Immunohistochemistry analysis of CD39 and CD73 expression. (A-B) Anti-CD39 immunostaining of 2 representative LN tissue sections using anti-mouse HRP-conjugated secondary Ab and 3,3′-diaminobenzidine (brown signal). CLL lymphocytes stain uniformly positive, even if with different intensity levels. Original magnification ×20. (C-D) Anti-CD73 immunostaining of a representative LN from a CLL patient using anti–rabbit HRP-conjugated secondary Ab and 3,3′-diaminobenzidine (brown signal). Low magnification shows CD73 expression around the paler areas corresponding to the proliferation centers. Original magnification ×4 (C) and ×10 (D). (E-F) Higher magnification shows that CD73⁺ cells have a prolymphocytic morphology and that perivascular areas are enriched in CD73⁺ cells. Endothelial cells are CD73⁺, as expected. Original magnification ×40. (G-H) Quantification of the percentage of CD73⁺ areas (brown pixels) in at least 15 different ×20 fields obtained from 5 independent sections depicting proliferation centers (PC), vessels, or the rest of the section (other). Images were analyzed using a DMI 3000 B optical microscope (Leica Microsystems), equipped with a DCF 310 FX digital camera and LAS Version 3.8 software.