Chalkley method in the angiogenesis research and its automation via computer simulation.

The aim of this study was to develop a computer simulation evaluating microvessel density according to the Chalkley method on digital images taken from neovascular hot spots. An image analysis algorithm has been developed using ImageJ, an extensible, open source image processing and analysis software. The idea was to create a virtual Chalkley point array graticule, and to calculate Chalkley counts automatically by stepwise angular rotation of it on the superimposed images containing properly segmented microvessels. This eliminates the necessity of having the Chalkley graticule, an accessory that has to be mounted on the microscope's ocular. The proposed method is a faithful simulation of the original Chalkley counting procedure. It gives pathologists who do not have the Chalkley graticule an opportunity to evaluate microvessels quantitatively according to the basic principles underlying Chalkley counting. Evaluating microvessel densities in solid tumors is a frequent procedure in angiogenesis research. A few standard methods, including Chalkley counting, are used for the estimation of microvessel density. Several independent studies have shown that the Chalkley counting is more consistent and may provide useful data on prognosis. The obvious disadvantages lie in the facts that this method is time-consuming and requires a special hardware. Computer simulation may overcome these obstacles.