Influence of cell cycle synchronization on digital image analysis of HL-60 granulopoiesis

Retinoic acid (RA)-treated HL-60 cells subjected to density arrest (DA) and double thymidine block (TB) synchronization demonstrated image feature changes associated with cellular proliferation and differentiation. RA-treated TB cells demonstrated an increased level of morphologic differentiation (assessed by differential counts and quantitation of nuclear shape) and more rapid functional differentiation (assessed by superoxide production and expression of complement receptors) than RA-treated DA cells. By comparison to DA cells, TB cells had less variation in virtually all image features values. A Kruskal-Wallis test of image features ranked total optical density (TOD) of Feulgen-stained cells, nuclear area and shape factor as the top three features regardless of synchronization method. Statistically significant changes in image feature values of RA-treated cells were first noted on day 1. The computer-assisted ability to discriminate RA-treated cells on a given day after induction from paired controls by means of an unsupervised learning algorithm increased over a seven-day period for both DA and TB cells. However, in the dichotomous (RA-treated versus untreated) classification scheme employed, which did not account for continuous levels of morphologic differentiation, there was no advantage in the use of the TB over DA procedure.