Splenic micro-anatomical localization of small lymphocytic lymphoma/chronic lymphocytic leukemia using a novel combined silver nitrate and immunoperoxidase technique

Small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) may be histologically difficult to differentiate from reactive lymphoid hyperplasia (RLH) in the spleen. Because routine hematoxylin and eosin (H&E) staining delineates splenic microanatomy poorly, we have developed a method that simultaneously stains reticulin fibers and B-lymphocytes. B3 or formalin-fixed, paraffin-embedded archival splenic tissue with diagnoses of SLL/CLL (11 cases), RLH (10 cases), and trauma (seven cases) were studied using a novel silver nitrate immunoperoxidase (SNIP) double-staining technique. Gordon and Sweet's reticulin stain was followed by immunoperoxidase staining for B-lineage marker CD20 (Dakopatts, Carpinteria, CA) using the avidin-biotin method. This allowed us to clearly localize B cells to Malpighian bodies, periarteriolar lymphoid sheaths, sinuses, or cords. Features identified by SNIP found only in SLL/CLL, but not in RLH or traumatized spleens, were trabecular infiltration (eight of 11 cases), subendothelial infiltration (seven of 11 cases), and prominent sinus involvement (seven of 11 cases). One or more of these features were seen in 10 of 11 cases of SLL/CLL. Other distinguishing features were the percentage area occupied by B-lymphocytes in each section (SLL/CLL = 74%; RLH = 46%; traumatized spleens = 36%); and mean spleen weight (SLL/CLL = 1,603 g; RLH = 287 g; traumatized spleens = 126 g). We have found the SNIP technique to be superior to traditional H&E staining in identifying B cells in the context of splenic microanatomy.