We examined the influence of different staining techniques (three-step immunoperoxidase technique (IP): alkaline phosphatasendash;antindash;alkaline phosphatase technique (APAAP) on the quantitative evaluation of Ki-67ndash;labeled nuclei. We studied five melanocytic skin tumors. From each case, five parallel sections were prepared and stained using the peroxidase-antiperoxidase (PAP) technique (slide 1) and the APAAP technique once (slide 2). Slide 3 consisted of a single repetition of the APAAP technique, slide 4 was a double repetition, and slide 5 was a third repetition. We assessed the volume fraction (VV) of Ki-67ndash;positive nuclei using computer-assisted image analysis. For each staining group, the mean value and standard deviation of VV were calculated. Comparing VV values obtained from the different staining groups we did not find a statistically significant difference between the IP and the various APAAP steps (Wilcoxon test, p = 0.05). However, the staining procedure influenced the quantitative results to some extent. The mean VV of the five staining groups ranged in our study from 0.10 to 0.17percnt;, which is narrow compared with the overall variability among different cases (dermal melanocytic nevus, 0.01percnt;; metastatic malignant melanoma. 0.43percnt;). Therefore, we can state that for a rough evaluation of Ki-67ndash;positive nuclei, the influence of different staining methods is negligible; for a subtle quantitative analysis, however, it would nevertheless be preferable to always apply the same staining technique.
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