Apoptosis - Selected Detection Methods of Apoptosis and AssociatedRegulatory Factors in Histologic Slides of Tumors

Overview

Methods for identification of apoptotic (AP) cells in tissue sections include light and electronmicroscopy and immunohistochemical (IHC) detection of apoptotic antigens. Bcl-2 at many tumorsinversely correlates with AP and is an indirect marker of AP index. Transglutaminase isoften expressed in nonapoptotic cells, and thus represents a non specific marker of AP. Likewise,expression of FAS does not necessarily represent a transformation into AP. IHC detection ofcaspases does not distinguish between active and nonactive forms of the proteases. Immunolabelingof biotin-conjugated Annexin V is used for the identification of phosphatidylserine residuesexposed on the surface of AP cells. Annexin V immuno-gold labeling by means of electron microscopywill allow a more refined description of the morphological events occurring during apoptosis.TUNEL, ISEL and ISNTA methods detect DNA breaks. The rate of AP detected by TUNEL isabout 20 % higher then by apoptotic figure counting. DNA strand breaks can also occur duringDNA repair, electrocoagulation, autolysis, fixation and paraffin embedding. With Apostain, DNA isselectively denaturated by heating with formamide and stained by monoclonal antibody specificto single-strand DNA. It specifically stains condensed chromatin of apoptotic cells. M30 IHC usesa monoclonal antibody binding to the product resulting from cleavage of cytokeratin 18 by activatedcaspases. M30 is negative in necrotic cells and in progressively degraded cells (AP bodies). Incontrast to some pilot studies, we have not reached sufficient sensitivity and specificity of IHCdetection with M30 (Roche) in breast carcinomas.

Key words:
apoptosis - regulation - detection