Figure 5.

Tissue Illustration of BRIM. This figure shows images of: (A) H and E image, (B) CD74 (numerator), (C) CD59 (denominator), and (D) CD74/CD59 (ratio). The micrograph in (A) was obtained using H&F staining followed by imaging with a color camera. This tissue sample was derived from a patient diagnosed with invasive breast cancer. As H&F staining should not be performed on the same slide as immunofluorescence imaging, the micrographs of (A–C) were taken from serial sections. Hence, (A) is similar to, but not identical with, (B–D). The images of (B,C) were autoscaled for clarity of illustration, but not in the calculation of the ratio in (D). This image was chosen for pedagogical purposes because it illustrates the principles of BRIM. Due to the brightness in each channel, cell subtypes are easily discerned. (B) shows the numerator, CD74. Two cells are designated as 1 and 2. Cell 1 is a CD74^lo/CD59^hi cell whereas Cell 2 is a CD74^hi/CD59^lo cell. This illustrates the fact that different cell populations are present. As the differences in CD74 and CD59 levels are very large, a considerable improvement in gain can be observed. As anticipated by genomic studies (Porter et al., 2003), large differences in CD74 and CD59 levels can be observed in invasive breast cancer cells, as physically manifested in the BRIM micrograph of (D). In the case of DCIS, a large range of ratios is observed (Clark and Petty, 2016). (D) shows a field containing many CD74^hi/CD59^lo cells. Such high numbers of CD74^hi/CD59^lo cells are not found in normal breast tissue, fibroadenoma of the breast, and a sub-population of DCIS samples (Clark and Petty, 2016). A ratio scale bar is given along the right hand side of (D) (20x objective).