Aperio image analysis system

Formalin-fixed and paraffin-embedded sections of TMA were deparaffinized in xylene and were rehydrated with gradually decreasing concentrations of alcohol. Sections were immunostained after antigen retrieval using the Bond-max automated immunostainer (Leica Microsystems, Newcastle, UK). Primary antibodies used were the anti-CD8 polyclonal antibody (1:100, Neomarkers, Fremont, CA, USA), the anti-CD45RO monoclonal antibody (1:50, Neomarkers) and the anti-FOXP3 monoclonal antibody (1:50, Abcam, Cambridge, MA, USA). Antibody binding was detected by using the Bond Polymer Refine Detection kit (Leica Microsystems).
After immunohistochemical staining for each of the T cell markers, TMA slides were scanned by the Aperio image analysis system (Leica Biosystems, New Castle, UK) (Fig 2). The software counted the number of immunopositive nuclei in each tissue core. The average density (cells/mm²) of each lymphocyte subset was calculated in a whole TMA core. T cell subset densities were divided into two groups (high versus low) according to a median-split.

TAMs were defined as intratumoral or peritumoral cells that stained positive for both CD68 (cytoplasmic DAB staining) and CD163 (membranous HistoGreen staining). An entire image of each case was acquired using an Aperio image analysis system (Leica Biosystems, New Castle, UK). In each case, bright- and dark-field images were taken in at least five fields [19 (link),25 (link)] (with 196 × 147 µm in size) using a fluorescence microscope (BX63, Olympus, Tokyo, Japan) connected to a DP80 CCD camera (Olympus). In each field, the number of TAMs was counted, and the number of PID particles per TAM was measured using a software for analyzing PID (PID analyzer, Konica Minolta, Tokyo, Japan), as described previously [18 (link)]. Five fields were randomly selected, and the numbers of TAMs and PID particles per TAM in the five fields were averaged and used to score each case. The upper tertiles of the average numbers of TAMs and PID particles per TAM were defined as a high number of TAMs and a high expression level of CSF1R in each case, respectively. The lower and middle tertiles were defined as a low–moderate number of TAMs and a low–moderate expression level of CSF1R in each case, respectively.