Development of a quantitative method for the study of apoptosis in peripheral blood

In vitro and ex vivo many methods are effective for the quantitation of apoptotic cells. These methods cannot be applied for the study of apoptosis in peripheral blood (PB) samples, because of the disappearance of circulating apoptotic cells and the heterogeneity of cell populations. In the present study we describe a nuclear chromatin staining method using ethidium bromide (EtBr), by which we were able to identify and quantify morphologically apoptotic cells in an early stage of the apoptotic process, before their disappearance. The application of the EtBr nuclear stain in CCRF-CEM cells incubated with epirubicin, provides a reliable and reproducible estimation of apoptosis compared with other assays. The main advantage of this method is that it permits the study of four cellular subpopulations by their morphological characteristics (normal cells, early apoptotic, late apoptotic and cells which died from necrosis). Furthermore, we utilized the EtBr nuclear stain to identify and quantify the circulating apoptotic cells directly in the PB of patients with acute lymphoblastic leukemia during multidrug chemotherapy. Circulating apoptotic cells were detectable after 24 hours treatment followed by the decrease of peripheral blood mononuclear cells. Thus staining of nuclei with EtBr provides a rapid and useful method for the quantitative study of apoptotic cells in somatic fluids during clinical trials.