Blood and Spinal Fluid Markers of Alzheimer’s Track Brain Plaques in a Strict Sequence

Alzheimer’s disease has long been defined by what pathologists see under the microscope: plaques of misfolded amyloid-beta protein accumulating between neurons, and tangles of hyperphosphorylated tau protein spreading through the brain’s memory circuits. In recent years, however, diagnosis has shifted decisively toward biology measured in living people. Fluids drawn from the spine and the bloodstream now carry molecular signatures of these same pathologies, and clinical criteria increasingly rely on them to identify and stage the disease. Yet a crucial question has remained surprisingly under-validated: at exactly what point in the accumulation of brain pathology does each of these fluid markers actually begin to change? A new study published in Acta Neuropathologica provides one of the most detailed answers to date, mapping eight fluid biomarkers against semi-quantitative measures of amyloid and tau burden in 250 autopsied brains.

The research team, led by Andrea Mastrangelo, Simone Baiardi and Piero Parchi at the University of Bologna and the Institute of Neurological Sciences of Bologna, exploited a rare and powerful resource. Their cohort consisted of participants whose cerebrospinal fluid or plasma samples had been collected shortly before death, with a median sampling-to-death interval of just 1.5 months for cerebrospinal fluid and one month for plasma. This proximity matters enormously. Most neuropathological validation studies suffer from long gaps between the fluid measurement and the autopsy, during which the underlying disease may have progressed substantially, blurring the relationship between what the biomarker showed and what the brain actually contained. By anchoring the measurements to the final weeks of life, the Italian team effectively froze the correspondence between fluid chemistry and brain pathology.

The cohort included 230 participants with antemortem cerebrospinal fluid and 101 with plasma samples, the majority affected by prion disease, a group whose rapidly progressive syndromes prompt autopsy through the Italian surveillance program. The researchers measured five cerebrospinal fluid markers, namely the Aβ42/Aβ40 ratio, p-tau181, p-tau217, and the hybrid ratios Aβ42/p-tau181 and Aβ42/p-tau217, together with three plasma markers, p-tau217, p-tau217/Aβ42 and Aβ42/Aβ40. All assays were run on a single automated chemiluminescent platform, ensuring analytical consistency. Because prion disease itself can elevate tau biomarkers, analyses involving phosphorylated tau were restricted to the 88 participants with non-prion conditions.

On the pathology side, the team went beyond conventional categorical staging. In addition to Thal amyloid phases, Braak neurofibrillary stages and ABC scores of Alzheimer’s neuropathologic change, two blinded evaluators scored amyloid plaques and cerebral amyloid angiopathy across nine brain regions, from neocortex to cerebellum, yielding a cumulative amyloid score from 0 to 90. Tau pathology, including neuropil threads, neurofibrillary tangles and thick neurites, was graded across six cortical regions to produce a cumulative score from 0 to 54. This continuous, region-weighted approach captures the actual regional burden of disease in a way that categorical stages cannot, allowing the researchers to ask precisely how much pathology is required before each biomarker begins to move.

The results reveal a strikingly sequential pattern. The cerebrospinal fluid Aβ42/Aβ40 ratio was the earliest mover, declining significantly already at the second quartile of amyloid burden, corresponding to mild-to-moderate plaque deposition across neocortical and limbic regions, and falling progressively further in the third and fourth quartiles. By contrast, cerebrospinal fluid p-tau181 and p-tau217 rose significantly only from the third quartile of amyloid burden, indicating that tau phosphorylation in the fluid lags behind the initial phases of plaque accumulation. The hybrid ratios behaved like early amyloid markers in terms of onset, with Aβ42/p-tau217 decreasing significantly from the second amyloid quartile, yet their abnormalities in relation to standard staging emerged only at intermediate levels of Alzheimer’s neuropathologic change, unlike the Aβ42/Aβ40 ratio, which was already reduced at low levels.

Sequential receiver operating characteristic analyses sharpened this picture by testing how well each biomarker discriminated progressively higher pathology thresholds. The cerebrospinal fluid Aβ42/Aβ40 ratio achieved its peak accuracy at low-to-intermediate amyloid burden, with an area under the curve of 0.984 at a threshold above 16 in the non-prion subgroup, before showing a modest plateau at more advanced stages. The p-tau markers and their ratios performed best at higher amyloid thresholds, with areas under the curve ranging from 0.889 to 0.980, and at intermediate tau burden, where p-tau217 and Aβ42/p-tau217 reached extraordinary values of 0.994 and 0.995 respectively at a tau score above 15. Across both amyloid and tau continua, p-tau217 consistently outperformed its p-tau181 counterpart, reinforcing a growing consensus that phosphorylation at threonine 217 is the more informative tau epitope.

