Detection of the M30 neoepitope as a new tool to quantify liver apoptosis: timing and patterns of positivity on frozen and paraffin-embedded sections.

One of the first stages of apoptosis is cytokeratin cleavage mediated by caspases, which is associated with the expression of a neoepitope, the cleavage site of cytokeratin 18, identifiable by the M30 monoclonal antibody. The aim of this study was to evaluate the timing of neoantigen expression and its modifications in the various morphologic stages of apoptosis on frozen and paraffin-embedded sections from liver biopsies of patients with chronic hepatitis or transplanted liver. The appearance of this neoepitope coincides with the gradual disappearance of cytokeratins, with the appearance of nuclear DNA fragmentation, and with the presence of Councilman bodies. The staining patterns on paraffin-embedded sections of liver specimens were similar to those found in frozen sections, with a reduced sensitivity. The M30 antibody is correlated with apoptosis, and its specificity for epithelial cells makes this method the first choice for routine evaluation of apoptosis in liver epithelial cells. Cell death by apoptosis is considered an important regulator of cell numbers in normal and diseased tissues. Quantitative data for normal liver tissue are scarce, but the process is considered exceptional, while in viral chronic hepatitis, 1,2 alcoholic hepatitis, 3,4 and transplanted liver, 5,6 the important role of apoptotic phenomena is well demonstrated. Apoptosis has been shown during rejection regarding biliary cells, 7 sinusoidal cells, 8 and hepatocytes. 9-12 In particular, Ballardini et al 12 have shown that apoptosis and regeneration are relevant phenomena in the livers of patients who have undergone transplantation because of hepatitis C virus (HCV)-related cirrhosis. The methods presently used to evaluate apoptosis have some intrinsic limitations. The terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method is particularly sensitive to fixation conditions and requires highly standardized procedures. Differentiation of apoptosis from necrosis is not complete, 13