Multi-analyte proteomic analysis identifies blood-based neuroinflammation, cerebrovascular and synaptic biomarkers in preclinical Alzheimer's disease.

Background: Blood-based biomarkers are gaining grounds for Alzheimer's disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte assessments and the need for markers of neuroinflammation, vascular, and synaptic dysfunction. Here, we evaluated a novel multi-analyte biomarker platform, NULISAseq CNS disease panel, a multiplex NUcleic acid-linked Immuno-Sandwich Assay (NULISA) targeting ∼120 analytes, including classical AD biomarkers and key proteins defining various disease hallmarks. Methods: The NULISAseq panel was applied to 176 plasma samples from the MYHAT-NI cohort of cognitively normal participants from an economically underserved region in Western Pennsylvania. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aß40, and Aß42, were also measured using Single Molecule Array (Simoa). Amyloid pathology, tau pathology, and neurodegeneration were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and MRI, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA biomarkers and AD pathologies. Spearman correlations were used to compare NULISA and Simoa. Results: NULISA concurrently measured 116 plasma biomarkers with good technical performance, and good correlation with Simoa measures. Cross-sectionally, p-tau217 was the top hit to identify Aß pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aß-PET+ participants, including TIMP3, which regulates brain Aß production, the neurotrophic factor BDNF, the energy metabolism marker MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aß PET-dependent yearly increases in Aß-PET+ participants. Markers with tau PET-dependent longitudinal changes included the microglial activation marker CHIT1, the reactive astrogliosis marker CHI3L1, the synaptic protein NPTX1, and the cerebrovascular markers PGF, PDGFRB, and VEFGA; all previously linked to AD but only reliably measured in cerebrospinal fluid. SQSTM1, the autophagosome cargo protein, exhibited a significant association with neurodegeneration status after adjusting age, sex, and APOE ε4 genotype. Conclusions: Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit 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). In this study, we 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 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h 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 improves the stability of Aß42 and Aß40 across all approaches. However, the Aß42/Aß40 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 Aß42 and Aß40, and the Aß42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Alzheimer blood biomarkers: practical guidelines for study design, sample collection, processing, biobanking, measurement and result reporting.

Alzheimer's disease (AD), the most common form of dementia, remains challenging to understand and treat despite decades of research and clinical investigation. This might be partly due to a lack of widely available and cost-effective modalities for diagnosis and prognosis. Recently, the blood-based AD biomarker field has seen significant progress driven by technological advances, mainly improved analytical sensitivity and precision of the assays and measurement platforms. Several blood-based biomarkers have shown high potential for accurately detecting AD pathophysiology. As a result, there has been considerable interest in applying these biomarkers for diagnosis and prognosis, as surrogate metrics to investigate the impact of various covariates on AD pathophysiology and to accelerate AD therapeutic trials and monitor treatment effects. However, the lack of standardization of how blood samples and collected, processed, stored analyzed and reported can affect the reproducibility of these biomarker measurements, potentially hindering progress toward their widespread use in clinical and research settings. To help address these issues, we provide fundamental guidelines developed according to recent research findings on the impact of sample handling on blood biomarker measurements. These guidelines cover important considerations including study design, blood collection, blood processing, biobanking, biomarker measurement, and result reporting. Furthermore, the proposed guidelines include best practices for appropriate blood handling procedures for genetic and ribonucleic acid analyses. While we focus on the key blood-based AD biomarkers for the AT(N) criteria (e.g., amyloid-beta [Aß]40, Aß42, Aß42/40 ratio, total-tau, phosphorylated-tau, neurofilament light chain, brain-derived tau and glial fibrillary acidic protein), we anticipate that these guidelines will generally be applicable to other types of blood biomarkers. We also anticipate that these guidelines will assist investigators in planning and executing biomarker research, enabling harmonization of sample handling to improve comparability across studies.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

INTRODUCTION: 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. METHODS: 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. RESULTS: Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improved the stability of Aß42 and Aß40 across all approaches. Additionally, the Aß42/Aß40 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. CONCLUSION: Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß ratio for IP-MS assay. This has crucial implications for preanalytical procedures, particularly in resource-limited settings.