Identification of HIV-1 in a positive specimen. (A) Double immunofluorescence using fluorescein isothiocyanate-conjugated antibody to CD68 in association with HIV-1 p24 (labeled with Texas Red). HIV-1–producing macrophages showing characteristic mixed (orange) fluorescence (white arrow) account for a small percentage of the total CD68⁺ cell population (<0.5%). Slides were scanned and analyzed with an Olympus BX61 fluorescent microscope. (B) Control tissue (persistent generalized lymphadenopathy) stained with p24 immunoperoxidase (i). The bulk of p24 is associated with follicular dendritic cell processes whereas the tingible-body macrophages (arrows) are negative (original magnification, ×100). In AR-DLBCL tissue (ii), few cells react with p24. The arrow indicates an infected macrophage and the arrowhead points to a lymphocyte (original magnification, ×400). Staining of the same AR-DLBCL biopsy for the HIV-1 regulatory tat protein (iii) revealed a higher number of infected macrophages (original magnification, ×400). (iv) The total macrophage population in this case is highlighted by the marker CD68. (C) Double staining using VIP (purple) for CD68 and DAB (brown) for CD34 (top) showing a ring of perivascular macrophages (original magnification, ×400). On double staining, p24 purple and CD34 brown (bottom), HIV-1–expressing macrophages (arrows) are not specifically seen perivascularly (original magnification, ×200). Images were obtained with a Leica DM2500 microscope.