Following an overview scan, 12 matching 20× HPFs were selected on the 60 tonsil serial sections: four from the cortex, four from the crypt/mantel, and four from the follicle. These microanatomic regions were selected to capture areas enriched for the markers of interest, that is, cortex: CD8 and FoxP3; crypt: PD-L1 and CK; and the follicle: CD68 and PD-1. Cells phenotyped as ‘positive’ for each marker per HPF were aggregated, and the average of the top quartile of signal intensity was determined. This approach was chosen for its sensitivity in highlighting potential variability in staining performance.

Intersite and intrasite percent coefficients of variation (%CV) were determined for each marker. First, an average cell number/HPF for each marker was calculated for four HPFs on each slide. The average cell numbers per slide were then used to calculate intersite and intrasite %CVs. The intersite %CV for each marker was determined by first calculating the %CV of average cell numbers in six serial sections distributed across the six sites (one slide per site), for a total of five groups. The %CVs for each marker were then averaged across the five groups, and an intersite %CV was calculated for each marker (online supplemental figure S1A). Intrasite %CV for each marker was determined by first calculating the %CV for average cell number per HPF across five serial sections from each site. The %CVs from each site were then averaged (online supplemental figure S1B).

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