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Blood platelets are highly sensitive circulating subcellular sensors that respond to a multitude of stimuli with a change of their resting discoid shape into more rounded structures processing blebs and pseudopodia. The platelet outline can be automatically segmented from digital images and shows a fractal behaviour. Due to the sensitivity of platelet shape change to preanalytical influencing factors, a highly standardized examination procedure is necessary in order to obtain reliable and reproducible results. In the current work, a completely standardized analytical procedure comprised of standardized sample preparation, robotic image acquisition and algorithmic image analysis is described and validated using calibration objects of different size. The system allows for the user independent examination of fractal and Euclidean geometrical properties from unfixed and unstained samples of soluble objects. The results presented demonstrate the large intra and inter slide reproducibility and the ability of the system to clearly identify and separate smaller and larger particles as well as aggregates and objects out of focus. As the system is ideally suited for the examination of slides at higher throughput, it will enable and expedite the examination of the platelet shape features of larger cohorts of patients. Hence, it might provide an ideal basis for the examination of possible preanalytical constraints and therefore help to improve our knowledge about the physiological and pathophysiological meaning of platelet shape change.

Automated microscopy for the standardized and reproducible quantitative morphometry of platelet shape