Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis)

Abstract

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology.