We have investigated the use of a novel technique, in situ end-labelling, as a means of the specific identification of apoptotic cells in formalin-fixed, paraffin-processed tissue sections. The technique relies on the presence of DNA strand breaks in apoptotic cells, caused by activation of endogenous nuclease activity during the process of cell death. These strands are labelled with a non-isotopic reporter molecule in the presence of a DNA polymerase, and labelled DNA is identified immunohistochemically. We show that in situ end-labelling stains cells with the morphological characteristics of apoptosis, and greatly simplifies their identification. Furthermore, in two model systems, the number of labelled cells parallels the number of cells undergoing apoptosis as measured by alternative techniques. The ability of the Klenow fragment of DNA polymerase to label apoptotic nuclei suggests that the characteristic DNA fragmentation seen during this process involves the formation of DNA breaks with a 5' overhang. In situ end-labelling will be valuable for the identification and quantitation of apoptosis in a range of normal tissues and in a variety of pathological states. However, the technique is not specific for programmed cell death, and results must be interpreted with caution and correlated with morphological criteria of apoptosis.

Patients with a preoperative diagnosis of DCIS should be considered for SLN biopsy.

T1 and T2 breast cancers located closer to the skin and those located closer to the nipple have a higher incidence of metastases to axillary lymph nodes. Distance from the skin and distance from the nipple should be considered when estimating a patient's likelihood of axillary nodal positivity.

We describe the development and application of in situ end labelling (ISEL) to identity sites of damaged DNA in the nuclei of individual cells. In cell culture, exposure to a variety of genotoxic agents induced a dose and time-dependent increase in nuclear labelling. In addition, examination of histological sections of human skin exposed to solar-stimulated UV light showed ISEL in both keratinocytes and superficial dermal cells, with the same spatial and temporal distribution as that of a marker of DNA repair, PCNA (proliferating cell nuclear antigen). Using co-localization techniques and confocal microscopy, we found increased levels of p53 in many ISEL-positive cells in vitro, with a similar distribution of labelling in the nucleus. This observation provides further evidence for a direct role of p53 in the recognition of damaged DNA. Thus, ISEL should prove a convenient method for demonstrating genotoxic insult in individual cells and in histological material, and may have value in toxicological screening. This high-resolution microscopy technique can also be used to compare the spatial distribution of various proteins implicated in the response to DNA damage with the sites of the lesion.