Clarification of SEA-AD MTG layer-level IBA1–6E10 colocalization percentage

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Hello SEA-AD team,

I am preparing a donor-level secondary analysis using the publicly released June 2026 SEA-AD quantitative neuropathology summary and processed MTG endothelial pseudobulk data. I would appreciate clarification of one measurement before defining the analysis.

I reviewed the public standardization code and confirmed that the released MTG Grey matter colocalization percentage is an area-weighted average of the layer-level percentages, while object counts are summed across layers. This reproduces the Grey matter percentages for all 84 donors, so I am not treating the difference between a Grey matter percentage and a ratio of summed counts as a data error.

The remaining question concerns the original layer-level percentage. For example, in the archived MTG table, donor H21.33.045, Layer2 has:

  • 1,569 IBA1–6E10 colocalized objects
  • 1,216 6E10-positive objects
  • a reported colocalization percentage of approximately 20.1438%

Could someone clarify:

  1. What exact HALO export fields and formula define the layer-level percentage?
  2. Are the colocalized objects object pairs, overlap segments, IBA1 objects, or 6E10 objects? Can one object contribute multiple matches?
  3. Are the IBA1 objects in the Object Colocalization module segmented in the same way as the IBA1-positive cells reported by the separate Microglia module?
  4. How should a layer with zero 6E10-positive objects be interpreted when a percentage is reported?
  5. Is the original I6-MTG-Summary data-08-10-22.csv export, a field dictionary, or a worked example available?
  6. For the June 2026 endothelial expression release, should donors carrying the older “Severely Affected Donor” flag be excluded, or is that issue handled by the newer nucleus-level QC pipeline?

I am not assuming that the released values are erroneous. I would like to avoid describing this measure as a simple fraction of plaques contacted or engulfed by IBA1-positive cells unless that interpretation is supported by the original image-analysis definition.

Thank you for any guidance.

— Alexy