Biopsies were stained with the following antibodies; T cells were labelled with rabbit antihuman CD3 polyclonal (A0452, Dako, Cambridge, UK), B cells with mouse antihuman CD20 (clone L26, DAKO), and HEV with rat antihuman PNAd (clone MECA‐79, PharMingen, Oxford, UK), anti-CD68 (macrophages, IgG1 KP1 clone; Dako), anti-CD117 (mast cells, rabbit antihuman polyclonal antibody; Dako), anti-CD15 (neutrophils, clone BY87; Novocastra), and anti-CD31 (endothelial cells, clone JC70A).
The entire area of each tissue specimen was photographed and digitized using a SPOT RT CCD camera and SPOT 4.0.4 software (Diagnostic Instruments) on an Axioplan 2 fluorescence microscope (Zeiss). For the quantitative evaluation (cells/mm2), tissues were sequentially photographed using Digital Image Analysis (Olympus), as previously described (10). As mentioned above, the presence of LN + is identified as radial aggregates of T and B lymphocytes with dendritic cells associated with the development of HEV with PNAd positive around a blood vessel. The highest grade of LN within each sample was determined according to a previously described scoring method [16 (link)] based on the number of radial cells count: grade 1 = 2–5 radial cells, grade 2 = 6–10 radial cells, and grade 3 ≥ 10 radial cells. LN was defined histologically as follicular aggregates grade ≥ 2 with T/B cell segregation.
The entire area of each tissue specimen was photographed and digitized using a SPOT RT CCD camera and SPOT 4.0.4 software (Diagnostic Instruments) on an Axioplan 2 fluorescence microscope (Zeiss). For the quantitative evaluation (cells/mm2), tissues were sequentially photographed using Digital Image Analysis (Olympus), as previously described (10). As mentioned above, the presence of LN + is identified as radial aggregates of T and B lymphocytes with dendritic cells associated with the development of HEV with PNAd positive around a blood vessel. The highest grade of LN within each sample was determined according to a previously described scoring method [16 (link)] based on the number of radial cells count: grade 1 = 2–5 radial cells, grade 2 = 6–10 radial cells, and grade 3 ≥ 10 radial cells. LN was defined histologically as follicular aggregates grade ≥ 2 with T/B cell segregation.