Figure 3 (a) Cells positively staining with antibodies to activated caspase-3 in a case of hepatocellular carcinoma. nearly Note that cell labelled with closed arrow shows nuclear pyknosis, but cells labelled with open arrows show nuclear features not sufficiently different … None of the cases of steatohepatitis showed positive staining with M30, although apoptotic cells were identified on H&E slides and with activated caspase-3. Figure 4 illustrates control tissue showing no apoptosis. Figure 4 Control liver showing no apoptosis. (a) H&E, (b) activated caspase-3 and (c) M30 (��400). Histological activity of chronic viral hepatitis cases The 32 cases were placed into two groups according to their Knodell necroinflammatory activity score, the sum of scores for categories (1) periportal inflammation and necrosis, (2) lobular inflammation and (3) portal inflammation.
Seven cases were in group 1, activity score 0�C4 (mean 2.43 �� 0.48) and 25 cases were in group 2 with activity scores of 5 and above, ranging from 5 to 14 (mean 7.80 �� 0.49). An activity score cut-off of 4 excludes cases with bridging necrosis from group 1. All three apoptotic indices were higher in group 2 than in group 1, with no apoptotic cells identified with H&E or M30 staining in any of the cases in group 1 (Table 2) (Figure 5). The H&E and activated caspase-3 apoptotic indices of the two groups were significantly different, P = 0.015 and P = 0.030, respectively (Mann�CWhitney U-test). The difference between the groups was not significant for the M30 apoptotic index (P = 0.
207), as many cases in group 2 had much lower apoptotic indices with M30 than with H&E or activated caspase-3, as was seen generally with chronic viral hepatitis, steatohepatitis and control cases. Table 2 Apoptotic indices (mean �� SEM) for cases of chronic viral hepatitis divided into two groups based on histological necroinflammatory activity Figure 5 Apoptotic indices (mean �� SEM) for chronic viral hepatitis cases divided into two groups according to Knodell activity grade. Note that H&E and M30 apoptotic indices are zero for group 1. The difference between the two groups is significant … Discussion Two important conclusions can be drawn from this study. Firstly, we have shown that the use of antibodies to activated caspase-3 and M30 enables the identification of apoptotic hepatocytes more frequently than by the use of H&E morphological features alone. Activated Cilengitide caspase-3 identified more cells than M30 in all of the different disease groups tested in this study. This may reflect the fact that activated caspase-3 identifies an earlier stage of apoptosis, as caspase-3 must be activated to cleave cytokeratin 18 and reveal the M30 epitope (Leers et al. 1999).