Chen Yijun, Zeng Xuemei, Lee Jihui, Sehrawat Anuradha, Lafferty Tara K, Boslett James J, Klunk William E, Pascoal Tharick A, Villemagne Victor L, Cohen Annie D, Lopez Oscar, Yates Nathan A, Karikari Thomas K
medRxiv. 2024 Mar 7:2024.03.05.24303504. doi: 10.1101/2024.03.05.24303504.
The reliability of plasma Alzheimer's disease (AD) biomarkers can be compromised by protease-induced degradation. This limits the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). This study conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions.
We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 hours. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0h or 24h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA or P100 tubes, followed by storage at RT for 0h or 24h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays.
Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improved the stability of Aβ42 and Aβ40 across all approaches. Additionally, the Aβ42/Aβ40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches.
Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aβ42 and Aβ40, and the Aβ ratio for IP-MS assay. This has crucial implications for preanalytical procedures, particularly in resource-limited settings.
蛋白酶诱导的降解会损害血浆阿尔茨海默病（AD）生物标志物的可靠性。这限制了在缺乏即时处理和适当储存血样能力的环境中进行血浆生物标志物研究的可行性。我们假设在采血管中添加蛋白酶抑制剂可以提高血浆生物标志物在室温（RT）下的稳定性。本研究对传统乙二胺四乙酸（EDTA）管与BD™ P100采血管中的血液生物标志物稳定性进行了比较分析，后者涂有蛋白酶抑制剂混合物。在室温条件下，对六种血浆AD生物标志物的稳定性进行了长期评估。
我们评估了三种实验方法。方法1中，混合血浆样本在室温下储存长达96小时。方法2中，预先从采集到EDTA或P100管中的全血中分离出的血浆样本，在进行生物标志物测量前，在室温下储存0小时或24小时。方法3中，将全血样本采集到配对的EDTA或P100管中，然后在室温下储存0小时或24小时，再分离血浆进行分析。使用单分子阵列（Simoa）和免疫沉淀-质谱（IP-MS）测定法测量生物标志物。
IP-MS和Simoa方法均显示，使用P100管在所有方法中均显著提高了Aβ42和Aβ40的稳定性。此外，仅在方法3的IP-MS测定中，Aβ42/Aβ40比值水平显著稳定。在任何方法中，使用两种管型采集的样本的血浆p-tau181、GFAP和NfL水平均未观察到显著差异。
在采血管中添加蛋白酶抑制剂可减少蛋白酶诱导的血浆Aβ42和Aβ40降解以及IP-MS测定中的Aβ比值。这对分析前程序具有至关重要的意义，特别是在资源有限的环境中。
