Donor cell quantification via XY FISH analysis

Scanned images were analyzed using TissueQuest (TissueGnostics, Vienna, Austria). DAPI-stained nuclei were segmented based on size to create nuclear masks. Background thresholds were defined to detect the Y probe (red) and X probe (green) FISH signals within the nuclear mask. A small sample region (5 mm^2; ∼1000 cells) was initially analyzed to ensure that automated detection using TissueQuest yielded accurate and comparable data to manual quantification. Following this validation, the number of donor and FISH-positive cells was quantified in a larger area (30-40 mm² encompassing 10 000-15 000 cells). The identified donor cells were confirmed via blinded manual inspection. The percentage of donor cells was defined as the number of cells with a Y chromosome divided by the number of FISH-positive cells (XX, XY, X, or Y cells). Donor cell quantification was presumably underestimated because some donor cells were not identified, given that the tissue section did not include the Y chromosome (partial sampling of the nucleus). On average, ∼66% of the cells had a positive FISH signal (X or Y), 33% had a single X or Y signal, and 33% had 2 signals (XX or XY).