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Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death. [ABSTRACT FROM AUTHOR]

Antibodies (Abs) to CD20 and MHC class II antigen were found to exhibit a novel processing pathway after binding to the surface of RL B-lymphoma cells. The Abs were 'excreted' as a part of large cytoplasmic fragments. These fragments formed at cell-cell junctions, but gentle dispersal of the cells, to form a single-cell suspension of high viability, caused the release of most of the fragments. This process also occurred in Raji cells and in three other B-lymphoma cell lines (of seven tested). Six B-lymphoblastoid cell lines tested did not form these objects. Once they were recognised, the fragments could be identified in cell preparations by phase contrast microscopy or after staining with Wright's stain. They were induced by the binding of certain Abs, but not by most Abs bound to the cell surface. The mode of formation, detailed morphology and function of these cytoplasmic fragments remain to be determined. They are similar in many respects to the lymphoglandular bodies that have been described by pathologists for many years, which are characteristic of B-cell lymphoma, but which have not previously been described in cell lines. This type of Ab processing, if it occurs in patients, will have an impact on the therapeutic use of these Abs. [ABSTRACT FROM AUTHOR]