分析。通过受试者工作特征（ROC）曲线分析评估诊断性能，并使用布兰德-奥特曼图评估方法间的一致性。
ExcCu测定法显示出优异的线性、精密度（CV < 6%）和实验室间重现性。在AD患者中，ExcCu水平与对照组相比显著升高（< 0.001）。ExcCu区分AD与对照组的曲线下面积（AUC）为0.80，特异性为95%。与非Cp Cu相比，ExcCu没有产生负值，并且偏差更小。相对可交换铜（REC）指数在区分AD与WD方面更有效（AUC = 0.88）。
经过验证的ExcCu测定法克服了传统非Cp Cu计算方法的局限性，为与铜相关的AD亚型提供了一种可靠的生物标志物。其高特异性支持将其用于患者分层，可能有助于AD诊断和治疗的个性化方法。
