Department of Biomedical and Organic Chemistry, Laboratoire National de Métrologie et d'Essais (LNE), Paris, France.
LBPC-PPC, Univ Montpellier, IRMB CHU Montpellier, INM INSERM, Montpellier, France.
Clin Chem Lab Med. 2023 Feb 24;61(7):1235-1244. doi: 10.1515/cclm-2022-1250. Print 2023 Jun 27.
In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF).
To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode.
A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high).
The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs.
在临床实践中，tau 蛋白的测量通常依赖于免疫分析（IA），其主要缺点是由于选择性和/或校准的差异，结果缺乏可比性。这凸显了建立 tau 蛋白（t-tau）在脑脊液（CSF）中绝对定量的可追溯性链的重要性。本工作的目的是开发一种更高阶的候选参考测量程序（RMP），用于 CSF 中 t-tau 的绝对定量。
为了校准候选 RMP 并建立与 SI 单位的计量可追溯性，我们采购了一种由高度纯化的重组蛋白组成的初级校准品。其纯度通过液相色谱-高分辨质谱（LC-HRMS）进行评估，溶液中的蛋白质量分数通过氨基酸分析（AAA）进行认证。为了通过同位素稀释质谱法（IDMS）开发 CSF 中 t-tau 绝对定量的候选 RMP，我们获得了一种同位素标记的同源物。通过重量法制备校准混合物和质控（QC）材料，并按照与 CSF 样品相同的制备工作流程进行处理，然后在平行反应监测（PRM）模式下进行 LC-HRMS 分析。
我们开发了一种初级校准品，并验证了用于 CSF t-tau 的 IDMS 候选 RMP。候选 RMP 用于认证三个 CSF 池（低、中、高）中的 t-tau 浓度。
候选 RMP 将为 CSF t-tau 测量的全球标准化铺平道路。与可互换的认证参考材料（CRM）一起，它将允许评估和提高 IA 提供的结果的准确性和可比性。
