The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL‐60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only Sphase HL‐60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: (a) Activation of an endonuclease in apoptotic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA‐specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity oaf the apoptotic process. (b) Plasma membrane integrity, which is lost in necrotic but not in apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early‐ and late‐apoptotic cells. (c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. (d) The ATP‐dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. (e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. (f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. (g) The decrease in forward light shatter, paralleled either by no change (HL‐60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. (h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at love pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. (i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis. The data presented indicate that flow cytometry can be applied in basic research on molecular and biochemical mechanisms of apoptosis, as well as in the clinic, where the ability to monitor early signs of apoptosis in samples from patients' tumors may be predictive of the outcome of some treatment protocols. © 1992 Wiley‐Liss, Inc.

Fine-needle aspiration cytology is a valuable adjunct to preoperative assessment of parotid masses. Preoperative recognition of malignant tumors may help prepare both the surgeon and patient for an appropriate surgical procedure.

The aim of this study was to compare three methods of detection of apoptotic cells: (1) the method based on elution of low molecular weight DNA from the ethanol fixed cells followed by cell staining with DAPI (diamidino‐2–phenylindole) or propidium iodide as the DNA fluorochromes, (2) the method of in situ labeling of DNA strand breaks with biotinylated dUTP, utilizing exogenous terminal deoxyribonucleotide transferase, and (3) the method of analysis of DNA denaturation in situ using acridine orange to differentially stain denatured and doublestranded DNA sections following cell exposure to 0.1 M HCl. Cells of the human promyelocytic HL‐60 line, treated in vitro with the DNA topoisomerase I inhibitor camptothecin, which selectively triggers apoptosis of S‐phase cells, were chosen as a model. The method based on analysis of changes in DNA denaturability was the most sensitive in terms of detection of the earliest changes in chromatin of cells undergoing apoptosis; the increased sensitivity of DNA to denaturation in Sphase cells was measured as early as 100 min after addition of camptothecin. DNA cleavage, assayed either by the univariate measurement of DNA content following extraction of low molecular weight DNA, or by labeling DNA strand breaks with biotinylated dUTP, was detected in S‐phase cells after 120 min incubation with camptothecin. The percentage of apoptotic cells at the late stage of apoptosis, the kinetics of cell transition to apoptosis, and kinetics of the loss of S phase cells were all essentially similar when measured by any method. All three methods can be used to estimate the cell cycle phase specificity of apoptosis, although the method based on DNA strand labeling with biotinylated dUTP by terminal deoxynucleotidyl transferase has the advantage of making it possible to estimate the cell cycle distribution of both the apoptotic and unaffected cell populations. The latter method also appears to be the most specific in terms of detection of apoptosis. © 1994 Wiley‐Liss, Inc.