Tangle density (after square root transformation) is based on historical and/or digital data and is the mean of the square root transformation of the tangle score in 8 regions:

1. Angular gyrus
2. Anterior cingulate
3. Calcarine cortex
4. Entorhinal cortex
5. Hippocampus
6. Inferior temporal
7. Midfrontal gyrus
8. Superior frontal

Historical measures of tau-tangles were obtained from 20-micron sections from the 8 brain regions. Immunohistochemistry was performed using an antibody specific to phosphorylated tau, AT8 (1:2000, ThermoScientific). Using the Stereo Investigator software, regions of interest were manually outlined, and the systematic random sampling (SRS) grid was applied for manual counting. Manual counts of tau-tangles within a defined area were calculated as a density (count) per mm². A square root transformation was applied.

Digital scanning of AT8-stained slides began in 2019 using the Aperio AT2 whole slide brightfield scanning system. Briefly, paraformaldehyde-fixed brain regions are sampled, embedded, sectioned at 6-microns, and subsequently stained using an AT8 antibody (1:2000, ThermoScientific). Pathology glass slides are loaded into the Aperio AT2 scanner console and processed to generate digital whole slide images. Digital image analysis for tau-tangles was quantified using the Aperio Image Analysis Toolbox Nuclear algorithm. The algorithm is applied to whole slide images from the 8 brain regions. Scoring parameters are set to specify weak, medium, and strong thresholds for tau-tangle staining intensity. Overall tau-tangle burden is calculated for each slide by adding up the nuclei with medium and strong threshold, and dividing by the analysis area, resulting in a value of nuclei in counts per mm². A square root transformation is applied. Digital scores are reported as density of tangle counts per mm², after square root transformation.

This variable harmonizes historical data with the new digital data for tau-tangles. The variable uses the new digital scores for tangles. If digital data is not available for a particular region, a digital score is predicted from the historical data. The mean of the 8 region values is calculated if data for at least 4 of the 8 regions are available.

This variable replaces "tangles" as the measure of tangle density.