Geometric complexity identifies platelet activation in familial hypercholesterolemic patients

Abstract

Familial hypercholesterolemia (FH), a genetic disease, is associated with a severe incidence of athero-thrombotic events, related, also, to platelet hyperreactivity. A plethora of methods have been proposed to identify those activated circulating platelets, none of these has proved really effective. We need efficient methods to identify the circulating platelet status in order to follow the patients after therapeutic procedures. We propose the use of computerized fractal analysis for an objective characterization of the complexity of circulating platelet shapes observed by means of transmission electron microscopy in order to characterize the in vivo hyperactivated platelets of familial hypercholesterolemic patients, distinguishing them from the in vivo resting platelets of healthy individuals. Platelet boundaries were extracted by means of automatically image analysis. Geometric complexity (fractal dimension, D) by box counting was automatically calculated. The platelet boundary observed by electron microscopy is fractal, the shape of the circulating platelets is more complex in FH (n = 6) than healthy subjects (n = 5, P < 0.01), with 100% correct classification in selected individuals. In vitro activated platelets from healthy subjects show an analogous increase of D. The observed high D in the platelet boundary in FH originates from the in vivo platelet activation. Computerized fractal analysis of platelet shape observed by transmission electron microscopy can provide accurate, quantitative data to study platelet activation in familial hypercholesterolemia and after administration of drugs or other therapeutic procedures.

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

“…We have shown that parameters characterising the platelet shape allow for the sensitive characterisation of different activation states of platelets. This research is supported by the findings of Bianciardi and coworkers who have shown that platelets activated with thrombin [15] as well as platelets from diabetic patients [14] and from patients with primary hypercholesterolemia [13] showed significant differences in the fractal dimension of their outlines on electron microscopic images. Furthermore Canault et al [28] found on fluorescence microscopic images that platelets from RASGRP2 knockout mice show a significantly reduced formation of filopodia during spreading on fibrinogen as compared to healthy subjects.…”

“…PSC can be quantitatively described by applying different geometrical descriptors to the platelet outline (PO) [12]. It has been found that the PO of platelets imaged by high-resolution darkfield microscopy [12] and electron microscopy [13][14][15][16] shows fractal behaviour and therefore can be described using the language of fractal geometry. Fractal geometry has gained increasing interest in life sciences as it enables a simple numerical description of complex and irregular forms [18][19][20][21][22][23][24][25].…”