Validating B Cell Deconvolution by IHC

In silico deconvolution results regarding B cells were validated by anti-CD20 immunohistochemistry analyses. Double-blinded labeled microscopic slides were prepared from the FFPE tumor samples, processed in the BenchMark ULTRA IHC System, incubated with CONFIRM anti-CD20 L26 primary antibody (Ventana Medical Systems, Oro Valley AZ, USA) and ultraView Universal DAB Detection Kit as a secondary antibody (Ventana Medical Systems, Oro Valley AZ, USA).
Following whole slide scanning on the Aperio LV1 System (Leica Biosystems, Richmond IL, USA), tumor and normal areas were defined by two pathologists. The slides were then quantified by two separate methods: (1) An independent intratumoral and peritumoral CD20 ${}^{+}$ percentage estimation by two experienced pathologists, with the joint assessment of a third senior pathologist in cases of discrepancy; (2) Independent digital image analysis and quantification using immunohistochemistry profiler software [44 (link)]. Digitally analyzed tumor, normal, and total CD20 ${}^{+}$ percentages were calculated by dividing the CD20 ${}^{+}$ pixel count by the number of total pixels.