Linear quantification of lymphocytes (LQL) was performed on virtual slides (A) on one to eight rectangles of 1x4 mm placed across the tumor invasive front. Each selected area was perpendicular to the invasive front, and the 2 mm of tumor were always to the right (B). CD3 positive cells (green stain) were identified by image analysis (C). Control was performed by comparing both images (B and C). Automated quantification of lymphocyte density was then performed every 5 μm from 2 mm outside to 2 mm inside the tumor, on 2 to 8 selected areas of the tumor. Each value corresponded to a surface of 500 μm2, and was associated with the distance from the tumor invasive front, corresponding to the x and y axis of the curve respectively. Each of the different colored curves (blue, green, red) is issued from one area of measurement (D). These different curves lead to a mean curve, which was used for the identification of the LQL pattern (E). The number of area of measurement varies from two to eight depending on the size of the tumor invasive front.
Allard et al. Diagnostic Pathology 2012 7:156 doi:10.1186/1746-1596-7-156