Tissue samples were fixed in 10% formalin and embedded in paraffin. An automated BOND-MAX stainer (Leica Biosystems, Melbourne, Vic., Australia) was used to stain the paraffin sections. The following primary antibodies were used (clone, dilutions): CD20 (L26, 1:200), CD3 (LN10, 1:200), CD5 (4C7, 1:100), CD7 (56C6, 1:50), CD8 (C8/144B, 1:100), CD4 (1F6, 1:40), TIA-1 (2G9, 1:500; Beckman Coulter, Brea, CA, USA) and GranzymeB (GrB-7, ready to use) (Nichirei, Tokyo, Japan). Primary monoclonal antibodies raised against the human TCRβ (βF1; TCR1151, 8A3, 1:50 [Thermo Scientific, Waltham, MA, USA]) and TCR-γ chain constant region (TCR1153, γ3.20, 1:80 [Thermo Scientific]) were used to detect the αβ and γδ TCR, respectively.(12 (link)) In situ hybridization with Epstein–Barr virus (EBV)-encoded small RNA (EBER) probes (INFORM EBER; Leica Biosystems) was used to detect EBV.
The samples of CD20, CD3, CD10, CD5, CD7, CD8, CD4, TIA-1,(13 (link)) granzyme B(14 (link),15 (link)) and EBER antigens were scored as positive when ≥30% of the lymphoma cells were positively stained. The samples of TCR γ, TCR δ and TCR β antigens were scored as positive when ≥50% of the lymphoma cells were positively stained.
The samples of CD20, CD3, CD10, CD5, CD7, CD8, CD4, TIA-1,(13 (link)) granzyme B(14 (link),15 (link)) and EBER antigens were scored as positive when ≥30% of the lymphoma cells were positively stained. The samples of TCR γ, TCR δ and TCR β antigens were scored as positive when ≥50% of the lymphoma cells were positively stained.