Annexin V expression in apoptotic peripheral blood lymphocytes: an electron microscopic evaluation

Loss of plasma membrane asymmetry, resulting in the exposure of phosphatidylserine (PS), is considered to be an 'early' event in apoptosis. It is generally accepted to precede nuclear condensation, independent of the apoptosis inductive agent. In the present study we focus on 2 apoptotic parameters: PS exposure in comparison with morphological alterations. Peripheral blood lymphocytes were irradiated in vitro (5 Gy Co-gamma-rays) or incubated with staurosporine (1 microM, 6 hours). PS exposure was measured flow cytometrically using FITC-labelled annexin V, combined with PI. Morphological alterations were evaluated by electron microscopy (EM). Results are based on 3 independent experiments. For the irradiated lymphocytes the amount of viable cells (annexin V-/PI-) as scored by flow cytometry was comparable or slightly lower than the number of viable cells as scored by EM (75% compared to 79%). However, for the staurosporine treated lymphocytes only about 24% of the cells were designated as viable by EM, whereas by flow cytometry about 65% of the cells were annexin V-/PI-. Examination by EM showed about 40% cells with a morphology distinct from that of a normal viable cell, but without the clear-cut characteristics of apoptotic cells. Time studies revealed that these cells went into apoptosis after prolonged incubation times up to 18 hours. Application of biotinilated annexin V for EM detection with gold-conjugated anti-biotin, showed that only clear-cut apoptotic, apoptotic necrotic and oncotic cells showed the gold-label at their membranes. Cells that could be detected under the EM as 'non-viable' but without the clear-cut characteristics of apoptotic cells, were not labelled. Data indicate that, dependent on the apoptosis inductive mechanism, morphological alterations can occur before PS exposure.