CONSTANT DETECTION OF CD2, CD3, CD4, AND CD5 IN FIXED AND PARAFFIN-EMBEDDED TISSUE USING THE PEROXIDASE-MEDIATED DEPOSITION OF BIOTIN-TYRAMIDE

Immunohistochemical methods are widely used for diagnostic purposes in histopathology. However, the use of most monoclonal anti-leukocyte an tibodies is limited to frozen tissues. Initially, it was believed that formalin fixation in particular, which is the gold standard for morph ological tissue preservation, destroys most of the antigen binding sit es. In recent years, protease digestion and the introduction of microw ave techniques have significantly enhanced the sensitivity of immunohi stochemical techniques, and a variety of hidden antigen sites in forma lin-fixed tissue have been retrieved for initially unreactive antibodi es. It therefore became clear that many of the leukocyte antigens are not irreversibly destroyed but are most probably masked during the fix ation process. We developed a technique combining optimized pretreatme nt of formalin-fixed tissue with a dramatic enhancement of the immunoh istochemical sensitivity and named it the ImmunoMax method. The Immuno Max method proves that by optimizing the technique at the following th ree levels it is possible to detect formalin-sensitive leukocyte antig ens: (a) standard fixation of the tissue; (b) sufficient antigen unmas king; and (c) increasing the substrate turnover by multiplication of b inding sites with subsequent enhancement of the immunohistochemical re action. Using this optimized ImmunoMax method, we were able to detect CD2, CD3, CD4, and CD5 with conventional monoclonal antibodies in form alin-fixed, paraffin-embedded tissue specimens of various lymphoid tis sues.