2002). The completed recipient blocks were sectioned at 4 ��m and transferred to silanized glass slides. A total of five recipient blocks were made. Histological Evaluation The whole section slides that were used for selecting representative TMA cores were used for histological evaluation of morphologic characteristics. Sunitinib purchase Mitotic figures were counted in 50 consecutive high-power fields (HPFs). Additional variables recorded included the presence of spindled and/or epithelioid cell morphology. Focal or diffuse atypia was defined according to Miettinen et al. (2006). Other variables such as necrosis, ulceration, and hemorrhage were also assessed. Immunohistochemical Staining and Scoring Sections from the arrays were stained with H&E to confirm the presence of representative tumor in each core.

Further sections were stained using a Ventana (Tucson, AZ) automated immunohistochemical stainer according to manufacturer’s guidelines. The antibody used for detecting KIT-positive cases was c-KIT (polyclonal, dilution 1:200; Dako, Carpinteria, CA). The immunostaining was performed with an avidin-biotin detection system. Diaminobenzidine hydrochloride solution with hydrogen peroxide (Ventana Gen II, Dab basic) was the chromogen. Morphometric Scoring Digital images of sections from the TMA stained with H&E were used for morphometric analyses, captured using a BLISS scanner (Bacus Laboratories; Lombard, IL). The slides were scanned at ��40 objective magnifications. A plug in for the open source program ImageJ (http://rsb.info.nih.

gov/ij/) was created to combine the separate images stored by BLISS scanner and form the individual images of the tissue cores (http://rsb.info.nih.gov/ij/plugins/stitch-bliss.html). The resulting pixel dimensions of the individual core images were 3760 �� 3360. A separate Nuclear Stain Analyzer plug in was created for assessment of morphometric parameters of the tumor nuclei (Figure 1). The following characteristics/dimensions of the nuclei were thereby quantified: area, intensity, optical density, perimeter, width and height of bounding rectangle, ellipse major and minor, ellipse ratio, ellipse angle, circularity, Feret diameter, and roundness. In addition, number of nuclei in each core was registered. The formula used for roundness was 4 �� area/�� �� square(major axis).

Ellipse major and minor were the primary and secondary axes of best-fitting ellipse, and the ratio between shortest and longest axis was used to evaluate the ratio between the two (SL ratio). Figure 1 Core with outlined nuclei. All the images are publicly available at the companion site: www.gpecimage.ubc.ca/tma/web/viewer.php. The site was constructed at Genetic Carfilzomib Pathology Evaluation Centre (GPEC) (Vancouver, Canada) using a GPEC database and a Java applet provided by Bacus Laboratories.