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Absolute numbers of FOXP3-positive, Treg lymphocytes in assessable 1-mm diameter invasive tumour cores were counted manually.
Absolute numbers of Foxp3-positive cells in assessable 1-μm invasive tumor cores were counted manually using an eyepiece reticule without any prior knowledge of specimen identity.
A total of 65 cores were counted independently by two observers (BE and ZM) blinded to patient outcome and the other observer's score, giving an interclass correlation coefficient (ICCC) of 0.94, indicating excellent agreement.
All cores were counted by one of us (SD) and at least 25% of the cases were counted by a second observer (ZM) and interclass correlation coefficient for CD34+ was 0.83.
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For CD31 and CD105, the total number of positive vessels per TMA core were counted.
All cells within each core were counted and the percentage of cells staining for each core determined.
This is a very stringent assumption since for instance suboptimal peptides sharing the ligand binding-core are counted as negatives even though they could be presented on the HLA molecule.
A total of 50 nuclei of each of the three cores per case were counted.
Uptake signal sequence (USS) 9-mer cores [ 23] were counted, and their surrounding sequences were reported using our perl script, 200804USS.pl.
Ring widths from the two cores from each tree were counted to determine the approximate ages/number of cohorts and averaged to analyse the growth history of each cohort.
Digitized images were overlaid with a square grid, and the cross sections overlapping with viral core and entire virions were counted.
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