Qwin based analysis system

Immunohistochemistry was performed on ST sections with a primary mouse mAb against human PRLR (1A2B1; Invitrogen, Camarillo, CA, USA) using a three-step immunoperoxidase method, as previously described [33 (link)]. Further, as a negative control, irrelevant isotype-matched immunoglobulins were applied to the sections instead of the primary antibody. Two independent observers (V.C. and D.C.) unaware of the clinical data performed the semi-quantitative analysis, image acquisition and analysis. The images were analysed using a computer-assisted image analysis Syndia algorithm on a Qwin-based analysis system (Leica, Cambridge, UK), as previously described in detail [34 (link)]. Values of integrated optical density/square millimetre were obtained and corrected for the total number of nucleated cells per square millimetre, representing the intensity of staining nucleus per square millimetre [35 (link)].

Sections (5 μm each) were cut and mounted on StarFrost adhesive glass slides (Knittelgläser, Braunschweig, Germany). Sealed slides were stored at −80°C until further use. LN tissue sections were stained using mouse monoclonal antibodies against T cells (anti-CD3, clone SK7; Becton Dickinson, Breda, the Netherlands), B cells (anti-CD22, clone RFB4; Millipore, Amsterdam, the Netherlands) and plasma cells (anti-CD138, clone MI15; Dako, Heverlee, Belgium).
Staining was performed using a three-step immunoperoxidase method to detect bound anti-CD138 antibodies, as described previously [17 (link)]. For anti-CD3 and anti-CD22, we used a two-step immunoperoxidase method with a secondary polymer—horseradish peroxidase—conjugated anti-mouse antibody (EnVision+ System; Dako). As a negative control, irrelevant isotype-matched immunoglobulins, instead of the primary antibody, were applied to the sections. Staining was analysed by digital image analysis in a blinded fashion using a Syndia algorithm on a Qwin-based analysis system (Leica, Cambridge, UK) as described previously [18 (link)]. The number of positive cells was calculated for each section as the number of positive cells per square millimetre of tissue.