Summary
Flow chambers are common tools used for studying thrombus formation in vitro. However, the use of such devices is not standardised and there is a large diversity
among the flow chamber systems currently used, and also in the methods used for quantifying
the thrombus development. It was the study objective to evaluate a new method for
analysis and quantification of platelet thrombus formation that can facilitate comparison
of results between research groups. Whole blood was drawn over a collagen patch in
commercial Ibid or in-house constructed PDMS flow chambers. Five percent of the platelets
were fluorescently labelled and z-stack time-lapse images were captured during thrombus
formation. Images were processed in a Python script in which the number of platelets
and their respective x-, yand z-positions were obtained. For comparison with existing
methods the platelets were also labelled and quantified using fluorescence intensity
and thrombus volume estimations by confocal microscopy. The presented method was found
less sensitive to microscope and image adjustments and provides more details on thrombus
development dynamics than the methods for measuring fluorescence intensity and thrombus
volume estimation. The platelet count method produced comparable results with commercial
and PDMS flow chambers, and could also obtain information regarding the stability
of each detected platelet in the thrombus. In conclusion, quantification of thrombus
formation by platelet count is a sensitive and robust method that enables measurement
of platelet accumulation and platelet stability in an absolute scale that could be
used for comparisons between research groups.
Keywords
Platelet aggregation - microfluidics - thrombosis - fluorescence microscopy - computer-assisted
image processing
