Immunohistochemical analyses were performed on paraffin blocks. Conventional 6-μm-thick histological sections were obtained with a microtome and mounted on slides pretreated with poly-l-lysine (Sigma Chemicals, St Louis, MO). Slides were then deparaffinised and rehydrated in ethanol and xylene. Two antigen retrieval methods were applied, 1) by incubating sections with a Tris-EDTA solution, pH 9.0, at 95-98°C for 25 min before staining with antibodies against BCL2, BCL6, CD10, and 2) by incubating sections with 0.01 M citrate buffer solution, pH 6.0, for 15 min before staining with antibodies against Ki67 and HGAL. After washing in H2O and then in Tris-buffer solution, sections were incubated with the following monoclonal antibodies: anti-BCL2 (clone 124, dil. 1:100, Dakocytomation, Milan, Italy); anti-BCL6 (clone PG-B6p, dil. 1:10, Dakocytomation, Milan, Italy); anti-CD10 (clone 56C6, dil. 1:30, Novocastra, Laboratories, New Castle, UK); anti-Ki67-antigen (clone Mib-1, dil. 1:80, Dakocytomation, Milan, Italy), and with the polyclonal anti-HGAL (dil. 1:400, Sigma-Aldrich, Milan, Italy). Incubations were carried out in humidified atmosphere at room temperature for 1 hour. The reaction was revealed using the streptavidin-biotin-peroxidase technique (Dako-Envision Plus/HRP peroxidase kit, Dakocytomation, Milan, Italy). Sections were then incubated with 3,3'-diaminobenzidine (0.05 diaminobenzidine in 0.05 M Tris buffer, pH 7.6 and 0.01% hydrogen peroxide; Sigma-Aldrich, Milan, Italy), counterstained with Mayer hematoxylin (Bio-Optica SpA, Milano, Italy) and cover-slipped with Paramount. Reactive tonsil tissue was used as positive controls for GC-markers and staining with omission of the primary antibody was performed as negative control.
The original slides immunostained for commonly used markers in lymphoma diagnostics, like CD20, CD3, CD5, BCL1/Cyclin D1, immunoglobulin light chains, CD21, CD23, were reviewed; when not available or not easily interpreted, the immunostaining was done or repeated.
The number of HGAL, BCL6, BCL2, CD10, and Ki67-antigen immunoreactive cells was counted among at least 500 cells in the more representative fields by using a light microscope at 250× magnification and expressed as percentage. Counting was repeated three times; the obtained mean values were considered for further analysis. For Ki67-antigen a three-point scale was used: low proliferative index for positive values ≤ 25%, intermediate proliferative index for > 25 and ≤ 50%, high proliferative index for values > 50%. HGAL, BCL6, BCL2, and CD10 positivity of immunostaining was then classified with a two-point scale: negative (-) for absence of immunostaining or positivity in less than 20% of positive cells; positive (+) for immunostaining in more than 20%. For BCL2 and BCL6, the immunostaining was considered only in the B-cell population, by comparing it with the CD3 staining in order to avoid the T-cell component marked by BCL2 and by BCL6.