Condensing the Aβ protocol to reduce the effort and cost of NIA-AA guidelines for neuropathologic assessment of Alzheimer disease

To the Editor: We refer to the article “Performance of a Condensed Protocol That Reduces Effort and Cost of NIAAA Guidelines for Neuropathologic Assessment of Alzheimer Disease” that uses 4 tissue blocks for Thal staging of amyloid-b (Ab) (1). In this protocol, the variable occipital visual cortex is recommended as the cortical region for examination of A1 (Thal phases 1 and 2), with the striatum being used to assess stage A2 (Thal phase 3) and midbrain and cerebellum used to assess stage A3 (Thal phase 4 for midbrain and phase 5 for cerebellum) (2–4). Indeed, according to Thal et al, Ab deposition occurs in the neocortex in 100% of phase 1 cases with only minor variability (temporal and/or occipital cortex affected in 5 of 6 cases, followed closely by frontal in 4 cases and parietal cortex in 2 cases) (4). However, in vivo amyloid imaging shows that amyloid is most consistently found in the inferior temporal, the anterior cingulate and the parietal opercular cortices followed by wide parts of temporal, frontal and parietal cortices (5). Sensory-motor and occipital cortices and anterior medial temporal cortices occur later, followed by the striatum (5). These data suggest that perhaps the choice of the occipital visual cortex as the only cortical region for the evaluation of Ab staining using the reduced tissue blocks should be reconsidered. We analyzed Ab deposition in a cohort of 50 cases (age range 62– 103 years) with variable Alzheimer disease (AD) neuropathological change (56% not/low, 44% intermediate/high) and compared the recommended sampling of the original NIAAA guidelines (2,3) and the inferior temporal and superomedial frontal cortices in a subset of cases with limited Ab deposition, then validated the reduction of blocks in the entire cohort. Consistent with the imaging findings (5), we show that the inferior temporal and superomedial frontal cortices were equally and reliably affected (13/13 cases affected, 100%) at the earliest stages of Ab deposition (A1) compared with the middle temporal (10/12 cases affected, 83%), middle frontal (10/12 cases affected, 83%), inferior parietal (5/7 cases affected, 71%), superior temporal (7/12 cases affected, 58%), and occipital visual cortices (4/7 cases affected, 57%) (Fig. 1A), using either the NovoLink Polymer Detection System (Leica Biosystems, Newcastle Ltd, Newcastle Upon Tyne, UK) according to the manufacturer’s instructions, with antibodies against b-amyloid (1:50 000, 6E10 clone, SIG-39320, Covance, Princeton, NJ, USA) or the Discovery XT autostainer (Ventana Medical Systems—a member of the Roche group, Tucson, AZ, USA) with antibodies against b-amyloid (1:500, 6F/ 3D clone, M 0872, Dako, Glostrup, Denmark). As expected, the hippocampus and entorhinal cortex was less consistently affected at stage A1 than neocortices in the entire cohort (Fig. 1B), and would only be necessary if complete Thal staging is required (4). The assessment of Ab in the basal ganglia was confirmed as appropriate for the determination of hierarchical Thal phase 3 (4) and A2 score (2,3) (Fig. 1B). Consistent with Thal staging, only 41% of cases demonstrated Ab in the cerebellum at A3 stage compared with 100% positivity in the midbrain (Fig. 1B), suggesting that the cerebellum would only be needed if comprehensive Thal staging was required (4). This would reduce the resources required to determine the Montine stages of Ab deposition to only 3 blocks; neocortex (with inferior temporal recommended from imaging studies (5) and validated in this study), basal ganglia, and midbrain. The occipital visual cortex gave more variable results and the cerebellum is unnecessary. Neuropathological diagnostic protocols are costly, and the development of reliable, reproducible and costeffective protocols are welcome. Our findings suggest an alternate condensed protocol from Flanagan et al (1) for the assessment of Ab pathology and provide evidence to support a further reduction of the regions necessary if Thal staging is not required.