Granzyme b clone grb 7

Immunohistochemistry on cell block and FFPE capsular specimens was performed using an automated immunostainer (Dako, Glostrup, Denmark) with the following primary antibodies: CD30 (clone Ber-H2), CD3 (clone F7.2.38), CD4 (clone 4B12), CD8 (clone C8/144B), CD68 (clone PG1), CD15 (clone Car-b), Granzyme B (clone GrB-7), IRF-4 (clone MUM1) (Dako), CD25 (clone 4C9, Novocastra, Newcastle Upon Tyne, UK), PAX5 (clone SP34, Thermo Scientific, Waltham, USA). Paraffin sections were pretreated using EnVision^™ FLEX Target Retrieval Solution (Dako) and incubated with an optimal dilution of the primary antibody. The reaction was visualized with the EnVision Detection Kit (Dako) using 3–3’-diaminobenzidine chromogenic substrate. Sections were counterstained with EnVision FLEX Hematoxylin (Link) (Dako).
For every marker, positive cells were counted out of 10 non-overlapping randomly selected high-power microscopic fields (HPFs, 40×10), and the mean number of positively stained cells per HPF was recorded. The percentage of positive cells was calculated as the ratio between the mean number of stained cells per HPF and the mean number of total cells per HPF.

Immunohistochemical studies were carried out in 38 of 45 cases using sections of decalcified paraffin-embedded bone marrow biopsy specimens. The following antibodies were used at the dilutions suggested by the manufacturers: CD3 (polyclonal, Dako), CD4 (clone 4B12, Dako), CD8 (clone C8/144B, Dako), CD20 (clone L26, Dako), CD56 (clone 123C3, Dako), CD57 (clone TB01, Dako), granzyme B (clone GrB-7, Dako), and T-cell restricted intracellular antigen 1 (TIA-1) (clone 2G9, Immunotech, France). After dewaxing and heat-induced antigen retrieval, immunostaining was performed on an Autostainer Link 48 (Dako, Denmark) according to the manufacturer’s instructions. All immunostained samples were counterstained with hematoxylin.

Immunohistochemical studies of the spleen (5 patients) and bone marrow (15 patients) were conducted using the formalin-fixed paraffin-embedded tissue. The following antibodies were used at dilutions suggested by the manufacturers: CD3 (polyclonal, Dako, Carpinteria, CA, United States); CD4 (clone 4B12, Dako); CD8 (clone C8/144B, Dako); CD16 (clone 2H7, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom); CD20 (clone L26, Dako); granzyme B (clone GrB-7, Dako); T-cell restricted intracellular antigen 1 (TIA-1) (clone 2G9, Immunotech, France); TCR-β F1 (clone 8A3, Thermo Scientific, Waltham, MA, United States); and TCR-γ (clone γ3.20, Thermo Scientific). After dewaxing and heat-induced antigen retrieval, immunostaining was performed using an Autostainer Link 48 (Dako, Denmark) according to the manufacturer’s instructions. All immunostained samples were counter-stained with hematoxylin.