William David Richards

Doubly stochastic Poisson distribution of platelet adhesion on material surfaces and its implication on fluorescence image analysis

An image based assay has been developed to quantify platelet adhesion on material surfaces. Briefly, citrated platelet rich plasma (PRP) is incubated with materials for 2 h to allow platelet adhesion on the surface, followed by fluorescence labeling of platelets with Celltracker Green. Multiple images are acquired by an automatic fluorescence microscope, IN Cell Analyzer 1000. Platelets are identified and counted by an automatic image analysis algorithm. We have observed that the variance of the counts is considerably greater than expected from simple distribution laws. Statistical analysis of that difference shows that these measurements will often follow a doubly stochastic Poisson process in which the variance is inherently very large. To overcome this, multiple images (n > or = 8 images/well, about 3% of total area) are necessary to achieve accurate counting. This method has been compared to the commonly used enzyme based platelet adhesion assay, lactate dehydrogenase (LDH) assay. It is concluded that the present method is only effective in quantifying adherent platelets when a large number of samples are used. However, this method does provide additional information on platelet morphology and spatial distribution, which is lacking in the LDH assay.