The plasma results told a more sobering story. Plasma p-tau217 and p-tau217/Aβ42 rose significantly only in the highest quartiles of both amyloid and tau burden, and their discriminatory power peaked at the most advanced thresholds, with areas under the curve between 0.893 and 0.928. Plasma Aβ42/Aβ40 showed only weak associations with pathology and poor discrimination across the entire amyloid continuum, a finding the authors attribute to possible peripheral contributions to circulating amyloid peptides, systemic confounders and blood-brain barrier effects. When both pathologies were modeled simultaneously, plasma p-tau217 remained independently associated only with tau burden, consistent with evidence that a marked surge in soluble p-tau217 release accompanies the spread of tau pathology into the neocortex.

These findings carry practical weight for the clinic and for drug development. The team showed that a cerebrospinal fluid Aβ42/Aβ40 cut-off below 0.074, slightly higher than values commonly used in clinical practice, achieved 95 percent specificity for identifying subjects with at least low Alzheimer’s neuropathologic change, supporting its use for early identification within the disease continuum. The staged behavior of the markers also aligns with therapeutic evidence suggesting that anti-amyloid antibodies such as donanemab and lecanemab exert greater clinical benefit at earlier pathological stages, and that plaque clearance proceeds faster in brains with lower amyloid load. Knowing which fluid marker corresponds to which pathological window could therefore help clinicians time interventions and interpret biomarker panels more rationally.

Equally important is what the study could not find. Even the best-performing cerebrospinal fluid marker, the Aβ42/Aβ40 ratio, showed limited accuracy at the very earliest phases of plaque formation, when only sparse neocortical deposits are present. This implies an initial window in the Alzheimer’s continuum that current fluid biomarkers do not fully capture, a gap that may matter for prevention trials aiming to intervene before substantial pathology accumulates. The authors also caution that their cohort, enriched for rapidly progressive syndromes and dominated by participants with no or low Alzheimer’s neuropathologic change, may limit generalizability, that kidney function data were largely unavailable, and that reliance on a single assay platform precludes direct comparison with other technologies.

Nevertheless, the study delivers a coherent and clinically actionable model of biomarker behavior across the Alzheimer’s pathological continuum. Cerebrospinal fluid Aβ42/Aβ40 emerges as the sentinel of early amyloid deposition, cerebrospinal fluid p-tau markers and hybrid ratios as indicators of advancing combined pathology, and plasma p-tau217 measures as signals of heavy, late-stage burden. As blood-based testing moves toward primary care and anti-amyloid therapies become routine, anchoring these tests to neuropathological ground truth, with sampling intervals measured in weeks rather than years, provides the kind of validation the field has long needed. The sequential model also sets a clear benchmark for the next generation of markers, which must reach further back into the disease process if the earliest, most treatable phases of Alzheimer’s pathology are to be caught in a tube of fluid.

Subject of Research: Neuropathological validation of cerebrospinal fluid and plasma Alzheimer's disease biomarkers across increasing brain amyloid-beta and tau pathology burden

Article Title: Changes in five cerebrospinal fluid and three plasma Alzheimer’s disease biomarkers across increasing brain amyloid-beta and tau pathology burden

Article References: Mastrangelo, A., Baiardi, S., Ruggeri, E., Bentivenga, G. M., Vargiu, C. M., Mammana, A., Sbriccoli, M., Polischi, B., Carlà, B., Capellari, S., & Parchi, P. (2026). Changes in five cerebrospinal fluid and three plasma Alzheimer’s disease biomarkers across increasing brain amyloid-beta and tau pathology burden. Acta Neuropathologica, 152(1), Article 28. https://doi.org/10.1007/s00401-026-03076-5

Image Credits: AI Generated

DOI: 10.1007/s00401-026-03076-5

Keywords: Alzheimer's disease, biomarkers, amyloid-beta, tau pathology, cerebrospinal fluid, plasma p-tau217, neuropathology, autopsy validation, Aβ42/Aβ40 ratio, p-tau181, diagnostic accuracy, neurodegeneration