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A Ready-To-Adopt LC-MS/MS Method for the Measurement of Serum Thyroglobulin in Clinical Laboratory

Updated: Jul 16, 2023

Author: Junyan Shi, PhD, DABCC, FCACB, Clinical Chemist, Vancouver General Hospital, Vancouver Coastal Health; Clinical Assistant Professor, Department of Pathology and Laboratory Medicine, University of British Columbia, Canada



Thyroglobulin (Tg) is synthesized exclusively by the follicular cells in the thyroid gland and serves as a specific marker of thyroid tissue. Post-surgical monitoring of serum Tg levels is performed regularly to evaluate the effectiveness of treatment for differentiated thyroid cancer (DTC) and to monitor for disease recurrence, persistence, or metastasis. Immunoassays are prone to interferences from anti-Tg autoantibodies and result in falsely low Tg concentrations, which may mask DTC persistence or relapse. Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) Tg assay starts with protein digestion and utilizes a specific antibody to pulldown the signature peptide of Tg by immunoaffinity purification, making the assay free of interferences from the anti-Tg autoantibodies. Despite the availability in numerous clinical laboratories, the adoption of LC-MS/MS assays has been slow due to the challenge of achieving comparable sensitivity to modern immunoassays for large molecule analysis.


In the September 2022 issue of the Journal of Mass Spectrometry and Advances in the Clinical Lab, Dr. Junyan Shi and her colleagues from the University of Washington published a distributable LC-MS/MS method for the measurement of serum Tg. The study aims to bridge the gap by sharing a high-sensitivity Tg assay with full validation information, as well as a standard operation protocol that is routinely used at the University of Washington Medical Center laboratory for Tg testing. The features of the study include:

  1. The addition of 2% DMSO in the mobile phase, which dramatically boosts the signal (up to 7.6-fold increase of the peak area of the signature peptide).

  2. Validation of the assay in both micro-centrifuge tubes and 96-well plates to accommodate low- and high-volume testing, respectively. The validation on 96-well plates also provides a basis for further automation.

  3. A cost-saving strategy by reusing magnetic beads for up to five cycles.

  4. Development of a harmonization approach by using a novel, serum-based 5-point distributable reference material (Husky Ref, freely available from the authors).

  5. Follow-up of 34 patients for an average of 3.56 years, all of whom had serum Tg below 0.15 ng/mL at their original blood draw. All patients stay clinically disease-free, while three of them have had detectable Tg during their follow-up.

The described method, along with its standard operating procedure and associated reference material, aims to address the needs of laboratories that are interested in transitioning to mass spectrometry-based thyroglobulin measurement.


可推广的LC-MS/MS方法用于临床实验室的甲状腺球蛋白测定

甲状腺球蛋白(Tg)仅由甲状腺中的滤泡细胞合成,因此成为甲状腺组织的特异性标志物。术后定期监测血清Tg水平可用于评估分化型甲状腺癌(DTC)的治疗效果,并及时发现DTC的持续存在或复发,以及可能的癌细胞转移。基于免疫测定法的Tg检测容易受到Tg自身抗体的干扰,并导致Tg浓度低的假象,这可能会掩盖持续/复发性DTC。液相色谱串联质谱(LC-MS/MS)方法的 Tg检测从蛋白质酶切开始,利用特异性抗体通过免疫亲和纯化法提取Tg的特征肽段,使检测不受Tg自身抗体的干扰。然而,尽管许多临床实验室都具备LC-MS/MS检测平台,基于LC-MS/MS 方法的Tg检测在临床上推广速度仍然非常缓慢。这主要是因为在大分子分析上,LC-MS/MS方法想要实现与现今免疫测定法相当的灵敏度会具有一定的挑战性。


在2022年九月的Journal of Mass Spectrometry and Advances in the Clinical Lab杂志中,Junyan Shi博士及其在华盛顿大学医学院工作期间的同事发表了一个可推广的LC-MS/MS方法用于Tg的临床测定。该研究旨在介绍一个高灵敏度的LC-MS/MS Tg测定法,以及通过分享该方法完整的验证数据和临床实验室中常规的标准操作程序来提高LC-MS/MS Tg 测定的可推广性。该研究的特点包括:


  1. 在流动相中添加2% 二甲亚砜(DMSO)可显著增强信号(特征肽的峰面积增加多达7.6倍);

  2. 在微量离心管和 96 孔板中验证了检测方法,以分别适用于低容量和大容量测试;在96孔板上的验证还为进一步实现自动化奠定了基础;

  3. 通过多次重复使用磁珠来达到节省成本的目的,每次可重复使用达五个循环;

  4. 通过使用新型的可分配五点参考物质血清样品(Husky Ref参考物质,可从作者处免费获得)统一校准方法;

  5. 一共有34名患者进行了平均3.56年的随访,所有患者的原始血清Tg含量均小于0.15 ng / mL。虽然所有患者仍然处于临床无病期,但其中3人在随访期间查出可检测Tg。


该研究作者希望所描述的方法及其标准操作程序和相关参考材料将满足有兴趣实验室的需求并帮助他们将甲状腺球蛋白测定成功过渡到LC-MS/MS平台。



References:

Shi, J., Phipps, W. S., Owusu, B. Y., Henderson, C. M., Laha, T. J., Becker, J. O., Razavi, M., Emrick, M. A., & Hoofnagle, A. N. A distributable LC-MS/MS method for the measurement of serum thyroglobulin. J Mass Spectrom Adv Clin Lab. 2022;26:28-33. https://doi.org/10.1016/j.jmsacl.2022.09.005

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