A blood‐based diagnostic for Alzheimer’s disease using spectral microscopy of immuno‐enriched Aβ from frozen PBMCs

Abstract

AbstractBackgroundIn Alzheimer’s disease (AD), toxic Aβ peptides aggregate into higher molecular weight assemblies and accumulate not only in the extracellular space, but also in the walls of blood vessels in the brain, increasing their permeability, and promoting immune cell migration and activation. As immune cells contact these pathological brain materials, they may act as “sentinels” that are detectable once they return to the circulation. Previously, we have demonstrated that leukocytes, when stained with an amyloid sensitive probe, K114 (Tocris Bioscience), display distinct spectral changes in subjects with AD and mild cognitive impairment (MCI) (Black et al. 2022). Here we extend this work by introducing an enhanced method that combines the specificity of immunocapture of Aβ peptides together with spectral interrogation to reveal their amyloid character from easily obtainable frozen peripheral blood mononuclear cells (PBMCs).Methods30 frozen PBMC samples were obtained from the National Centralized Repository for Alzheimer’s Disease and Related Dementias (NCRAD) with 15 clinically‐ and histologically‐confirmed MCI/AD and 15 cognitively normal subjects based on the National Alzheimer’s Coordinating Center (NACC) database. To adapt our method to frozen samples, we used immunoprecipitation to purify target amyloid proteins by anti‐Aβ antibody (4G8)‐coated magnetic beads (Protein G SureBeads™, BioRad Laboratories), which were then labelled with K114, and imaged with a spectral confocal microscope.ResultsComparing subjects with neuropathologically‐confirmed AD (n=6), Braak stage for neurofibrillary degeneration (B score) >5 and density of neocortical neuritic plaques (C score) >2 to cognitively normal controls (n=6), our technique detected highly significant differences between groups (P<0.005, Fig. 1). Correlation between our AD Score and B scores was R = 0.824, and with cognitive status was R = 0.735 (Fig. 2).ConclusionsThese observations indicate that our method is capable of detecting AD from circulating human PBMCs, potentially mirroring the “Aβ load” in the brain. Our technique could constitute a reliable and inexpensive biomarker for early AD and AD‐related MCI.